CN108949772A - For generating the modification polynucleotides of biological agent relevant to human diseases and protein - Google Patents

For generating the modification polynucleotides of biological agent relevant to human diseases and protein Download PDF

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Publication number
CN108949772A
CN108949772A CN201810507242.8A CN201810507242A CN108949772A CN 108949772 A CN108949772 A CN 108949772A CN 201810507242 A CN201810507242 A CN 201810507242A CN 108949772 A CN108949772 A CN 108949772A
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China
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polynucleotides
optionally replaced
mmrna
protein
optionally
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斯蒂芬·邦塞尔
提尔塔·柴可拉博提
安东宁·德富热罗勒
S·M·伊巴希尔
马蒂亚斯·约翰
阿坦恩·洛依
苏珊·沃利斯基
K·M·伍德
保罗·哈塔拉
杰森·P·斯洛姆
凯内齐·伊杰贝
杰夫·L·埃尔斯沃思
贾斯汀·基尔德
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Modern Tex Co
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Modern Tex Co
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Abstract

The present invention relates to the compositions and method for polynucleotides, the preparation of primary transcript and mmRNA molecule, manufacture and therapeutical uses.

Description

For generating the modification multicore of biological agent relevant to human diseases and protein Thuja acid
The application is application No. is 201380028773.4, and the applying date is on March 9th, 2013, entitled " for producing The divisional application of the modification polynucleotides of raw relevant to human diseases biological agent and protein ".
Sequence table reference
The application submits together in electronic format together with sequence table.The sequence table file of entitled M300PCTSQLST.txt It is created on March 9th, 2013 and size is 49,417,315 bytes.Information in the electronic format of the sequence table is to quote Mode be integrally incorporated herein.
Cross reference to related applications
This application claims the priority of the following terms: on August 10th, the 2012 entitled Modified submitted Polynucleotides for the Production of Oncology-Related Proteins and Peptides The entitled Terminally Optimized that on December 14th, 61/681,742,2012 submits of U.S. Provisional Patent Application No. The U.S. Provisional Patent Application No. of Modified RNAs on December 14th, 61/737,224,2012 is submitted entitled The international application no PCT/ of Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions The entitled Modified Polynucleotides for that US2012/069610, on April 2nd, 2012 submit 61/618,862,2012 year August 10 of U.S. Provisional Patent Application No. of Production of Biologics is submitted entitled The U.S. Provisional Patent Application No. of Modified Polynucleotides for the Production of Biologics 61/681,645, the entitled Modified Polynucleotides for the submitted on December 14th, 2012 The U.S. Provisional Patent Application No. title submitted on April 2nd, 61/737,130,2012 of Production of Biologics For the U.S. Provisional Patent Application of Modified Polynucleotides for the Production of Antibodies The entitled Modified Polynucleotides for that number 61/618,866,2012 year August 10 is submitted The U.S. Provisional Patent Application No. name submitted on December 14th, 61/681,647,2012 of Production of Antibodies The referred to as US provisional patent Shen of Modified Polynucleotides for the Production of Antibodies The entitled Modified Polynucleotides for that please be submitted number on April 2nd, 61/737,134,2012 61/618,868,2012 year August 10 of U.S. Provisional Patent Application No. of Production of Vaccines is submitted entitled The U.S. Provisional Patent Application No. of Modified Polynucleotides for the Production of Vaccines 61/681,648, the entitled Modified Polynucleotides for the submitted on December 14th, 2012 The U.S. Provisional Patent Application No. of Production of Vaccines on April 2nd, 61/737,135,2012 is submitted entitled Modified Polynucleotides for the Production of Therapeutic Proteins and On August 10th, 61/618,870, the 2012 entitled Modified submitted of U.S. Provisional Patent Application No. of Peptides The U.S. of Polynucleotides for the Production of Therapeutic Proteins and Peptides The entitled Modified Polynucleotides that Provisional Patent Application No. is submitted on December 14th, 61/681,649,2012 The U.S. Provisional Patent Application No. of for the Production of Therapeutic Proteins and Peptides 61/737,139, the entitled Modified Polynucleotides for the submitted on April 2nd, 2012 U.S. Provisional Patent Application No. on August 10th, 61/618,873,2012 of Production of Secreted Proteins mention The entitled Modified Polynucleotides for the Production of Secreted Proteins's handed over The entitled Modified that U.S. Provisional Patent Application No. is submitted on December 14th, 61/681,650,2012 The U.S. Provisional Patent Application No. of Polynucleotides for the Production of Secreted Proteins 61/737,147, the entitled Modified Polynucleotides for the submitted on April 2nd, 2012 U.S. Provisional Patent Application No. 61/618,878,2012 year 8 of Production of Plasma Membrane Proteins The entitled Modified Polynucleotides for the Production of Plasma that the moon 10 is submitted The U.S. Provisional Patent Application No. of Membrane Proteins on December 14th, 61/681,654,2012 is submitted entitled The U.S. of Modified Polynucleotides for the Production of Plasma Membrane Proteins The entitled Modifed Polynucleotides for that Provisional Patent Application No. is submitted on April 2nd, 61/737,152,2012 The U.S. Provisional Patent Application No. of the Production of Cytoplasmic and Cytoskeletal Proteins 61/618,885, the entitled Modified Polynucleotides for the submitted on August 10th, 2012 The U.S. Provisional Patent Application No. 61/ of Production of Cytoplasmic and Cytoskeletal Proteins 681,658, the entitled Modified Polynucleotides for the Production submitted on December 14th, 2012 U.S. Provisional Patent Application No. 61/737,155,2012 year of of Cytoplasmic and Cytoskeletal Proteins The entitled Modified Polynucleotides for the Production of submitted April 2 U.S. Provisional Patent Application No. in July, 61/618,896,2012 of Intracellular Membrane Bound Proteins The entitled Modified Polynucleotides for the Production of Intracellular submitted for 5th 61/668,157,2012 year August 10 of U.S. Provisional Patent Application No. of Membrane Bound Proteins is submitted entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound The entitled Modified that the U.S. Provisional Patent Application No. of Proteins is submitted on December 14th, 61/681,661,2012 Polynucleotides for the Production of Intracellular Membrane Bound Proteins's The entitled Modified Polynucleotides that U.S. Provisional Patent Application No. is submitted on April 2nd, 61/737,160,2012 U.S. Provisional Patent Application No. 61/618,911,2012 year 8 of for the Production of Nuclear Proteins The entitled Modified Polynucleotides for the Production of Nuclear that the moon 10 is submitted The entitled Modified that the U.S. Provisional Patent Application No. of Proteins is submitted on December 14th, 61/681,667,2012 The U.S. Provisional Patent Application No. 61/ of Polynucleotides for the Production of Nuclear Proteins 737,168, the entitled Modified Polynucleotides for the Production submitted on April 2nd, 2012 On August 10th, 61/618,922, the 2012 entitled Modified submitted of U.S. Provisional Patent Application No. of of Proteins The U.S. Provisional Patent Application No. 61/681,675 of Polynucleotides for the Production of Proteins, The entitled Modified Polynucleotides for the Production of submitted on December 14th, 2012 The entitled Modified that the U.S. Provisional Patent Application No. of Proteins is submitted on April 2nd, 61/737,174,2012 Polynucleotides for the Production of Proteins Associated with Human Disease 61/618,935,2012 year August 10 of U.S. Provisional Patent Application No. submit entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease The entitled Modified that on December 14th, 61/681,687,2012 submits of U.S. Provisional Patent Application No. Polynucleotides for the Production of Proteins Associated with Human Disease The entitled Modified that on April 2nd, 61/737,184,2012 submits of U.S. Provisional Patent Application No. Polynucleotides for the Production of Proteins Associated with Human Disease On August 10th, 61/618,945, the 2012 entitled Modified submitted of U.S. Provisional Patent Application No. Polynucleotides for the Production of Proteins Associated with Human Disease The entitled Modified that on December 14th, 61/681,696,2012 submits of U.S. Provisional Patent Application No. Polynucleotides for the Production of Proteins Associated with Human Disease The entitled Modified that on April 2nd, 61/737,191,2012 submits of U.S. Provisional Patent Application No. Polynucleotides for the Production of Proteins Associated with Human Disease On August 10th, 61/618,953, the 2012 entitled Modified submitted of U.S. Provisional Patent Application No. Polynucleotides for the Production of Proteins Associated with Human Disease The entitled Modified that on December 14th, 61/681,704,2012 submits of U.S. Provisional Patent Application No. Polynucleotides for the Production of Proteins Associated with Human Disease The entitled Dosing Methods for that on April 2nd, 61/737,203,2012 submits of U.S. Provisional Patent Application No. The entitled Dosing that the U.S. Provisional Patent Application No. of Modified mRNA is submitted on May 17th, 61/618,961,2012 The U.S. Provisional Patent Application No. 61/648,286 of Methods for Modified mRNA, the respective content of the application with The mode of reference is integrally incorporated herein.
The application further relates to the entitled Modified Nucleosides submitted on October 3rd, 2012, The international publication number PCT/US2012/58519 of Nucleotides, and Nucleic Acids, and Uses Thereof and The entitled Modified Nucleoside that on December 14th, 2012 submits, Nucleotide, and Nucleic Acid The international publication number PCT/US2012/69610 of Compositions.
The application further relates to copending application, and copending application each comfortable 9 days March in 2013 is same with the application When submit and have attorney docket M301.20 (PCT/US13/XXXXX), entitled Modified Polynucleotides；Attorney docket M304.20 (PCT/US13/XXXXX), entitled Modified Polynucleotides for the Production of Secreted Proteins；Attorney docket M305.20 (PCT/US13/XXXXX), entitled Modified Polynucleotides for the Production of Membrane Proteins；Attorney docket M306.20 (PCT/US13/XXXXX), entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins； Attorney docket M308.20 (PCT/US13/XXXXX), entitled Modified Polynucleotides for the Production of Nuclear Proteins；Attorney docket M309.20 (PCT/US13/XXXXX), it is entitled Modified Polynucleotides for the Production of Proteins；Attorney docket M310.20 (PCT/US13/XXXXX), entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease；Attorney docket MNC1.20 (PCT/US13/XXXXX), title For Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides；And attorney docket MNC2.20 (PCT/US13/XXXXX), entitled Modified Polynucleotides For the Production of Oncology-Related Proteins and Peptides, the application are respective interior Appearance is incorporated herein in its entirety by reference.
Invention field
The present invention relates to for designing, preparing, manufacturing and/or preparing polynucleotides, primary construct and modification mRNA Composition, method, process, kit and the device of molecule (mmRNA).
Background of invention
There are various problems for the existing method opinion of influence protein expression.For example, the DNA introduced can be whole at some frequencies It closes in host cell gene group DNA, so as to cause the change and/or damage of host cell gene group DNA.Alternatively, being introduced into Heterologous DNA (DNA) in cell can by progeny cell (no matter whether allogeneic dna sequence DNA is integrated into chromosome) or by Offspring inherits.Moreover, it is assumed that appropriate delivering and not damaged or be integrated into host genome, then in the albumen that coding is made Multiple steps must occur before matter.Once portion in the cell, DNA must be transported in nucleus, its quilt in nucleus It is transcribed into RNA.Then the RNA transcribed from DNA has to enter into cytoplasm, it is translated into protein in cytoplasm.From application Multiple procedure of processings of DNA to protein delay time, but also each step are not only formed before generating functional protein Suddenly it also means error and causes the chance of damage to cell.Furthermore it is known that being difficult to obtain DNA expression in cell, because of DNA It frequently enters cell but does not express or do not expressed with reasonable rate or concentration.When DNA being introduced into primary cell or modification is thin When in born of the same parents system, this is particularly problematic.
At the twentieth century initial stage nineties, Bloom and colleague pass through the pitressin (vasopressin) that will be transcribed in vitro MRNA is injected to rat (the Science 255:996-998 that vasopressin deficiency has successfully been saved in hypothalamus；1992).So And low translation skill and the immunogenicity of molecule hinder development of the mRNA as therapeutic agent, and always from the work after it The alternate application that can use these defects on the contrary is concentrated on, i.e., is immunized with the mRNA of encoding cancer antigen.
Other people have studied using mRNA the certain chemical modifications for delivering target polypeptides and showing mRNA molecule, especially It is that pseudouridine and 5- methyl-cytosine reduce immunostimulation.
These researchs disclose in the following: such as Ribostem Limited submits (being now abandoned) on July 9th, 2003 That submits be published as the PCT Application No. of WO2005005622 UK Patent Application serial number on July 9th, 0316089.2,2004 PCT/GB2004/002981, on June 8th, 2006, that submits be published as the United States Patent (USP) Shen of US20060247195 (being now abandoned) Please thenational phase enter serial number 10/563,897 and that submits on July 9th, 2004 be published as EP1646714 (having recalled) European patent application thenational phase enter in serial number EP2004743322；Novozymes, Inc. were mentioned on December 19th, 2007 What the PCT Application No. PCT/US2007/88060 for being published as WO2008140615 of friendship, on July 2nd, 2009 submitted is published as The U.S. Patent application thenational phase of US20100028943 enters serial number 12/520,072 and submits on July 7th, 2009 The European patent application thenational phase for being published as EP2104739 enters in serial number EP2007874376；University of Rochester (University of Rochester) that submits be published as the PCT Application No. of WO2007064952 on December 4th, 2006 The U.S. patent application serial number 11/ for being published as US20070141030 that on December 1st, PCT/US2006/46120 and 2006 submits In 606,995；BioNTech AG submits the European patent application serial number of (being now abandoned) on December 14th, 2007 The PCT Application No. PCT/EP2008/ for being published as WO2009077134 that EP2007024312, on December 12nd, 2008 submit 01059, the European patent application thenational phase for being published as EP2240572 that on June 2nd, 2010 submits enters serial number The U.S. Patent application thenational phase for being published as US20110065103 that EP2008861423, on November 24th, 2010 submit into Enter on September 28th, 12/, 735,060, the 2005 German patent application serial number DE 10 2,005 046 490,2006 submitted of serial number Submit on September 28, the PCT application PCT/EP2006/0448 for being published as WO2007036366, on March 21st, 2012 announce What thenational phase European patent EP 1934345 and 2009 on August was submitted for 14 is published as the 20100129877 thenational phase U.S. Patent application serial number 11/992,638；Co., Ltd of immunological diseases research institute (Immune Disease Institute Inc.) In the U.S. patent application serial number 13/088,009 and 2011 year 4 for being published as US20120046346 that on April 15th, 2011 submits The PCT application PCT/US2011/32679 for being published as WO20110130624 that the moon is submitted on the 15th；Shire Human Genetic The U.S. patent application serial number 12/ for being published as US20110244026 that Therapeutics was submitted on November 20th, 2010 In 957,340；The PCT application PCT/ for being published as WO1999014346 that Sequitur Inc. was submitted on September 18th, 1998 In US1998/019492；What The Scripps Research Institute was submitted on 2 24th, 2010 is published as What the PCT Application No. PCT/US2010/00567 of WO2010098861 and on November 3rd, 2011 submitted is published as The U.S. Patent application thenational phase of US20120053333 enters in serial number 13/203,229；Ludwig-Maximillians The PCT Application No. PCT/EP2010/004681 for being published as WO2011012316 that University was submitted on July 30th, 2010 In；Cellscript Inc. is submitted on June 30th, 2008 and the U.S. Patent number 8,039 of authorization on October 18th, 2011, 214, U.S. patent application serial number on December 7th, 2010, that submits was published as 12/962,498,2010 year of US20110143436 What what what December 7 submitted be published as US20110143397 submitted on September 20th, 12/962,468,2011 is published as What 13/237,451 and the PCT application of US20120009649 was submitted on December 7th, 2010 is published as WO2011071931's What on December 7th, PCT/US2010/59305 and 2010 submitted is published as in the PCT/US2010/59317 of WO2011071936； What The Trustees of the University of Pennsylvania was submitted on August 21st, 2006 is published as What the PCT Application No. PCT/US2006/32372 of WO2007024708 and on March 27th, 2009 submitted is published as The U.S. Patent application thenational phase of US20090286852 enters in serial number 11/990,646；Curevac GMBH is in German special The DE10 2001 that the DE10 that sharp application serial is submitted on June 5th, 2001 is submitted on December 19th, 2,001 027 283.9,2001 DE 20 2,006 051 516 (all having abandoned), the european patent number 2005 that on October 31st, 062 480.8 and 2006 submits The EP1458410 of the authorization in 2, EP1392341 and 2008 on January of authorization on March 30, in, PCT Application No. are mentioned on June 5th, 2002 What the PCT/EP2002/06180 for being published as WO2002098443 of friendship, on December 19th, 2002 submitted is published as The PCT/EP2002/14577 of WO2003051401, on December 31st, 2007, that submits be published as the PCT/ of WO2008052770 The PCT/EP2008/03033 for being published as WO2009127230 that EP2007/09469, on April 16th, 2008 submit, 2005 years 5 What the PCT/EP2006/004784 for being published as WO2006122828 that submits for 19th of the moon, on January 9th, 2007 submitted is published as What the PCT/EP2008/00081 and U.S. patent application serial number of WO2008083949 was submitted on December 5th, 2003 is published as What on June 18th, 10/729,830,2004 of US20050032730 submitted be published as US20050059624 10/870,110, What on October 27th, 11/914,945,2009 that on July 7th, 2008 submitted be published as US20080267873 submitted is published as On January 4th, 12/446,912,2010 of US2010047261 (being now abandoned), that submits be published as the 12/ of US20100189729 522,214, on May 26th, 12/787,566,2010 that on May 26th, 2010 submitted be published as US20110077287 submits That submits be published as the 13/ of US20110269950 on the July 18th, 12/787,755,2011 for being published as US20100239608 On May 12nd, 185,119 and 2011, that submits be published as the 13/106 of US20110311472, in 548, the patent all with The mode of reference is integrally incorporated herein.
Despite the presence of be restricted to include the chemical modification of pseudouridine and 5- methyl-cytosine selection these report, But there remains a need to solve effective to adjust and coding polypeptide or the nucleic acid of its segment around what is translated into the cell in this field The therapeutic modality of a large amount of obstacles of processing.
For this purpose, inventors have established that it is more than only to escape, avoid or weaken that certain modification mRNA sequences, which have as benefit, The potentiality of the therapeutic agent of immune response.It is this kind of to study the copending application international application August 5 in 2011 for being described in detail in announcement Day submit 3, PCT/US2011/046861 and 2011 on October the PCT/US2011/054636 submitted, on October 3rd, 2011 In the international application no PCT/US2011/054617 of submission, the content of the application is incorporated herein in its entirety by reference.
The present invention solves this needs based on the compound of nucleic acid or polynucleotides by providing, and the polynucleotides are compiled Code target polypeptides (for example, modification mRNA or mmRNA) and have the structure for avoiding one or more problems in this field and/ Or chemical feature, such as preparation and delivering suitable for optimizing the therapeutic agent based on nucleic acid and keep structure and function complete simultaneously Property, overcome expression threshold value, improve expression rate, half-life period and/or protein concentration, optimization protein positioning and avoided The feature of evil biological response such as immune response and/or degradation pathway.
Summary of the invention
Be described herein for design, prepare, manufacture and/or prepare the modification composition of mRNA (mmRNA) molecule, method, Process, kit and device.
The details of various embodiments of the present invention illustrates in the following description.Other features of the invention, purpose and excellent Point will be clear from description of the invention and attached drawing and claim.
Brief description
Foregoing end other objects, feature and advantage will be from specific embodiments of the present invention as illustrated in the drawings Be described below clearly, in the accompanying drawings same reference numeral through different views refer to same section.Attached drawing is not necessarily to scale It draws, but focuses on and show in the principle of various embodiments of the present invention.
Fig. 1 is the schematic diagram of primary construct of the invention.
Fig. 2 shows be suitable for the invention lipid structure in the prior art.98N12-5 (TETA5-LAP), DLin- are shown DMA, DLin-K-DMA (2,2- bis- Asia oil base -4- dimethylaminomethyls-[1,3]-dioxolane), DLin-KC2- The structure of DMA, DLin-MC3-DMA and C12-200.
Fig. 3 is the representative liposome suitable for the IVT reaction instructed herein.Liposome includes to be designed by the present inventor Insert 64818.
Fig. 4 is the gel profiles for the modification mRNA being packaged in PLGA microballoon.
Fig. 5 is the histogram that IX factor protein matter generates PLGA preparation IX factor modification mRNA.
Fig. 6 is that the vegf protein matter shown after a series of modification mRNA of dosage is transfected in Human keratinocytes produces The histogram of amount.Fig. 6 A shows the protein output after the modification mRNA transfection comprising natural nucleus glycoside triphosphoric acid (NTP).Figure The protein that 6B shows after the modification mRNA transfection modified completely with pseudouridine (vacation-U) and 5-methylcytosine (5mC) produces Amount.Fig. 6 C is shown in the modification modified completely with N1- methyl-pseudouridine (N1- methyl-vacation-U) and 5-methylcytosine (5mC) Protein output after mRNA transfection.
Fig. 7 is the histogram of the vegf protein matter yield in HEK293 cell.
Fig. 8 is that the vegf expression that VEGF is modified after mRNA transfection in peripheral blood mononuclear cells (PBMC) and IFN-α lure The histogram led.Fig. 8 A shows vegf expression.Fig. 8 B shows IFN-α induction.
Fig. 9 is the histogram of the vegf protein matter yield from VEGF modification mRNA in Hela cell.
Figure 10 is the straight of the vegf protein matter yield from compound (lipoplexed) the VEGF modification mRNA of lipid in mouse Fang Tu.
Figure 11 is the histogram of the G-CSF protein output from G-CSF modification mRNA in Hela cell.
Figure 12 is the histogram of the G-CSF protein output from the compound G-CSF modification mRNA of lipid in mouse.
Figure 13 is the histogram of the IX factor protein matter yield from IX factor modification mRNA in Hela cell supernatant.
Figure 14 is in Hela cell from APOA1 wild type modification mRNA, APOA1 Milano modification mRNA or APOA1 Paris modifies the histogram of the APOA1 protein output of mRNA.
Figure 15 is the gel profiles of the APOA1 protein from APOA1 wild type modification mRNA.
Figure 16 is the gel profiles of the APOA1 protein from APOA1 Paris modification mRNA.
Figure 17 is the gel profiles of the APOA1 protein from APOA1 Milano modification mRNA.
Figure 18 is the gel profiles of fibrinogen α (FGA) protein from FGA modification mRNA.
Figure 19 is the histogram of the plasminogen protein matter yield from plasminogen modification mRNA in Hela cell supernatant Figure.
Figure 20 is the gel profiles of the plasminogen protein matter from plasminogen modification mRNA.
Figure 21 is that the gel of galactose-1-phosphate uridyl transferase (GALT) protein from GALT modification mRNA is general Condition.
Figure 22 is the gel profiles of argininosuccinase (ASL) protein from ASL modification mRNA.
Figure 23 is the gel profiles of tyrosine aminotransferase (TAT) protein from TAT modification mRNA.
Figure 24 is the gel profiles of glucan (Isosorbide-5-Nitrae-α -) branching enzyme 1 (GBE1) protein from GBE1 modification mRNA.
Figure 25 is the histogram for carrying out the factor protein output of autoprothrombin modification mRNA in Hela cell supernatant Figure.
Figure 26 is the histogram for carrying out the factor protein output of autoprothrombin modification mRNA in Hela cell supernatant Figure.
Figure 27 is the gel profiles of ceruloplasmin (CP or CLP) protein from CP modification mRNA.
Figure 28 is transforminggrowthfactor-β1 (TGF-β 1) albumen for modifying mRNA in Hela cell supernatant from TGF-β 1 The histogram of matter yield.
Figure 29 is the gel profiles of ornithine transcarbamylase (OTC) protein from OTC modification mRNA.
Figure 30 is the flow cytometry curves figure of LDL receptor (LDLR) modification mRNA.
Figure 31 is UDP glucuronyl transferase 1 family polypeptides A1 (UGT1A1) egg from UGT1A1 modification mRNA The gel profiles of white matter.
Figure 32 is the histogram of the XI factor protein matter yield in HEK293 cell.
Figure 33 is the gel profiles of the Aquaporin-5 protein from Aquaporin-5 modification mRNA.
Figure 34 is the histogram of the VII factor protein matter yield from VII factor modification mRNA in Hela cell.
Figure 35 is the histogram of the insulin glargine protein output from insulin glargine modification mRNA in Hela cell.
Figure 36 is the histogram of the tissue factor protein matter yield from tissue factor modification mRNA in Hela cell.
Figure 37 is the histogram of the XI factor protein matter yield from XI factor modification mRNA in Hela cell.
Figure 38 is the histogram of the XI factor protein matter yield from XI factor modification mRNA in Hela cell supernatant.
Figure 39 is the histogram of the insulin aspart protein output from insulin aspart modification mRNA in Hela cell.
Figure 40 is the histogram of the insulin lispro protein output from insulin lispro modification mRNA in Hela cell.
Figure 41 is to rely the paddy of insulin modification mRNA to rely the histogram of insulin protein yield from paddy in HeLa cell.
Figure 42 is the histogram of the human growth hormone (HGH) protein output from human growth hormone (HGH) modification mRNA in HeLa cell.
Figure 43 is the gel profiles of oncoprotein 53 (p53) protein from p53 modification mRNA.Figure 43 A shows p53's It is expected that size.Figure 43 B shows the expection size of p53.
Figure 44 is the gel profiles of tuftelin (tuftelin) (TUFT1) protein from TUFT1 modification mRNA.
Figure 45 is the gel profiles of galactokinase 1 (GALK1) protein from GALK1 modification mRNA.Figure 45 A is shown The expection size of GALK1.Figure 45 B shows the expection size of GALK1.
Figure 46 is the gel profiles of alexin β 103A (DEFB103A) protein from DEFB103A modification mRNA.
Figure 47 is the flow cytometry curves figure of LDLR modification mRNA.
Figure 48 is the histogram of Hela expression of vascular endothelial growth factor.
Figure 49 is the histogram of the cell viability of the Hela cell transfected with vascular endothelial growth factor mRNA.
Figure 50 is the histogram of insulin aspart protein expression.
Figure 51 is the histogram of insulin glargine protein expression.
Figure 52 is the histogram that paddy relies insulin protein expression.
Figure 53 is the histogram of interleukin-17 (IL-7) protein expression.
Figure 54 is the histogram of hematopoietin (EPO) protein expression.
Figure 55 is the gel profiles of the lysosomal acid lipase protein from lysosomal acid lipase modification mRNA.
Figure 56 is the gel profiles of the glucocerebroside zymoprotein from glucocerebroside enzyme modification mRNA.
Figure 57 is the iduronic acid 2- sulfatase protein matter from iduronic acid 2- sulfatase modification mRNA Gel profiles.
Figure 58 is the gel profiles of the luciferase protein matter from luciferase modification mRNA.
Figure 59 is the histogram of the IgG concentration after the Trastuzumab modification mRNA that application is prepared in mammal.
Figure 60 is the histogram of the IgG concentration (in terms of ng/ml) after with Trastuzumab modification mRNA transfection.
Figure 61 is the gel profiles of the Trastuzumab protein from Trastuzumab modification mRNA.
Figure 62 is the histogram of glucocerebrosidase enzymatic activity.
Figure 63 is the histogram of lysosomal acid lipase enzymatic activity.
Figure 64 is the histogram of VIII factor protein matter expression.
Figure 65 is the histogram of VIII factor Chomogenic activity.
Figure 66 is the chart of LDLR expression.Figure 66 A is shown compared with adding LDLR mRNA, the ldl receptor expression of cell. Figure 66 B shows the ldl receptor expression of Transfected cells.Figure 66 C is shownThe saturation degree of the LRL of label.Figure 66 D The binding affinity of BODIPY-LDL and cell is shown.
Figure 67 is the chart for showing the positive cell percentage for UGT1A1 expression.
Figure 68 is the chart for showing UGT1A1 protein accumulation.
Figure 69 is the gel profiles of UGT1A1 protein and OTC from UGT1A1 or OTC modification mRNA.
Figure 70 is the flow cytometry curves figure with the PAh or UGT1A1 HEK293 cell transfected.
Figure 71 is the gel profiles of the UGT1A1 protein from UGT1A1 modification mRNA.
Figure 72 is the gel profiles with the microsome extract of the mouse of the LNP processing containing UGT1A1.
It is described in detail
It, can be by nucleic acid (for example, ribonucleic acid in acology, diagnostics, reagent field and for bioassay (RNA)) be delivered to cell interior (no matter external, internal, in situ or in vitro) for example so as to cause nucleic acid it is intracellular translation and The generation of encoded target polypeptides is of great significance.The particularly importantly delivering and function of nonconformity polynucleotides.
It is described herein and encodes the polynucleotides of one or more target polypeptides for designing, preparing, manufacture and/or prepare Composition (including pharmaceutical composition) and method.It also provides for selecting, designing and/or using coding target described herein System, process, device and the kit of the polynucleotides of polypeptide.
According to the present invention, these polynucleotides are preferably modified to avoid other peptide coding molecules of this field Defect.Therefore, these polynucleotides, which are referred to as, modifies mRNA or mmRNA.
The present inventor explored modification polynucleotides antibody, virus, veterinary applications field and it is a variety of in vivo Purposes and these researchs in environment are disclosed in, for example, co-pending and jointly owned U.S. Provisional Patent Application serial number The vivo applications of mmRNA are instructed in 61/470,451 submitted on March 31st, 2011；61/ submitted on April 26th, 2011 517,784, instruct the nucleic acid for generating the engineering of antibody polypeptides；61/519,158 submitted on May 17th, 2011, Instruct the veterinary applications of mmRNA technology；September in 2011 submit within 12nd 61/533,537, introduction mmRNA technology resist it is micro- Biologic applications；September in 2011 submit within 12nd 61/533,554, instruct mmRNA technology virus applications；October 3 in 2011 61/542,533 submitted day, is instructed for the various chemical modifications used in mmRNA technology；On December 14th, 2011 mentions 61/570,690 handed over, introduction is for making or using movable fixture used in mmRNA technology；December 14 in 2011 Purposes of the mmRNA in acute care situation is instructed in 61/570,708 submitted day；61/ submitted on December 16th, 2011 576,651, instruct the end modified construction of mmRNA；61/576,705 submitted on December 16th, 2011, introduction use The delivering method of the liposome of mmRNA；61/578,271 submitted on December 21st, 2011, introduction are increased using mmRNA The method of the viability of organ or tissue；Codocyte penetrating peptide is instructed in 61/581,322 submitted on December 29th, 2011 MmRNA；61/581,352 submitted on December 29th, 2011, introduction in cytotoxic nucleoside be incorporated to and 2012 1 61/631,729 submitted for 10th moon, introduction is used for the method across blood-brain barrier using mmRNA；The patent application all with The mode of reference is integrally incorporated herein.
Polynucleotides, primary construct and/or the mmRNA of encoding target polypeptide, the multicore glycosides are partly provided herein Acid, primary construct and/or mmRNA have been designed to improve one or more in following: stability in the tissue and/ Or clearance rate, receptor intake and/or dynamics, composition cell pathway, with the engagement of machine translator, mRNA half-life period, turn over Translate efficiency, immune evasion, protein generate ability, secernment efficiency (where applicable), circulation accessibility, protein half life and/ Or cell state, function and/or active adjusting.
I. composition of the invention (mmRNA)
The present invention provides the nucleic acid molecules for encoding one or more target polypeptides, specifically polynucleotides, primary building Body and/or mmRNA.Term " nucleic acid " includes any compound and/or substance comprising nucleotide polymer in its broad sense.This A little polymer are commonly referred to as polynucleotides.Exemplary nucleic acid of the invention or polynucleotides include but is not limited to ribonucleic acid (RNA), DNA (DNA), threose nucleic acid (TNA), ethylene glycol nucleic acid (GNA), peptide nucleic acid (PNA), lock nucleic acid (LNA, Including with β-D-ribose configuration LNA, with α-L- ribo configuration α-LNA (diastereomer of LNA), have 2 '-amino Functionalized 2 '-amino-LNA and 2 '-amino-α-LNA with 2 '-aminofunctionals) or its heterozygote.
In preferred embodiments, nucleic acid molecules are mRNA (mRNA).As used herein, term " mRNA " (mRNA) refer to encoding target polypeptide and the target that can be translated to the coding of generation in vitro, in vivo, in situ or in vitro is more Any polynucleotides of peptide.
Traditionally, the basic component of mRNA molecule includes at least code area, 5 ' UTR, 3 ' UTR, 5 ' caps and poly-A tail. Based on this wild type modular structure, the present invention extends traditional mRNA points by providing polynucleotides or primary RNA construct Functional range of son, the polynucleotides or primary RNA construct maintenance module tissue but include one or more structures And/or chemical modification or change, the modifications and changes assign the polynucleotides useful characteristic, in some embodiments The characteristic includes being introduced into the shortage of the substantive induction of innate immune response of the cell of polynucleotides.In this way, this hair Bright modification mRNA molecule is referred to as " mmRNA ".As used herein, " structure " feature or modification are that two of them or more connect Connect nucleotide in polynucleotides, primary construct or mmRNA insertion, missing, duplication, reversion or randomization and without to the core The feature or modification of the significant chemical modification of thuja acid itself.Because realizing structural modification just and must making chemical bond rupture and again It is formed, so structural modification has chemical property and is therefore chemical modification.However, structural modification will generate different nucleotide Sequence.For example, polynucleotides " ATCG " can chemical modification at " AT-5meC-G ".Same polynucleotides can be repaired from " ATCG " structure Adorn into " ATCCCG ".Here, dinucleotides " CC " has been inserted into, to generate the structural modification to polynucleotides.
MmRNA construction
MmRNA of the invention in its function and/or structural design features be different from wild type mRNA, the function and/ Or structure feature such as existing issue of this paper proof for overcoming effective polypeptide using the therapeutic agent based on nucleic acid to generate.
Fig. 1 shows representative polynucleotides primary construct 100 of the invention.As used herein, term " primary building Body " or " primary mRNA construct " refer to the one or more target polypeptides of coding and retain enough structures and/or chemical feature To allow to translate the polynucleotides transcript of the target polypeptides wherein encoded.Primary construct can be multicore glycosides of the invention Acid.When in structure or chemically modifying, primary construct is referred to alternatively as mmRNA.
Referring to Fig. 1, first area 102 of the primary construct 100 herein comprising connection nucleotide, firstth area 102 is by the One flanking region 104 and the second flanking region 106 flank.As used herein, " the firstth area " is referred to alternatively as " code area " or " compiles The area of code ... " is or simply " the firstth area ".This firstth area may include but be not limited to the target polypeptides of coding.Target polypeptides It can include the one or more signal sequences encoded by signal sequence area 103 in its 5 ' end.Flanking region 104 may include connection core The area comprising one or more complete or imperfect 5 ' UTR sequences of thuja acid.Flanking region 104 also may include 5 ' end caps 108.The Two flanking regions 106 may include the area comprising one or more complete or imperfect 3 ' UTR for connecting nucleotide.Flanking region 106 is also It may include 3 ' tailing sequences 110.
First operating space 105 makes the 5 ' of the first area 102 to hold and the first flanking region 104 bridge joint.Traditionally this operating space packet Containing initiation codon.Operating space may include any translation initiation sequence or signal containing initiation codon.
Second operating space 107 makes the 3 ' of the first area 102 to hold and the second flanking region 106 bridge joint.Traditionally this operating space packet Containing terminator codon.Operating space may include any translation initiation sequence or signal containing terminator codon.According to this hair It is bright, multiple continuous terminator codons also can be used.
In general, the shortest length in the firstth area of primary construct of the invention can be enough to encode dipeptides, tripeptides, Tetrapeptide, pentapeptide, hexapeptide, heptapeptide, the length of the nucleic acid sequence of octapeptide, nonapeptide or decapeptide.In another embodiment, the length Degree can be enough to encode 2 to 30 amino acid, such as 5 to 30,10 to 30,2 to 25,5 to 25,10 to 25 or 10 to 20 amino acid Peptide.The length can be enough to encode the peptide of at least 11,12,13,14,15,17,20,25 or 30 amino acid, or be no more than 40 A amino acid, such as the peptide no more than 35,30,25,20,17,15,14,13,12,11 or 10 amino acid.Polynucleotide sequence The example of the dipeptides of codified includes but is not limited to carnosine and anserine (anserine).
In general, the length for encoding the firstth area of target polypeptides of the invention is greater than about 30 nucleotide (for example, at least or greatly In about 35,40,45,50,55,60,70,80,90,100,120,140,160,180,200,250,300,350,400,450, 500、600、700、800、900、1,000、1,100、1,200、1,300、1,400、1,500、1,600、1,700、1,800、1, 900,2,000,2,500 and 3,000,4,000,5,000,6,000,7,000,8,000,9,000,10,000,20,000,30, 000,40,000,50,000,60,000,70,000,80,000,90,000 or up to and including 100,000 nucleotide).Such as Used herein, " the firstth area " is referred to alternatively as " code area " or " coding ... area " or simply " the firstth area ".
In some embodiments, polynucleotides, primary construct or mmRNA include about 30 to about 100,000 nucleosides Acid (for example, 30 to 50,30 to 100,30 to 250,30 to 500,30 to 1,000,30 to 1,500,30 to 3,000,30 to 5, 000,30 to 7,000,30 to 10,000,30 to 25,000,30 to 50,000,30 to 70,000,100 to 250,100 to 500, 100 to 1,000,100 to 1,500,100 to 3,000,100 to 5,000,100 to 7,000,100 to 10,000,100 to 25, 000,100 to 50,000,100 to 70,000,100 to 100,000,500 to 1,000,500 to 1,500,500 to 2,000,500 To 3,000,500 to 5,000,500 to 7,000,500 to 10,000,500 to 25,000,500 to 50,000,500 to 70, 000,500 to 100,000,1,000 to 1,500,1,000 to 2,000,1,000 to 3,000,1,000 to 5,000,1,000 to 7,000,1,000 to 10,000,1,000 to 25,000,1,000 to 50,000,1,000 to 70,000,1,000 to 100,000, 1,500 to 3,000,1,500 to 5,000,1,500 to 7,000,1,500 to 10,000,1,500 to 25,000,1,500 to 50, 000,1,500 to 70,000,1,500 to 100,000,2,000 to 3,000,2,000 to 5,000,2,000 to 7,000,2,000 To 10,000,2,000 to 25,000,2,000 to 50,000,2,000 to 70,000 and 2,000 to 100,000).
According to the present invention, the length of the first flanking region and the second flanking region can be independently in 15 to 1,000 nucleotide models In enclosing (for example, be greater than 30,40,45,50,55,60,70,80,90,100,120,140,160,180,200,250,300,350, 400,450,500,600,700,800 and 900 nucleotide, or at least 30,40,45,50,55,60,70,80,90,100, 120,140,160,180,200,250,300,350,400,450,500,600,700,800,900 and 1,000 nucleotide).
According to the present invention, the length of tailing sequence can there is no in 500 nucleotide ranges (for example, at least 60, 70,80,90,120,140,160,180,200,250,300,350,400,450 or 500 nucleotide).It is in tailing area In the case where polyA tail, what the length can be combined using the unit of polyA binding protein combination or as polyA binding protein Function determines.In this embodiment, polyA tail long enough is to combine the protein-bonded at least four monomer of polyA. PolyA binding protein monomer combines the section of about 38 nucleotide.So, it has been observed that about 80 nucleotide and 160 nucleosides The polyA tail of acid is functional.
According to the present invention, capped area may include single cap or a series of nucleotide for forming cap.In this embodiment, The length in capped area can be 1 to 10, such as 2 to 9,3 to 8,4 to 7,1 to 5,5 to 10 or at least 2 or 10 or less nucleosides Acid.In some embodiments, cap is not present.
According to the present invention, the first operating space and the second operating space can be 3 to 40, such as 5 to 30,10 to 20,15 or at least 4 Or in the length range of 30 or less nucleotide, and starting and/or terminator codon, one or more letters can be additionally comprised Number sequence and/or limitation sequence.
Cyclic annular mmRNA
According to the present invention, primary construct can be made or mmRNA is cyclized or concatemerization (concatemerized), to have The molecule of translation ability come help poly- A binding protein and 5 ' hold binding proteins between interaction.The machine of cyclisation or concatemerization System can pass through at least three kinds of different approach and occur: 1) chemical, 2) enzyme and 3) ribozyme catalysis.5 ' newly formed -/3 '-are bonded It can be intramolecular or intermolecular.
In the first approach, the 5 ' ends and 3 ' ends of nucleic acid are new comprising being formed between 5 ' ends of molecule and 3 ' ends when close Covalent bonded chemically reactive group.5 ' ends may include NHS- ester reactive group and 3 ' ends may include 3 '-amino envelope The nucleotide at end, so that the 3 '-amino-terminated nucleotide synthesized on 3 ' ends of mRNA molecule in organic solvent will be undergone Nucleophillic attack on 5 '-NHS- ester moieties, to form 5 ' -/3 '-new amido bonds.
In a second approach, T4 RNA ligase can be used for the nucleic acid molecules enzyme of 5 '-phosphorylations being connected to the 3 '-of nucleic acid Hydroxyl terminal, so that it is bonded to form new di-phosphate ester.In example reaction, according to the scheme of manufacturer by 1 μ g nucleic acid molecules and 1 T4 RNA ligase (New England Biolabs, Ipswich, MA) to 10 units is incubated for 1 hour at 37 DEG C.Connection Reaction can have the separation oligonucleotides that can be reacted with the base pairing of both arranged side by side 5 '-and 3 '-areas to help enzyme to connect In the case where occur.
In third approach, 5 '-or 3 '-end coding ligase ribozyme sequences of cDNA template, so that transcribing in vitro In the process, gained nucleic acid molecules may include the active ribozyme sequence that 5 ' ends of nucleic acid molecules can be connected to 3 ' ends of nucleic acid molecules Column.Ligase ribozyme may originate from I group introne, I group introne, Hepatitis D virus, hairpin ribozymes or can pass through SELEX (the Fas lignand system evolution technology of index concentration) selection.Ribozyme connection enzyme reaction can carry out 1 at a temperature of between 0 DEG C with 37 DEG C To 24 hours.
MmRNA polymer
According to the present invention, a variety of different polynucleotides, primary construct or mmRNA may be used at 3 ' terminal modified nucleosides Acid is linked together by 3 ' ends.It is chemically conjugated to can be used for controlling the Chemical Measurement being delivered in cell.For example, can be by acetaldehyde Sour cyclophorase, i.e. isocitrate lyase and malate synthase are supplied in HepG2 cell with 1: 1 ratio to change cellular fat Acid metabolic.This ratio can be blocked by using 3 '-azidos on a polynucleotides, primary construct or mmRNA species Nucleotide and opposite polynucleotides, primary construct or mmRNA species on the nucleotide containing C5- acetenyl or alkynyl Polynucleotides, primary construct or mmRNA is connected chemically to be controlled.Terminal enzyme (DNA) (New is used according to the scheme of manufacturer England Biolabs, Ipswich, MA) modified nucleoside acid is added after transcription.After adding 3 '-modified nucleosides acid, two A polynucleotides, primary construct or mmRNA species can be combined presence or absence of copper in aqueous solution, so as to It is formed via the click chemistry mechanism as described in document new covalent bonded.
In another example, functionalized linkers can be used that more than two polynucleotides link together.Example Such as, functionalized glycan molecule can be modified by sulphation into comprising multiple chemically reactive group (SH-, NH2-、N3Deng) so as to 3 '- Analogous parts (i.e. 3 '-maleimide esters, 3 '-NHS- esters, alkynyl) reaction on functionalized mRNA molecule.The sugar modified On the number of reactive group can be controlled with stoichiometric manner, to directly control polynucleotides, the primary of conjugation The stoichiometric ratio of construct or mmRNA.
MmRNA conjugate and combination
Generated to further enhance protein, primary construct of the invention or mmRNA be designed to it is following Conjugation: other polynucleotides, dyestuff, intercalator (intercalating agent) (such as acridine), crosslinking agent (such as are mended Bone fat element (psoralene), mitomycin C), porphyrin (TPPC4, texaphyrin (texaphyrin), thiophene quinoline (Sapphyrin)), polycyclic aromatic hydrocarbons (PAH) (such as azophenlyene, dihydrophenazine), artificial endonuclease (such as EDTA), alkylating agent, phosphorus Acid esters, amino, sulfydryl, PEG (such as PEG-40K), MPEG, [MPEG]2, poly- amino, alkyl, substituted alkyl, radioactivity mark The marker of note, enzyme, haptens (such as biotin), transhipment/sorbefacient (such as aspirin, vitamin E, folic acid), Synthetic rna enzyme, protein (such as glycoprotein) or peptide (such as with molecule for the specific affinity of ligand altogether) Or antibody (such as in conjunction with designated cell type such as antibody of cancer cell, endothelial cell or osteocyte), hormone and hormone receptor, it is non- Peptide species such as lipid, agglutinin, carbohydrate, vitamin, confactor or drug.
Conjugation can produce increased stability and/or half-life period and may be particularly useful for making polynucleotides, primary building Specific site in body or mmRNA targeting cell, tissue or organ.
According to the present invention, mmRNA or primary construct can be compiled with one of following or a variety of applications together or further Yard or less one of or it is multiple: RNAi agent, siRNA, shRNA, miRNA, miRNA binding site, antisense RNA, ribozyme, catalysis Property DNA, tRNA, induce RNA, aptamer or the carrier etc. of three spiralizations.
Difunctional mmRNA
It in one embodiment of the invention, is difunctional polynucleotides (for example, difunctional primary construct or double function Energy mmRNA).As its name suggests, difunctional polynucleotides are that have the function of or can have those of at least two polynucleotides.These Molecule can also be traditionally referred to as multi-functional.
The multi-functional of difunctional polynucleotides can be encoded that (function may be turned over until the product of coding by RNA Translate and just show) or characteristic itself that can be polynucleotides.It can be structure or chemical.Difunctional modification polynucleotides May include with polynucleotides covalently or Electrostatic association function.In addition, two kinds of functions can be in the compound of mmRNA and another molecule It is provided under the background of object.
The peptide of difunctional polynucleotides codified anti proliferative.These peptides can be linear, cricoid, constraint or nothing Rule curling.They may act as aptamer, signal transduction molecule, ligand or its analogies or mimetic.Antiproliferative peptide is in translation Length can be 3 to 50 amino acid.They can be 5 to 40,10 to 30 or about 15 amino acid longs.They can be list Chain, multichain or branch and be once translated and can form compound, aggregation or any more unit structures.
Non-coding polynucleotide and primary construct
As described herein, providing to have can translate or generally not interpretable sequence for part, such as with non-volume The polynucleotides in code area and primary construct.This noncoding region can be " the firstth area " of primary construct.Alternatively, non-coding Area can be the area different from firstth area.This kind of molecule is not translated usually, but can by combine and chelate it is a kind of or One of a variety of machine translator components (such as ribosomal protein or transfer RNA (tRNA)) or a variety of pairs of protein, which generate to play, to be made With to efficiently reduce one of protein expression or adjusting cell in cell or number of ways or cascade, this is in turn Change protein level.Polynucleotides or primary construct may include or encode the non-coding RNA (lncRNA of one or more length Or lincRNA) or part thereof, little nucleolar RNA (sno-RNA), Microrna (miRNA), siRNA (siRNA) or Piwi- It interacts RNA (piRNA).
Target polypeptides
According to the present invention, primary construct is designed to encode one or more target polypeptides or its segment.Target polypeptides It may include but be not limited to the segment of full polypeptide, multiple polypeptides or polypeptide, it independently can be by one or more nucleic acid, Duo Zhonghe Acid, the segment of nucleic acid or any of above variant coding.As used herein, term " target polypeptides ", which refers to, is selected in this hair Any polypeptide encoded in bright primary construct.As used herein, " polypeptide " means to link together most often by peptide bond Amino acid residue (naturally or not natural) polymer.As used herein, the term refers to any size, structure Or protein, polypeptide and the peptide of function.In some cases, the polypeptide of coding is less than about 50 amino acid and the polypeptide is right It is referred to as peptide afterwards.If polypeptide is peptide, it will be that at least about 2,3,4 or at least five amino acid residue are long.Therefore, polypeptide Including gene product, naturally occurring polypeptide, synthesis polypeptide, homologue, ortholog, lateral homologue, segment above-mentioned With other equivalents, variant and analog.Polypeptide can be individual molecule or can be multi-molecular complex such as dimer, trimerization Object or tetramer.They also may include single-stranded or multi-chain polypeptides such as antibody or insulin and can be association or connection. Most commonly, disulfide bond is found in multi-chain polypeptides.Term polypeptide applies also for amino acid polymer, wherein one or more Amino acid residue is the artificial chemical analogue of corresponding naturally occurring amino acid.
Term " polypeptide variants " refers to molecule in terms of its amino acid sequence and natural or different reference sequences.With it is natural Or reference sequences are compared, amino acid sequence variation can have substitution at certain positions in amino acid sequence, missing and/or Insertion.In general, variant will have at least about 50% identity (homology) with natural or reference sequences, and preferably they will With natural or reference sequences at least about 80%, more preferably at least about 90% identical (homologous).
In some embodiments, " variant analogies " are provided.As used herein, term " variant analogies " is comprising inciting somebody to action Simulate a kind of variant analogies of one or more amino acid of activated sequence.For example, glutamic acid can be used as phosphorus-threonine And/or phosphorus-serine analogies.Alternatively, variant analogies can cause deactivation or the inactivation product containing analogies, for example, The inactivation that phenylalanine may act as tyrosine replaces；Or alanine may act as the inactivation substitution of serine.
" homology " be defined as when it is applied to amino acid sequence in aligned sequences and if necessary introducing vacancy with It is identical with the residue in the amino acid sequence of the second sequence in candidate amino acid sequence after realizing largest percentage homology Residue percentage.Method and computer program for comparison is well known in the art.It should be understood that homology depends on percentage The calculating of identity, but different values is likely to be obtained due to being introduced into the vacancy in calculating and point penalty.
" homologue " means there is Substantial identity with the second sequence of the second species when it is applied to polypeptide sequence The corresponding sequence of other species.
" analog " be intended to include because one or more amino acid changes (for example, the substitution of amino acid residue, addition or lack Lose) and the polypeptide variants of one or more characteristics that are different but still maintaining parent or starting polypeptide.
The present invention is thought of as the composition of the several types based on polypeptide, including variant and derivative.These include replace, Insertion, missing and covalent variant and derivative.Term " derivative " is synonymously used with term " variant ", but is typically referred to The molecule modified and/or changed in any way relative to reference molecule or starting molecule.
In this way, coding is relative to reference sequences, polypeptide sequence specifically disclosed herein containing substituted, insertion and/ Or the mmRNA of the polypeptide of addition, missing and covalent modification is included within the scope of the invention.For example, sequence label or amino acid (such as one or more lysines) may be added to that peptide sequence of the invention (for example, at N-terminal or C-terminal).Sequence label can For peptide purification or positioning.Lysine can be used for increasing peptide solubility or allow biotinylation.Alternatively, being located at peptide or protein matter The carboxyl and the amino acid residue at amino terminal area of amino acid sequence are optionally lacked, to provide truncated sequence.Certain A little amino acid (for example, C-terminal or N-terminal residue) are alternatively lacked, this depends on the use of sequence, such as solvable Or it is connected to the expression of the sequence of a part of the larger sequence of solid support.
When referring to polypeptide " replace variant " be natural or homing sequence at least one amino acid residue be removed simultaneously And the polypeptide of different amino acid is inserted into the position at its same position.Substitution can be it is single, wherein in molecule Only one amino acid has been substituted；Or replace can be it is multiple, wherein two or more amino acid in same molecule are It is substituted.
As used herein, term " conserved amino acid substitution " refers to the amino acid being typically found in sequence by with similar Size, charge or polar different aminoacids replace.The example of conservative substitution includes for example different bright ammonia of nonpolar (hydrophobicity) residue Acid, valine and leucine replace another non-polar residue.Equally, the example of conservative substitution includes a kind of polarity (hydrophily) Residue replace it is another, as between arginine and lysine, between glutamine and asparagine and glycine and serine it Between.In addition, alkaline residue such as lysine, arginine or histidine replace another alkaline residue or a kind of acidic residues such as day Aspartic acid or glutamic acid replace another acidic residues to be the other example of conservative substitution.The example of non-conservative substitutions includes non-pole Property (hydrophobicity) amino acid residue for example isoleucine, valine, leucine, alanine, methionine replace polarity (hydrophily) it is residual Base such as cysteine, glutamine, glutamic acid or lysine and/or polar residues replace non-polar residue.
When referring to polypeptide, " insertion variant " is with the amino acid close to the specific location in natural or homing sequence Those of one or more amino acid of insertion variant." close to " amino acid means the α-carboxyl for being connected to the amino acid or α- Amido functional group.
When referring to polypeptide " deletion mutants " be natural or original amino acid sequence in one or more amino acid gone Except those of variant.In general, deletion mutants lack one or more amino acid in the given zone of molecule.
When referring to polypeptide, " covalence derivative " includes natural with organic protein or the modification of nonprotein derivatization reagent Or initiation protein and/or posttranslational modification.Conventionally by make the target amino acid residue of protein with can be with selected side Organic derivatization reagent reaction of chain or terminal residue reaction, or by being turned over using what is worked in selected recombinant host cell The mechanism modified after translating introduces covalent modification.Gained covalence derivative is suitable for being intended to identifying for bioactivity, for exempting from Epidemic disease measurement or the journey for preparing residue important for the anti-protein antibody for the immunoaffinity purification of recombinant glycoprotein In sequence.This kind of modification carries out in the range of ordinary skill and in the case where no excessive experiment.
Certain posttranslational modifications are the results of effect of the recombinant host cell to expressed polypeptide.Glutaminyl and day Winter amide acyl residue often sloughs acylamino- into corresponding glutamyl and aspartyl residue upon translation.Alternatively, this A little residues slough acylamino- under mildly acidic conditions.Any form of these residues may be present in generate according to the present invention it is more In peptide.
Other posttranslational modifications include the hydroxylating of proline and lysine, the hydroxyl of seryl- or threonyl residues Phosphorylation, lysine, arginine and histidine side chains alpha-amino methylation (T.E.Creighton, Proteins: Structure and Molecular Properties, W.H.Freeman & Co., San Francisco, the 79-86 pages (1983))。
When referring to polypeptide, " feature " is defined as the different component based on amino acid sequence of molecule.By of the invention The feature of the polypeptide of mmRNA coding include surface apparent, local conformation shape, folding, ring, semi-ring, structural domain, half domain, Site, end or any combination thereof.
As used herein when referring to polypeptide, term " surface is apparent " refers to the base of protein appeared in outmost surface In the component of polypeptide.
As used herein, when referring to polypeptide term " local conformation shape " refer to protein be located at its can limit space The interior structure based on polypeptide is apparent.
As used herein when referring to polypeptide, term " folding " refers to the gained structure of the amino acid sequence in energy minimization As.Folding can issue life in the second level or three-level level of folding process.The example that two grades of horizontal folds includes β-pleated sheet and α spiral. The example that three-level folds includes the structural domain formed due to the aggregation or separation of ability and area.Area's packet formed in this way Include hydrophobic pocket and hydrophilic bag etc..
As used herein, term " corner " means to change the main chain direction of peptide or polypeptide when it is related to protein conformation Bending and can be related to one, two, three or more amino acid residue.
As used herein, when referring to polypeptide, term " ring ", which refers to, can be used for making the main chain direction of peptide or polypeptide reversed The structure feature of polypeptide.When ring exists in polypeptide and only changes the direction of main chain, it may include four or more ammonia Base acid residue.Oliva et al. authenticated at least 5 proteinoid rings (J.Mol Biol 266 (4): 814-830；1997). Ring can be open or closure.The ring of closure or " ring-type " ring can between bridging part comprising 2,3,4,5,6,7,8,9, 10 or more amino acid.This kind of bridging part may include typical-half Guang ammonia of cysteine in the polypeptide with disulphide bridges Sour bridge (Cys-Cys), or alternatively bridging part can be based on nonprotein, as used herein Dibromozylyl reagent.
As used herein, when referring to polypeptide, term " semi-ring " refers to a part of identified ring, and the part has The amino acid residue of at least half number of its ring being derived from.It should be understood that ring may not always include the ammonia of even number Base acid residue.Therefore, in the case of ring includes or is identified as the amino acid comprising odd number, the ring of odd number Semi-ring by the integer part comprising the ring or next integer part (number of the amino acid of the ring/2+/- 0.5 ammonia Base acid).For example, the ring for being identified as 7 amino acid rings can produce the semi-ring (7/2=3.5 of 3 amino acid or 4 amino acid +/- 0.5 is 3 or 4).
As used herein, when referring to polypeptide, term " structural domain " refers to one or more identifiable structures or function The motif of the polypeptide of energy feature or characteristic (for example, binding ability, site as protein-protein interaction).
As used herein, when referring to polypeptide term " half domain " mean identify structural domain a part, the portion Divide the amino acid residue of at least half number with its structural domain being derived from.It should be understood that structural domain may not always include The amino acid residue of even number.Therefore, in structural domain include or be identified as those of the amino acid feelings comprising odd number Under condition, the half domain of the structural domain of odd number is by the integer part comprising the structural domain or next integer part (institute State number/2+/- 0.5 amino acid of the amino acid of structural domain).For example, being identified as the structural domain of 7 amino acid domains It can produce the half domain of 3 amino acid or 4 amino acid (7/2=3.5+/- 0.5 is 3 or 4).It should also be understood that can be in structure Identify subdomain in domain or half domain, these subdomains possess in the structural domain or half domain being derived from less than it All structure or function characteristics identified.It should also be understood that the amino acid of any domain type comprising this paper needs not be edge The main chain of polypeptide is continuous (that is, non-conterminous amino acid can be folded in structure to generate structural domain, half domain or substructure Domain).
As used herein, when referring to polypeptide term " site " when it is related to the embodiment based on amino acid with " ammonia Base acid residue " and " amino acid side chain " synonymously use.Site indicate in peptide or polypeptide can be of the invention based on polypeptide Intramolecular modification manipulation, changes, the position of derivatization or variation.
As used herein, term " end (termini) " or " end (terminus) " refer to when referring to polypeptide peptide or The end of polypeptide.This end is not limited only to first or last site of peptide or polypeptide, and may include another in end region Outer amino acid.Molecule based on polypeptide of the invention is characterized by with N-terminal (by the amino with free amino (NH2) Both acid blocked) and C-terminal (by the amino acid blocked with free carboxyl group (COOH)).Protein of the invention is in some cases Under be made of the multiple polypeptide chains to gather together by disulfide bond or by noncovalent force (polymer, oligomer).These kinds The protein of class will have multiple N-terminals and C-terminal.Alternatively, the end of polypeptide may be trimmed so that they according to circumstances Started with the part (such as organic conjugate) based on non-polypeptide or is terminated.
Once any feature has been authenticated or has been defined as to by the polypeptide of primary construct of the invention or mmRNA coding Required component, can by movement, exchange, reversion, missing, randomization or duplication come carry out these features several manipulations and/ Or any one of modification.Further, it should be understood that the manipulation of feature can produce result identical with the modification to molecule of the present invention. For example, being related to the manipulation for lacking structural domain will generate as modification of nucleic acids is as encoding and be less than full-length molecule for generation The change of molecular length.
Modification and manipulation can be realized by methods known in the art, such as, but not limited to direct mutagenesis.Then can make It is surveyed with measurement in vitro or in vivo (as described herein those) or any other suitable screening test known in the art Try the activity of obtained decorating molecule.
According to the present invention, polypeptide may include the consensus sequence by more taking turns experiment discovery.As used herein, " shared " sequence It is the single sequence for indicating to allow the arrangement set group of the variability at one or more sites.
As is appreciated by those skilled in the art, protein fragments, functional protein structural domain and homologous protein It is considered in the range of target polypeptides of the invention.For example, provided herein is length be 10,20,30,40,50,60,70,80, 90,100 or (mean to be as short as than reference polypeptide sequence few greater than any protein fragments of the reference protein of 100 amino acid One amino acid residue but in other aspects identical polypeptide sequence).In another example, can used according to the invention include It is about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95% or about with any sequence described herein Any protein of the 100% identical section with about 20, about 30, about 40, about 50 or about 100 amino acid.In certain implementations It include 2,3,4,5,6,7,8,9,10 or more mutation to polypeptide used according to the invention, as provided herein in scheme Or shown in any sequence referred to.
The polypeptide of coding
Primary construct of the invention or mmRNA are designed to coding selected from any one of several target classifications Target polypeptides, the target classification include but is not limited to biological agent, antibody, vaccine, therapeutic protein or peptide, cell-penetrating Peptide, secreted protein, plasmalemma protein, cytoplasm or cytoskeletal protein, intercellular membrane binding protein, nucleoprotein and mankind's disease The relevant protein of disease, targeting moiety those of encode protein by human genome, do not identify also for the protein It treats indication but the protein has effectiveness still in research and discovery field.
In one embodiment, primary construct or mmRNA codified and reference polypeptide sequence have certain identity Variant polypeptide.As used herein, " reference polypeptide sequence " refers to starting polypeptide sequence.Reference sequences can be wild-type sequence Or designing any sequence referenced in another sequence." reference polypeptide sequence " can be SEQ for example as disclosed herein ID NO:769 to any one of 1392, for example, SEQ ID NO 769,770,771,772,773,774,775,776, 777、778、779、780、781、782、783、784、785、786、787、788、789、790、791、792、793、794、795、 796、797、798、799、800、801、802、803、804、805、806、807、808、809、810、811、812、813、814、 815、816、817、818、819、820、821、822、823、824、825、826、827、828、829、830、831、832、833、 834、835、836、837、838、839、840、841、842、843、844、845、846、847、848、849、850、851、852、 853、854、855、856、857、858、859、860、861、862、863、864、865、866、867、868、869、870、871、 872、873、874、875、876、877、878、879、880、881、882、883、884、885、886、887、888、889、890、 891、892、893、894、895、896、897、898、899、900、901、902、903、904、905、906、907、908、909、 910、911、912、913、914、915、916、917、918、919、920、921、922、923、924、925、926、927、928、 929、930、931、932、933、934、935、936、937、938、939、940、941、942、943、944、945、946、947、 948、949、950、951、952、953、954、955、956、957、958、959、960、961、962、963、964、965、966、 967、968、969、970、971、972、973、974、975、976、977、978、979、980、981、982、983、984、985、 986、987、988、989、990、991、992、993、994、995、996、997、998、999、1000、1001、1002、1003、 1004、1005、1006、1007、1008、1009、1010、1011、1012、1013、1014、1015、1016、1017、1018、 1019、1020、1021、1022、1023、1024、1025、1026、1027、1028、1029、1030、1031、1032、1033、 1034、1035、1036、1037、1038、1039、1040、1041、1042、1043、1044、1045、1046、1047、1048、 1049、1050、1051、1052、1053、1054、1055、1056、1057、1058、1059、1060、1061、1062、1063、 1064、1065、1066、1067、1068、1069、1070、1071、1072、1073、1074、1075、1076、1077、1078、 1079、1080、1081、1082、1083、1084、1085、1086、1087、1088、1089、1090、1091、1092、1093、 1094、1095、1096、1097、1098、1099、1100、1101、1102、1103、1104、1105、1106、1107、1108、 1109、1110、1111、1112、1113、1114、1115、1116、1117、1118、1119、1120、1121、1122、1123、 1124、1125、1126、1127、1128、1129、1130、1131、1132、1133、1134、1135、1136、1137、1138、 1139、1140、1141、1142、1143、1144、1145、1146、1147、1148、1149、1150、1151、1152、1153、 1154、1155、1156、1157、1158、1159、1160、1161、1162、1163、1164、1165、1166、1167、1168、 1169、1170、1171、1172、1173、1174、1175、1176、1177、1178、1179、1180、1181、1182、1183、 1184、1185、1186、1187、1188、1189、1190、1191、1192、1193、1194、1195、1196、1197、1198、 1199、1200、1201、1202、1203、1204、1205、1206、1207、1208、1209、1210、1211、1212、1213、 1214、1215、1216、1217、1218、1219、1220、1221、1222、1223、1224、1225、1226、1227、1228、 1229、1230、1231、1232、1233、1234、1235、1236、1237、1238、1239、1240、1241、1242、1243、 1244、1245、1246、1247、1248、1249、1250、1251、1252、1253、1254、1255、1256、1257、1258、 1259、1260、1261、1262、1263、1264、1265、1266、1267、1268、1269、1270、1271、1272、1273、 1274、1275、1276、1277、1278、1279、1280、1281、1282、1283、1284、1285、1286、1287、1288、 1289、1290、1291、1292、1293、1294、1295、1296、1297、1298、1299、1300、1301、1302、1303、 1304、1305、1306、1307、1308、1309、1310、1311、1312、1313、1314、1315、1316、1317、1318、 1319、1320、1321、1322、1323、1324、1325、1326、1327、1328、1329、1330、1331、1332、1333、 1334、1335、1336、1337、1338、1339、1340、1341、1342、1343、1344、1345、1346、1347、1348、 1349、1350、1351、1352、1353、1354、1355、1356、1357、1358、1359、1360、1361、1362、1363、 1364、1365、1366、1367、1368、1369、1370、1371、1372、1373、1374、1375、1376、1377、1378、 1379, any in 1380,1381,1382,1383,1384,1385,1386,1387,1388,1389,1390,1391,1392 One.
Term " identity " as known in the art refers to the sequence of two or more peptides as determined by comparing sequence Relationship between column.In the art, identity is still meant that such as by between the string with two or more amino acid residues Matching number determine peptide between serial correlation degree.Identity is estimated through specific mathematical model or computer program The identical match that smaller between two or more sequences of (if present) is compared with vacancy that (i.e. " algorithm ") solves Percentage.The identity of related peptide can be readily calculated by known method.Such methods include but is not limited to retouch in following Those of state: Computational Molecular Biology, Lesk, A.M. are edited, Oxford University Press, New York, 1988；Biocomputing:Informatics and Genome Projects, Smith, D.W. are compiled Volume, Academic Press, New York, 1993；Computer Analysis of Sequence Data, part 1, Griffin, A.M. and Griffin, H.G. are edited, Humana Press, New Jersey, and 1994；Sequence Analysis In Molecular Biology, von Heinje, G., Academic Press, 1987；Sequence Analysis Primer, Gribskov, M. and Devereux, J. are edited, M.Stockton Press, New York, and 1991；And Carillo Et al., SIAM J.Applied Math.48,1073 (1988).
In some embodiments, polypeptide variants can have and the same or similar activity of reference polypeptide.Alternatively, variant can There is the activity (for example, increased or reduction) changed relative to reference polypeptide.In general, specific polynucleotides of the invention or The variant of polypeptide will with it is described have at least about 40% with particular reference to polynucleotides or polypeptide, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% but Sequence identity less than 100%, such as by alignment programs described herein and well known by persons skilled in the art with What parameter determined.This kind of tool for comparison includes those of BLAST external member (Stephen F.Altschul, Thomas L.Madden, Alejandro A.Jinghui Zhang, Zheng Zhang, Webb Miller and David J.Lipman (1997), " Gapped BLAST and PSI-BLAST:a new generation of protein Database search programs ", Nucleic Acids Res.25:3389-3402.).Other tools are in this paper, tool It is described in the definition of " identity " to body.
Default parameter in BLAST algorithm includes for example, it is contemplated that threshold value 10, word length 28, matching/mispairing score 1, -2, empty Bit-loss is linear.It can be using any filter and selection species specificity repetitive sequence, such as homo sapiens.
Biological agent
Polynucleotides, primary construct or the one or more biological agents of mmRNA codified disclosed herein.As herein Used, " biological agent " is to be generated by method provided herein and can be used for treating, cured, mitigate, prevent or examine The molecule of the gene polypeptide for serious or life-threatening disease or the medical condition of breaking.Biological agent includes but not according to the present invention It is limited to, Allergen extract (such as allergy vaccine injection (allergy shot) and testing), blood constitutent, gene therapy The tissue of people used in product, transplanting or cellular products, vaccine, monoclonal antibody, cell factor, growth factor, enzyme, thrombus dissolving Agent and immunomodulator etc..
According to the present invention, current commercial or in research and development one of or a variety of biological agents can be by multicore glycosides of the invention Acid, primary construct or mmRNA coding.While not wishing to it is bound by theory, it is believed that by the coding polypeptide of known biological agent Improved therapeutic efficiency will be generated by being incorporated into primary construct or mmRNA of the invention, and this improved therapeutic efficiency is at least It is partly due to the specificity, purity and/or selectivity of construct designs.
Antibody
Primary construct disclosed herein or the one or more antibody of mmRNA codified or its segment.Term " antibody " Including monoclonal antibody (including the full length antibody with immunoglobulin fc region), the antibody combination with multi-epitope specificity Object, multi-specificity antibody (for example, bispecific antibody, double antibody and single chain molecule) and antibody fragment.Term " immune globulin It is white " (Ig) this paper can be used interchangeably with " antibody ".As used herein, term " monoclonal antibody " refers to anti-from generally homogeneity The antibody that the group of body obtains, that is, constitute the independent antibody of the group be it is identical, in addition to may be with micro existing possibility Naturally occurring mutation and/or posttranslational modification (for example, isomerization, amidation).Monoclonal antibody is high degree of specificity, To be directed to single antigen site.
The monoclonal antibody of this paper specifically includes " chimeric " antibody (immunoglobulin), wherein heavy chain and/or light chain A part with from particular species or to belong to corresponding sequence in the antibody of specific antibodies classification or subclass identical or homologous, and institute It states the remainder of chain and is originated from another species or belongs to the antibody of another antibody isotype or subclass and the segment of this kind of antibody In corresponding sequence it is identical or homologous, as long as they show required bioactivity.The target chimeric antibody of this paper include but It is not limited to variable domains antigen-binding subsequences and people comprising being originated from non-human primate (for example, old world monkey, ape etc.) " primatized " antibody of constant-region sequences.
" antibody fragment " includes a part of complete antibody, the antigen binding domain of the preferably described complete antibody and/or can Become area.The example of antibody fragment includes Fab, Fab ', F (ab ')2With Fv segment；Double antibody；Linear antibodies；Nano antibody (nanobodies)；The single-chain antibody molecules and multi-specificity antibody formed from antibody fragment.
Any one of five immunoglobulin like protein IgA, IgD, IgE, IgG and IgM can be encoded by mmRNA of the invention, Heavy chain including being respectively designated as α, δ, ε, λ and μ.It further include the polynucleotide sequence for encoding subclass γ and μ.Therefore Subclass of antibody Any one of can partially or entirely be encoded and including following subclass: IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2。
According to the present invention, current commercial or can be by multicore of the invention in one of research and development or Multiple Antibodies or segment Thuja acid, primary construct or mmRNA coding.While not wishing to it is bound by theory, it is believed that being incorporated into primary building of the invention To generate improveds therapeutic efficiency in body, this improved therapeutic efficiency be at least partially attributed to the specificity that mmRNA designs, Purity and selectivity.
The antibody encoded in polynucleotides of the invention, primary construct or mmRNA can be used for treating many treatment necks Symptom or disease in domain, the therapy field be such as, but not limited to blood, angiocarpy, CNS, poisoning (including antivenin), Dermatology, endocrinology, stomach and intestine, medical imaging, muscle skeleton, oncology, immunology, respiratory system, sensory system and Anti-infectious therapy field.
In one embodiment, primary construct or mmRNA codified monoclonal antibody disclosed herein and/or its Variant.The variant of antibody may also include but be not limited to, replace variant, conserved amino acid replace, insertion variant, deletion mutants and/ Or covalent variant.In one embodiment, primary construct disclosed herein and/or mmRNA codified immunoglobulin The area Fc.In another embodiment, primary construct and/or the area mmRNA codified variant immunoglobulin Fc.As non-limit Property example processed, primary construct and/or mmRNA codified have the antibody in the area variant immunoglobulin Fc, are such as described in draw Mode is integrally incorporated in the U.S. Patent number 8,217,147 of this paper.
Vaccine
Primary construct disclosed herein or the one or more vaccines of mmRNA codified.As used herein, " vaccine " is Improve the biological agent to the immunity of specified disease or infectious agent.According to the present invention, current commercial or in research and development one Kind or a variety of vaccines can be encoded by polynucleotides of the invention, primary construct or mmRNA.While not wishing to it is bound by theory, It is believed that improved therapeutic efficiency, this improved treatment function will be generated by being incorporated into primary construct or mmRNA of the invention Effect is at least partially attributed to the specificity, purity and selectivity of construct designs.
The vaccine encoded in polynucleotides of the invention, primary construct or mmRNA can be used for treating many treatment necks Symptom or disease in domain, the therapy field be such as, but not limited to angiocarpy, CNS, dermatology, endocrinology, oncology, Immunology, respiratory system and anti-infectious therapy field.
Therapeutic protein or peptide
Primary construct disclosed herein or mmRNA codified are one or more verified or " in test " Therapeutic protein or peptide.
According to the present invention, current commercial or can be by the present invention in one of research and development or a variety of therapeutic proteins or peptide Polynucleotides, primary construct or mmRNA coding.While not wishing to bound by theory, it is believed that be incorporated into it is of the invention just Improved therapeutic efficiency will be generated in grade construct or mmRNA, this improved therapeutic efficiency is at least partially attributed to construct Specificity, purity and the selectivity of body design.
The therapeutic protein and peptide encoded in polynucleotides of the invention, primary construct or mmRNA can be used for controlling The symptom or disease in many therapy fields are treated, the therapy field is such as, but not limited to blood, angiocarpy, CNS, poisoning (packet Include antivenin), dermatology, endocrinology, science of heredity, urogenital, stomach and intestine, muscle skeleton, oncology and immunology, exhale Desorption system, sensory system and anti-infectious therapy field.
Cell penetrating peptide
Primary construct disclosed herein or the one or more cell penetrating peptides of mmRNA codified.As used herein, " cell penetrating peptide " or CPP refer to the polypeptide that can help to the cellular uptake of molecule.Cell penetrating peptide of the invention can wrap Containing one or more detectable labels.The polypeptide can be marked partly or all fully be marked.The polynucleotides, just Grade construct or mmRNA can complete, partial coding or not encoded detectable labels completely.Cell-penetrating peptides may also include signal sequence Column.As used herein, " signal sequence " refers at the amino terminal for being incorporated in nascent protein during protein translation The sequence of amino acid residue.Signal sequence can be used for the secretion of signal transduction cell-penetrating peptides.
In one embodiment, the polynucleotides, primary construct or mmRNA also codified fusion protein.Fusion Albumen can be formed by the way that electrically charged protein is operably coupled to therapeutic protein.As used herein, it " can operate Ground connection " refers to that therapeutic protein and electrically charged protein are when being introduced in cell to allow to express compound in this way A kind of mode connects.As used herein, " electrically charged protein " refers to the albumen for carrying positive and negative or overall neutral charge Matter.Preferably, therapeutic protein can be covalently coupled to electrically charged protein in the formation of fusion protein.Surface charge And total or sour surface amino groups ratio can be about 0.1,0.2,0.3,0.4,0.5,0.6,0.7,0.8 or 0.9.
It can be formed after being translated by the cell penetrating peptide of polynucleotides, primary construct or mmRNA coding compound Object.The compound may include connecting (such as covalent linkage) to the electrically charged protein of cell penetrating peptide." therapeutic egg White matter " refers to the biology and/or medicine when being applied to cell with treatment, diagnosis and/or prophylactic action and/or needed for drawing The protein of Neo-Confucianism effect.
In one embodiment, cell penetrating peptide may include first structure domain and the second structural domain.First structure domain It may include the polypeptide of hypercharge.Second structural domain may include protein binding partner.As used herein, " protein, which combines, matches Even body " includes but is not limited to antibody and its functional fragment, scaffolding protein or peptide.Cell penetrating peptide can further include albumen The intracellular binding partner of matter binding partners.Cell penetrating peptide can be from can introduce polynucleotides, primary construct Or the cell secretion of mmRNA.Cell penetrating peptide may also be able to penetrate the first cell.
In another embodiment, cell penetrating peptide can penetrate the second cell.Second cell may be from first carefully The identical region of born of the same parents or it may be from different regions.The region may include but be not limited to tissue and organ.Second cell is also The proximal end or distal end of the first cell can be located at.
In one embodiment, the polynucleotides, primary construct or mmRNA codified may include that protein combines The cell penetrating peptide of gametophyte.Protein binding partner may include but be not limited to the antibody or functionality of antibody, hypercharge Segment.The polynucleotides, primary construct or mmRNA can be introduced to and be introduced into the cell comprising protein binding partner It penetrates in the cell of polypeptide.
Secreted protein
People is separated into the different compartment of many functions with other eukaryocyte envelopes.The compartment or organelle that each film defines Include different proteins necessary for the function of organelle.Cell uses " sorting signals ", and (sorting signals are positions In the amino acid motif in protein) come make protein target specific cells device.
Referred to as a type of sorting signals of signal sequence, signal peptide or leader sequence by class protein guide to The referred to as organelle of endoplasmic reticulum (ER).
It can be used as secreted protein by the protein that signal sequence targets ER to be released into extracellular space.It is similar Ground, the protein rested on cell membrane can also be secreted and protein to be retained to the proteolytic cleavage of " connector " of film Into extracellular space.While not wishing to which molecule bound by theory but of the invention can be used for using cell described above Transport.In this way, in some embodiments of the present invention, polynucleotides, primary construct or mmRNA are provided to expression secretion Property albumen.Secreted protein can be secreted those of from those of described herein or U.S. Patent Publication 20100255574, institute The content for stating patent disclosure is incorporated herein in its entirety by reference.
In one embodiment, these can be used for manufacturing a large amount of valuable people's gene products.
Plasmalemma protein
In some embodiments of the present invention, polynucleotides, primary construct or mmRNA are provided to expression plasma membrane Protein.
Cytoplasm or cytoskeletal protein
In some embodiments of the present invention, polynucleotides, primary construct or mmRNA are provided to expression cell matter Or cytoskeletal protein.
Intercellular membrane binding protein
In some embodiments of the present invention, polynucleotides, primary construct or mmRNA are provided in expression cell Embrane-associated protein.
Nucleoprotein
In some embodiments of the present invention, polynucleotides, primary construct or mmRNA are provided to expression core egg It is white.
Protein relevant to human diseases
In some embodiments of the present invention, polynucleotides, primary construct or mmRNA are provided to expression and the mankind The relevant protein of disease.
Other oroteins
In some embodiments of the present invention, polynucleotides, primary construct or mmRNA, which are provided to expression, has mesh The protein of preceding unknown treatment function.
Targeting moiety
In some embodiments of the present invention, polynucleotides, primary construct or mmRNA are provided to expression targeting portion Point.These include the receptor on protein binding partner or cell surface, are used to that cell to be made to target spy in vivo or in vitro Determine organization space or interacts with specific part.Suitable protein binding partner includes but is not limited to antibody and its function Property segment, scaffolding protein or peptide.In addition, polynucleotides, primary construct or mmRNA can be used for instructing lipid, carbohydrate Or the synthesis of other biological moieties or biomolecule is positioned with extracellular.
Polypeptide libraries
In one embodiment, the polynucleotides, primary construct or mmRNA can be used for generating polypeptide libraries.This A little libraries can be generated by the generation of the group of polynucleotides, primary construct or mmRNA, respectively contain different structure or change Learn modification design.In this embodiment, the group of polynucleotides, primary construct or mmRNA may include a variety of codings Polypeptide, including but not limited to antibody or antibody fragment, protein binding partner, scaffolding protein and teaching herein or ability Other polypeptides known to domain.In preferred embodiments, polynucleotides are primary constructs of the invention, including are applicable to straight Meet the mmRNA being introduced in target cell or culture, the target cell or culture so that can composite coding polypeptide.
In certain embodiments, a variety of variants of the respectively protein with different amino acid modifications be can produce, and And it is tested to determine with regard to pharmacokinetics, stability, biocompatibility and/or bioactivity or biological object Optimal variant for characteristic of science such as expression.This library may include 10,102、103、104、105、106、107、108、 109Or more than 109(including but not limited to, the substitution of one or more residues, missing and one or more are residual for a possible variant The insertion of base).
Antimicrobial polypeptide and antiviral polypeptide
Polynucleotides, primary construct and mmRNA of the invention may be designed to encode one or more antimicrobial peptides (AMP) or antiviral peptide (AVP).From broad range of animal be such as, but not limited to microorganism, invertebrate, plant, Amphibian, bird, fish and mammal separate and describe AMP and AVP (Wang et al., Nucleic Acids Res.2009；37 (Database issue): D933-7).For example, antimicrobial polypeptide is described in the following terms: resisting micro- life Object peptide database (http://aps.unmc.edu/AP/main.php；Wang et al., Nucleic Acids Res.2009；37 (Database issue): D933-7), CAM P:Collection of Anti-Microbial Peptides (http: // www.bicnirrh.res.in/an timicrobial/)；Thomas et al., Nucleic Acids Res.2010；38 D774-80), US 5221732, US 5447914, US 5519115, US 5607914, US (Database i ssue): 5714577、US 5734015、US 5798336、US 5821224、US 5849490、US 5856127、US 5905187、US 5994308、US 5998374、US 6107460、US 6191254、US 6211148、US 6300489、US 6329504、US 6399370、US 6476189、US 6478825、US 6492328、US 6514701、US 6573361、US 6573361、US 6576755、US 6605698、US 6624140、US 6638531、US 6642203、US 6653280、US 6696238、US 6727066、US 6730659、US 6743598、US 6743769、US 6747007、US 6790833、US 6794490、US 6818407、US 6835536、US 6835713、US 6838435、US 6872705、US 6875907、US 6884776、US 6887847、US 6906035、US 6911524、US 6936432、US 7001924、US 7071293、US 7078380、US 7091185, US 7094759, US 7166769, US 7244710, US 7314858 and US 7582301, the document Content is incorporated herein in its entirety by reference.
Antimicrobial polypeptide described herein can be blocked carefully by one or more enveloped virus (such as HIV, HCV) Born of the same parents' fusion and/or cell entry.For example, antimicrobial polypeptide may include synthetic peptide or be made from it, the synthetic peptide corresponds to One area of the cross-film subunit of virus envelope protein (such as HIV-1 gp120 or gp41), for example, at least about 5,10,15,20, 25, the continuous sequence of 30,35,40,45,50,55 or 60 amino acid.The amino acid and nucleotide of HIV-1 gp120 or gp41 Sequence description is in such as Kuiken et al., (2008) " HIV Sequence Compendium, " Los Alamos National In Laboratory.
In some embodiments, antimicrobial polypeptide can with corresponding virus protein sequence have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology.In some embodiments, antimicrobial polypeptide can be with corresponding disease Poisonous protein sequence has at least about 75%, 80%, 85%, 90%, 95% or 100% sequence homology.
In other embodiments, antimicrobial polypeptide may include synthetic peptide or be made from it, and the synthetic peptide corresponds to One area of the protein-bonded binding structural domain of capsid, for example, at least about 5,10,15,20,25,30,35,40,45,50,55 Or the continuous sequence of 60 amino acid.In some embodiments, antimicrobial polypeptide can be with the protein-bonded corresponding sequence of capsid Arrange the sequence homology at least about 75%, 80%, 85%, 90%, 95% or 100%.
Antimicrobial polypeptide described herein can blocks protein enzyme dimerization and inhibit albumen before virus (for example, HIV Gag-pol processing) it is cracked into functional protein, to prevent one or more enveloped virus (for example, HIV, HCV) Release.In some embodiments, antimicrobial polypeptide can with corresponding virus protein sequence have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology.
In other embodiments, antimicrobial polypeptide may include synthetic peptide or be made from it, and the synthetic peptide corresponds to One area of the protein-bonded binding structural domain of protease, for example, at least about 5,10,15,20,25,30,35,40,45,50, The continuous sequence of 55 or 60 amino acid.In some embodiments, antimicrobial polypeptide can be protein-bonded right with protease Answer sequence that there is at least about 75%, 80%, 85%, 90%, 95% or 100% sequence homology.
Antimicrobial polypeptide described herein may include the polypeptide for the Evolution in vitro of viral pathogen.
Antimicrobial polypeptide
Antimicrobial polypeptide (AMP) is the small peptide with variable-length, sequence and structure, and the small peptide, which has to be directed to, includes But it is not limited to the broad range microorganism of bacterium, virus, fungi, protozoan, helminth, prion and lesion/cancer cell Broad spectrum of activity.(see, e.g. Zaiou, J Mol Med, 2007；85:317；It is incorporated herein in its entirety by reference).It has shown Show that AMP has the killing activity of quick acting of wide spectrum, together with potential low-level induction of resistance and adjoint extensive anti-inflammatory work With.
In some embodiments, antimicrobial polypeptide (for example, antibacterial polypeptide) can be less than 10kDa, such as less than 8kDa, 6kDa, 4kDa, 2kDa or 1kDa.In some embodiments, antimicrobial polypeptide (such as antibacterial polypeptide) is by about 6 To about 100 amino acid, for example, about 6 to about 75 amino acid, about 6 to about 50 amino acid, about 6 to about 25 amino acid, about 25 to about 100 amino acid, about 50 to about 100 amino acid or about 75 to about 100 amino acid compositions.In certain embodiment party In case, antimicrobial polypeptide (such as antibacterial polypeptide) can be made of about 15 to about 45 amino acid.In some embodiments, Antimicrobial polypeptide (such as antibacterial polypeptide) is generally cationic.
In some embodiments, antimicrobial polypeptide (such as antibacterial polypeptide) can be generally amphiphilic.? In certain embodiments, antimicrobial polypeptide (such as antibacterial polypeptide) can be generally cationic and amphiphilic. In some embodiments, antimicrobial polypeptide (such as antibacterial polypeptide) can be raw for cell for gram-positive bacterium Long inhibition.In some embodiments, antimicrobial polypeptide (such as antibacterial polypeptide) can be for Gram-positive Bacterium is cytotoxicity.In some embodiments, antimicrobial polypeptide (such as antibacterial polypeptide) can be blue for leather Family name's positive bacteria is cell growth inhibiting and cytotoxicity.In some embodiments, antimicrobial polypeptide is (such as anti- Bacterial peptide) can be for gramnegative bacterium be it is cell growth inhibiting.In some embodiments, antimicrobial It is cytotoxicity that polypeptide (such as antibacterial polypeptide), which can be for gramnegative bacterium,.In some embodiments, resist It is cell growth inhibiting and cell toxicant that antimicrobial polypeptide (such as antibacterial polypeptide), which can be for gram-positive bacterium, Property.In some embodiments, antimicrobial polypeptide can be for virus, fungi, protozoan, helminth, prion Or combinations thereof to be cell growth inhibiting.In some embodiments, antimicrobial polypeptide can be for virus, fungi, Protozoan, helminth, prion or combinations thereof are cytotoxicity.In certain embodiments, antimicrobial polypeptide can be with It is to be directed to virus, fungi, protozoan, helminth, prion or combinations thereof as cell growth inhibiting and cytotoxicity. In some embodiments, antimicrobial polypeptide can be is for tumour or cancer cell (such as human tumour and/or cancer cell) Cytotoxicity.In some embodiments, antimicrobial polypeptide can be for tumour or cancer cell (such as human tumour and/ Or cancer cell) it is cell growth inhibiting.In certain embodiments, antimicrobial polypeptide can be thin for tumour or cancer Born of the same parents' (such as human tumour or cancer cell) are cytotoxicity and cell growth inhibiting.In some embodiments, resist micro- life Object polypeptide (such as antibacterial polypeptide) can be secreted polypeptide.
In some embodiments, antimicrobial polypeptide includes alexin or is made from it.Exemplary alexin include but α-alexin is not limited to (for example, neutrophil leucocyte alexin 1, alexin α 1, neutrophil leucocyte alexin 3, neutrophil leucocyte are anti- Imperial element 4, alexin 5, alexin 6), beta-alexin is (for example, beta-alexin 1, beta-alexin 2, beta-alexin 103, beta-alexin 107, beta-alexin 110, beta-alexin 136) and θ-alexin.In other embodiments, antimicrobial polypeptide includes triumphant Sa Woods rhzomorph (cathelicidin) (for example, hCAP18) is made from it.
Antiviral polypeptide
Antiviral polypeptide (AVP) is the small peptide with variable-length, sequence and structure, and the small peptide, which has, is directed to extensive model The broad spectrum of activity for the virus enclosed.See, e.g. Zaiou, J Mol Med, 2007；85:317.AVP has been displayed with the fast of wide spectrum The killing activity that speed works, together with potential low-level induction of resistance and adjoint extensive anti-inflammatory effect.In some embodiments In, antiviral polypeptide is less than 10kDa, such as less than 8kDa, 6kDa, 4kDa, 2kDa or 1kDa.In some embodiments, resist Virus polypeptide include about 6 to about 100 amino acid, for example, about 6 to about 75 amino acid, about 6 to about 50 amino acid, about 6 to About 25 amino acid, about 25 to about 100 amino acid, about 50 to about 100 amino acid or about 75 to about 100 amino acid or It is made from it.In certain embodiments, antiviral polypeptide includes about 15 to about 45 amino acid or is made from it.In some realities It applies in scheme, antiviral polypeptide is generally cationic.In some embodiments, antiviral polypeptide is generally amphiphilic Property.In certain embodiments, antiviral polypeptide is generally cationic and amphiphilic.In some embodiments In, antiviral polypeptide is cell growth inhibiting for virus.In some embodiments, antiviral polypeptide is for disease Poison is cytotoxicity.In some embodiments, it is cell growth inhibiting and cell that antiviral polypeptide, which is for virus, Toxicity.In some embodiments, antiviral polypeptide be directed toward bacteria, fungi, protozoan, helminth, prion or its Group is combined into cell growth inhibiting.In some embodiments, antiviral polypeptide is directed toward bacteria, fungi, protozoan, posts Infested, prion or combinations thereof is cytotoxicity.In certain embodiments, antiviral polypeptide is directed toward bacteria, fungi, original Lively object, helminth, prion or combinations thereof are cell growth inhibiting and cytotoxicity.In some embodiments, Antiviral polypeptide is cytotoxicity for tumour or cancer cell (such as human cancer cell).In some embodiments, disease-resistant Malicious polypeptide is cell growth inhibiting for tumour or cancer cell (such as human cancer cell).In certain embodiments, resist It is cytotoxicity and cell growth inhibiting that virus polypeptide, which is for tumour or cancer cell (such as human cancer cell),.Some In embodiment, antiviral polypeptide is secreted polypeptide.
Cytotoxic nucleoside
In one embodiment, polynucleotides of the invention, primary construct or mmRNA are in combination with one or more thin Cellular toxicity nucleosides.For example, cytotoxic nucleoside may be incorporated into polynucleotides, primary construct or for example difunctional modification of mmRNA In RNA or mRNA.Cytotoxic nucleoside anticancer agent includes but is not limited to arabinosy ladenosine, cytarabine (cytarabine), arabinose Cytidine (cytosine arabinoside), 5 FU 5 fluorouracil, fludarabine, floxuridine,(Tegafur (tegafur) with the combination of uracil), Tegafur (the fluoro- 1- of (RS) -5- (tetrahydrofuran -2- base) pyrimidine -2,4 (1H, 3H)-two Ketone) and Ismipur.
A large amount of cytotoxic nucleosides are in clinical use like object as anticancer agent or the theme of always clinical test. The example of this kind of analog includes but is not limited to that cytarabine, troxacitabine, Decitabine, replaces and pricks his shore, 2 '-gemcitabine Deoxidation -2 '-methylene cytidine (DMDC), Cladribine, clofarabine, 5-azacitidine, 4 '-thio-cytarabines (4 ' - Thio-aracytidine), cyclopentenyl cytimidine and 1- (2-C- cyano -2- deoxidation-β-D- Arab-furan pentose base) - Cytimidine.Another example of this compound is fludarabine phosphate.These compounds can be applied systematically and can be had The typical side effects of cytotoxic agent, such as, but not limited to relative to proliferative normal cells to the little or no spy of tumour cell It is anisotropic.
This field also reported many prodrugs of the cytotoxic nucleoside like object.Example includes but is not limited to N4- behenyl acyl Oxy-1-β-D- arabino-furanosylcytosine, N4- octadecyl-1- β-D- arabino-furanosylcytosine, N4- palm fibre Palmitic acid acyl group -1- (2-C- cyano -2- deoxidation-β-D- Arab-furan pentose base) cytimidine and P-4055 (cytarabine 5 ' - Elaidic acid ester).In general, these prodrugs can be mainly converted in liver and system circulation active medicine and show seldom or Selectivity release of the active medicine in tumor tissues is not shown.For example, 5 '-deoxidation -5- fluorine cytidines (and be finally 5- fluorine urine Pyrimidine) a kind of prodrug, capecitabine is metabolized in both liver and tumor tissues.It is a series of containing " being easy in physiological conditions The capecitabine analogues of the group of hydrolysis " are claimed by Fujiu et al. (U.S. Patent number 4,966,891) and to draw Mode is incorporated herein.It include the N4 alkyl carbamate and ammonia of 5 '-deoxidation -5- fluorine cytidines by the series that Fujiu is described Base formic acid aralkyl ester and following hint: these compounds will be activated under normal physiological conditions by hydrolysis to provide 5 '- Deoxidation -5- fluorine cytidine.
A series of cytarabine N4- carbamates by Fadl et al. (Pharmazie.1995,50,382-7, to draw Mode is incorporated herein) report, wherein compound is designed to be converted to cytarabine in liver and blood plasma.With the side of reference The WO 2004/041203 that formula is incorporated herein discloses the prodrug of gemcitabine, and some of prodrugs are N4- carbamates.This A little compounds are designed to overcome the gastrointestinal toxicity of gemcitabine and are intended to pass through after absorbing complete prodrug from gastrointestinal tract Hydrolysis release is in liver and blood plasma to provide gemcitabine.Nomura et al. (Bioorg Med.Chem.2003,11,2453- 61, be hereby incorporated herein by) describe the acetal of 1- (3-C- acetenyl-β-D-ribose-furan pentose base) cytimidine Derivative, the acetal derivant generate needs in biological reducing and further hydrolyze in acid condition to generate cytotoxicity The intermediate of nucleoside compound.
The cytotoxic nucleoside that can be used as chemotherapeutant further includes but is not limited to, pyrazolo [3,4-D]-pyrimidine, not fast Purine alcohol, imuran, capecitabine, cytarabine, fluorouracil, purinethol, 6- thioguanine, acyclovir, arabinose gland Glycosides (ara-adenosine), virazole, 7- denitrogenation-adenosine, 7- denitrogenation-guanosine, 6- azepine-uracil, 6- azepine-cytidine, chest The chloro- purine of glycosides ribonucleotide, 5-bromouracil deoxyribose, 2- and inosine or combinations thereof.
Flanking region: non-translational region (UTR)
The non-translational region (UTR) of gene is transcribed but is not translated.5 ' UTR start from transcription initiation site and proceed to Initiation codon but do not include initiation codon；And 3 ' UTR follow terminator codon closely and start and be continued until that tanscription termination is believed Number.About the regulating and controlling effect that UTR rises for the stability of nucleic acid molecules and translation, there are more and more evidences.UTR's Regulation feature may be incorporated into polynucleotides of the invention, primary construct and/or mmRNA the stability for enhancing molecule.Turning Record object can also be incorporated into the special characteristic in the case where being misguided to undesirable organ sites to ensure transcript Controlled downward.
5 ' UTR and translation initiation
Natural 5 ' UTR carries the feature to work in translation initiation.They possess the signature sequence as Kozak sequence, The Kozak sequence is commonly known have during the translation of ribosomes starting many genes it is involved.Kozak sequence has Shared CCR (A/G) CCAUGG, wherein R is purine (adenine or the bird at the base of the upstream of initiation codon (AUG) three Purine), it is later another " G ".It it is known that 5 ' UTR are formed in secondary structure involved in elongation factors combination.
By being engineered the feature usually found in the gene of the abundant expression of specific target organ, can enhance of the invention The stability and protein of polynucleotides, primary construct or mmRNA generate.For example, introducing mRNA (such as white egg of liver expression White, serum amyloid A protein, aPoA/B/E, transferrins, alpha-fetoprotein, hematopoietin or Factor IX) 5 ' UTR can be used for enhancing expression of the nucleic acid molecules (such as mmRNA) in hepatic cell line or liver.Equally, using from other groups 5 ' UTR of specific mrna are knitted to improve the expression in the tissue be possible for the following terms: muscle (MyoD, Myosin, myoglobins, myogenin (Myogenin), power albumen (Herculin)), endothelial cell (Tie-1, CD36), bone marrow cell (C/EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leucocyte (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and pulmonary epithelial cells (SP-A/B/C/D).
Other non-UTR sequences may be incorporated into 5 ' (or 3 ' UTR) UTR.For example, the part of introne or intron sequences can be simultaneously In the flanking region for entering polynucleotides of the invention, primary construct or mmRNA.Protein generation can be increased by being incorporated to intron sequences And mRNA level in-site.
3 ' UTR and element rich in AU
Known 3 ' UTR have be embedded in they among adenosine (ADenosine) and uridine (URidine) section.These are rich in The signature sequence of AU is particularly common in the gene with Gao Gengxin rate.Based on its sequence signature and functional characteristic, it is rich in The element (ARE) of AU can be divided into three classes (Chen et al., 1995): I class ARE is several comprising AUUUA motif in the area rich in U The copy of dispersion.C-Myc and MyoD includes I class ARE.II class ARE has UUAUUUA (U/A) (U/ of two or more overlappings A) nonamer.Molecule containing such ARE includes GM-CSF and TNF-a.The definition of Group III ARE is less clear.These Area rich in U does not include AUUUA motif.C-Jun and myogenin are two examples sufficiently studied of this classification. Known most of protein for combining ARE keep courier unstable, and the member (most significantly HuR) of ELAV family has been demonstrated Increase the stability of mRNA.HuR combines the ARE of all three classes.HuR specific binding site is engineered to the 3 ' of nucleic acid molecules It will lead to HuR combination in UTR, and therefore lead to the stabilisation of internal courier.
Introducing, removal or the modification of element of the 3 ' UTR rich in AU can be used for adjusting polynucleotides of the invention, primary building The stability of body or mmRNA.When being engineered specific polynucleotides, primary construct or mmRNA, can introduce one of ARE or Multiple copies are less stable and thus reduction translation and reduce to make polynucleotides of the invention, primary construct or mmRNA The generation of gained protein.Equally, ARE can be authenticated and remove or be mutated to increase intracellular stability and therefore increase and to turn over Translate the generation with gained protein.Can be used polynucleotides of the invention, primary construct or mmRNA in relevant cell system into Row transfection experiment, and can after transfection different time points measurement protein generate.For example, can be engineered with different ARE Molecule transfects cell, and uses the 6th hour, the 12nd hour, the after the ELISA kit measurement transfection for related protein 24 hours, the 48th hour and 7 days protein generated.
It is incorporated to microRNA binding site
Microrna (or miRNA) is the long non-coding RNA of 19 to 25 nucleotide, the non-coding RNA combination nucleic acid point Son 3 ' UTR and by reduce nucleic acid molecules stability or by inhibit translation come down-regulation of gene expression.Multicore of the invention Thuja acid, primary construct or mmRNA may include one or more Microrna target sequences, microRNA seqeunce or Microrna seed. This kind of sequence can correspond to any of Microrna such as U.S. and announce US2005/0261218 and U.S.'s announcement US2005/ Those of taught in 0059005, the content of the announcement is incorporated herein in its entirety by reference.
MicroRNA seqeunce includes " seed " area, i.e., the sequence in the area of the position 2 to 8 of mature Microrna, the sequence Have perfect Watson-Crick (Watson-Crick) complementary with miRNA target sequence.Microrna seed may include maturation The position 2 to 8 or 2 to 7 of Microrna.In some embodiments, Microrna seed may include that 7 nucleotide are (such as mature The nucleotide 2 to 8 of Microrna), wherein the seed complementation site in corresponding miRNA target is by opposite with Microrna position 1 Adenine (A) flanks.In some embodiments, Microrna seed may include 6 nucleotide (such as core of mature Microrna Thuja acid 2 to 7), wherein the seed complementation site in corresponding miRNA target is by adenine (A) side opposite with Microrna position 1 It connects.See, for example, Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP； Mol Cell.2007 July 6；27 (1): 91-105；The document is respectively incorporated herein in its entirety by reference.It is small The base and target sequence of RNA seed have complete complementarity.By the way that Microrna target sequence to be engineered to multicore glycosides of the invention In 3 ' UTR of acid, primary construct or mmRNA, the molecule for degrading or reducing translation can be targeted, condition be discussed it is micro- Tiny RNA is obtainable.The harm of undershooting-effect when this method is by reduction nucleic acid molecules delivering.Have reported Microrna, Identification (Bonauer et al., Curr the Drug Targets of Microrna target area and its expression pattern and the effect in biology 2010 11:943-949；2011 18:171-176 of Anand and Cheresh Curr Opin Hematol；Contreras and 2012 26:404-413 of Rao Leukemia (.doi:10.1038/leu.2011.356 on December 20th, 2011)；Bartel 2009 136:215-233 of Cell；Landgraf et al., Cell, 2007 129:1401-1414；The document is respectively with reference Mode be integrally incorporated herein).
For example, if nucleic acid molecules are mRNA and are not intended to be delivered to liver but are finally delivered to liver, One or more target sites of miR-122 (a kind of Microrna abundant in liver) are engineered to polynucleotides, primary building In the case where in the 3 ' UTR of body or mmRNA, miR-122 can inhibit the expression of target gene.Different Micrornas can be engineered The introducing of one or more binding sites is to be further reduced service life, the stability of polynucleotides, primary construct or mmRNA And protein translation.
As used herein, term " Microrna site " refers to Microrna target site or Microrna recognition site or small Any nucleotide sequence that RNA is combined or associated.It should be understood that " in conjunction with " can follow traditional Watson-Crick hybridization rule or can Reflect Microrna and target sequence or neighbouring Microrna site at any stable association.
On the contrary, for the purpose of polynucleotides of the invention, primary construct or mmRNA, it can be by Microrna bound site Point engineering from their naturally occurring sequences comes out (that is, removal) to increase the protein expression in specific organization.Example Such as, miR-122 binding site can be removed to improve the protein expression in liver.The regulation of expression in Various Tissues can lead to It crosses and introduces or remove one or several microRNA binding sites to realize.
In wherein regulation mRNA and therefore the example of the tissue of regulation protein expression includes but unlimited known Microrna In liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cell (miR-17-92, miR-126), marrow Cell (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let- 7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204) and pulmonary epithelial cells (let-7,miR-133,miR-126).The also controllable complex biological process of Microrna such as angiogenesis (miR-132) (Anand With 2011 18:171-176 of Cheresh Curr Opin Hematol；It is incorporated herein in its entirety by reference).In this hair In bright polynucleotides, primary construct or mmRNA, can remove or introduce during this kind of involved in Microrna combine Site, so as to for the upper relevant cell type of biology or for background customization polynucleotides, the primary structure of related biological processes Build body or mmRNA expression.The list of Microrna, miR sequence and miR binding site is listed in the following terms: in January, 2013 The U.S. Provisional Application No. 61/ that the table for the U.S. Provisional Application No. 61/753,661 submitted for 17th is submitted on January 18th, 9,2013 The table 7 for the U.S. Provisional Application No. 61/758,921 that 754,159 table 9 and on January 31st, 2013 submit, the application are respective It is incorporated herein in its entirety by reference.
Finally, by understanding expression pattern of the Microrna in different cell types, polynucleotides, primary construct or MmRNA can be directed in particular cell types or only the expression of stronger targeting is engineered under the conditions of particular organisms.Pass through Tissue specificity microRNA binding site is introduced, can design will be for the protein table in tissue or in the background of biotic factor Optimal polynucleotides, primary construct or mmRNA for reaching.Microrna is listed for driving tissue or disease specific Example (Getner and Naldini, the Tissue Antigens.2012,80:393-403 of the purposes of gene expression；With reference Mode is integrally incorporated herein).In addition, Microrna seed site may be incorporated into mRNA to reduce the expression in certain cells, this Cause biological improvement.This example is incorporated to the site miR-142 into the slow virus carrier of expression UGT1A1.miR-142 The presence in seed site reduces the expression in hematopoietic cell, and therefore reduces the expression in antigen presenting cell, so as to cause not In the presence of immune response (Schmitt et al., the Gastroenterology 2010 of the UGT1A1 for expressing viral；139:999- 1007；Gonzalez-Asequinolaza et al. Gastroenterology 2010,139:726-729；Both with reference Mode be integrally incorporated herein).The site miR-142, which is incorporated in modification mRNA, can not only reduce the albumen encoded in hematopoietic cell The expression of matter, and the immune response of the protein to mRNA coding can be reduced or eliminated.By (one, miR-142 seed site Or multiple) be incorporated into mRNA in treatment with complete protein defect (UGT1A1 I type, the patient of LDLR- defect, CRIM- Negative Pang Pei Shi (Pompe) patient etc.) patient in the case where will be important.
Polynucleotides, primary construct or the mmRNA of engineering can be used to carry out transfection experiment in relevant cell system, and And can after transfection different time points measurement protein generate.For example, can be engineered with different microRNA binding sites Polynucleotides, primary construct or mmRNA transfect cell, and are turned using the ELISA kit measurement for related protein 6th hour, the 12nd hour, the 24th hour, the 48th hour, the 72nd hour and 7 days protein generated after dye.It also can be used micro- The molecule of tiny RNA binding site engineering carries out experiment in vivo, so as to detect prepared polynucleotides, primary construct or The variation of the tissue specific expression of mmRNA.
5 ' is capped
5 ' the cap structures of mRNA are related to core output, to increase mRNA stability, and combine mRNA cap binding protein (CBP), the mRNA cap binding protein associated by CBP and poly- (A) binding protein with form mature cyclic annular mRNA species come The mRNA stability and translation ability being responsible in cell.The cap further helps to remove in 5 ' proximal ends during mRNA montage Containing son.
Endogenous mRNA molecule can be 5 ' end it is capped, thus in 5 ' ends of end guanosine cap residue and mRNA molecule It is bonded that 5 '-ppp-5 '-triguaiacyl phosphate is generated between the ariyoshi nucleotide of transcription.Then this 5 '-guanylic acid cap can be methylated To generate N7- methyl-guanosine acid residue.The end at the 5 ' ends of mRNA and/or the core of front end (anteterminal) transcription The ribose of thuja acid is also optionally that 2 '-O- methylate.Being raised one's hat by the hydrolysis of guanylic acid cap structure and the 5 '-of cracking can target To the nucleic acid molecules for degradation, such as mRNA molecule.
It can produce the cap structure of non-hydrolysable to the modification of polynucleotides of the invention, primary construct and mmRNA, thus It prevents from raising one's hat and therefore increases mRNA half-life period.Because cap structure hydrolyzes the cracking for requiring 5 '-ppp-5 ' di-phosphate ester bonded, institute Modified nucleoside acid can be used during capping.For example, from New England Biolabs (Ipswich, MA) Cowpox capping enzyme can according to the manufacturer's instructions with α-it is thio-guanosine nucleotide is used together so as in 5 '-ppp-5 ' cap It is bonded to form thiophosphate.The guanosine nucleotide in addition modified can be used, such as Alpha-Methyl-phosphonate ester and seleno-phosphate core Thuja acid.
In addition modification includes but is not limited to 5 '-ends and/or 5 '-of the mRNA (as described above) on 2 '-hydroxyls of saccharide ring 2 '-O- of the ribose of front end nucleotide methylate.A variety of 5 ' different cap structures can be used for generating nucleic acid molecules such as mRNA points 5 ' caps of son.
Cap analog is also known as synthesis cap analog, chemical cap, chemical cap analog or structure or function cap herein Analog, 5 ' caps are different, while retaining cap function from natural (i.e. endogenous, wild type or physiological) in its chemical structure. It is that cap analog can be chemical (i.e. non-enzymatic) or enzymatic synthesis and/or be connected to nucleic acid molecules.
For example, anti-reversed cap analog (ARCA) cap includes fast by two birds of 5 ' -5 '-triguaiacyl phosphate group connection Purine, one of guanine include N7 methyl and 3 '-O- methyl (i.e. N7,3 '-O- dimethyl-guanosine -5 '-triguaiacyl phosphate - 5 '-guanosine (m7G-3′mppp-G；It can equally be appointed as 3 ' O-Me-m7G (5 ') ppp (5 ') G).Another unmodified bird is fast The 3 ' of purine-O atom becomes the 5 ' terminal nucleotides for being connected to capped nucleic acid molecules (such as mRNA or mmRNA).N7- and 3 '- The guanine of O- methylation provides the end section of capped nucleic acid molecules (such as mRNA or mmRNA).
Another exemplary cap is mCAP, it is similar with ARCA but on guanosine have 2 '-O- methyl (i.e. N7,2 '-O- two Methyl-guanosine -5 '-triguaiacyl phosphate -5 '-guanosine, m7Gm-ppp-G)。
Although cap analog allow nucleic acid molecules in vitro in responsive transcription with capped, up to 20% transcript It can still keep not being capped.The endogenous of such case and cap analog and the nucleic acid generated by endogenous cell transcriptional machinery The architectural difference of 5 ' cap structures can lead to the translation ability and reduced cell stability of reduction.
Polynucleotides, primary construct and mmRNA of the invention can also be capped using enzyme after transcription, truer to generate 5 ' cap structures.As used herein, phrase " more true " refers to and closely reflects or simulate endogenous in structure or function Or the feature of wild-type characteristics.That is, " more true " is characterized in and the phases such as the composite character of the prior art or the like Than, endogenous, wild type, natural or physiological cells function and/or structure more preferable expression, or surpass in one or more aspects Corresponding endogenous, wild type, natural or physiological characteristic feature.The non-limiting reality of more true 5 ' cap structure of the invention Example is to remove other business compared with 5 ' cap structures of synthesis known in the art (or with wild type, natural or 5 ' cap structure of physiology) Except the combination of enhancing with cap binding protein, increased half-life period, to the sensibility of 5 ' endonuclease reductions and/or subtract Few 5 ' those of are raised one's hat example.For example, recombined vaccinia virus capping enzyme and 2 '-O- transmethylases of recombination can be the 5 ' of mRNA 5 ' -5 '-triguaiacyl phosphate that specification is formed between terminal nucleotide and guanine cap nucleotide is bonded, wherein the cap guanine packet 5 ' the terminal nucleotides containing N7 methylation and mRNA include 2 '-O- methyl.This structure is referred to as 1 structure of cap.This cap is led Cause higher translation ability and cell stability and reduction compared with other 5 ' cap analog structures for example known in the art Cell proinflammatory cytokine activation.Cap structure includes but is not limited to 7mG (5 ') ppp (5 ') N, pN2p (cap 0), 7mG (5 ') Ppp (5 ') N1mpNp (cap 1) and 7mG (5 ')-ppp (5 ') N1mpN2mp (cap 2).
Because polynucleotides, primary construct or mmRNA can be capped after transcription, and because this method is more effective , so can be capped close to 100% polynucleotides, primary construct or mmRNA.This with during responsive transcription in vitro About 80% when cap analog is connected to mRNA is contrasted.
According to the present invention, 5 ' end caps may include endogenous cap or cap analog.According to the present invention, 5 ' end caps may include Guanine analog.Useful guanine analog includes but is not limited to that inosine, N1- methyl-guanosine, 2 ' fluoro- guanosines, 7- are de- Nitrogen-guanosine, 8- oxo-guanosine, 2- amino-guanosine, LNA- guanosine and 2- azido-guanosine.
Virus sequence
Other virus sequence, such as, but not limited to luteovirus (BYDV-PAV), pulmonary adenomatosis reverse transcription The translational enhancer sequence of viral (JSRV) and/or the intranasal tumour virus of region is (see, for example, international publication number WO2012129648；Be incorporated herein in its entirety by reference), can be engineered and be inserted into polynucleotides of the invention, just In the 3 ' UTR of grade construct or mmRNA, and the translation of construct can be stimulated in vitro and in vivo.It can be in relevant cell system It carries out transfection experiment and ELISA can be passed through within the 12nd hour, the 24th hour, the 48th hour, the 72nd hour and the 7th day after transfection Protein is measured to generate.
IRES sequence
Further it is provided that may include polynucleotides, primary construct or the mmRNA of internal ribosome entry site (IRES). Feature picornavirus RNA, IRES is identified as first to synthesize in the case where 5 ' cap structure is not present in protein Starting in play an important role.IRES can be used as unique ribosome bind site or can be used as multiple ribosome binding sites of mRNA One of point.Polynucleotides containing more than one functional ribosomes binding site, primary construct or mmRNA codified are independent Several peptides or polypeptide (" polycistron nucleic acid molecules ") that ground is translated by ribosomes.When polynucleotides, primary construct or When mmRNA has IRES, optionally further providing second can translated region.The example of IRES sequence that can be used according to the invention Including but not limited to from those of following virus: picornavirus (such as FMDV), insect viruses (CFFV), spinal cord Poliovirus (PV), encephalomyocarditis virus (ECMV), foot and mouth disease virus (FMDV), Hepatitis C Virus (HCV), classical swine fever Viral (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV) or cricket paralysis virus (CrPV).
Poly-A tail
In RNA process, long-chain adenylic acid (poly-A tail) can be added to polynucleotides such as mRNA points Son is to increase stability.After following transcription closely, 3 ' ends of transcript can be cleaved to discharge 3 ' hydroxyls.Then poly- A polymerase Adenylic acid chain is added to RNA.Referred to as polyadenylation process addition can such as about 100 and 250 it Between the long poly-A tail of residue.
It has been found that unique poly-A tail length degree is certain to polynucleotides of the invention, primary construct or mmRNA offer Advantage.
In general, it is long to be greater than 30 nucleotide for the length of poly-A tail of the invention.In another embodiment, Poly-A tail be greater than in length 35 nucleotide (for example, at least or greater than about 35,40,45,50,55,60,70,80,90, 100、120、140、160、180、200、250、300、350、400、450、500、600、700、800、900、1,000、1,100、 1,200,1,300,1,400,1,500,1,600,1,700,1,800,1,900,2,000,2,500 and 3,000 nucleosides Acid).In some embodiments, polynucleotides, primary construct or mmRNA include about 30 to about 3,000 nucleotide (example Such as, 30 to 50,30 to 100,30 to 250,30 to 500,30 to 750,30 to 1,000,30 to 1,500,30 to 2,000,30 to 2,500,50 to 100,50 to 250,50 to 500,50 to 750,50 to 1,000,50 to 1,500,50 to 2,000,50 to 2, 500,50 to 3,000,100 to 500,100 to 750,100 to 1,000,100 to 1,500,100 to 2,000,100 to 2,500, 100 to 3,000,500 to 750,500 to 1,000,500 to 1,500,500 to 2,000,500 to 2,500,500 to 3,000,1, 000 to 1,500,1,000 to 2,000,1,000 to 2,500,1,000 to 3,000,1,500 to 2,000,1,500 to 2,500, 1,500 to 3,000,2,000 to 3,000,2,000 to 2,500 and 2,500 to 3,000).
In one embodiment, it is designed relative to the length of overall polynucleotides, primary construct or mmRNA Poly-A tail.This design can be length, the length of special characteristic or area (such as the firstth area or flanking region) based on code area Or the length based on the final product expressed from polynucleotides, primary construct or mmRNA.
In this background, poly-A tail can be greater than polynucleotides, primary construct or mmRNA or its feature in length 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%.Poly-A tail is also designed to it A part of affiliated polynucleotides, primary construct or mmRNA.In this background, poly-A tail can be construct Total length or construct subtract the total length of poly-A tail 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% or more.In addition, the bound site of polynucleotides, primary construct or mmRNA for the engineering of poly- A conjugated protein Point and conjugation can Enhanced expressings.
In addition, the modification of 3 ' ends of poly-A tail can be used in multiple and different nucleotide, primary construct or mmRNA Nucleotide is connected to PABP (poly- A binding protein) by 3 ' ends together.Can be carried out in relevant cell system transfection experiment and Protein can be measured by ELISA within the 12nd hour, the 24th hour, the 48th hour, the 72nd hour and the 7th day after transfection to generate.
In one embodiment, polynucleotides primary construct of the invention is designed to include polyA-G tetrad. G tetrad is that the cycloalkyl hydroperoxide for four guanylic acids that can be formed by the sequence rich in G in both DNA and RNA is bonded Array.In this embodiment, G tetrad is incorporated in the end of poly-A tail.It is directed to stability, albumen in different time points Matter generates and the other parameters including half-life period are measured gained mmRNA construct.It has been found that polyA-G tetrad makes It obtains protein and generates the generation of protein observed by the poly-A tail for being equivalent to and 120 nucleotide being used alone at least 75%.
Quantization
In one embodiment, multicore glycosides of the invention can be quantified in the allochthon for being originated from one or more body fluid Acid, primary construct or mmRNA.As used herein, " body fluid " includes peripheral blood, serum, blood plasma, ascites, urine, celiolymph (CSF), phlegm, saliva, marrow, synovia, aqueous humor, amniotic fluid, earwax, breast milk, BAL fluid, sperm, prostatic fluid, examine Amber liquid (cowper ' s fluid) or pre-firing sperm, sweat, fecal matter, hair, tear, cyst fluid, pleura and peritoneal fluid, pericardium Liquid, lymph, chyme, chyle, bile, interstitial fluid, menses, fester, sebum, vomitus, vaginal fluid, mucosal secretion, Watery stools, pancreatic juice, the eluate from sinus cavities, broncho-pulmonary aspirate, blastocyst cavity liquid and Cord blood.Alternatively, can be from Organ selected from the group being made up of captures allochthon: lung, heart, pancreas, stomach, intestines, bladder, kidney, ovary, spermary, skin, Colon, breast, prostate, brain, esophagus, liver and placenta.
In quantization method, the sample no more than 2mL is obtained from subject and by size exclusion chromatography, density level bands Degree centrifugation, differential centrifugation, nanometer film ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation or combinations thereof are outer to separate Carry out body.In analysis, the level or concentration of polynucleotides, primary construct or mmRNA can be the table of applied construct Up to level, exists, is not present, truncates or changes.Advantageously, make it is described it is horizontal with one or more clinical phenotypes or be used for The measurement of human diseases biomarker is related.It is described measure can be used construct specific probe, cell count, qRT-PCR, Real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrography or combinations thereof carry out, while immunohistochemical method such as enzyme can be used Linked immunosorbent assay (ELISA) method separates allochthon.Size exclusion chromatography, density gradient centrifugation, differential can also be passed through Centrifugation, nanometer film ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation or combinations thereof separate allochthon.
These methods give polynucleotides, primary construct or the mmRNA that researcher's real-time monitoring is remaining or is delivered Horizontal ability.This be it is possible because polynucleotides of the invention, primary construct or mmRNA due to structural modification or Chemical modification and be different from endogenous form.
The design and synthesis of II.mmRNA
It can be according to any available skill for polynucleotides used according to the invention, primary construct or mmRNA Prepared by art, the technology includes but is not limited to chemical synthesis, enzymatic synthesis, the commonly known as in-vitro transcription of longer precursor (IVT) or enzyme or chemical cracking.The method for synthesizing RNA is known in the art (see, for example, Gait, M.J. (editor) Oligonucleotide synthesis:a practical approach, Oxford [Oxfordshire], Washington, DC:IRL Press, 1984；And Herdewijn, P. (editor) Oligonucleotide synthesis: Methods and applications, Methods in Molecular Biology, volume 288 (Clifton, N.J.) Totowa, N.J.:Humana Press, 2005；It is both hereby incorporated herein by).
The method of design and synthesis primary construct of the invention include gene constructed, mRNA generate (with or without modification) and The step of purifying.In enzymatic synthesis method, the target polynucleotide sequence of selection encoding target polypeptide is for being incorporated to carrier first In, the carrier will be expanded to generate cDNA template.Optionally, target polynucleotide sequence and/or any flanking sequence can Carry out codon optimization.Then mRNA is generated by the way that (IVT) is transcribed in vitro using cDNA template.After generation, mRNA can be undergone Purifying and purification process.The step provides in further detail below.
It is gene constructed
Gene constructed step may include but be not limited to gene chemical synthesis, vector amplification, plasmid purification, plasmid linearization and net Change and cDNA templated synthesis and purification.
Gene chemical synthesis
Once target polypeptides or target are selected for generating, primary construct is just designed.In primary construct, coding The open reading frame (ORF) of selected nucleic acid (DNA or RNA) transcript can be used to carry out for firstth area of the connection nucleosides of target polypeptides Building.ORF may include wild type ORF, its isoform, variant or segment.As used herein, " open reading frame " or " ORF " anticipates Think of is the nucleic acid sequence (DNA or RNA) for referring to encoding target polypeptide.ORF often begins with initiation codon ATG and terminates In nonsense or terminator codon or signal.
In addition, the nucleotide sequence in the firstth area can carry out codon optimization.Codon optimization method is known in the art And it is applicable in the work for realizing one or more of several targets.These targets include matching target and host organism Codon frequency in body is to ensure correctly to fold；Biasing G/C content is to increase mRNA stability or reduce secondary structure；It is minimum Gene constructed or expression tandem sequence repeats codon or base operation may be damaged by changing, and customized transcription and translation control area, inserted Enter or remove protein import sequence, in the protein of coding removal/addition posttranslational modification site (such as glycosylation position Point), addition, removal or shuffled proteins matter structural domain are inserted into or lack restriction site, modify ribosome bind site and mRNA Degradation site, to adjust translation rate to allow the different structure territory of protein correctly to fold, or to reduce or to disappear Except the problem secondary structure in mRNA.Codon optimization tool, algorithm and service are known in the art, non-limiting example packets Include service, DNA2.0 (Menlo Park CA) and/or proprietary method from GeneArt (Life Technologies).? In one embodiment, optimize ORF sequence using optimization algorithm.Codon selection for every kind of amino acid is given in Table 1.
The selection of 1. codon of table
The feature that can be considered as beneficial in some embodiments of the present invention can be encoded by primary construct and can Positioned at the side ORF as first or second flanking region.The flanking region can be incorporated to primary before or after the optimization of ORF In construct.Not requiring primary construct includes both 5 ' and 3 ' flanking regions.The example of this category feature includes but is not limited to non-turns over It translates area (UTR), Kozak sequence, oligo (dT) sequence and detectable label and may include more grams for can having XbaI to identify Grand site.
In some embodiments, 5 ' UTR and/or 3 ' UTR may be provided as flanking region.Multiple 5 ' or 3 ' UTR may include In flanking region and it can have identical or different sequence.Any part (including no any part) of flanking region can be into Row codon optimization and any part can be before or after codon optimizations independently comprising one or more different Structural modification or chemical modification.The combination of feature may include that in the first flanking region and the second flanking region and may be included in other In feature.For example, ORF can be by may include 5 ' UTR of strong Kozak translation initiation signal and/or may include adding for templating 3 ' the UTR of oligo (dT) sequence of poly-A tail are flanked.5 ' UTR may include from identical and/or different genes the first multicore Acid fragments and the second polynucleotide passage, the 5 ' UTR as described in U.S. Patent Application Publication No. 20100293625, institute Patent application is stated to be incorporated herein in its entirety by reference.
Table 2 and table 3 provide the list that can be used as flanking region for the exemplary UTR in primary construct of the invention.Table 2 In the lists of 5 ' non-translational regions of the invention is shown.The variant of 5 ' UTR can be used, wherein one or more nucleotide are added into End or removal, including A, T, C or G.
2. 5 ' non-translational region of table
The exemplary lists of 3 ' non-translational regions of the invention are shown in table 3.The variant of 3 ' UTR can be used, one of them or Multiple nucleotide are added into end or removal, including A, T, C or G.
3. 3 ' non-translational region of table
It should be understood that those listed in above-mentioned table is example and any UTR from any gene may be incorporated into primary In the corresponding first or second flanking region of construct.In addition, multiple wild type UTR of any known gene can be used.It provides not Artificial UTR for the variant of wild type gene is also within the scope of the invention.These UTR or part thereof can be placed in they selected by From transcript in it is identical orientation or can be changed in orientation or position.Therefore, 5 ' or 3 ' UTR can be inverted, shorten, be prolonged It is long, chimeric with one or more of the other 5 ' UTR or 3 ' UTR.As used herein, term " change " is when it is related to UTR sequence Mean that the UTR changes relative to reference sequences in some aspects.For example, 3 ' or 5 ' UTR can be taken by what is as above instructed To or position variation and change relative to wild type or natural UTR or can by other nucleotide include, nucleotide lacks It loses, the exchange of nucleotide or indexing change.Any of these variations for generating " change " UTR (either 3 ' or 5 ') include Variant UTR.
In one embodiment, dual, triple or quadruple UTR such as 5 ' or 3 ' UTR can be used.As used herein, " double Weight " UTR is two copy series connection of wherein same UTR or a kind of UTR of generally tandem coding.For example, can be such as United States Patent (USP) Dual 3 ' UTR of beta-globin is used described in announcing 20100129877, disclosure is whole by reference simultaneously Enter herein.
It is also within the scope of the invention with patterned UTR.As used herein, " patterned UTR " is that reflection repeats Or alternating pattern is as being repeated once, twice or the ABABAB more than 3 times or AABBAABBAABB or ABCABCABC or its variant Those UTR.In these patterns, each letter A, B or C indicate the different UTR on nucleotide level.
In one embodiment, flanking region be selected from its protein share common function, structure, property feature transcript Family.For example, target polypeptides can belong to the albumen sometime expressed in specific cells, tissue or in growth course The family of matter.Any other UTR of the commutative identical or different protein families of UTR from any of these genes is to form New chimeric primary transcript.As used herein, " protein families " are in a broad sense for referring to shared at least one function, knot Structure, feature, positioning, origin or expression pattern two or more target polypeptides group.
After optimization (if necessary), primary construct component is reconstructed and is transformed into carrier such as, but not limited to, matter In grain, virus, clay and artificial chromosome.For example, the construct of optimization can be reconstructed and to be transformed into Competent big In enterobacteria, yeast, Neurospora, maize, Drosophila etc., wherein high copy number plasmid sample or chromosome structure pass through this paper institute The method of description occurs.
Non-translational region may also include translational enhancer element (TEE).As non-limiting examples, TEE may include U.S. Shen Please be those of described in numbers 20090226470 and those of known in the art, the application is incorporated hereby Herein.
Terminator codon
In one embodiment, primary construct of the invention can include at least two before 3 ' non-translational regions (UTR) A terminator codon.Terminator codon can be selected from TGA, TAA and TAG.In one embodiment, primary construct of the invention Include terminator codon TGA and an other terminator codon.In another embodiment, terminator codon in addition can be with It is TAA.In another embodiment, primary construct of the invention includes three terminator codons.
Vector amplification
Then the carrier containing primary construct is expanded and using methods known in the art such as, but not limited to use Invitrogen PURELINKTMA large amount of preparations (maxi prep) of HiPure Maxiprep kit (Carlsbad, CA) To separate and plasmid purification.
Plasmid linearization
Then methods known in the art can be used such as, but not limited to use restriction enzyme and buffer by plasmid Linearisation.The method including such as the following terms can be used to carry out purified linearization reaction: Invitrogen ' s PURELINKTM The small kit of PCR (Carlsbad, CA) and the purification process based on HPLC, such as, but not limited to, strong anion exchange HPLC, weak Anion exchange HPLC, reversed-phase HPLC (RP-HPLC) and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen Standard PURELINKTMPCR kit (Carlsbad, CA).Purification process may depend on the size of carried out linearisation reaction It modifies.Then the cDNA for (IVT) reaction to be transcribed in vitro is generated using the plasmid of linearisation.
CDNA templated synthesis
CDNA template can be synthesized by making plasmid experience polymerase chain reaction (PCR) of linearisation.Table 4 is applicable The list of primer and probe in PCR reaction of the invention.It should be understood that the list is not detailed and for any expansion The primer-probe of increasing designs in the range of those skilled in the art.Probe also may include the base of chemical modification to increase Base pairing fidelity and base pairing intensity to target molecule.This kind of modification may include 5- Methyl-Cytidine, 2,6-, bis--amino- Purine, 2 '-fluorine, thiophosphate or lock nucleic acid.
4. primer and probe of table
* UFP is general forward primer；URP is general reverse primer.
In one embodiment, cDNA can be submitted before experience transcription for sequencing analysis.
MRNA is generated
The method of mRNA or mmRNA may include but be not limited to, be transcribed in vitro, cDNA template removal and RNA purification and MRNA is capped and/or tailings reactions.
It is transcribed in vitro
(IVT) system of in-vitro transcription can be used to be transcribed for the cDNA generated in step before.The system is usually wrapped Include transcription buffer, nucleotide triphosphoric acid (NTP), RNase inhibitor and polymerase.NTP can make manufacture by oneself, can be selected from supplying Quotient can synthesize as described herein.NTP may be selected from but not limited to those of described herein including natural and non-natural (modification) NTP.Polymerase may be selected from but not limited to T7 RNA polymerase, T3 RNA polymerase and mutated polymerase as but not It is limited to that the polymerase of modification of nucleic acids can be incorporated to.
RNA polymerase
In the design of any amount of RNA polymerase or variant primary construct for use in the present invention.
RNA polymerase can be modified by being inserted into or lacking the amino acid of RNA polymerase sequence.As non-limiting Example, RNA polymerase may be trimmed to show increased to be incorporated to 2 ' modified nucleosides compared with unmodified RNA polymerase The ability of sour triphosphoric acid is (referring to international publication WO2008078180 and United States Patent (USP) 8,101,385；By reference integrally simultaneously Enter herein).
It can be by evolution RNA polymerase, optimization RNA polymerase amino acid and/or nucleic acid sequence and/or by using ability Other methods known to domain obtain variant.As non-limiting examples, T7 RNA polymerase variant can be used by Esvelt etc. People (Nature (2011) 472 (7344): 499-503；Be incorporated herein in its entirety by reference) propose continuously-directional evolve System is evolved, wherein at least one mutation of clone's codified of T7 RNA polymerase, such as, but not limited to, relying at position 93 Propylhomoserin be substituted by threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S、G175R、H176L、Y178H、F182L、L196F、G198V、D208Y、E222K、S228A、Q239R、T243N、 G259D、M267I、G280C、H300R、D351A、A354S、E356D、L360P、A383V、Y385C、D388Y、S397R、 M401T、N410S、K450R、P451T、G452V、E484A、H523L、H524N、G542V、E565K、K577E、K577M、 N601S, S684Y, L699I, K713E, N748D, Q754R, E775K, A827V, D851N or L864F.It is unrestricted as another Property example, T7 RNA polymerase variant codified as described in U.S. Publication No 20100120024 and 20070117112 extremely A few mutation；The announcement is incorporated herein in its entirety by reference.The variant of RNA polymerase may also include but be not limited to, Variant, conserved amino acid is replaced to replace, be inserted into variant, deletion mutants and/or covalent variant.
In one embodiment, primary construct may be designed to be identified by wild type or variant RNA polymerase.It does so When, primary construct can be modified into site or area comprising the sequence variation from wild type or parent's primary construct.
In one embodiment, primary construct be designed to RNA polymerase combine or recognition site upstream, RNA polymerase combines or the downstream of recognition site, the upstream of TATA box sequence, primary construct TATA box sequence downstream but The upstream of the code area of primary construct, include in 5 ' UTR, before 5 ' UTR and/or after 5 ' UTR at least one replace and/or Insertion.
In one embodiment, 5 ' UTR of primary construct can pass through at least one area of the nucleotide of same base And/or string insertion and be replaced.The area of nucleotide and/or string may include but be not limited at least three, at least four, at least five, At least six, at least seven or at least eight nucleotide and the nucleotide can be natural and/or non-natural.As non- Limitative examples, the group of nucleotide may include 5 to 8 adenines, cytimidine, thymidine, disclosed herein any other String of nucleotide and/or combination thereof.
In one embodiment, 5 ' UTR of primary construct can pass through at least the two of the nucleotide of two different bases The insertion of a area and/or string and be replaced, the base is such as, but not limited to that adenine, cytimidine, thymidine, institute is public herein Any other nucleotide opened and/or combination thereof.For example, 5 ' UTR can be by being inserted into 5 to 8 adenine bases, being then inserted into 5 It is replaced to 8 cytosine bases.In another example, 5 ' UTR can be by being inserted into 5 to 8 cytosine bases, being then inserted into 5 to 8 adenine bases are replaced.
In one embodiment, primary construct may include under the transcription initiation site that can be identified by RNA polymerase At least one of trip replaces and/or insertion.As non-limiting examples, at least one replaces and/or insertion can be tight by replacing At least one nucleic acid in the area of transcription initiation site downstream (such as, but not limited to,+1 to+6) comes under transcription initiation site Trip occurs.Variation against the area of the neucleic acid in transcription initiation site downstream can influence initial rate, increase apparent nucleotide three Phosphoric acid (NTP) reacts steady state value and increases the dissociation of short transcript and transcription complex initially to be turned to eliminate (curing) Record (Brieba et al., Biochemistry (2002) 41:5144-5149；It is incorporated herein in its entirety by reference).At least one Modification, substitution and/or the insertion of a nucleic acid can cause the silent mutation of nucleic acid sequence or can cause the mutation in amino acid sequence.
In one embodiment, primary construct may include at least one in transcription initiation site downstream, at least two, At least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least 11, extremely The substitution of few 12 or at least 13 guanine bases.
In one embodiment, primary construct may include against transcription initiation site downstream area at least one, The substitution of at least two, at least three, at least four, at least five or at least six guanine base.As non-limiting examples, such as Nucleotide in area described in fruit is GGGAGA, then guanine base can be by least one, at least two, at least three or at least four Adenylic acid replaces.In another non-limiting example, if the nucleotide in the area is GGGAGA, bird is fast Purine base can be replaced by least one, at least two, at least three or at least four cytosine base.In another non-limiting example In, if the nucleotide in the area is GGGAGA, guanine base can be by least one, at least two, at least three or extremely Few 4 thymidines and/or any nucleotide described herein replace.
In one embodiment, primary construct may include at least one substitution of upstream from start codon and/or insert Enter.For clarity, it will be appreciated by those skilled in the art that initiation codon is first codon of protein coding region, and turn Record initiation site is the site that transcription starts.Primary construct may include but be not limited at least one of nucleotide base, at least 2 A, at least three, at least four, at least five, at least six, at least seven or at least eight replace and/or insertion.Nucleotide base can It is inserted into or takes at 1 of upstream from start codon, at least one, at least two, at least three, at least four or at least five position Generation.The nucleotide for being inserted into and/or replacing can be identical base (for example, entirely A or entirely C or entirely T or whole G), two different bases (for example, A and C, A and T or C and T), three kinds of different bases (for example, A, C and T or A, C and T) Or at least four different base.As non-limiting examples, the guanine base of the upstream of coding region in primary construct can Replaced by adenine, cytimidine, thymidine or any nucleotide described herein.In another non-limiting example, The substitution of guanine base in primary construct can be designed to be retained in the area in transcription initiation site downstream and A guanine base before initiation codon is (referring to Esvelt et al. .Nature (2011) 472 (7344): 499- 503；It is incorporated herein in its entirety by reference).As non-limiting examples, at least five nucleotide can be inserted into transcription initiation position At point downstream but 1 position of upstream from start codon and at least five nucleotide can be same base type.
The removal of cDNA template and purification
Usable methods known in the art are such as, but not limited to, handled with deoxyribonuclease I (DNA enzymatic I) to remove CDNA template.RNA purification may also include purification process, such as, but not limited to, from Beckman Coulter (Danvers, MA) System, the exchange of the purification process based on HPLC, such as, but not limited to, strong anion HPLC, weak anionic exchange HPLC, reversed-phase HPLC (RP-HPLC) and hydrophobic interaction HPLC (HIC-HPLC).
Capped and/tailings reactions
Primary construct or mmRNA can also undergo capped and/or tailings reactions.Capping can be by known in the art Method come carry out so as to by 5 ' caps be added to primary construct 5 ' end.It include but is not limited to use cowpox for capped method Capping enzyme (New England Biolabs, Ipswich, MA).
Poly- A tailings reactions can be by methods known in the art such as, but not limited to, 2 ' O- transmethylases and such as this paper institute The method of description carries out.It, can be it is beneficial that first in purification if the primary construct from cDNA generation does not include poly- T Poly- A tailings reactions are carried out before grade construct.
MRNA purifying
Primary construct or mmRNA purifying may include but be not limited to mRNA or mmRNA purification, quality assurance and quality control System.MRNA or mmRNA purification can be by methods known in the art such as, but not limited to,Bead (Beckman Coulter Genomics, Danvers, MA), poly- T bead, LNATMOligo-T capture probe (Inc, Vedbaek, Denmark), or the such as, but not limited to, strong anion exchange of the purification process based on HPLC HPLC, weak anionic exchange HPLC, reversed-phase HPLC (RP-HPLC) and hydrophobic interaction HPLC (HIC-HPLC) to carry out. Term " purifying " about polynucleotides when using as " mRNA or mmRNA of purifying " refers to and at least one separated from contaminants Polynucleotides.As used herein, " pollutant " is to make any substance that another substance is inappropriate, impure or low-quality.Cause This, the polynucleotides (such as DNA and RNA) of purifying are in the form of being different from finding it in nature or in the form of arrangement or cloth Set or different from make it be subject to processing purification process before existing form or arrangement form or arrangement exist.
Such as, but not limited to gel electrophoresis, UV absorbance or analytic type can be used in quality assurance and/or quality control checking The method of HPLC carries out.
In another embodiment, mRNA or mmRNA can be surveyed by including but is not limited to the method for reverse transcriptase PCR Sequence.
In one embodiment, such as, but not limited to uv-vis spectra (UV/Vis) can be used in mRNA or mmRNA Method quantifies.The non-limiting example of UV/Vis spectrometer isSpectrometer (ThermoFisher, Waltham, MA).The mRNA or mmRNA of quantization can be analyzed to determine whether the mRNA or mmRNA can have suitably Size checks that there is no the degradations of the mRNA or mmRNA.The degradation of mRNA or mmRNA can check by the following method, Such as, but not limited to agarose gel electrophoresis, the such as, but not limited to, strong anion of the purification process based on HPLC exchange HPLC, weak yin Ion exchange HPLC, reversed-phase HPLC (RP-HPLC) and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrography (LCMS), Capillary Electrophoresis (CE) and capillary gel electrophoresis (CGE).
Signal sequence
Primary construct or mmRNA are also encoded with the other feature for helping transport polypeptide treatment related locus.Side Such a feature for helping protein import is signal sequence.As used herein, " signal sequence " or " signal peptide " is long respectively Degree is the polynucleotides or polypeptide of about 9 to 200 nucleotide (3 to 60 nucleic acid), and the polynucleotides or polypeptide are respectively incorporated into Code area or 5 ' (or ends N-) of encoded polypeptide.Adding these sequences makes encoded polypeptide by one or more Secretory pathway is transported to endoplasmic reticulum.Some signal sequences pass through signal peptidase from protein cleavage after protein is transported.
Table 5 is that can be incorporated into that for being believed by the protein of polynucleotides of the invention, primary construct or mmRNA coding The exemplary lists of number sequence.
5. signal sequence of table
In the table, SS is secretion signal and MLS is mitochondria targeting signal.Primary construct of the invention or MmRNA may be designed to any signal sequence or its segment or variant of coding SEQ ID NO 94 to 155.These sequences may include In the beginning of polypeptid coding area, centre or end or alternatively into flanking region.In addition, any polynucleotides of the invention Primary construct also may include the one or more sequences defined by SEQ ID NO 32-93.These can be in the firstth area or any In flanking region.
The other signal sequence that can be used in the present invention includes for example in database such as http: // Www.signalpeptide.de/ or http://proline.bic.nus.edu.sg/spdb/ is instructed in those of finding Those of.It those of is described in United States Patent (USP) 8,124,379,7,413,875 and 7,385,034 also in the scope of the present invention It is interior, and the respective content of the patent is incorporated herein in its entirety by reference.
Target selection
According to the present invention, primary construct includes to encode at least the firstth area of the connection nucleosides of at least one target polypeptides. Target polypeptides or " target " of the invention are listed in table 6.In addition to the Name and Description of the gene of encoding target polypeptide, table The transcript for showing ENSEMBL transcript ID (ENST), ENSEMBL protein ID (ENSP) in 6 and optimizing when can get Serial ID (Optim Trans SEQ ID) or the open reading frame sequence ID (Optim ORF SEQ ID) of optimization.For any One or more variants or isoform may be present in specific gene.In the presence of these, they are also shown in table.This field The skilled person will understand that being potential flanking region disclosed in the table.These 5 ' (upstreams) of ORF or code area or 3 ' (under Trip) each ENST transcript in encode.By instructing ENSP sequence clearly and disclosing particularly code area.Therefore, it is taught The sequence of the side for the sequence in coding protein led is considered as flanking region.It is also possible to can by using one or more 5 ' and 3 ' flanking regions are further characterized for the database that uses or algorithm.Database annotation is contained in the side of ENST transcript Feature in pterion and these be obtainable in this field.
6. target of table
Protein cleavage signal and site
In one embodiment, polypeptide of the invention may include at least one containing at least one protein cleavage site A protein cleavage signal.Protein cleavage site can be located at the end N-, and the end C- is any between the end N- and the end C- At space such as, but not limited to, the centre between the end N- and the end C-, between the end N- and intermediate point, in intermediate point and C- Between end, with and combinations thereof.
Polypeptide of the invention may include but be not limited to proprotein convertases (or prohormone convertase), fibrin ferment or Xa factor Protein cleavage signal.Proprotein convertases are the families of nine kinds of protease, and the protease includes related with yeast kexin Seven kinds of basic amino acid specificity bacillus subtilis protein sample serine proteases, are referred to as 1/3 (PC1/ of prohormone convertase 3), PC2, furin, PC4, PC5/6, pairs of basic amino acid lyases 4 (PACE4) and PC7；With it is residual in non-alkaline Two kinds of other novel subtilases of Ji Chu cracking, are referred to as subtilopeptidase A kexin isoazyne 1 (SKI-1) and preceding egg White invertase subtilopeptidase A kexin 9 (PCSK9).The non-limiting example of protein cleavage signal amino acid sequence exists It is listed in table 7.In table 7, " X " refers to any amino acid, and " n " can be 0,2,4 or 6 amino acid and " * " is finger protein Matter cracking site.In table 7, SEQ ID NO:21426 refers to as n=4 and SEQ ID NO:21427 refers to and works as n=6 When.
7. protein cleavage site sequence of table
In one embodiment, primary construct of the invention and mmRNA can be engineered so that the primary structure It builds body or mmRNA includes the protein cleavage signal of at least one coding.The protein cleavage signal of the coding can be located at Before beginning codon, after initiation codon, before code area, in code area be such as, but not limited to code area among, starting it is close Between numeral and intermediate point, between intermediate point and terminator codon, after code area, before terminator codon, two terminations are close Between numeral, after terminator codon and combinations thereof.
In one embodiment, primary construct of the invention or mmRNA may include splitting containing at least one protein Solve the protein cleavage signal of at least one coding in site.Before the protein cleavage signal of the coding may include but be not limited to Convertase (or prohormone convertase), fibrin ferment and/or Xa factor protein cleavage signal.Those skilled in the art can make The protein cleavage signal of coding appropriate is determined with the above table 1 or other known method so as to be included in it is of the invention just Grade construct or mmRNA in.For example, starting and considering the codon of table 1 with the signal in table 7, may be configured to can be in institute Obtain the signal that the primary construct of protein signal is generated in polypeptide.
In one embodiment, polypeptide of the invention includes at least one protein cleavage signal and/or site.
As non-limiting examples, U.S. Patent number 7,374,930 and U.S. Publication No 20090227660 (with reference Mode is integrally incorporated herein) it is cracked using Furin cleavage site in the expression product of the golgiosome from cell The N-terminal methionine of GLP-1.In one embodiment, polypeptide of the invention include at least one protein cleavage signal and/ Or site, condition are that the polypeptide is not GLP-1.
In one embodiment, primary construct of the invention or mmRNA include that the protein of at least one coding is split Solve signal and/or site.
In one embodiment, primary construct of the invention or mmRNA include that the protein of at least one coding is split Signal and/or site are solved, condition is that the primary construct or mmRNA do not encode GLP-1.
In one embodiment, primary construct of the invention or mmRNA may include more than one code area.Multiple In the case that code area is present in primary construct or mmRNA of the invention, the multiple code area can be by the albumen that encodes Matter cracking site separates.As non-limiting examples, primary construct or mmRNA can the form of orderly pattern indicate.In this way A kind of pattern follows AXBY form, wherein A and B be can be identical or different code area and/or the identical or different polypeptide of codified Code area, and X and Y are the protein cleavage signals of the coding of the identical or different protein cleavage signal of codified.Second This pattern follows form AXYBZ, wherein A and B be can be identical or different code area and/or the identical or different polypeptide of codified Code area, and X, Y and Z are the protein cleavage signals of the coding of the identical or different protein cleavage signal of codified.The Three patterns follow form ABXCY, wherein A, B and C be can be identical or different code area and/or the identical or different polypeptide of codified Code area, and X and Y are the protein cleavage signals of the coding of the identical or different protein cleavage signal of codified.
In one embodiment, polypeptide, primary construct and mmRNA also may include the sequence of coding protein cracking site Column, so that the polynucleotides, primary construct and mmRNA can be by with the specificity for being directed to the protein cleavage site Protease Treatment to discharge from carrier area or fusion partner.
In one embodiment, polypeptide of the invention, primary construct and mmRNA may include the sequence for encoding 2A peptide. In one embodiment, this sequence can be used for separating the code area of two or more target polypeptides.As non-limiting The sequence of example, coding 2A peptide can be between code area A and code area B (A-2Apep-B).The presence of 2A peptide will lead to one long Protein cleavage is at a-protein, PROTEIN B and 2A peptide.A-protein and PROTEIN B can be identical or different target polypeptides. In another embodiment, in 2A peptide polynucleotides for use in the present invention, primary construct and/or mmRNA, to generate Two, three, four, five, six, seven, eight, nine, ten or more protein.
It is incorporated to post-transcriptional control regulator
In one embodiment, polynucleotides of the invention, primary construct and/or mmRNA may include at least one Post-transcriptional control regulator.These post-transcriptional control regulators can be but not limited to small molecule, compound and regulating and controlling sequence.Make For non-limiting example, post-transcriptional control can be used is reflected by PTC Therapeutics Inc. (South Plainfield, NJ) Other small molecule uses its GEMSTM(the Gene Expression Modulation from Small-Moleclues) screens skill Art is realized.
Post-transcriptional control regulator can be gene expression regulator, pass through institute in international publication number WO2006022712 The method of detailed description or described gene expression regulator screening, the announcement are incorporated herein in its entirety by reference.Identify The method of RNA regulating and controlling sequence involved in translation control is described in international publication number WO2004067728, with reference Mode is integrally incorporated herein；Identify the method for adjusting the compound of non-translational region dependent expression of gene and is described in international publication In number WO2004065561, it is incorporated herein in its entirety by reference.
In one embodiment, polynucleotides of the invention, primary construct and/or mmRNA may include at least one Post-transcriptional control regulator, the post-transcriptional control regulator be located at polynucleotides of the invention, primary construct and/or In the 5 ' of mmRNA and/or 3 ' non-translational regions.
In another embodiment, polynucleotides of the invention, primary construct and/or mmRNA may include at least one Kind post-transcriptional control regulator terminates to adjust premature translation.Post-transcriptional control regulator can be international publication number Described in WO2004010106, WO2006044456, WO2006044682, WO2006044503 and WO2006044505 Compound or the compound of the discovery of the method by summarizing in the international publication, the international publication is respectively by reference It is integrally incorporated herein.As non-limiting examples, the compound is adjusted in combination with the area of 28S rRNA turns over too early Translate termination (see, for example, WO2004010106, being incorporated herein in its entirety by reference).
In one embodiment, polynucleotides of the invention, primary construct and/or mmRNA may include at least one Post-transcriptional control regulator is to change protein expression.As non-limiting examples, international publication number can be used in the expression of VEGF Compound described in WO2005118857, WO2006065480, WO2006065479 and WO2006058088 passes through The compound of the discovery of method described in the international publication regulates and controls, the international publication respectively it is whole by reference simultaneously Enter herein.
Polynucleotides of the invention, primary construct and/or mmRNA may include at least one post-transcriptional control regulator with Just control translation.In one embodiment, post-transcriptional control regulator can be RNA regulating and controlling sequence.As non-limiting reality Example, RNA regulating and controlling sequence can be identified by method described in international publication number WO2006071903, and the international publication is to draw Mode is integrally incorporated herein.
III. it modifies
Herein, it in polynucleotides (such as primary construct or mRNA molecule), term " modification " or " repairs in due course Decorations " refer to modification relative to A, G, U or C ribonucleotide.In general, herein, these terms are not intended to refer to day So ribonucleotide modification in existing 5 ' end mRNA cap portions.In polypeptide, term " modification " refer to such as with 20 ammonia The modification that the specification group of base acid moieties is compared).
The modification can be a variety of different modifications.In some embodiments, code area, flanking region and/or end Area may include one, two or more (optionally different) nucleosides or nucleotide modification.In some embodiments, draw Entering to the modification polynucleotides of cell, primary construct or mmRNA can show and unmodified polynucleotides, primary construct Or mmRNA is compared to the degradation of reduction in cell.
Polynucleotides, primary construct and mmRNA may include any useful modification, such as to sugar, nucleobase or nucleosides Between bonded (such as to connection phosphate/bonded to di-phosphate ester/to phosphodiester backbone) modification.One of pyrimidine nucleobase Or amino, the mercaptan optionally replaced, the alkyl (such as methyl or ethyl) that optionally replaces that multiple atoms can be optionally substituted Or halogeno-group (such as chlorine or fluorine) is replaced or is replaced.In certain embodiments, modification (for example, one or more modifications) can deposit It is in each of sugar and internucleoside linkage connection.Modification according to the present invention can be ribonucleic acid (RNA) and be modified into deoxidation core Ribosomal ribonucleic acid (DNA), threose nucleic acid (TNA), ethylene glycol nucleic acid (GNA), peptide nucleic acid (PNA), lock nucleic acid (LNA) or its heterozygote). In addition modification is being described herein.
As described herein, polynucleotides of the invention, primary construct and mmRNA generally do not induce mRNA introduced To the innate immune response of cell therein.The feature of the innate immune response of induction includes 1) increased proinflammatory cytokine The expression of the factor, 2) activation and/or 3 of intracellular PRR (RIG-I, MDA5 etc.)) protein translation termination or reduction.
It in certain embodiments, may be it is desirable that intracellular degradation be introduced into the modification of nucleic acids in cell point Son.For example, if protein generate accurate timing be it is required, the degradation of modified nucleic acid molecule may be preferred. Therefore, in some embodiments, the present invention provides a kind of modified nucleic acid molecule containing degrading texture domain, the degrading texture Domain can be applied in a directional manner in the cell.On the other hand, the disclosure provides the multicore glycosides comprising nucleosides or nucleotide Acid, the nucleosides or nucleotide can destroy major groove effect (such as in conjunction with) gametophyte and the polynucleotides combination (for example, Wherein compared with unmodified nucleotide, modified nucleoside acid has the binding affinity of the reduction to major groove effect gametophyte).
Polynucleotides, primary construct and mmRNA optionally include other doses (for example, RNAi- inducer, RNAi agent, SiRNA, shRNA, miRNA, antisense RNA, ribozyme, catalytic DNA, tRNA, RNA, aptamer, the carrier for inducing three spiralizations Deng).In some embodiments, polynucleotides, primary construct or mmRNA may include one or more mRNAs (mRNA) With one or more modified nucleosides or nucleotide (for example, mmRNA molecule).These polynucleotides, primary construct and mmRNA Details then provides.
Polynucleotides and primary construct
Polynucleotides of the invention, primary construct and mmRNA include the connection nucleosides of encoding target polypeptide the firstth area, Positioned at the first flanking region of the 5 ' ends in firstth area and the second flanking region of the 3 ' ends positioned at firstth area.
In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, firstth area, first Flanking region or the second flanking region) include n number connection nucleosides, the nucleosides have formula (Ia) or formula (Ia-1):
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
U is O, S, N (RU)nuOr C (RU)nu, integer and each R that wherein nu is 0 to 2UIndependently be H, it is halogenated or appoint Choose the alkyl in generation；
--- for singly-bound or it is not present；
R1’、R2′、R1”、R2”、R1、R2、R3、R4And R5It is each independently (if present) H, halogeno-group, hydroxyl, sulphur Alcohol, the alkoxy optionally replaced, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, optionally replaces the alkyl optionally replaced Aminoalkoxy, the alkyloxy-alkoxy, the hydroxy alkoxy base optionally replaced, the amino, the nitrine that optionally replace that optionally replace Base, the aryl optionally replaced, the aminoalkyl optionally replaced, the aminoalkenyl optionally replaced, the aminoalkynyl optionally replaced or It is not present；Wherein the combination of one or more of R3 and R1 ', R1 ", R2 ', R2 " or R5 are (for example, the combination of R1 ' and R3, R1 " Combination, R2 ' and the combination of R3, the combination of R2 " and R3 or the combination of R5 and R3 with R3) it can be connected together to be formed and optionally be taken The alkylidene in generation or the miscellaneous alkylidene optionally replaced, and carbon connected to them provides the heterocycle optionally replaced together (for example, bicyclic, tricyclic or Fourth Ring heterocycle)；Wherein combination (the example of one or more of R5 and R1 ', R1 ", R2 ' or R2 " Such as, the combination of the combination of R1 ' and R5, R1 " and R5, R2 ' and the combination of R5 or the combination of R2 " and R5) it can be connected together with shape At the alkylidene optionally replaced or the miscellaneous alkylidene optionally replaced, and carbon connected to them provides optionally replace together Heterocycle (for example, bicyclic, tricyclic or Fourth Ring heterocycle)；And wherein R4With R1’、R1”、R2′、R2”、R3Or R5In one or more A combination can be connected together to form the alkylidene that optionally replaces or the optional miscellaneous alkylidene that replaces, and with their companies of institute The carbon connect provides the heterocycle (for example, bicyclic, tricyclic or Fourth Ring heterocycle) optionally replaced together；M ' and m " is each independently The integer of 0 to 3 (for example, 0 to 2,0 to 1,1 to 3 or 1 to 2)；
Y1、Y2And Y3It is each independently O, S, Se ,-NRN1, the optionally alkylidene that replaces or the miscellaneous alkylene optionally replaced Base, wherein RN1For H, optionally the alkyl that replaces, the alkenyl optionally replaced, the alkynyl optionally replaced, the aryl optionally replaced or not In the presence of；
Each Y4It independently is H, hydroxyl, mercaptan, boryl, the alkyl optionally replaced, the alkenyl optionally replaced, optionally takes The alkynyl in generation, the alkoxy optionally replaced, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, the thio alkane optionally replaced Oxygroup, the alkyloxy-alkoxy optionally replaced or the amino optionally replaced；
Each Y5It independently is O, S, Se, the alkylidene (for example, methylene) optionally replaced or the miscellaneous alkylene optionally replaced Base；
The integer that n is 1 to 100,000；And
B is nucleobase (for example, purine, pyrimidine or derivatives thereof), wherein B and R1’Combination, B and R2′Combination, B with R1”Combination or B and R2”Combination can carbon connected to them be optionally formed bicyclic radicals together (for example, bicyclic heterocycle Base) or in which B, R1”And R3Combination or B, R2”And R3Combination may be optionally formed tricyclic or four cyclic groups (for example, tricyclic or Fourth Ring heterocycle, such as in the formula of this paper (IIo)-(IIp)).In some embodiments, the polynucleotides, primary building Body or mmRNA include modification ribose.In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, institute State the firstth area, first flanking region or second flanking region) include n number connection nucleosides, the nucleosides have formula (Ia-2) to (Ia-5)；Or its pharmaceutically acceptable salt or stereoisomer.
In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, firstth area, described First flanking region or second flanking region) include n number connection nucleosides, the nucleosides have formula (Ib) or formula (Ib-1):
Or its is pharmaceutically acceptable Salt or stereoisomer,
Wherein
U is O, S, N (RU)nuOr C (RU)nu, integer and each R that wherein nu is 0 to 2UIndependently be H, it is halogenated or appoint Choose the alkyl in generation；
--- for singly-bound or it is not present；
R1、R3′、R3”And R4It is each independently H, halogeno-group, hydroxyl, the alkyl optionally replaced, the alcoxyl optionally replaced Base, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, the aminoalkoxy optionally replaced, the alkoxy alkane optionally replaced Oxygroup, the hydroxy alkoxy base optionally replaced, the amino optionally replaced, azido, the aryl optionally replaced, the amino optionally replaced Alkyl, the aminoalkenyl optionally replaced, the aminoalkynyl optionally replaced are not present；And wherein R1With R3′Combination or R1With R3”Combination can be combined to form the alkylidene that optionally replaces or the optionally miscellaneous alkylidene that replaces is (for example, lock core to generate Acid)；
Each R5It independently is H, halogeno-group, hydroxyl, the alkyl optionally replaced, the alkoxy optionally replaced, optionally replaces Alkenyloxy group, the alkynyloxy group optionally replaced, the aminoalkoxy optionally replaced, the alkyloxy-alkoxy that optionally replaces or do not deposit ?；
Y1、Y2And Y3It is each independently O, S, Se ,-NRN1, the optionally alkylidene that replaces or the miscellaneous alkylene optionally replaced Base, wherein RN1For H, optionally the alkyl replaced, the alkenyl optionally replaced, the alkynyl optionally replaced or the aryl optionally replaced；
Each Y4It independently is H, hydroxyl, mercaptan, boryl, the alkyl optionally replaced, the alkenyl optionally replaced, optionally takes The alkynyl in generation, the alkoxy optionally replaced, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, the alkoxy optionally replaced Alkoxy or the amino optionally replaced；
The integer that n is 1 to 100,000；And
B is nucleobase.
In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, firstth area, first Flanking region or the second flanking region) include n number connection nucleosides, the nucleosides have formula (Ic):
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
U is O, S, N (RU)nuOr C (RU)nu, integer and each R that wherein nu is 0 to 2UIndependently be H, it is halogenated or appoint Choose the alkyl in generation；
--- for singly-bound or it is not present；
B1、B2And B3Be each independently nucleobase (for example, purine, pyrimidine or derivatives thereof, as described herein), H, Halogeno-group, mercaptan, the alkyl optionally replaced, the alkoxy optionally replaced, the alkenyloxy group that optionally replaces, optionally replaces hydroxyl Alkynyloxy group, the aminoalkoxy optionally replaced, the alkyloxy-alkoxy optionally replaced, the hydroxy alkoxy base optionally replaced, optionally Substituted amino, azido, the aryl optionally replaced, the aminoalkyl optionally replaced, the aminoalkenyl optionally replaced or optionally Substituted aminoalkynyl, wherein B1、B2And B3In one and only one be nucleobase；
Rb1、Rb2、Rb3、R3And R5It is each independently H, halogeno-group, hydroxyl, mercaptan, the alkyl optionally replaced, optionally replaces Alkoxy, the alkenyloxy group optionally replaced, the alkynyloxy group, the aminoalkoxy optionally replaced, the alkane that optionally replaces that optionally replace Oxygroup alkoxy, the amino optionally replaced, azido, the aryl optionally replaced, optionally replaces the hydroxy alkoxy base optionally replaced Aminoalkyl, the aminoalkenyl optionally replaced or the aminoalkynyl optionally replaced；
Y1、Y2And Y3It is each independently O, S, Se ,-NRN1, the optionally alkylidene that replaces or the miscellaneous alkylene optionally replaced Base, wherein RN1For H, optionally the alkyl replaced, the alkenyl optionally replaced, the alkynyl optionally replaced or the aryl optionally replaced；
Each Y4It independently is H, hydroxyl, mercaptan, boryl, the alkyl optionally replaced, the alkenyl optionally replaced, optionally takes The alkynyl in generation, the alkoxy optionally replaced, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, the thio alkane optionally replaced Oxygroup, the alkyloxy-alkoxy optionally replaced or the amino optionally replaced；
Each Y5It independently is O, S, Se, the alkylidene (for example, methylene) optionally replaced or the miscellaneous alkylene optionally replaced Base；
The integer that n is 1 to 100,000；And
Ring including U may include one or more double bonds.
In a particular embodiment, the ring including U does not have U-CB3Rb3Between or CB3Rb3-CB2Rb2Between double bond.
In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, firstth area, first Flanking region or the second flanking region) include n number connection nucleosides, the nucleosides have formula (Id):
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
U is O, S, N (RU)nuOr C (RU)nu, integer and each R that wherein nu is 0 to 2UIndependently be H, it is halogenated or appoint Choose the alkyl in generation；
Each R3Independently be H, halogeno-group, hydroxyl, mercaptan, the alkyl optionally replaced, the alkoxy optionally replaced, optionally Substituted alkenyloxy group, the aminoalkoxy optionally replaced, the alkyloxy-alkoxy optionally replaced, is appointed at the alkynyloxy group optionally replaced It chooses the hydroxy alkoxy base in generation, the amino optionally replaced, azido, the aryl optionally replaced, the aminoalkyl optionally replaced, appoint The aminoalkynyl choosing the aminoalkenyl in generation or optionally replacing；
Y1、Y2And Y3It is each independently O, S, Se ,-NRN1, the optionally alkylidene that replaces or the miscellaneous alkylene optionally replaced Base, wherein RN1For H, optionally the alkyl replaced, the alkenyl optionally replaced, the alkynyl optionally replaced or the aryl optionally replaced；
Each Y4It independently is H, hydroxyl, mercaptan, boryl, the alkyl optionally replaced, the alkenyl optionally replaced, optionally takes The alkynyl in generation, the alkoxy optionally replaced, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, the thio alkane optionally replaced Oxygroup, the alkyloxy-alkoxy optionally replaced or the amino optionally replaced；
Each Y5It independently is O, S, optionally replace alkylidene (for example, methylene) or the miscellaneous alkylidene that optionally replaces；
The integer that n is 1 to 100,000；And
B is nucleobase (for example, purine, pyrimidine or derivatives thereof).
In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, firstth area, first Flanking region or the second flanking region) include n number connection nucleosides, the nucleosides have formula (Ie):
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
U ' and U " is each independently O, S, N (RU)nuOr C (RU)nu, integer and each R that wherein nu is 0 to 2UIt is independent Ground is H, halogenated or optional substituted alkyl；
Each R6Independently be H, halogeno-group, hydroxyl, mercaptan, the alkyl optionally replaced, the alkoxy optionally replaced, optionally Substituted alkenyloxy group, the aminoalkoxy optionally replaced, the alkyloxy-alkoxy optionally replaced, is appointed at the alkynyloxy group optionally replaced It chooses the hydroxy alkoxy base in generation, the amino optionally replaced, azido, the aryl optionally replaced, the aminoalkyl optionally replaced, appoint The aminoalkynyl choosing the aminoalkenyl in generation or optionally replacing；
Each Y5′It independently is O, S, the alkylidene (for example, methylene or ethylidene) that optionally replaces or optionally replaces miscellaneous Alkylidene；
The integer that n is 1 to 100,000；And
B is nucleobase (for example, purine, pyrimidine or derivatives thereof).
In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, firstth area, first Flanking region or the second flanking region) include n number connection nucleosides, the nucleosides have formula (If) or formula (If-1):
Or it pharmaceutically may be used The salt or stereoisomer of receiving,
Wherein
U ' and U " is each independently O, S, N, N (RU)nuOr C (RU)nu, integer and each R that wherein nu is 0 to 2USolely It is on the spot H, halogenated or optional substituted alkyl (for example, U ' is O and U " is N)；
--- for singly-bound or it is not present；
R1’、R2′、R1”、R2”、R3And R4It is each independently H, halogeno-group, hydroxyl, mercaptan, the alkyl optionally replaced, appoints The alkoxy for choosing generation, the alkynyloxy group optionally replaced, the aminoalkoxy optionally replaced, optionally takes the alkenyloxy group optionally replaced The alkyloxy-alkoxy in generation, the amino optionally replaced, azido, the aryl optionally replaced, is appointed at the hydroxy alkoxy base optionally replaced The aminoalkyl for choosing generation, the aminoalkenyl optionally replaced, the aminoalkynyl optionally replaced are not present；And wherein R1’With R3 Combination, R1”With R3Combination, R2’With R3Combination or R2”With R3Combination can be combined to form the alkylene that optionally replaces Base or the miscellaneous alkylidene optionally replaced (for example, to generate lock nucleic acid)；M ' and m " is each independently 0 to 3 (for example, 0 to 2,0 To 1,1 to 3 or 1 to 2) integer；
Y1、Y2And Y3It is each independently O, S, Se ,-NRN1, the optionally alkylidene that replaces or the miscellaneous alkylene optionally replaced Base, wherein RN1For H, optionally the alkyl that replaces, the alkenyl optionally replaced, the alkynyl optionally replaced, the aryl optionally replaced or not In the presence of；
Each Y4It independently is H, hydroxyl, mercaptan, boryl, the alkyl optionally replaced, the alkenyl optionally replaced, optionally takes The alkynyl in generation, the alkoxy optionally replaced, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, the thio alkane optionally replaced Oxygroup, the alkyloxy-alkoxy optionally replaced or the amino optionally replaced；
Each Y5It independently is O, S, Se, the alkylidene (for example, methylene) optionally replaced or the miscellaneous alkylene optionally replaced Base；
The integer that n is 1 to 100,000；And
B is nucleobase (for example, purine, pyrimidine or derivatives thereof).
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia), (Ia-1) to (Ia- 3), (Ib) to (If) and (IIa) to (IIp)) in, the ring including U has one or two double bond.
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IV1) and (IXa) to (IXr)) in, R1、R1’And R1”Respective (if present) is H.In other embodiments, R2、R2′With R2”Respective (if present) independently is H, halogenated (for example, fluorine), hydroxyl, the alkoxy optionally replaced (for example, methoxyl group or second Oxygroup) or the alkyloxy-alkoxy that optionally replaces.In a particular embodiment, alkyloxy-alkoxy is-(CH2)s2 (OCH2CH2)s1(CH2)s3OR ', wherein s1 is the integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are each independently 0 To the integer of 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and R ' is H or C1-20Alkyl).In some embodiment party In case, s2 0, s1 are 1 or 2, and s3 is 0 or 1, and R ' is C1-6Alkyl.
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, R2、R2’And R2”Respective (if present) is H.In other embodiments, R1、R1′With R1”Respective (if present) independently is H, halogenated (for example, fluorine), hydroxyl, the alkoxy optionally replaced (for example, methoxyl group or second Oxygroup) or the alkyloxy-alkoxy that optionally replaces.In a particular embodiment, alkyloxy-alkoxy is-(CH2)s2 (OCH2CH2)s1(CH2)s3OR ', wherein s1 is the integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are each independently 0 To the integer of 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and R ' is H or C1-20Alkyl).In some embodiment party In case, s2 0, s1 are 1 or 2, and s3 is 0 or 1, and R ' is C1-6Alkyl.
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, R3、R4And R5It is each independently H, halogenated (for example, fluorine), hydroxyl, optionally replaces Alkyl, the alkyloxy-alkoxy alkoxy optionally replaced (for example, methoxy or ethoxy) or optionally replaced.It is being embodied In scheme, R3For H, R4For H, R5For H or R3、R4And R5It is all H.In a particular embodiment, R3For C1-6Alkyl, R4For C1-6Alkane Base, R5For C1-6Alkyl or R3、R4And R5It is all C1-6Alkyl.In a particular embodiment, R3And R4The two is H, and R5For C1-6Alkyl.
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, R3And R5It is joined together to form the alkylidene optionally replaced or optionally replaces miscellaneous Alkylidene, and carbon connected to them provides the heterocycle optionally replaced (for example, bicyclic, tricyclic or Fourth Ring heterocycle together Base, such as trans- -3 ', 4 ' analogs, wherein R3And R5Miscellaneous alkylidene is joined together to form (for example,-(CH2)b1O(CH2)b2O (CH2)b3, wherein b1, b2 and b3 are each independently 0 to 3 integer).
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, R3With R1’、R1”、R2′、R2”Or R5One or more of be joined together to form appoint The miscellaneous alkylidene choosing the alkylidene in generation or optionally replacing, and carbon connected to them provides the heterocycle optionally replaced together Base is (for example, bicyclic, tricyclic or Fourth Ring heterocycle, R3With R1’、R1”、R2′、R2”Or R5One or more of link together with Miscellaneous alkylidene is formed (for example,-(CH2)b1O(CH2)b2O(CH2)b3, wherein b1, b2 and b3 be each independently 0 to 3 it is whole Number).
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, R5With R1’、R1”、R2′Or R2”One or more of be joined together to form optionally Substituted alkylidene or the miscellaneous alkylidene optionally replaced, and carbon connected to them provides the heterocycle optionally replaced together (for example, bicyclic, tricyclic or Fourth Ring heterocycle, R5With R1’、R1”、R2′Or R2”One or more of be joined together to form it is miscellaneous Alkylidene is (for example,-(CH2)b1O(CH2)b2O(CH2)b3, wherein b1, b2 and b3 are each independently 0 to 3 integer).
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, each Y2It independently is O, S or-NRN1, wherein RN1For H, optionally the alkyl that replaces is appointed Choose alkenyl, the alkynyl optionally replaced or the aryl optionally replaced in generation.In a particular embodiment, Y2For NRN1, wherein RN1 The alkyl for H or optionally replaced is (for example, C1-6Alkyl, such as methyl, ethyl, isopropyl or n-propyl).
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, each Y3It independently is O or S.
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, R1For H；Each R2It independently is H, halogenated (for example, fluorine), hydroxyl, the alkane optionally replaced Oxygroup (for example, methoxy or ethoxy) or the alkyloxy-alkoxy optionally replaced are (for example,-(CH2)s2(OCH2CH2)s1 (CH2)s3OR ', wherein s1 is the integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are each independently 0 to 10 (for example, 0 To 4,0 to 6,1 to 4,1 to 6 or 1 to 10) integer, and R ' be H or C1-20Alkyl, it is 1 or 2, s3 that such as wherein s2, which is 0, s1, It is 0 or 1, and R ' is C1-6Alkyl)；Each Y2It independently is O or-NRN1, wherein RN1For H, optionally the alkyl that replaces, optionally Substituted alkenyl, the alkynyl optionally replaced or the aryl optionally replaced is (for example, wherein RN1Alkyl (the example for H or optionally replaced Such as, C1-6Alkyl, such as methyl, ethyl, isopropyl or n-propyl))；And each Y3It independently is O or S (for example, S).Other In embodiment, R3For H, halogenated (for example, fluorine), hydroxyl, optionally replace alkyl, the optionally alkoxy that replaces is (for example, methoxy Base or ethyoxyl) or the alkyloxy-alkoxy that optionally replaces.In other embodiments again, each Y1Independently be O or- NRN1, wherein RN1For H, optionally the alkyl replaced, the alkenyl optionally replaced, the alkynyl optionally replaced or the aryl optionally replaced (for example, wherein RN1The alkyl for H or optionally replaced is (for example, C1-6Alkyl, such as methyl, ethyl, isopropyl or n-propyl))；And And each Y4It independently is H, hydroxyl, mercaptan, the alkyl optionally replaced, the alkoxy optionally replaced, the thio alkane optionally replaced Oxygroup, the alkyloxy-alkoxy optionally replaced or the amino optionally replaced.
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IV1) and (IXa) to (IXr)) in, each R1It independently is H, halogenated (for example, fluorine), hydroxyl, the alkoxy optionally replaced (for example, methoxy or ethoxy) or the alkyloxy-alkoxy optionally replaced are (for example,-(CH2)s2(OCH2CH2)s1(CH2)s3OR ', wherein s1 is the integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are each independently 0 to 10 (for example, 0 to 4,0 To 6,1 to 4,1 to 6 or 1 to 10) integer, and R ' be H or C1-20Alkyl, such as wherein s2 be 0, s1 is 1 or 2, s3 for 0 or 1, and R ' is C1-6Alkyl)；R2For H；Each Y2It independently is O or-NRN1, wherein RN1For H, optionally the alkyl that replaces, optionally Substituted alkenyl, the alkynyl optionally replaced or the aryl optionally replaced is (for example, wherein RN1Alkyl (the example for H or optionally replaced Such as, C1-6Alkyl, such as methyl, ethyl, isopropyl or n-propyl))；And each Y3It independently is O or S (for example, S).Other In embodiment, R3For H, halogenated (for example, fluorine), hydroxyl, optionally replace alkyl, the optionally alkoxy that replaces is (for example, methoxy Base or ethyoxyl) or the alkyloxy-alkoxy that optionally replaces.In other embodiments again, each Y1Independently be O or- NRN1, wherein RN1For H, optionally the alkyl replaced, the alkenyl optionally replaced, the alkynyl optionally replaced or the aryl optionally replaced (for example, wherein RN1The alkyl for H or optionally replaced is (for example, C1-6Alkyl, such as methyl, ethyl, isopropyl or n-propyl))；And And each Y4It independently is H, hydroxyl, mercaptan, the alkyl optionally replaced, the alkoxy optionally replaced, the thio alkane optionally replaced Oxygroup, the alkyloxy-alkoxy optionally replaced or the amino optionally replaced.
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, the ring including U be in β-D (for example, β-D-ribose) configuration.
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, the ring including U be in α-L (for example, α-L- ribose) configuration.
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, one or more B are not pseudouridine (ψ) or 5- Methyl-Cytidine (m5C).In some realities It applies in scheme, the B nucleobase of the n number of about 10% to about 100% is not ψ or m5C (for example, 10% to 20%, 10% to 35%, 10% to 50%, 10% to 60%, 10% to 75%, 10% to 90%, 10% to 95%, 10% to 98%, 10% to 99%, 20% to 35%, 20% to 50%, 20% to 60%, 20% to 75%, 20% to 90%, 20% to 95%, 20% to 98%, 20% to 99%, 20% to 100%, 50% to 60%, 50% to 75%, 50% to 90%, 50% to 95%, 50% To 98%, 50% to 99%, 50% to 100%, 75% to 90%, 75% to 95%, 75% to 98%, 75% to 99%, with And 75% to 100% B of n number is not ψ or m5C).In some embodiments, B is not ψ or m5C。
Polynucleotides, primary construct or mmRNA some embodiments (for example, formula (Ia) to (Ia-5), (Ib) are extremely (If-1), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) be extremely (IVl) and (IXa) to (IXr)) in, when B be selected from cytimidine, guanine, uracil and adenine unmodified core alkali When base, then Y1、Y2Or Y3At least one of be not O.
In some embodiments, the polynucleotides, primary construct or mmRNA include modification ribose.In some realities It applies in scheme, the polynucleotides, primary construct or mmRNA are (for example, firstth area, first flanking region or described Second flanking region) include n number connection nucleosides, the nucleosides have formula (IIa) to (IIc):
Or its pharmaceutically acceptable salt or stereoisomer.In a particular embodiment, U For O or C (RU)nu, integer and each R that wherein nu is 0 to 2UH, halogenated or optional substituted alkyl independently are (for example, U For-CH2Or-CH-).In other embodiments, R1、R2、R3、R4And R5It is each independently H, halogeno-group, hydroxyl, sulphur Alcohol, the alkoxy optionally replaced, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, optionally replaces the alkyl optionally replaced Aminoalkoxy, the alkyloxy-alkoxy, the hydroxy alkoxy base optionally replaced, the amino, the nitrine that optionally replace that optionally replace Base, the aryl optionally replaced, the aminoalkyl optionally replaced, the aminoalkenyl optionally replaced, the aminoalkynyl optionally replaced or There is no (for example, each R1And R2It independently is H, halogeno-group, hydroxyl, the alkyl optionally replaced or the alkoxy optionally replaced； Each R3And R4The alkyl for independently being H or optionally replacing；And R5For H or hydroxyl), andFor singly-bound or double bond.
In a particular embodiment, the polynucleotides or mmRNA include the connection nucleosides of n number, and the nucleosides has Formula (IIb-1) to (IIb-2):
Or it can pharmaceutically connect The salt or stereoisomer received.In some embodiments, U is O or C (RU)nu, integer and each R that wherein nu is 0 to 2U H, halogenated or optional substituted alkyl independently are (for example, U is-CH2Or-CH-).In other embodiments, R1And R2Respectively From independently being H, halogeno-group, hydroxyl, mercaptan, the optionally alkyl, the optionally alkoxy, the optional alkene oxygen that replaces that replace that replace Base, the alkynyloxy group optionally replaced, the aminoalkoxy optionally replaced, the alkyloxy-alkoxy optionally replaced, the hydroxyl optionally replaced Base alkoxy, the amino optionally replaced, azido, the aryl optionally replaced, the aminoalkyl optionally replaced, the ammonia optionally replaced Base alkenyl, the aminoalkynyl optionally replaced are not present (for example, each R1And R2It independently is H, halogeno-group, hydroxyl, optionally takes The alkyl in generation or the alkoxy optionally replaced, for example, H, halogeno-group, hydroxyl, alkyl or alkoxy).In a particular embodiment, R2The alkoxy (for example, methoxyl group, ethyoxyl or any alkoxy described herein) for hydroxyl or optionally replaced.
In a particular embodiment, the polynucleotides, primary construct or mmRNA include the connection nucleosides of n number, institute Nucleosides is stated with formula (IIc-1) to (IIc-4):
Or its is pharmaceutically acceptable Salt or stereoisomer.In some embodiments, U is O or C (RU)nu, integer and each R that wherein nu is 0 to 2UIt is independent Ground is H, halogenated or optional substituted alkyl is (for example, U is-CH2Or-CH-).In some embodiments, R1、R2And R3Respectively From independently being H, halogeno-group, hydroxyl, mercaptan, the optionally alkyl, the optionally alkoxy, the optional alkene oxygen that replaces that replace that replace Base, the alkynyloxy group optionally replaced, the aminoalkoxy optionally replaced, the alkyloxy-alkoxy optionally replaced, the hydroxyl optionally replaced Base alkoxy, the amino optionally replaced, azido, the aryl optionally replaced, the aminoalkyl optionally replaced, the ammonia optionally replaced Base alkenyl, the aminoalkynyl optionally replaced are not present (for example, each R1And R2It independently is H, halogeno-group, hydroxyl, optionally takes The alkyl in generation or the alkoxy optionally replaced, for example, H, halogeno-group, hydroxyl, alkyl or alkoxy；And each R3It independently is H or the alkyl optionally replaced)).In a particular embodiment, R2It is the alkoxy that optionally replaces (for example, methoxy or ethoxy Or any alkoxy described herein).In a particular embodiment, R1For the alkyl optionally replaced, and R2For hydroxyl.? In other embodiments, R1For hydroxyl, and R2For the alkyl optionally replaced.In other embodiments, R3Optionally replace Alkyl.
In some embodiments, the polynucleotides, primary construct or mmRNA include the modification ribose of non-annularity. In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, firstth area, first flank Area or second flanking region) include n number connection nucleosides, the nucleosides have formula (IId) to (IIf):
Or its pharmaceutically acceptable salt or stereoisomer.
In some embodiments, the polynucleotides, primary construct or mmRNA include the modification hexose of non-annularity Alcohol.In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, firstth area, first side Pterion or second flanking region) include n number connection nucleosides, the nucleosides have formula (IIg) to (IIj):
Or its pharmaceutically acceptable salt Or stereoisomer.
In some embodiments, the polynucleotides, primary construct or mmRNA include with shrink or expansion The saccharide part of ribose ring.In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, described first Area, first flanking region or second flanking region) include n number connection nucleosides, the nucleosides have formula (IIk) extremely (IIm):
Or its pharmaceutically acceptable salt or stereoisomer, wherein R1’、R1”、R2′ And R2”It is each independently H, halogeno-group, hydroxyl, the alkyl optionally replaced, the alkoxy optionally replaced, the alkene optionally replaced Oxygroup, the alkynyloxy group optionally replaced, the aminoalkoxy optionally replaced, the alkyloxy-alkoxy optionally replaced are not present；And And wherein R2′With R3Combination or R2”With R3Combination can be combined to form the alkylidene that optionally replaces or optionally replace Miscellaneous alkylidene.
In some embodiments, the polynucleotides, primary construct or mmRNA include the modification ribose of locking.? In some embodiments, the polynucleotides, primary construct or mmRNA are (for example, firstth area, first flanking region Or second flanking region) include n number connection nucleosides, the nucleosides have formula (IIn):
Or its pharmaceutically acceptable salt or stereoisomer, wherein R3′For O, S Or-NRN1, wherein RN1For H, optionally the alkyl replaced, the alkenyl optionally replaced, the alkynyl optionally replaced or the virtue optionally replaced Base, and R3”It is the alkylidene that optionally replaces (for example,-CH2-、-CH2CH2Or-CH2CH2CH2) or the miscellaneous alkylene that optionally replaces Base is (for example,-CH2NH-、-CH2CH2NH-、-CH2OCH2Or-CH2CH2OCH2) (for example, R3′For O and R3”Optionally to replace Alkylidene (for example,-CH2-、-CH2CH2Or-CH2CH2CH2-))。
In some embodiments, the polynucleotides, primary construct or mmRNA include the connection nucleosides of n number, institute Nucleosides is stated with formula (IIn-1) to (IIn-2):
Or it can pharmaceutically connect The salt or stereoisomer received, wherein R3′For O, S or-NRN1, wherein RN1For H, optionally the alkyl replaced, the alkene optionally replaced Base, the alkynyl optionally replaced or the aryl optionally replaced, and R3”It is the alkylidene that optionally replaces (for example,-CH2-、-CH2CH2- Or-CH2CH2CH2) or the miscellaneous alkylidene that optionally replaces (for example,-CH2NH-、-CH2CH2NH-、-CH2OCH2Or- CH2CH2OCH2) (for example, R3′For O and R3”It is the alkylidene that optionally replaces (for example,-CH2-、-CH2CH2Or- CH2CH2CH2-))。
In some embodiments, the polynucleotides, primary construct or mmRNA include the lock to form Fourth Ring heterocycle Fixed modification ribose.In some embodiments, the polynucleotides, primary construct or mmRNA (for example, firstth area, First flanking region or second flanking region) include n number connection nucleosides, the nucleosides have formula (IIo):
Or its pharmaceutically acceptable salt, wherein R12a、R12c、T1’、T1”、T2′、 T2”、V1And V3As described herein.
Any formula of the polynucleotides, primary construct or mmRNA may include one or more cores described herein Base (for example, formula (b1) to (b43)).
In one embodiment, the method that offer of the present invention prepares polynucleotides, primary construct or mmRNA, wherein The polynucleotides include n number as herein defined with the nucleosides of formula (Ia):
The method includes making formula as herein defined (IIIa) compound:
With RNA polymerase and cDNA template reaction.
In another embodiment, it includes at least one nucleotide (for example, mmRNA molecule) that the present invention, which provides amplification, The method of polynucleotides, primary construct or mmRNA, which comprises make the chemical combination of formula as herein defined (IIIa) Object is reacted with primer, cDNA template and RNA polymerase.
In one embodiment, it includes the more of at least one nucleotide (for example, mmRNA molecule) that the present invention, which provides preparation, The method of nucleotide, primary construct or mmRNA, wherein the polynucleotides include having as herein defined for n number The nucleosides of formula (Ia):
The method includes making formula as herein defined (IIIa-1) compound:
With RNA polymerase and cDNA template reaction.
In another embodiment, it includes at least one nucleotide (for example, mmRNA molecule) that the present invention, which provides amplification, The method of polynucleotides, primary construct or mmRNA, which comprises
React formula as herein defined (IIIa-1) compound with primer, cDNA template and RNA polymerase.
In one embodiment, the present invention provides preparation repairing comprising at least one nucleotide (for example, mmRNA molecule) The method for adoring mRNA, wherein the polynucleotides include n number as herein defined with the nucleosides of formula (Ia-2):
The method includes making formula as herein defined (IIIa-2) compound:
With RNA polymerase and cDNA template reaction.
In another embodiment, it includes at least one nucleotide (for example, mmRNA molecule) that the present invention, which provides amplification, The method for modifying mRNA, which comprises
React formula as herein defined (IIIa-2) compound with primer, cDNA template and RNA polymerase.
In some embodiments, the reaction repeatable 1 to about 7,000 time.In any embodiment of this paper, B It can be the nucleobase of formula (b1) to (b43).
The polynucleotides, primary construct and mmRNA optionally include 5 ' and/or 3 ' flanking regions, the flanking region It is being described herein.
Modify RNA (mmRNA) molecule
The invention also includes the structural units of modification RNA (mmRNA) molecule, for example, modification ribonucleotide, modification ribose core Thuja acid.For example, these structural units can be used conveniently to prepare polynucleotides of the invention, primary construct or mmRNA.
In some embodiments, the structural unit molecule has formula (IIIa) or (IIIa-1):
Or its Pharmaceutically acceptable salt or stereoisomer, wherein substituent group is as described herein (for example, come formula (Ia) and (Ia-1) Say), and wherein when B is selected from the unmodified nucleobase of cytimidine, guanine, uracil and adenine, then Y1、Y2 Or Y3At least one of be not O.
In some embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA has Formula (IVa) to (IVb):
Or its pharmaceutically acceptable salt Or stereoisomer, wherein B is (for example, any of (b1) to (b43)) as described herein.In a particular embodiment, By formula (IVa) or (IVb) and modification uracil (for example, formula (b1) to (b9), (b21) to (b23) and (b28) is into (b31) Either one or two of, such as formula (b1), (b8), (b28), (b29) or (b30)) combination.In a particular embodiment, by formula (IVa) or (IVb) with modification cytimidine (for example, any of formula (b10) to (b14), (b24), (b25) and (b32) to (b36), Such as formula (b10) or (b32)) combination.In a particular embodiment, by formula (IVa) or (IVb) and modification guanine (for example, formula Any of (b15) to (b17) and (b37) to (b40)) combination.In a particular embodiment, by formula (IVa) or (IVb) with Modify adenine (for example, any of formula (b18) to (b20) and (b41) to (b43)) combination.
In some embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA has Formula (IVc) to (IVk):
Or it pharmaceutically may be used The salt or stereoisomer of receiving, wherein B is (for example, any of (b1) to (b43)) as described herein.It is being embodied In scheme, by formula (IVc) one into (IVk) and modification uracil (for example, formula (b1) to (b9), (b21) to (b23) with And any of (b28) to (b31), such as formula (b1), (b8), (b28), (b29) or (b30)) combination.In specific embodiment In, by formula (IVc) one into (IVk) and modification cytimidine (for example, formula (b10) to (b14), (b24), (b25) and Any of (b32) to (b36), such as formula (b10) or (b32)) combination.In a particular embodiment, extremely by formula (IVc) (IVk) one in is combined with modification guanine (for example, any of formula (b15) to (b17) and (b37) to (b40)).? In specific embodiment, by formula (IVc) one into (IVk) and modification adenine (for example, formula (b18) to (b20) and Any of (b41) to (b43)) combination.
In other embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA has Formula (Va) or (Vb):
Or its pharmaceutically acceptable salt or stereoisomer, wherein B is as described herein (for example, (b1) is into (b43) Any one).
In other embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA has Formula (IXa) to (IXd):
Or its pharmaceutically acceptable salt or Stereoisomer, wherein B is (for example, any of (b1) to (b43)) as described herein.In a particular embodiment, will One and modification uracil of the formula (IXa) into (IXd) are (for example, formula (b1) to (b9), (b21) to (b23) and (b28) is extremely Any of (b31), such as formula (b1), (b8), (b28), (b29) or (b30)) combination.In a particular embodiment, by formula One and modification cytimidine of (IXa) into (IXd) are (for example, formula (b10) to (b14), (b24), (b25) and (b32) is extremely Any of (b36), such as formula (b10) or (b32)) combination.In a particular embodiment, by formula (IXa) into (IXd) one It is a to be combined with modification guanine (for example, any of formula (b15) to (b17) and (b37) to (b40)).In specific embodiment party In case, by formula (IXa) one into (IXd) with modification adenine (for example, formula (b18) to (b20) and (b41) are into (b43) Either one or two of) combination.
In other embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA has Formula (IXe) to (IXg):
Or its pharmaceutically acceptable salt or stereoisomer, wherein B is such as retouched herein State (for example, any of (b1) to (b43)).In a particular embodiment, one by formula (IXe) into (IXg) with repair Adorn uracil (for example, any of formula (b1) to (b9), (b21) to (b23) and (b28) to (b31), as formula (b1), (b8), (b28), (b29) or (b30)) combination.In a particular embodiment, one by formula (IXe) into (IXg) and modification Cytimidine (for example, any of formula (b10) to (b14), (b24), (b25) and (b32) to (b36), such as formula (b10) or (b32)) it combines.In a particular embodiment, one by formula (IXe) into (IXg) and modification guanine are (for example, formula (b15) Any of to (b17) and (b37) to (b40)) combination.In a particular embodiment, by formula (IXe) into (IXg) one It is a to be combined with modification adenine (for example, any of formula (b18) to (b20) and (b41) to (b43)).
In other embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA has Formula (IXh) to (IXk):
Or its pharmaceutically acceptable salt Or stereoisomer, wherein B is (for example, any of (b1) to (b43)) as described herein.In a particular embodiment, By formula (IXh) one into (IXk) with modification uracil (for example, formula (b1) to (b9), (b21) to (b23) and (b28) To any of (b31), such as formula (b1), (b8), (b28), (b29) or (b30)) combination.In a particular embodiment, by formula One and modification cytimidine of (IXh) into (IXk) are (for example, formula (b10) to (b14), (b24), (b25) and (b32) is extremely Any of (b36), such as formula (b10) or (b32)) combination.In a particular embodiment, by formula (IXh) into (IXk) one It is a to be combined with modification guanine (for example, any of formula (b15) to (b17) and (b37) to (b40)).In specific embodiment party In case, by formula (IXh) one into (IXk) with modification adenine (for example, formula (b18) to (b20) and (b41) are into (b43) Either one or two of) combination.
In other embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA has Formula (IXl) to (IXr):
Or its pharmaceutically acceptable salt or stereoisomer, wherein each r1 and r2 is independent Ground is the integer of 0 to 5 (for example, 0 to 3,1 to 3 or 1 to 5) and B is as described herein (for example, (b1) appointing into (b43) One).In a particular embodiment, one by formula (IXl) into (IXr) and modification uracil are (for example, formula (b1) is extremely (b9), any of (b21) to (b23) and (b28) to (b31), such as formula (b1), (b8), (b28), (b29) or (b30)) Combination.In a particular embodiment, one by formula (IXl) into (IXr) and modification cytimidine are (for example, formula (b10) is extremely (b14), any of (b24), (b25) and (b32) to (b36), such as formula (b10) or (b32)) combination.It is being embodied In scheme, by formula (IXl) one into (IXr) with modification guanine (for example, formula (b15) to (b17) and (b37) to (b40) Any of) combination.In a particular embodiment, one by formula (IXl) into (IXr) and modification adenine are (for example, formula Any of (b18) to (b20) and (b41) to (b43)) combination.
In some embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA is optional Free group consisting of:
AndOr its pharmaceutically acceptable salt or stereoisomer, wherein often Integer of a r independently 0 to 5 (for example, 0 to 3,1 to 3 or 1 to 5).
In some embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA is optional Free group consisting of:
AndOr it pharmaceutically may be used The salt or stereoisomer of receiving, wherein integer and s1 of each r independently 0 to 5 (for example, 0 to 3,1 to 3 or 1 to 5) As described herein.
In some embodiments, it may be incorporated into nucleic acid (for example, RNA, mRNA, polynucleotides, primary construct or mmRNA) In structural unit molecule be modify uridine (for example, selected from the group that is made up of:
Or its pharmaceutically acceptable salt or stereoisomer, wherein Y1、Y3、Y4、Y6And r (example as described herein Such as, each r is independently 0 to 5, such as 0 to 3,1 to 3 or 1 to 5 integer)).
In some embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA is to repair Decorations cytidine (for example, selected from the group being made up of:
AndOr its pharmaceutically acceptable salt or three-dimensional different Structure body, wherein Y1、Y3、Y4、Y6And r is as described herein (for example, each r is independently 0 to 5, such as 0 to 3,1 to 3 or 1 to 5 Integer)).For example, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA may is that
Or its Pharmaceutically acceptable salt or stereoisomer, wherein each r is independently 0 to the whole of 5 (for example, 0 to 3,1 to 3 or 1 to 5) Number.
In some embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA is to repair Decorations adenosine (for example, selected from the group being made up of:
AndOr its is pharmaceutically acceptable Salt or stereoisomer, wherein Y1、Y3、Y4、Y6And r as described herein (for example, each r is independently 0 to 5, such as 0 to 3, 1 to 3 or 1 to 5 integer)).
In some embodiments, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA is to repair Decorations guanosine (for example, selected from the group being made up of:
AndOr its pharmaceutically acceptable salt or stereoisomer, wherein Y1、 Y3、Y4、Y6And r is as described herein (for example, each r independently 0 to 5, such as 0 to 3,1 to 3 or 1 to 5 integer)).
In some embodiments, chemical modification may include using the C group at the C-5 of N D-loop (for example, for pyrimidine Nucleosides, for cytimidine or uracil) (for example, with > NRN1> CH group at group displacement C-5, wherein RN1 for H or appoint Choose the alkyl in generation).For example, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA may is that
Or its pharmaceutically acceptable salt or stereoisomer, wherein each r is independently 0 to 5 (for example, 0 to 3,1 to 3 Or 1 to 5) integer.
In another embodiment, chemical modification may include with halogenated (for example, Br, Cl, F or I) or optionally replacing Alkyl (for example, methyl) replaces the hydrogen at the C-5 of cytimidine.For example, may be incorporated into polynucleotides, primary construct or mmRNA Structural unit molecule may is that
Or its pharmaceutically acceptable salt or stereoisomer, wherein each r is independently 0 to 5 (for example, 0 to 3,1 to 3 Or 1 to 5) integer.
In another embodiment again, chemical modification may include by the NH of the position C-42It is former with the carbon at the position C-5 The condensed ring that son is formed.For example, the structural unit molecule that may be incorporated into polynucleotides, primary construct or mmRNA may is that
Or its pharmaceutically acceptable salt or stereoisomer, wherein often Integer of a r independently 0 to 5 (for example, 0 to 3,1 to 3 or 1 to 5).
Modification on sugar
It may be incorporated into the modification in polynucleotides, primary construct or mmRNA (for example, RNA or mRNA, as described herein) Nucleosides and nucleotide (for example, structural unit molecule) can be modified on the sugar of ribonucleic acid.For example, 2 ' hydroxyls (OH) can be with By multiple and different substituent group modifications or displacement.Illustrative substituents at 2 '-positions include but is not limited to H, halogeno-group, optionally Substituted C1-6Alkyl；The C optionally replaced1-6Alkoxy；The C optionally replaced6-10Aryloxy group；The C optionally replaced3-8Naphthenic base；Appoint Choose the C in generation3-8Cycloalkyloxy；The C optionally replaced6-10Aryloxy group；The C optionally replaced6-10Aryl-C1-6Alkoxy optionally replaces C1-12(heterocycle) oxygroup；Sugared (for example, ribose, pentose or any sugar described herein)；Polyethylene glycol (PEG) ,-O (CH2CH2O)nCH2CH2OR, wherein R is H or the alkyl optionally replaced, and n is 0 to 20 (for example, 0 to 4,0 to 8,0 to 10,0 To 16,1 to 4,1 to 8,1 to 10,1 to 16,1 to 20,2 to 4,2 to 8,2 to 10,2 to 16,2 to 20,4 to 8,4 to 10,4 to 16 and 4 to 20) integer；" lock " nucleic acid (LNA), wherein 2 '-hydroxyls pass through C1-6Alkylidene or C1-6Miscellaneous alkylidene bridging connects To 4 '-carbon of same ribose, wherein exemplary bridge includes methylene, propylidene, ether or amino bridge；Amino as herein defined Alkyl；Aminoalkoxy as herein defined；Amino as herein defined；And amino acid as herein defined.
In general, RNA includes glycosyl ribose, and the glycosyl ribose is 5 member rings with oxygen.It is exemplary, non-limiting Modified nucleoside acid include oxygen in ribose displacement (for example, being replaced with S, Se or alkylidene such as methylene or ethylidene)；Add Add double bond (for example, to use cyclopentenyl or cyclohexenyl group displacement ribose)；The ring contracting reaction of ribose is (for example, to form ring 4 member rings of butane or propylene oxide)；The ring expansion of ribose is (for example, to form with additional carbon or heteroatomic also to have 6 yuan or 7 member rings of phosphoramidate main chain, such as anhydrohexitol, altritol, mannitol, cyclohexyl, cyclohexenyl group And for morpholino)；Polycyclic form is (for example, tricyclic；And " non-locking " form, if ethylene glycol nucleic acid (GNA) is (for example, R- GNA or S-GNA, wherein ribose is connected to the ethylene glycol replacement unit of phosphodiester bond), threose nucleic acid (TNA, wherein ribose Replaced by α-L- threo form furyl glycosyl-(3 ' → 2 ')) and peptide nucleic acid (PNA, wherein 2- amino-ethyl-bonded displacement of glycine Ribose and phosphodiester backbone).Glycosyl also may include having the three-dimensional opposite with the three-dimensional chemical configuration for corresponding to carbon in ribose Learn one or more carbon of configuration.Therefore, polynucleotides, primary construct or mmRNA molecule may include containing for example Arabic Nucleotide of the sugar as sugar.
Modification in nucleobase
The disclosure provides modified nucleoside and nucleotide.As described herein, " nucleosides " is defined as containing glycan molecule (example Such as, pentose or ribose) or derivatives thereof (be also known as " core herein with organic base (for example, purine or pyrimidine) or derivatives thereof Base ") combined compound.As described herein, " nucleotide " is defined as comprising phosphate-based nucleosides.Modified nucleoside Acid can be by any useful method as described herein (for example, chemical method, enzyme method or recombination method are so as to including one A or multiple modifications or non-natural nucleosides) it synthesizes.
Standard adenosine-thymidine, adenosine-uracil or guanosine-cytimidine are not only covered in the base pairing of modified nucleoside acid Base-pair, and cover in nucleotide and/or the base-pair comprising being formed between non-standard or modified base modified nucleoside acid, The wherein arrangement of hydrogen bond donor and hydrogen bond receptor allows between non-standard bases and standard base or two complementary non-standard alkali Hydrogen bonding between based structures.One example of this non-standard bases pairing is that modified nucleoside acid inosine and adenine, born of the same parents are phonetic Base pairing between pyridine or uracil.
Modified nucleoside and nucleotide may include modification nucleobase.The example of the nucleobase found in RNA includes but is not limited to Adenine, guanine, cytimidine and uracil.The example of the nucleobase found in DNA includes but is not limited to that adenine, bird are fast Purine, cytimidine and thymidine.These nucleobases can be modification or complete displacement in order to provide have enhancing characteristic The polynucleotides of (for example, the resistance of nuclease is enhanced by the combination for destroying major groove binding partners), primary construct Or mmRNA molecule.Following table 8 identifies the chemical face of every kind of canonical nucleotide acid.Circle identifies the atom comprising corresponding chemical area.
Table 8
In some embodiments, B is modification uracil.Illustrative modification uracil includes having formula (b1) to (b5) Those of:
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
It is singly-bound or double bond；
T1’、T1”、T2′And T2”It is each independently H, the alkyl that optionally replaces, the alkoxy optionally replaced or optionally takes The thio alkoxy or T in generation1’With T1”Combination or T2′With T2”Combination link together (for example, such as T2In) to form O (oxygen Dai Ji), S (thio group) or Se (seleno base)；
V1And V2It is each independently O, S, N (RVb)nvOr C (RVb)nv, integer and each R that wherein nv is 0 to 2VbSolely On the spot it is H, halogeno-group, the amino acid optionally replaced, the alkyl optionally replaced, the halogenated alkyl optionally replaced, optionally replaces Alkenyl, the alkoxy optionally replaced, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, optionally takes the alkynyl optionally replaced The hydroxy alkyl in generation, the hydroxyalkenyl group optionally replaced, the hydroxyalkynyl optionally replaced, the aminoalkyl optionally replaced (for example, by Any N-protected group (such as trifluoroacetyl group) replaces N-protected group as described herein), the amino alkene that optionally replaces Base, the aminoalkynyl optionally replaced, the acylaminoalkyl optionally replaced are (for example, as described herein by N-protected group Any N-protected group (such as trifluoroacetyl group) replaces), the alkoxy carbonyl alkyl that optionally replaces, the alkoxy optionally replaced Carbonyl alkenyl, the alkoxycarbonylalkynyl optionally replaced or the alkynyloxy group optionally replaced are (for example, optionally described herein Any substituent group (selected from those of (1) to (21) such as alkyl) replaces)；
R10For H, halogeno-group, optionally replace amino acid, hydroxyl, optionally replace alkyl, optionally replace alkenyl, optionally Substituted alkynyl, the hydroxy alkyl optionally replaced, the hydroxyalkenyl group optionally replaced, optionally replaces the aminoalkyl optionally replaced Hydroxyalkynyl, optionally replace aminoalkenyl, optionally replace aminoalkynyl, optionally replace alkoxy, optionally replace Alkoxy carbonyl alkyl, the alkoxycarbonylalkenyl optionally replaced, the alkoxycarbonylalkynyl optionally replaced, the alkane optionally replaced Epoxide carbonyl alkoxy, the Carboxyalkoxy optionally replaced, the carboxyalkyl optionally replaced or the carbamyl alkane optionally replaced Base；
R11The alkyl for H or optionally replaced；
R12aFor H, optionally the alkyl that replaces, the hydroxyalkenyl group optionally replaced, optionally replaces the hydroxy alkyl optionally replaced Hydroxyalkynyl, the aminoalkyl optionally replaced, the aminoalkenyl optionally replaced or the aminoalkynyl optionally replaced, optionally take The carboxyalkyl (for example, being optionally optionally substituted by a hydroxyl group) in generation, the Carboxyalkoxy optionally replaced, the carboxyaminoalkyl optionally replaced Or the carbamyl alkyl optionally replaced；And
R12cFor H, halogeno-group, optionally replace alkyl, optionally replace alkoxy, optionally replace thio alkoxy, appoint The amino for choosing generation, the hydroxyalkenyl group optionally replaced, the hydroxyalkynyl optionally replaced, optionally takes the hydroxy alkyl optionally replaced The aminoalkyl in generation, the aminoalkenyl optionally replaced or the aminoalkynyl optionally replaced.
The modification uracil of other examples includes having those of formula (b6) to (b9):
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
It is singly-bound or double bond；
T1’、T1”、T2′And T2”It is each independently H, the alkyl that optionally replaces, the alkoxy optionally replaced or optionally takes The thio alkoxy or T in generation1’With T1”Combination link together (for example, such as T1In) or T2′With T2”Combination link together (for example, such as T2In) to form O (oxo base), S (thio group) or Se (seleno base) or each T1And T2It independently is O (oxo Base), S (thio group) or Se (seleno base)；
W1And W2It is each independently N (RWa)nwOr C (RWa)nw, integer and each R that wherein nw is 0 to 2WaIndependently For H, optionally the alkyl replaced or the alkoxy optionally replaced；
Each V3It independently is O, S, N (RVa)nvOr C (RVa)nv, integer and each R that wherein nv is 0 to 2VaIndependently For H, halogeno-group, the optionally amino acid, the optionally alkyl, the optionally hydroxy alkyl, the optional hydroxyl that replaces that replace that replace that replace Alkenyl, the alkenyl optionally replaced, the alkynyl optionally replaced, the heterocycle optionally replaced, optionally takes the hydroxyalkynyl optionally replaced The alkane heterocycle in generation, the alkoxy optionally replaced, the alkenyloxy group optionally replaced or the alkynyloxy group optionally replaced, the ammonia optionally replaced Base alkyl is (for example, by N-protected group any N-protected group (such as trifluoroacetyl group or sulfo group alkane as described herein Base) replace), the aminoalkenyl that optionally replaces, the aminoalkynyl optionally replaced, the acylaminoalkyl optionally replaced (for example, by Any N-protected group (such as trifluoroacetyl group) replaces N-protected group as described herein), the alkoxy that optionally replaces Carbonylic alkyl, the alkoxycarbonylalkenyl optionally replaced, the alkoxycarbonylalkynyl optionally replaced, the alkoxy carbonyl optionally replaced Base acyl group, the Alkoxycarbonylalkoxy optionally replaced, the carboxyalkyl optionally replaced by hydroxyl and/or O- (for example, optionally protected Group is protected to replace), the Carboxyalkoxy optionally replaced, the carboxyaminoalkyl optionally replaced or the carbamyl alkane optionally replaced Base (for example, optionally being replaced by any substituent group (selected from those of (1) to (21) such as alkyl) described herein), And wherein RVaAnd R12cCarbon atom connected to them can form the naphthenic base optionally replaced, the aryl optionally replaced together Or the heterocycle (for example, 5- or 6-membered ring) optionally replaced；
R12aFor H, optionally the alkyl that replaces, the hydroxyalkenyl group optionally replaced, optionally replaces the hydroxy alkyl optionally replaced Hydroxyalkynyl, optionally replace aminoalkyl, optionally replace aminoalkenyl, optionally replace aminoalkynyl, optionally replace Carboxyalkyl (for example, optionally being replaced by hydroxyl and/or O- blocking group), optionally replace Carboxyalkoxy, optionally replace Carboxyaminoalkyl, the carbamyl alkyl that optionally replaces or be not present；
R12bFor H, optionally the alkyl that replaces, the alkenyl optionally replaced, the alkynyl optionally replaced, the hydroxyl alkane optionally replaced Base, the hydroxyalkenyl group optionally replaced, the hydroxyalkynyl optionally replaced, the aminoalkyl optionally replaced, the amino alkene optionally replaced Base, the alkaryl optionally replaced, the heterocycle optionally replaced, the alkane heterocycle optionally replaced, is appointed at the aminoalkynyl optionally replaced The amino acid for choosing generation, the Alkoxycarbonylalkoxy optionally replaced, optionally replaces the alkoxy carbonyl acyl group optionally replaced Alkoxy carbonyl alkyl, the alkoxycarbonylalkenyl optionally replaced, the alkoxycarbonylalkynyl optionally replaced, the alkane optionally replaced Epoxide carbonyl alkoxy, optionally takes the carboxyalkyl optionally replaced (for example, optionally being replaced by hydroxyl and/or O- blocking group) The Carboxyalkoxy in generation, the carboxyaminoalkyl optionally replaced or the carbamyl alkyl optionally replaced,
Wherein R12bWith T1’Combination or R12bWith R12cCombination can be connected together to form the heterocycle that optionally replaces； And
R12cFor H, halogeno-group, optionally replace alkyl, optionally replace alkoxy, optionally replace thio alkoxy, appoint Choose the amino in generation, the aminoalkyl optionally replaced, the aminoalkenyl optionally replaced or the aminoalkynyl optionally replaced.
The modification uracil of other examples includes having those of formula (b28) to (b31):
Or its pharmaceutically acceptable salt or alloisomerism Body,
Wherein
T1And T2It is each independently O (oxo base), S (thio group) or Se (seleno base)；
Each RVb’And RVb”It independently is H, halogeno-group, the amino acid optionally replaced, the alkyl optionally replaced, optionally replaces Halogenated alkyl, optionally replace hydroxy alkyl, optionally replace hydroxyalkenyl group, optionally replace hydroxyalkynyl, optionally replace Alkenyl, optionally replace alkynyl, optionally replace alkoxy, optionally replace alkenyloxy group, optionally replace alkynyloxy group, optionally Substituted aminoalkyl (for example, by N-protected group as described herein any N-protected group (for example, trifluoroacetyl group Or sulfonyl alkyl) replace), the aminoalkenyl that optionally replaces, the aminoalkynyl optionally replaced, the acylaminoalkyl optionally replaced (for example, by N-protected group, any N-protected group (for example, trifluoroacetyl group) replaces as described herein) optionally replaces Alkoxy carbonyl alkyl, optionally replace alkoxycarbonylalkenyl, optionally replace alkoxycarbonylalkynyl, optionally replace Alkoxy carbonyl acyl group, the Alkoxycarbonylalkoxy optionally replaced, the carboxyalkyl optionally replaced are (for example, optionally by hydroxyl And/or O- blocking group replaces), the Carboxyalkoxy that optionally replaces, the carboxyaminoalkyl optionally replaced or optionally replace Carbamyl alkyl is (for example, optionally by any substituent group described herein (as being selected from (1) to (21) for alkyl Those) replace) (for example, RVb’For alkyl, the optionally alkenyl that replaces or the optional aminoalkyl that replaces optionally replaced, for example, By N-protected group, any N-protected group (for example, trifluoroacetyl group or sulfonyl alkyl) replaces as described herein)；
R12aFor H, optionally the alkyl that replaces, the carboxyaminoalkyl optionally replaced, the aminoalkyl optionally replaced (for example, By N-protected group, any N-protected group (for example, trifluoroacetyl group or sulfonyl alkyl) replaces as described herein), optionally Substituted aminoalkenyl or the aminoalkynyl optionally replaced；And
R12bFor H, optionally the alkyl that replaces, the alkenyl optionally replaced, the alkynyl optionally replaced, the hydroxyl alkane optionally replaced Base, the hydroxyalkenyl group optionally replaced, the hydroxyalkynyl optionally replaced, the aminoalkyl optionally replaced, the amino alkene optionally replaced Base, the aminoalkynyl optionally replaced are (for example, by N-protected group any N-protected group (such as trifluoro as described herein Acetyl group or sulfonyl alkyl) replace),
The alkoxy carbonyl acyl group that optionally replaces, the Alkoxycarbonylalkoxy optionally replaced, the alkoxy optionally replaced Carbonylic alkyl, the alkoxycarbonylalkenyl optionally replaced, the alkoxycarbonylalkynyl optionally replaced, the alkoxy carbonyl optionally replaced Base alkoxy, the Carboxyalkoxy optionally replaced, the carboxyalkyl optionally replaced or the carbamyl alkyl optionally replaced.
In a particular embodiment, T1For O (oxo base), and T2For S (thio group) or Se (seleno base).In other realities It applies in scheme, T1For S (thio group), and T2For O (oxo base) or Se (seleno base).In some embodiments, RVb’For H, The alkyl optionally replaced or the alkoxy optionally replaced.
In other embodiments, each R12aAnd R12bIndependently be H, the alkyl that optionally replaces, the alkenyl optionally replaced, The alkynyl optionally replaced or the hydroxy alkyl optionally replaced.In a particular embodiment, R12aFor H.In other embodiments, R12aAnd R12bThe two is H.
In some embodiments, R12bEach RVb’The aminoalkyl optionally replaced independently is (for example, by N-protected Any N-protected group (for example, trifluoroacetyl group or sulfonyl alkyl) replaces group as described herein), the ammonia that optionally replaces Base alkenyl, the aminoalkynyl optionally replaced or the acylaminoalkyl optionally replaced are (for example, by N-protected group as this paper is retouched Any N-protected group (for example, trifluoroacetyl group) stated replaces).In some embodiments, the aminoalkyl optionally replaced Amino and/or alkyl by one of the following or multiple substitutions: the alkyl that optionally replaces, optionally takes the alkenyl optionally replaced The sulfonyl alkyl in generation, the carboxyl optionally replaced (for example, being replaced by O- blocking group), the hydroxyl optionally replaced by O- (for example, protected Shield group replaces), optionally replace carboxyalkyl (for example, being replaced by O- blocking group), the alkoxy carbonyl alkane that optionally replaces Base (for example, being replaced by O- blocking group) or N-protected group.In some embodiments, the aminoalkyl optionally replaced is appointed The alkenyl choosing the sulfonyl alkyl in generation or optionally replacing replaces.In a particular embodiment, R12aAnd RVb”The two is H.Having In body embodiment, T1For O (oxo base), and T2For S (thio group) or Se (seleno base).
In some embodiments, RVb’The carbamyl alkane for the alkoxy carbonyl alkyl that optionally replaces or optionally replaced Base.
In a particular embodiment, R12a、R12b、R12cOr RVaOptional substituent group be polyethylene group (for example,- (CH2)s2(OCH2CH2)s1(CH2)s3OR ', wherein s1 is the integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are respectively independent Ground is the integer of 0 to 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and R ' is H or C1-20Alkyl)；Or amino- Polyethylene group is (for example,-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 be 1 to 10 (for example, 1 to 6 or 1 to 4) integer, s2 and s3 are each independently the integer of 0 to 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and Each RN1The C for independently being hydrogen or optionally replacing1-6Alkyl).
In some embodiments, B is modification cytimidine.Illustrative modification cytimidine includes formula (b10) to (b14) Compound:
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
T3′And T3”It is each independently H, the alkyl optionally replaced, the alkoxy optionally replaced or optionally replaces thio Alkoxy or T3′With T3”Combination link together (for example, such as T3In) to form O (oxo base), S (thio group) or Se (selenium Dai Ji)；
Each V4It independently is O, S, N (RVc)nvOr C (RVc)nv, integer and each R that wherein nv is 0 to 2VcIndependently For H, halogeno-group, optionally replace amino acid, optionally replace alkyl, optionally replace alkenyl, optionally replace alkynyl, optionally Substituted alkoxy, the alkenyloxy group optionally replaced, the heterocycle optionally replaced, the alkane heterocycle optionally replaced optionally replace Alkynyloxy group by any substituent group (selected from those of (1) to (21) such as alkyl) described herein (for example, optionally taken Generation), wherein R13bWith RVcCombination can be altogether to form the heterocycle that optionally replace；
Each V5It independently is N (RVd)nvOr C (RVd)nv, integer and each R that wherein nv is 0 to 2VdIndependently be H, Halogeno-group, the alkyl optionally replaced, the alkenyl optionally replaced, the alkynyl optionally replaced, optionally replaces the amino acid optionally replaced Alkoxy, the alkenyloxy group optionally replaced, the heterocycle optionally replaced, the alkane heterocycle optionally replaced or the alkynes oxygen optionally replaced Base (for example, optionally being replaced by any substituent group (selected from those of (1) to (21) such as alkyl) described herein) (for example, V5For-CH or N)；
R13aAnd R13bIt is each independently H, the acyl group that optionally replaces, the acyloxyallcyl optionally replaced, optionally replaces Alkyl or the alkoxy optionally replaced, wherein R13bWith R14Combination can be altogether to form the heterocycle that optionally replace；
Each R14Independently be H, halogeno-group, hydroxyl, mercaptan, the acyl group optionally replaced, the amino acid optionally replaced, optionally Substituted alkyl, the halogenated alkyl optionally replaced, the alkenyl optionally replaced, the alkynyl optionally replaced, the hydroxyl alkane optionally replaced Base (for example, being replaced by O- blocking group), the hydroxyalkenyl group optionally replaced, the hydroxyalkynyl optionally replaced, the alkane optionally replaced Oxygroup, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, the aminoalkoxy optionally replaced, the alkoxy optionally replaced (for example,-NHR, wherein R is H, alkyl, aryl or phosphinylidyne for alkoxy, the acyloxyallcyl optionally replaced, the amino optionally replaced Base), azido, the aryl optionally replaced, the heterocycle optionally replaced, the alkane heterocycle optionally replaced, the amino optionally replaced Alkyl, the aminoalkenyl optionally replaced or the aminoalkyl optionally replaced；And
R15And R16It is each independently H, the alkyl optionally replaced, the alkenyl optionally replaced or the alkynyl optionally replaced.
The modification cytimidine of other examples includes having those of formula (b32) to (b35):
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
T1And T3It is each independently O (oxo base), S (thio group) or Se (seleno base)；
R13aAnd R13bIt is each independently H, the acyl group that optionally replaces, the acyloxyallcyl optionally replaced, optionally replaces Alkyl or the alkoxy optionally replaced, wherein R13bWith R14Combination can be altogether to form the heterocycle that optionally replace；
Each R14Independently be H, halogeno-group, hydroxyl, mercaptan, the acyl group optionally replaced, the amino acid optionally replaced, optionally Substituted alkyl, the halogenated alkyl optionally replaced, the alkenyl optionally replaced, the alkynyl optionally replaced, the hydroxyl alkane optionally replaced Base (for example, being replaced by O- blocking group), the hydroxyalkenyl group optionally replaced, the hydroxyalkynyl optionally replaced, the alkane optionally replaced Oxygroup, the alkenyloxy group optionally replaced, the alkynyloxy group optionally replaced, the aminoalkoxy optionally replaced, the alkoxy optionally replaced (for example,-NHR, wherein R is H, alkyl, aryl or phosphinylidyne for alkoxy, the acyloxyallcyl optionally replaced, the amino optionally replaced Base), azido, the aryl optionally replaced, the heterocycle optionally replaced, the alkane heterocycle optionally replaced, the amino optionally replaced Alkyl (for example, hydroxy alkyl, alkyl, alkenyl or alkynyl), the aminoalkenyl optionally replaced or the aminoalkynyl optionally replaced；And And
R15And R16It is each independently H, the alkyl optionally replaced, the alkenyl optionally replaced or the alkynyl (example optionally replaced Such as, R15For H, and R16The alkyl for H or optionally replaced).
In some embodiments, R15For H, and R16The alkyl for H or optionally replaced.In a particular embodiment, R14 For H, acyl group or hydroxy alkyl.In some embodiments, R14It is halogenated.In some embodiments, R14And R15The two is H.In some embodiments, R15And R16The two is H.In some embodiments, R14And R15And R16Respectively H.? In other embodiments, R13aAnd R13bThe alkyl for being each independently H or optionally replacing.
Other non-limiting examples of modification cytimidine include the compound of formula (b36):
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
Each R13bIndependently be H, the acyl group that optionally replaces, the acyloxyallcyl optionally replaced, the alkyl optionally replaced or The alkoxy optionally replaced, wherein R13bWith R14bCombination can be altogether to form the heterocycle that optionally replace；
Each R14aAnd R14bIt independently is H, halogeno-group, hydroxyl, mercaptan, the acyl group optionally replaced, the amino optionally replaced Acid, the halogenated alkyl optionally replaced, the alkenyl optionally replaced, the alkynyl optionally replaced, optionally replaces the alkyl optionally replaced Hydroxy alkyl (for example, being replaced by O- blocking group), the alkoxy optionally replaced, optionally replaces the hydroxyalkenyl group optionally replaced Alkenyloxy group, optionally replace alkynyloxy group, optionally replace aminoalkoxy, optionally replace alkyloxy-alkoxy, optionally take (for example,-NHR, wherein R is H, alkyl, aryl, phosphoryl, optionally replaces for the acyloxyallcyl in generation, the amino optionally replaced Aminoalkyl or the carboxyaminoalkyl optionally replaced), azido, the aryl optionally replaced, the heterocycle optionally replaced, optionally Substituted alkane heterocycle, the aminoalkyl optionally replaced, the aminoalkenyl optionally replaced or the aminoalkynyl optionally replaced；And
R15It is each independently H, the alkyl optionally replaced, the alkenyl optionally replaced or the alkynyl optionally replaced.
In a particular embodiment, R14bFor the amino acid (for example, the lysine optionally replaced) optionally replaced.Some In embodiment, R14aFor H.
In some embodiments, B is modification guanine.Illustrative modification guanine includes formula (b15) to (b17) Compound:
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
T4’、T4”、T5′、T5”、T6’And T6”It is each independently H, the alkyl optionally replaced or the alkoxy optionally replaced, And wherein T4’With T4”Combination (for example, such as T4In) or T5′With T5”Combination (for example, such as T5In) or T6’With T6”Combination (for example, such as T6In) it is joined together to form O (oxo base), S (thio group) or Se (seleno base)；
V5And V6It is each independently O, S, N (RVd)nvOr C (RVd)nv, integer and each R that wherein nv is 0 to 2VdSolely It is on the spot H, halogeno-group, mercaptan, the amino acid optionally replaced, cyano, amidine, the aminoalkyl optionally replaced, the ammonia optionally replaced Base alkenyl, the alkyl optionally replaced, the alkenyl optionally replaced, the alkynyl optionally replaced, optionally takes the aminoalkynyl optionally replaced The alkoxy in generation, the alkenyloxy group optionally replaced or the alkynyloxy group optionally replaced are (for example, optionally by any substitution described herein Base those of (such as alkyl selected from (1) to (21)) replaces), the thio alkoxy optionally replaced or the ammonia optionally replaced Base；And
R17、R18、R19a、R19b、R21、R22、R23And R24It is each independently H, halogeno-group, mercaptan, the alkane optionally replaced Base, the alkenyl optionally replaced, the alkynyl optionally replaced, the thio alkoxy optionally replaced, the amino optionally replaced optionally take The amino acid in generation.
Illustrative modification guanosine includes the compound of formula (b37) to (b40):
Or its pharmaceutically acceptable salt or Stereoisomer,
Wherein
T4’It is each independently H, the alkyl optionally replaced or the alkoxy optionally replaced, and each T4It independently is O (oxo base), S (thio group) or Se (seleno base)；
R18、R19a、R19bAnd R21It is each independently H, halogeno-group, mercaptan, the alkyl that optionally replaces, optionally replaces Alkenyl, the alkynyl optionally replaced, the thio alkoxy optionally replaced, the amino optionally replaced or the amino acid optionally replaced.
In some embodiments, R18The alkyl for H or optionally replaced.In other embodiments, T4For oxo.One In a little embodiments, R19aAnd R19bThe alkyl for being each independently H or optionally replacing.
In some embodiments, B is modification adenine.Illustrative modification adenine includes formula (b18) to (b20) Compound:
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
Each V7It independently is O, S, N (RVe)nvOr C (RVe)nv, integer and each R that wherein nv is 0 to 2VeIndependently For H, halogeno-group, optionally replace amino acid, optionally replace alkyl, optionally replace alkenyl, optionally replace alkynyl, optionally Substituted alkoxy, the alkenyloxy group optionally replaced or the alkynyloxy group optionally replaced by described herein (for example, optionally any taken Dai Ji (selected from those of (1) to (21) such as alkyl) replaces)；
Each R25It independently is H, halogeno-group, mercaptan, the alkyl optionally replaced, the alkenyl that optionally replaces, optionally replaces Alkynyl, the thio alkoxy optionally replaced or the amino optionally replaced；
R26aAnd R26bIt is each independently H, the acyl group that optionally replaces, the amino acid optionally replaced, the ammonia first optionally replaced Acyl, the alkyl optionally replaced, the alkenyl optionally replaced, the alkynyl optionally replaced, the hydroxy alkyl optionally replaced, optionally Substituted hydroxyalkenyl group, the hydroxyalkynyl optionally replaced, the alkoxy optionally replaced or polyethylene group are (for example,-(CH2)s2 (OCH2CH2)s1(CH2)s3OR ', wherein s1 is the integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are each independently 0 To the integer of 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and R ' is H or C1-20Alkyl)；Or the poly- second of amino- Glycol group is (for example,-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is 1 to 10 (for example, 1 to 6 or 1 to 4) Integer, s2 and s3 are each independently the integer of 0 to 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and each RN1The C for independently being hydrogen or optionally replacing1-6Alkyl)；
Each R27It independently is H, the alkyl that optionally replaces, the alkenyl optionally replaced, the alkynyl optionally replaced, optionally replaces Alkoxy, the thio alkoxy optionally replaced or the amino optionally replaced；
Each R28It independently is H, the alkyl optionally replaced, the alkenyl optionally replaced or the alkynyl optionally replaced；And
Each R29It independently is H, the acyl group that optionally replaces, the amino acid optionally replaced, the carbamyl alkane optionally replaced Base, the alkenyl optionally replaced, the alkynyl optionally replaced, the hydroxy alkyl that optionally replaces, optionally replaces the alkyl optionally replaced Hydroxyalkenyl group, the alkoxy optionally replaced or the amino optionally replaced.
Illustrative modification adenine includes the compound of formula (b41) to (b43):
Or its pharmaceutically acceptable salt or stereoisomer,
Wherein
Each R25It independently is H, halogeno-group, mercaptan, the alkyl optionally replaced, the alkenyl that optionally replaces, optionally replaces Alkynyl, the thio alkoxy optionally replaced or the amino optionally replaced；
R26aAnd R26bIt is each independently H, the acyl group that optionally replaces, the amino acid optionally replaced, the ammonia first optionally replaced Acyl, the alkyl optionally replaced, the alkenyl optionally replaced, the alkynyl optionally replaced, the hydroxy alkyl optionally replaced, optionally Substituted hydroxyalkenyl group, the hydroxyalkynyl optionally replaced, the alkoxy optionally replaced or polyethylene group are (for example,-(CH2)s2 (OCH2CH2)s1(CH2)s3OR ', wherein s1 is the integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are each independently 0 To the integer of 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and R ' is H or C1-20Alkyl)；Or the poly- second of amino- Glycol group is (for example,-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is 1 to 10 (for example, 1 to 6 or 1 to 4) Integer, s2 and s3 are each independently the integer of 0 to 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and each RN1The C for independently being hydrogen or optionally replacing1-6Alkyl)；And
Each R27It independently is H, the alkyl that optionally replaces, the alkenyl optionally replaced, the alkynyl optionally replaced, optionally replaces Alkoxy, the thio alkoxy optionally replaced or the amino optionally replaced.
In some embodiments, R26aFor H, and R26bFor the alkyl optionally replaced.In some embodiments, R26a And R26bIt is each independently the alkyl optionally replaced.In a particular embodiment, R27For optionally replace alkyl, optionally replace Alkoxy or the thio alkoxy that optionally replaces.In other embodiments, R25For optionally replace alkyl, optionally replace Alkoxy or the thio alkoxy optionally replaced.
In a particular embodiment, R26a、R26bOr R29Optional substituent group be polyethylene group (for example,-(CH2)s2 (OCH2CH2)s1(CH2)s3OR ', wherein s1 is the integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are each independently 0 To the integer of 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and R ' is H or C1-20Alkyl)；Or the poly- second of amino- Glycol group is (for example,-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1, wherein s1 is 1 to 10 (for example, 1 to 6 or 1 to 4) Integer, s2 and s3 are each independently the integer of 0 to 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and each RN1The C for independently being hydrogen or optionally replacing1-6Alkyl).
In some embodiments, B can have formula (b21):
Wherein X12The alkylidene (for example, methylene) for independently being O, S, optionally replacing Or the miscellaneous alkylidene optionally replaced, the integer that xa is 0 to 3, and R12aAnd T2As described herein.
In some embodiments, B can have formula (b22):
Wherein R10’Independently be the alkyl optionally replaced, the alkenyl optionally replaced, The alkynyl that optionally replaces, the heterocycle optionally replaced, the aminoalkyl that optionally replaces, optionally replaces the aryl optionally replaced Aminoalkenyl, the alkoxy optionally replaced, the alkoxy carbonyl alkyl optionally replaced, optionally takes the aminoalkynyl optionally replaced The alkoxycarbonylalkenyl in generation, the Alkoxycarbonylalkoxy optionally replaced, optionally takes the alkoxycarbonylalkynyl optionally replaced The Carboxyalkoxy in generation, the carboxyalkyl optionally replaced or the carbamyl alkyl optionally replaced, and R11、R12a、T1And T2 As described herein.
In some embodiments, B can have formula (b23):
Wherein R10For optionally replace heterocycle (for example, the furyl optionally replaced, appoint Choose the thienyl in generation or the pyrrole radicals that optionally replaces), the aryl that optionally replaces is (for example, the phenyl that optionally replaces or optionally take The naphthalene in generation) or any substituent group described herein (for example, for R10For)；And wherein R11(for example, H or this paper institute Description any substituent group), R12a(for example, any substituent group of H or described herein), T1(for example, oxo or this paper are retouched Any substituent group stated) and T2(for example, oxo or any substituent group described herein) is as described herein.
In some embodiments, B can have formula (b24):
Wherein R14’Independently be the alkyl optionally replaced, the alkenyl optionally replaced, The alkynyl that optionally replaces, the aryl optionally replaced, the heterocycle optionally replaced, the alkaryl optionally replaced, the alkane optionally replaced Heterocycle, the aminoalkyl optionally replaced, the aminoalkenyl optionally replaced, the aminoalkynyl optionally replaced, the alkane optionally replaced Oxygroup, the alkoxycarbonylalkenyl optionally replaced, the alkoxycarbonylalkynyl optionally replaced, the alkoxy carbonyl alkane optionally replaced Base, the Alkoxycarbonylalkoxy optionally replaced, the Carboxyalkoxy optionally replaced, the carboxyalkyl optionally replaced optionally take The carbamyl alkyl in generation, and R13a、R13b、R15And T3As described herein.
In some embodiments, B can have formula (b25):
Wherein R14’It is the heterocycle that optionally replaces (for example, the furan optionally replaced Mutter base, the thienyl optionally replaced or the pyrrole radicals optionally replaced), the aryl that optionally replaces (for example, the phenyl that optionally replaces or The naphthalene optionally replaced) or any substituent group described herein (for example, for R14Or R14’For)；And wherein R13a(example Such as, any substituent group of H or described herein), R13b(for example, any substituent group of H or described herein), R15(for example, H or Any substituent group described herein) and T3(for example, oxo or any substituent group described herein) is as described herein.
In some embodiments, B is the core selected from the group being made of cytimidine, guanine, adenine and uracil Base.In some embodiments, B may is that
In some embodiments, modification nucleobase is modification uracil.Exemplary nucleobase with modification uracil It include pseudouridine (ψ), pyridine -4- ketone ribonucleotide, 5- azepine-uridine, 6- azepine-uridine, the thio -5- azepine-of 2- with nucleosides Uridine, 2- be thio-uridine (s2U), 4- it is thio-uridine (s4U), 4- it is thio-pseudouridine, 2- be thio-pseudouridine, 5- hydroxyl-uridine (ho5U), 5- aminoallyl-uridine, 5- it is halogenated-uridine (for example, the iodo- uridine of 5- or the bromo- uridine of 5-), 3- methyl-uridine (m3U), 5- methoxyl group-uridine (mo5U), uridine 5- fluoroacetic acid (cmo5U), uridine 5- fluoroacetic acid methyl esters (mcmo5U), 5- carboxylic first Base-uridine (cm5U), 1- carboxymethyl-pseudouridine, 5- carboxyl hydroxymethyl-uridine (chm5U), 5- carboxyl hydroxymethyl-uridine methyl esters (mchm5U), 5- Methoxycarbonylmethyl-uridine (mcm5U), 5- Methoxycarbonylmethyl -2- it is thio-uridine (mcm5s2U)、5- Amino methyl -2- is thio-uridine (nm5s2U), 5- Methylaminomethyl-uridine (mnm5U), 5- Methylaminomethyl -2- it is thio - Uridine (mnm5s2U), 5- Methylaminomethyl -2- seleno-uridine (mnm5se2U), 5- carbamoyhnethyl-uridine (ncm5U)、 5- carboxymethylamino methyl-uridine (cmnm5U), 5- carboxymethylamino methyl -2- it is thio-uridine (cmnm5s2U), 5- propinyl- Uridine, 1- propinyl-pseudouridine, 5- taurine methyl-uridine (τ m5U), 1- taurine methyl-pseudouridine, 5- taurine first Base -2- is thio-uridine (τ m5s2U), 1- taurine methyl -4- it is thio-pseudouridine, 5- methyl-uridine (m5U has nucleobase Deoxythymidine), 1- methyl pseudouridine (m1ψ), 5- methyl -2- it is thio-uridine (m5s2U), 1- methyl -4- it is thio-pseudouridine (m1s4ψ), the thio -1- methyl-pseudouridine of 4-, 3- methyl-pseudouridine (m3ψ), the thio-1- methyl-pseudouridine of 2-, 1- methyl-1- Denitrogenation-pseudouridine, the thio-1- methyl-1-denitrogenation-pseudouridine of 2-, dihydrouridine (D), dihydro pseudouridine, 5,6- dihydrouridine, 5- methyl-dihydro uridine (m5D), 2- it is thio-dihydrouridine, 2- be thio-dihydro pseudouridine, 2- methoxyl group-uridine, 2- methoxy Base -4- is thio-and uridine, 4- methoxyl group-pseudouridine, 4- methoxyl group -2- be thio-pseudouridine, N1- methyl-pseudouridine (also known as 1- Methyl pseudouridine (m1ψ)), 3- (3- amino -3- carboxylic propyl) uridine (acp3U), the false urine of 1- methyl -3- (3- amino -3- carboxylic propyl) Glycosides (acp3ψ), 5- (isopentene group amino methyl) uridine (inm5U), 5- (isopentene group amino methyl) -2- it is thio-uridine (inm5s2U), α-it is thio-uridine, 2 '-O- methyl-uridines (Um), 5,2 '-O- dimethyl-uridine (m5Um), 2 '-O- methyl-vacation Thio -2 '-O- methyl-uridine (s of uridine (ψ m), 2-2Um), -2 '-O- of 5- Methoxycarbonylmethyl methyl-uridine (mcm5Um)、 5- carbamoyhnethyl -2 '-O- methyl-uridine (ncm5Um), 5- carboxymethylamino methyl -2 '-O- methyl-uridine (cmnm5Um), 3,2 '-O- dimethyl-uridine (m3Um), 5- (isopentene group amino methyl) -2 '-O- methyl-uridine (inm5Um), 1- it is thio-uridine, deoxythymidine, 2 '-F-ara- uridines, 2 '-F- uridines, 2 '-OH-ara- uridines, 5- (2- first Oxygen carbonyl vinyl) uridine and 5- [3- (1-E- allylamino) uridine.
In some embodiments, modification nucleobase is modification cytimidine.Exemplary nucleobase with modification cytimidine It include 5- azepine-cytidine, 6- azepine-cytidine, false different cytidine, 3- Methyl-Cytidine (m with nucleosides3C), N4- acetyl group-cytidine (ac4C), 5- formoxyl-cytidine (f5C), N4- Methyl-Cytidine (m4C), 5- Methyl-Cytidine (m5C), 5- it is halogenated-cytidine (for example, The iodo- cytidine of 5-), 5- methylol-cytidine (hm5C), 1- methyl-different cytidine of vacation, pyrrolo--cytidine, the different cytidine of pyrrolo--vacation, 2- Thio-cytidine (s2C), thio-false different cytidine of the thio -5- Methyl-Cytidine of 2-, 4-, the thio -1- methyl of 4--different cytidine of vacation, 4- sulphur Generation-1- methyl-1-different cytidine of denitrogenation-vacation, the 1- methyl-1-different cytidine of denitrogenation-vacation, Ze Bulaen (zebularine), 5- azepine- Ze Bulaen, 5- methyl-Ze Bulaen, the thio-Ze Bulaen of 5- azepine -2-, the thio-Ze Bulaen of 2-, 2- methoxyl group-born of the same parents Glycosides, 2- methoxyl group -5- Methyl-Cytidine, the 4- methoxyl group-different cytidine of vacation, 4- methoxyl group -1- methyl-different cytidine of vacation, lysidine (k2C), α-it is thio-cytidine, 2 '-O- Methyl-Cytidines (Cm), 5,2 '-O- dimethyl-cytidine (m5Cm), -2 '-O- of N4- acetyl group Methyl-Cytidine (ac4Cm), N4,2 '-O- dimethyl-cytidine (m4Cm), -2 '-O- of 5- formoxyl Methyl-Cytidine (f5Cm), N4, N4,2 '-O- trimethyls-cytidine (m4 2Cm), 1- it is thio-cytidine, 2 '-F-ara- cytidines, 2 '-F- cytidines and 2 '-OH-ara- born of the same parents Glycosides.
In some embodiments, modification nucleobase is modification adenine.The exemplary core alkali of adenine with modification Base and nucleosides include 2- Amino-purin, 2,6- diaminopurine, 2- amino -6- it is halogenated-purine is (for example, 2- amino -6- is chloro- fast Purine), 6- it is halogenated-purine (for example, the chloro- purine of 6-), 2- amino -6- methyl-Purine, 8- azido-adenosine, 7- denitrogenation-gland be fast Purine, 7- denitrogenation -8- azepine-adenine, 7- denitrogenation -2- Amino-purin, 7- denitrogenation -8- azepine -2- Amino-purin, 7- denitrogenation - 2,6- diaminopurines, 7- denitrogenation -8- azepine -2,6- diaminopurine, 1- methyl-adenosine (m1A), 2- methyl-adenine (m2A), N6- methyl-adenosine (m6A), 2- methyl mercapto-N6- methyl-adenosine (ms2m6A), N6- isopentenyl-adenosine (i6A)、2- Methyl mercapto-N6- isopentenyl-adenosine (ms2i6A), N6- (cis-hydroxyl groups isopentene group) adenosine (io6A), 2- methyl mercapto-N6- (cis-hydroxyl groups isopentene group) adenosine (ms2io6A), N6- glycyl carbamyl-adenosine (g6A), N6- Threonyl ammonia first Acyl group-adenosine (t6A), N6- methyl-N6- Threonyl carbamyl-adenosine (m6t6A), 2- methyl mercapto-N6- Threonyl ammonia Formoxyl-adenosine (ms2g6A), N6, N6- dimethyl-adenosine (m6 2A), the positive valyl base carbamyl-adenosine of N6- hydroxyl (hn6A), the positive valyl base carbamyl-adenosine (ms of 2- methyl mercapto-N6- hydroxyl2hn6A), N6- acetyl group-adenosine (ac6A)、 7- methyl-adenine, 2- methyl mercapto-adenine, 2- methoxyl group-adenine, α-be thio-adenosine, 2 '-O- methyl-adenosine (Am), N6,2 '-O- dimethyl-adenosine (m6Am) ,-O- of N6, N6,2 ' trimethyl-adenosine (m6 2Am), 1,2 '-O- dimethyl-adenosine (m1Am), 2 '-O- ribosyl adenosines (phosphate) (Ar (p)), 2- amino-N6- methyl-Purine, 1- it is thio-adenosine, 8- nitrine Base-adenosine, 2 '-F-ara- adenosines, 2 '-F- adenosines, 2 '-OH-ara- adenosines and N6- (- five oxa- nonadecane of 19- amino Base)-adenosine.
In some embodiments, modification nucleobase is modification guanine.The exemplary core alkali of guanine with modification Base and nucleosides include inosine (I), 1- methyl-inosine (m1I), Y nucleosides (wyosine) (imG), methyl Y nucleosides (mimG), 4- are de- Methyl-Y nucleosides (imG-14), different Y nucleosides (imG2), bosom fourth glycosides (yW), peroxide cherish fourth glycosides (o2YW), hydroxyl cherishes fourth glycosides (OHyW), it modifies insufficient hydroxyl and cherishes fourth glycosides (OHyW*), 7- denitrogenation-guanosine, pigtail glycosides (Q), epoxy pigtail glycosides (oQ), galactosyl- Pigtail glycosides (galQ), mannose group-pigtail glycosides (manQ), 7- cyano -7- denitrogenation-guanosine (preQ0), 7- amino methyl -7- denitrogenation-bird Glycosides (preQ1), ancient fast glycosides (G+), 7- denitrogenation -8- azepine-guanosine, 6- it is thio-guanosine, the thio -7- denitrogenation-guanosine of 6-, 6- be thio - 7- denitrogenation -8- azepine-guanosine, 7- methyl-guanosine (m7G), the thio -7- methyl-guanosine of 6-, 7- methyl-inosine, 6- methoxyl group - Guanosine, 1- methyl-guanosine (m1G), N2- methyl-guanosine (m2G), N2, N2- dimethyl-guanosine (m2 2G), N2,7- dimethyl-bird Glycosides (m2,7G), N2, N2,7- dimethyl-guanosine (m2,2,7G), 8- oxo-guanosine, 7- methyl -8- oxo-guanosine, 1- methyl -6- Thio-guanosine, N2- methyl -6- be thio-guanosine, N2, N2- dimethyl -6- is thio-and guanosine, α-be thio-guanosine, 2 '-O- methyl - Guanosine (Gm), N2- methyl -2 '-O- methyl-guanosine (m2Gm), N2, N2- dimethyl -2 '-O- methyl-guanosine (m2 2Gm), 1- first Base -2 '-O- methyl-guanosine (m1Gm), N2,7- dimethyl -2 '-O- methyl-guanosine (m2,7Gm), 2 '-O- methyl-inosine (Im), 1,2 '-O- dimethyl-inosine (m1) and 2 '-O- ribosyl guanosine (phosphate) (Gr (p)) Im.
The nucleobase of nucleotide can be independently selected from purine, pyrimidine, purine or pyrimidine analogue.For example, nucleobase can be each From independently selected from adenine, cytimidine, guanine, uracil or hypoxanthine.In another embodiment, nucleobase may be used also Including, for example, the both naturally occurring and synthetic derivative of base, including pyrazolo [3,4-d] pyrimidine, 5-methylcytosine The 6- methyl of (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2- aminoadenine, adenine and guanine and The 2- propyl and other alkyl derivatives of other alkyl derivatives, adenine and guanine, 2- thiouracil, the thio thymus gland of 2- are phonetic Pyridine and the thio cytimidine of 2-, 5- propynyluracil and cytimidine, 6- azo uracil, cytimidine and thymidine, 5- urine are phonetic Pyridine (pseudouracil), 4- thiouracil, 8- halogenated (for example, 8- bromine), 8- amino, 8- mercaptan, 8- alkylthio, 8- hydroxyl and its The adenine and guanine that its 8- replaces, the uracil that 5- halogeno-group, specifically 5- bromine, 5- trifluoromethyl and other 5- replace And cytimidine, 7- methyl guanine and 7- methyl adenine, guanozola and 8- azaadenine, deazaguanine, 7- Deazaguanine, 3- deazaguanine, denitrogenation adenine, 7- denitrogenation adenine, 3- denitrogenation adenine, pyrazolo [3,4-d] are phonetic Pyridine, 1,3,5 triazinone of imidazo [1,5-a], 9- deazapurine, imidazo [4,5-d] pyrazine, thiazole simultaneously [4,5-d] pyrimidine, pyrrole Piperazine -2- ketone, 1,2,4- triazine, pyridazine；And 1,3,5 triazines.When A, G, C, T or U are write a Chinese character in simplified form in use describes nucleotide, each word Mother refers to its representative base and/or derivative, for example, A includes adenine or Adenine derivatives, for example, 7- denitrogenation gland is fast Purine).
Modification on internucleoside linkage connection
The modified nucleoside acid that may be incorporated into polynucleotides, primary construct or mmRNA molecule can join (example in internucleoside linkage Such as, phosphate backbone) on be modified.Under the background of this paper polynucleotides main chain, phrase " phosphate " and " di-phosphate ester " can It is used interchangeably.Backbone phosphates group can be modified by replacing one or more oxygen atoms with different substituent groups.In addition, repairing Decorations nucleosides and nucleotide may include all replacing unmodified phosphate portion with another internucleoside linkage connection as described herein Point.The example of the bound phosphate groups of modification includes but is not limited to thiophosphate, phosphoroselenoate, borane phosphonate (boranophosphate), borane phosphonate (boranophosphate ester), hydrogen phosphonate ester, phosphoramidate, diamino Base phosphate, alkyl or aryl phosphonate ester and phosphotriester.Two disconnected oxygen that phosphorodithioate has are by sulphur Displacement.Phosphate connector can also be by with nitrogen (bridging phosphoramidate), sulphur (bridging thiophosphate) and carbon (bridging methylene Base-phosphonate ester) oxygen of connection is replaced to modify.
The thio substituted phosphonate moiety of α-is provided with will pass through non-natural phosphorothioate backbone it is bonded come to RNA and DNA polymer assigns stability.Phosphorothioate dna and RNA have increased nuclease resistant and therefore in cellular environments In have longer half-life period.It is expected that the polynucleotides of thiophosphate connection, primary construct or mmRNA molecule also pass through carefully Relatively weak binding/activation of born of the same parents' congenital immunity molecule reduces innate immune response.
In a particular embodiment, modified nucleoside include α-it is thio-nucleosides is (for example, 5 '-O- (1- thiophosphate)-gland Glycosides, 5 '-O- (1- thiophosphate)-cytidine (α-thio-cytidine), 5 '-O- (1- thiophosphate)-guanosine, 5 '-O- (1- sulphur Substituted phosphate)-uridine or 5 '-O- (1- thiophosphate)-pseudouridine).
It is described below the other internucleoside linkages connection that can be used according to the present invention herein, including between the nucleosides without phosphorus atoms It is bonded.
The combination of the sugar, nucleobase and internucleoside linkage connection of modification
Polynucleotides, primary construct and mmRNA of the invention may include joining to sugar, nucleobase and/or internucleoside linkage The combination of modification.These combinations may include any one or more modifications described herein.For example, this paper formula (Ia), (Ia- 1) to (Ia-3), (Ib) to (If), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), any nucleotide described in (IVa) to (IVl) and (IXa)-(IXr) can be with any core described herein Base composition (for example, formula (b1) to (b43) or it is described herein it is any other in).
The synthesis of polypeptide, primary construct and mmRNA molecule
It can be appointed according to as described herein for polypeptide used according to the invention, primary construct and mmRNA molecule It is prepared by what useful technology.For repairing in the synthesis of polynucleotides disclosed herein, primary construct and mmRNA molecule Following conventional method and process can be used from readily available starting material to prepare in decorations nucleosides and nucleotide.It is typical providing Or in the case where preferred process conditions (for example, reaction temperature, time, the molar ratio of reactant, solvent, pressure etc.), it is ripe Experienced insider will optimize and develop other process conditions.Optimum reaction condition can be according to used specific reaction Object or solvent change, but this kind of condition can be determined by those skilled in the art by optimization routine process.
Procedures described herein can be monitored according to any suitable method as known in the art.For example, can lead to Cross spectrum means such as nuclear magnetic resonance spectroscopy (for example,1H or13C), infra-red sepectrometry, spectrophotometry are (for example, UV-- can See) or mass spectrography or formed by chromatography such as high performance liquid chromatography (HPLC) or thin-layered chromatography to monitor product.
The preparation of polypeptide, primary construct and mmRNA molecule of the invention can be related to the protection of various chemical groups and take off Protection.The selection of the needs and suitable protecting group of protection and deprotection can be readily determined by those skilled in the art. The chemistry of blocking group can be in such as Greene et al., Protective Groups in Organic Synthesis, and the 2nd Version, Wiley & Sons are found in 1991, and the document is incorporated herein in its entirety by reference.
The reaction of procedures described herein can carry out in a suitable solvent, and the solvent can be by organic synthesis field Technical staff be readily selected.Suitable solvent can be in the carried out temperature of reaction (that is, can be in the cryogenic temperature of solvent Temperature to the boiling temperature of solvent) under it is generally nonreactive with starting material (reactant), intermediate or product. Given reaction can carry out in the mixture of a kind of solvent or more than one solvent.It, can be for tool depending on specific reaction step Precursor reactant step selects suitable solvent.
The fractionation of the racemic mixture of modified nucleoside and nucleotide can be by a variety of methods as known in the art Any one carries out.Illustrative methods include using the fractional recrystallization of " chiral resolution acid ", and chiral resolution acid is a kind of light Learn active, salt-forming organic acid.Suitable resolving agent for fractional recrystallization method is such as optically active acid, such as D and L shape Tartaric acid, acetyl tartaric acid, dibenzoyl tartaric acid, mandelic acid, malic acid, the lactic acid or various optically active of formula Camphorsulfonic acid.The fractionation of racemic mixture can also be by being filled with optical activity resolving agent (for example, dinitrobenzoyl Phenylglycine) column on elution to carry out.Suitable eluting solvent composition can be determined by those skilled in the art.
It is prepared by modified nucleoside and nucleotide (for example, the structural unit molecule) synthetic method described in following: Ogata et al., J.Org.Chem.74:2585-2588 (2009)；Purmal et al., Nucl.Acids Res.22 (1): 72- 78, (1994)；Fukuhara et al., Biochemistry, 1 (4): 563-568 (1962)；And Xu et al., Tetrahedron, 48 (9): 1729-1740 (1992), the document are respectively incorporated herein in its entirety by reference.
Polypeptide of the invention, primary construct and mmRNA can be or can not be the whole length along molecule and uniformly repair Decorations.For example, one or more or all types of nucleotide are (for example, purine or any one of pyrimidine or A, G, U, C It is multiple or whole) it can be or can not be in polynucleotides of the invention or give predetermined sequence area at it It is uniformly modified in (for example, the one or more sequence areas indicated in Fig. 1).In some embodiments, multicore of the invention All nucleotide X in thuja acid (or in its given sequence area) are modified, and wherein X can be any in nucleotide A, G, U, C Any of a or combination A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
Different sugar-modified, nucleotide is modified and/or internucleoside linkage connection (for example, backbone structure) may be present in multicore glycosides At different location in acid, primary construct or mmRNA.Those skilled in the art will be appreciated that nucleotide analog or its It is modified at any position that can be located at polynucleotides, primary construct or mmRNA, so that polynucleotides, primary construct Or the function of mmRNA is not reduced generally.Modification can also be that 5 ' or 3 ' is end modified.Polynucleotides, primary construct or MmRNA can include about the modified nucleoside acid of 1% to about 100% (relative to overall nucleotide content or relative to one or more The nucleotide of type, that is, any one or more of A, G, U or C) or any intermediate percentage (for example, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% To 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100% and 95% to 100%).
In some embodiments, polynucleotides, primary construct or mmRNA include modification pyrimidine (for example, modification urine is phonetic Pyridine/uridine/U or modification cytimidine/cytidine/C).In some embodiments, polynucleotides, primary construct or mmRNA molecule In uracil or uridine (usually: U) can with the modification uracil of about 1% to about 100% or modification uridine (for example, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% To 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100% and 95% to 100% modification Uracil or modification uridine) displacement.It modifies uracil or uridine can be by the compound with single unique texture or by with not The multiple compounds displacement of same structure (for example, 2,3,4 or more unique textures, as described herein).In some embodiment party In case, polynucleotides, primary construct or cytimidine in mmRNA molecule or cytidine are (usually: C) can use about 1% to about 100% Modification cytimidine or modification cytidine (for example, 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% To 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% To the modification cytimidine or modification cytidine of 95%, 90% to 100% and 95% to 100%) displacement.Modify cytimidine or cytidine Can by with single unique texture compound or by with different structure (for example, 2,3,4 or more unique textures, such as this Described by text) multiple compounds displacement.
In some embodiments, the disclosure provides the polynucleotides for the connection nucleosides that synthesis includes n number, primary building The method of body or mmRNA (for example, firstth area, the first flanking region or second flanking region), the nucleosides have formula (Ia-1):
The method includes
A) nucleotide of formula (IV-1):
It is reacted with the phosphoramidite compound of formula (V-1):
Wherein Y9For H, hydroxyl, phosphoryl, pyrophosphate, sulfuric ester, amino, mercaptan, the optionally amino acid or peptide that replace (e.g., including 2 to 12 amino acid)；And each P1、P2And P3It independently is suitable blocking group；AndIt refers to solid Phase support；
In order to provide the polynucleotides of formula (VI-1), primary construct or mmRNA:
And
B) make polynucleotides, primary construct or the mmRNA oxidation of formula (V) or vulcanize more so as to production (VII-1) Nucleotide, primary construct or mmRNA:
And
C) removal blocking group is so as to the polynucleotides of production (Ia), primary construct or mmRNA.
In some embodiments, it is repeated 1 times by step a) and b) to about 10,000 times.In some embodiments, institute The method of stating further comprises the nucleotide (example selected from the group being made of A, C, G and U adenosine, cytimidine, guanosine and uracil Such as, mmRNA molecule).In some embodiments, nucleobase can be pyrimidine or derivatives thereof.In some embodiments, institute It is interpretable for stating polynucleotides, primary construct or mmRNA.
Other components of polynucleotides, primary construct and mmRNA are optional, and are had in some embodiments Benefit.For example, providing 5 ' non-translational regions (UTR) and/or 3 ' UTR, any one of them or both can be independently comprising a kind of or more The different nucleotide modification of kind.In this kind of embodiment, nucleotide modification also may be present in can be in translated region.It also provides and contains The polynucleotides of Kozak sequence, primary construct and mmRNA.
The exemplary synthesis of the modified nucleoside acid in modification of nucleic acids or mmRNA (for example, RNA or mRNA) is incorporated to lower section Case 1 is provided into scheme 11.Scheme 1 provides the conventional method for phosphorylated nucleosides sour (including modified nucleoside).
Scheme 1
Various blocking groups can be used to control reaction.For example, scheme 2 provide using it is multiple protection and deprotection steps with Just promote sugar 5 ' positions at rather than the phosphorylation of 2 ' and 3 ' hydroxyls.
Scheme 2
Modified nucleoside acid can synthesize any useful manner.Scheme 3,4 and 7, which is provided, has modification purine for synthesizing The illustrative methods of the modified nucleoside acid of nucleobase；And scheme 5 and 6, which is provided, is respectively provided with modification pseudouridine or vacation for synthesizing The illustrative methods of the modified nucleoside acid of different cytidine.
Scheme 3
Scheme 4
Scheme 5
Scheme 6
Scheme 7
The exemplary synthesis of the offer modified nucleoside acid of scheme 8 and 9.Scheme 10 is provided for generating the non-limiting of nucleotide Biocatalysis method.
Scheme 8
Scheme 9
Scheme 10
Scheme 11 provides the exemplary synthesis of modification uracil, wherein the position the N1 R provided such as other places12bModification, and 5 ' positions of ribose are phosphorylated.T1、T2、R12a、R12bAnd r is as provided herein.The pattern of this synthesis and its optimization can For modify other pyrimidine nucleobases and purine nucleobase (see, e.g. formula (b1) to (b43)) and/or for be arranged one or Multiple bound phosphate groups (for example, at 5 ' positions of sugar).This alkylated reaction can also be used in described herein any Any reactive group (for example, amino) in nucleobase is (for example, the Watson-of cytimidine, uracil, adenine and guanine Amino in Crick base pairing face) at include one or more alkyl optionally replaced.
Scheme 11
The combination of nucleotide in mmRNA
Modified nucleoside acid and other examples of modified nucleoside acid combination are provided in following table 9.These modified nucleosides acid Combination can be used to form polypeptide of the invention, primary construct or mmRNA.Unless otherwise specified, modified nucleoside acid can be with Replace the natural nucleotide of modification of nucleic acids or mmRNA of the invention completely.As non-limiting examples, natural nucleotide uridine can To be replaced by modified nucleoside described herein.In another non-limiting example, natural nucleotide uridine can be by herein Disclosed at least one modified nucleoside partly replace (for example, about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%).
Table 9
Other examples of modified nucleoside acid combination are provided in following table 10.The combination of these modified nucleosides acid can be used for shape At polypeptide of the invention, primary construct or mmRNA.
Table 10
In some embodiments, at least 25% cytimidine is replaced by the compound of formula (b10) to (b14) (for example, extremely Few about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, extremely Few about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or About 100%).
In some embodiments, at least 25% uracil is replaced by the compound of formula (b1) to (b9) (for example, at least About 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least About 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100%).
In some embodiments, at least 25% cytimidine is replaced by formula (b10)-(b14) compound, and at least 25% uracil by formula (b1)-(b9) compound replace (for example, at least about 30%, at least about 35%, at least about 40%, At least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, At least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100%).
IV. pharmaceutical composition
It prepares, application, deliver and be administered
The present invention provide with the polynucleotides of one or more pharmaceutically acceptable excipient compositions, primary construct and MmRNA composition and compound.Pharmaceutical composition optionally including one or more other active materials, such as treatment and/ Or prophylactic activity substance.General Consideration in terms of preparing and/or manufacturing medicament is found in such as Remington:The Science and Practice of Pharmacy the 21st edition, Lippincott Williams & Wilkins, 2005 (with The mode of reference is incorporated herein) in.
In some embodiments, to people, i.e. human patient or subject applies composition.For the purpose of this disclosure, short Language " active constituent " typically refers to the polynucleotides delivered as described herein, primary construct and mmRNA.
Although the description of pharmaceutical composition provided herein relates generally to the pharmaceutical composition for being suitable for administering to the human, It is for example (such as inhuman to non-human animal to any other animal that skilled people in the industry will be understood that such composition is commonly available to Mammal) application.Change the pharmaceutical composition suitable for administering to the human to make composition be suitable for applying to various animals With for it is well known that and common veterinary pharmacology man can be designed only by routine experimentation (if necessary) and/or into Such change of row.Being expected includes but is not limited to that people and/or other primates are dynamic to its subject for applying pharmaceutical composition Object；Mammal, including commercially relevant mammal such as ox, pig, horse, sheep, cat, dog, mouse and/or rat；And/or Birds, including commercially relevant birds such as poultry, chicken, duck, goose and/or turkey.
The preparation of pharmaceutical composition described herein can pass through any method that is known in area of pharmacology or hereafter developing To prepare.In general, such preparation method is the following steps are included: make active constituent and excipient and/or one or more other auxiliary Co-ingredients association, and then if necessary and/or wishing, make product divide, shape and/or be packaged as desired single dose or Multi-dose unit.
Pharmaceutical composition according to the present invention can be made with single unit dose and/or with multiple single unit dose high-volume Standby, packaging and/or sale.As used herein, " unit dose " is the medicine group of the active constituent comprising predetermined quantities Close the discrete amount of object.The amount of active constituent be generally equal to will to subject apply active constituent dosage and/or this dose The suitable score of amount, a half or thirds of such as this dosage.
Active constituent, pharmaceutically acceptable excipient in pharmaceutical composition according to the present invention and/or it is any in addition The relative quantity of ingredient will depend on identity, size and/or the symptom of treated subject and further depend on to be administered The approach of composition and change.As an example, composition may include between 0.1% and 100%, such as between .5% and 50%, Between 1% to 30%, between 5% and 80%, the active constituent of at least 80% (w/w).
Preparation
Polynucleotides of the invention, primary construct and mmRNA one or more excipient can be used prepare so as to: (1) increase stability；(2) increase cell transfecting；(3) allow to continue or sustained release is (for example, from polynucleotides, primary building The depot formulation of body or mmRNA release)；(4) change bio distribution (for example, making polynucleotides, primary construct or mmRNA target To specific organization or cell type)；(5) increase the translation of the protein encoded in vivo；And/or (6) change the egg encoded in vivo The release overview of white matter.In addition to for example any and all solvents of conventional excipients, decentralized medium, diluent or other liquid medias Except object, dispersion or suspension aids, surfactant, isotonic agent, thickener or emulsifier, preservative, excipient of the invention It may include but be not limited to lipoids (lipidoid), liposome, lipidic nanoparticles, polymer, lipid complex, core-shell structure copolymer to receive Rice grain, peptide, protein, the cell transfected with polynucleotides, primary construct or mmRNA are (for example, for being transplanted to subject In), hyaluronidase, nano particle analogies with and combinations thereof.Therefore, preparation of the invention may include respectively with a certain amount Existing one or more excipient, the amount increase polynucleotides, the stability of primary construct or mmRNA, increasing altogether Add the cell transfecting carried out by polynucleotides, primary construct or mmRNA, increase polynucleotides, primary construct or mmRNA volume The expression of the protein of code and/or the release overview for changing the protein that polynucleotides, primary construct or mmRNA are encoded.This Outside, self assembly nucleic acid nano particle can be used to prepare for primary construct of the invention and mmRNA.
The preparation of pharmaceutical composition described herein can pass through any method that is known in area of pharmacology or hereafter developing To prepare.In general, such preparation method includes that active constituent and excipient and/or one or more other auxiliary elements is made to associate The step of.
It can be made with single unit dose and/or with multiple single unit dose high-volume according to the pharmaceutical composition of the disclosure Standby, packaging and/or sale.As used herein, " unit dose " refers to the drug of the active constituent comprising predetermined quantities The discrete amount of composition.The amount of active constituent can generally equal to will to subject apply active constituent dosage and/or this The suitable score of kind dosage, a half or thirds of including but not limited to this dosage.
According in the pharmaceutical composition of the disclosure active constituent, pharmaceutically acceptable excipient and/or it is any in addition The relative quantity of ingredient may depend on identity, size and/or the symptom of treated subject and further depend on to be administered The approach of composition and change.For example, composition may include the active constituent between 0.1% and 99% (w/w).
In some embodiments, preparation described herein can contain at least one mmRNA.As a non-limiting reality Example, preparation can contain 1,2,3,4 or 5 kind of mmRNA.In one embodiment, preparation is containing coding selected from following classification The modification mRNA of protein, the protein classification are such as, but not limited to human protein, protein for animals, bacterio protein, life Object protein, antibody, immunogenic protein, therapeutic peptide and protein, secretory protein, plasmalemma protein, cytoplasmic protein and cell Skelemin, intercellular membrane binding protein, nucleoprotein, protein relevant to human diseases and/or related with non-human disease Protein.In one embodiment, preparation contains the modification mRNA of at least three kinds coding proteins.In an embodiment In, preparation contains the modification mRNA of at least five kinds coding proteins.
Pharmaceutical preparation can additionally comprise pharmaceutically acceptable excipient, and as used herein excipient includes but unlimited In any and all solvents, decentralized medium, diluent or the other liquid vehicles, dispersion that are suitable for desired specific dosage form Or suspension aids, surfactant, isotonic agent, thickener or emulsifier, preservative etc..For the various of compounding pharmaceutical composition Excipient and the technology for being used to prepare composition are as known in the art (referring to Remington:The Science and Practice of Pharmacy, the 21st edition, A.R.Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006；It is incorporated herein in its entirety by reference).The use of conventional excipients medium can be covered in this public affairs In the range of opening, in addition to as by generate any undesirable biological effect or in addition in harmful manner with pharmaceutical composition The interaction of any other component and may be incompatible with substance or derivatives thereof any conventional excipients medium.
In some embodiments, it can increase and/or reduce the granularity of lipidic nanoparticles.The variation of granularity can be helped It helps counteracting biological respinse to be such as, but not limited to inflammation or the biological effect for being delivered to the modification mRNA of mammal can be increased.
The pharmaceutically acceptable excipient used in the manufacture of pharmaceutical composition include but is not limited to inert diluent, Surfactant and/or emulsifier, preservative, buffer, lubricant and/or oil.Such excipient can be optionally included in this hair In bright pharmaceutical preparation.
Lipoids
The synthesis of lipoids and the preparation containing these compounds have been generally described especially suitable for delivering multicore glycosides Acid, primary construct or mmRNA are (referring to Mahon etc., Bioconjug Chem.2010 21:1448-1454；Schroeder Deng J Intern Med.2010 267:9-21；Akinc etc., Nat Biotechnol.2008 26:561-569；Love etc., Proc Natl Acad Sci U S A.2010 107:1864-1869；Siegwart etc., Proc Natl Acad Sci U S A.2011 108:12996-3001；All bibliography are integrally incorporated herein).
Although these lipoids have been used in rodent and non-human primate body effective delivering double stranded small interference RNA molecule is (referring to Akinc etc., Nat Biotechnol.2008 26:561-569；Frank-Kamenetsky etc., Proc Natl Acad Sci U S A.2008 105:11915-11920；Akinc etc., Mol Ther.2009 17:872-879； Love etc., Proc Natl Acad Sci U S A.2010 107:1864-1869；Leuschner etc., Nat Biotechnol.2011 29:1005-1010；All bibliography are integrally incorporated herein), but the present disclosure describes It is prepared and the purposes in delivering single stranded polynucleotide, primary construct or mmRNA.Compound, micella, liposome or particle It can be prepared into containing these lipoids and therefore the effective delivering that can produce polynucleotides, primary construct or mmRNA, it is such as logical It crosses part and/or systemic application approach injection lipoids preparation generates what the protein encoded was judged later.Polynucleotides, just The lipoids compound of grade construct or mmRNA can be applied by various modes, including but not limited to intravenous route, intramuscular way Diameter or subcutaneous route.
The internal delivering of nucleic acid can be included but is not limited to the property that preparation forms, particle is PEGylated, load journey by many parameters Degree, oligonucleotides and drugrlipid ratio and biophysical parameters such as, but not limited to, granularity influence (Akinc etc., Mol Ther.2009 17:872-879；It is incorporated herein in its entirety by reference).As an example, poly(ethylene glycol) (PEG) The small variation of the length of chain cable of lipid can make a significant impact in vivo efficacy.The body of the preparation with different lipoids can be tested Interior activity, the lipoids include but is not limited to five [3- (1- lauryl amino propiono)]-trien hydrochlorides (TETA-5LAP；Also known as 98N12-5, referring to Murugaiah etc., Analytical Biochemistry, 401:61 (2010)； It is incorporated herein in its entirety by reference), C12-200 (including derivative and variant) and MD1.
The herein referred as lipoids of " 98N12-5 " is by Akinc etc., and Mol Ther.200917:872-879 is disclosed and it It is incorporated hereby.(referring to fig. 2)
The herein referred as lipoids of " C12-200 " is by Love etc., Proc Natl Acad Sci U S A.2010 107: 1864-1869 (referring to fig. 2) and Liu and Huang, Molecular Therapy.2010 669-670 (referring to fig. 2) are open； The bibliography is incorporated herein in its entirety by reference.Lipoids preparation may include in addition to polynucleotides, primary building Particle except body or mmRNA also comprising 3 or 4 kind or more component.As an example, the preparation with certain lipoids Including but not limited to 98N12-5 and 42% lipoids, 48% cholesterol and 10%PEG (C14 long alkyl chains) can be contained.Make For another example, the preparation with certain lipoids includes but is not limited to C12-200 and can contain 50% lipoids, 10% Distearoyl Phosphatidylcholine, 38.5% cholesterol and 1.5%PEG-DMG.
In one embodiment, with the polynucleotides for being applied in systemic veins of lipoids preparation, primary building Body or mmRNA can target liver.For example, the iv formulation of final optimization pass can cause preparation to be greater than 90% distribution to liver, institute The iv formulation of final optimization pass is stated using polynucleotides, primary construct or mmRNA, and includes 42%98N12-5,48% The molar lipid of cholesterol and 10%PEG- lipid composition, about 7.5 to 1 total lipid than polynucleotides, primary construct or The final weight ratio of mmRNA, and the C14 long alkyl chains on PEG lipid, and average particle size is about 50-60nm.(ginseng See, Akinc etc., Mol Ther.2009 17:872-879；It is incorporated herein in its entirety by reference).In another example In, using C12-200 (referring to U.S. Provisional Application 61/175,770 and the international application WO2010129709 announced, it is described specially Benefit is respectively incorporated herein in its entirety by reference) iv formulation of lipoids can have the C12- of 50/10/38.5/1.5 200/ Distearoyl Phosphatidylcholine/cholesterol/PEG-DMG molar ratio, 7 to 1 total lipid is than polynucleotides, primary building The weight ratio of body or mmRNA, and average particle size is 80nm, it can be effective in polynucleotides, primary construct or mmRNA be delivered To liver cell (referring to Love etc., Proc Natl Acad Sci U S A.2010 107:1864-1869, with the side of reference Formula is integrally incorporated herein).In another embodiment, the preparation of the lipoids containing MD1 can be used in vivo effectively by multicore Thuja acid, primary construct or mmRNA are delivered to liver cell.Spy for intramuscular route or the optimization lipoids preparation of subcutaneous route Sign may depend on target cell type and preparation diffuse through extracellular matrix enter blood flow ability and significant changes.Although due to The size of endothelium fenestra (endothelial fenestrae), it may be desirable to be passed less than the granularity of 150nm for effective liver cell It send (referring to Akinc etc., Mol Ther.2009 17:872-879 are incorporated herein in its entirety by reference), but uses Polynucleotides, primary construct or the mmRNA that lipoids is prepared are by formulation delivered to other cell types (in including but not limited to Chrotoplast, bone marrow cell and myocyte) it can not be by similarly sized limitation.Having reported will be passed using lipoids preparation in siRNA body Send to the cell such as bone marrow cell and endothelium of other non-liver cells (referring to Akinc etc., Nat Biotechnol.2008 26: 561-569；Leuschner etc., Nat Biotechnol.2011 29:1005-1010；Cho etc. Adv.Funct.Mater.2009 19:3112-3118；8th International Judah Folkman Conference, Cambridge, MA October 8-9,2010；The bibliography respectively it is whole by reference simultaneously Enter herein).The lipoids preparation for being effectively delivered to bone marrow cell such as monocyte can have similar component molar ratio.Lipoids It can be used to optimize from the different ratios that other components include but is not limited to Distearoyl Phosphatidylcholine, cholesterol and PEG-DMG Polynucleotides, primary structure for delivery to different cell types (including but not limited to liver cell, bone marrow cell, myocyte etc.) Build body or the preparation of mmRNA.For example, component molar ratio may include but be not limited to 50%C12-200,10% distearoylphosphatidyl Choline, 38.5% cholesterol and %1.5PEG-DMG are (referring to Leuschner etc., 2011 29:1005- of Nat Biotechnol 1010；It is incorporated herein in its entirety by reference).Used by subcutaneously or intramuscularly delivering for by nucleic acid local delivery extremely The lipoids preparation of cell (such as, but not limited to fat cell and myocyte) can not need system and deliver the desired ownership Agent component, and therefore can be only comprising lipoids and polynucleotides, primary construct or mmRNA.
The protein that the combination of different lipoids can be used to improve polynucleotides, primary construct or mmRNA guidance generates The effect of, because lipoids can increase the cell transfecting carried out by polynucleotides, primary construct or mmRNA；And/or increase Add the translation of the protein of coding (referring to Whitehead etc., Mol.Ther.2011,19:1688-1694, with the side of reference Formula is integrally incorporated herein).
Liposome, lipid complex and lipidic nanoparticles
Can be used one or more liposomes, lipid complex or lipidic nanoparticles prepare polynucleotides of the invention, Primary construct and mmRNA.In one embodiment, the pharmaceutical composition of polynucleotides, primary construct or mmRNA includes Liposome.Liposome is the vesica manually prepared, can mainly be made of and can be used as applying nutrients double-layer of lipoid With the delivery vehicle of pharmaceutical preparation.Liposome can have a different size, such as, but not limited to diameter can for hundreds of nanometers and Contain the small list that 50nm is smaller than by a series of multi-layer vesicles (MLV) of the separated concentric bilayers of narrow aqueous compartment, diameter Cell vesicle (SUV) and diameter can big unicellular vesicas (LUV) between 50 and 500 nm (nanometers.Liposome designs But it is not limited to opsonin or ligand, to improve liposome to the attachment of unhealthy tissue or starting such as, but not limited to endocytosis Event.Liposome improves the delivering of pharmaceutical preparation containing low or high pH.
The formation of liposome may depend on physicochemical characteristic, the pharmaceutical preparation and liposome such as, but not limited to embedded at Point, wherein disperse the property of medium of lipid vesicle, the effective concentration of institute's embedding substance and its genotoxic potential, the application in vesica And/or it any other process involved in delivery process, the optimizing the size of vesica for intended application, polydispersity and guarantees the quality Phase, and the reproducibility and possibility between batches of safely and effectively liposomal product is mass produced.
In one embodiment, pharmaceutical composition described herein may include but be not limited to liposome, such as from following object Those of matter formation: 1,2- bis- oil base oxygroup-N, N- dimethylaminopropanecompounds (DODMA) liposome comes from Marina The sub- oil base oxygroup -3- dimethylaminopropanecompounds (DLin-DMA) of DiLa2 liposome, 1,2- bis- of Biotech (Bothell, WA), 2,2- bis- Asia oil base -4- (2- dimethyl aminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA) and MC3 (US20100324120；It is incorporated herein in its entirety by reference), and the liposome of small-molecule drug can be delivered, such as but It is not limited to from Janssen Biotech, Inc.'s (Horsham, PA)
In one embodiment, pharmaceutical composition described herein may include but be not limited to liposome, such as first from synthesis It is preceding to have described and the stable plasmid-lipid granule (SPLP) or stable nucleus suitable in vitro and in vivo oligonucleotide delivery are shown Sour lipid granule (SNALP) and formed those of (referring to the Gene Therapy.1999 such as Wheeler 6:271-281；Zhang Equal Gene Therapy.1999 6:1438-1447；The Pharm Res.2005 such as Jeffs 22:362-372；Morrissey etc., Nat Biotechnol.2005 2:1002-1007；Zimmermann etc., Nature.2006 441:111-114；The J such as Heyes Contr Rel.2005 107:276-287；The Nature Biotech.2010 such as Semple 28:172-176；The J such as Judge Clin Invest.2009 119:661-673；DeFougerolles Hum Gene Ther.2008 19:125-132；It is all The bibliography is integrally incorporated herein).The original manufacturing method of Wheeler etc. is detergent dialysis method, later quilt Jeffs etc. is improved and referred to as spontaneous vesica forming method.Other than polynucleotides, primary construct or mmRNA, liposome Preparation is mainly made of 3 to 4 kinds of lipid compositions.As an example, liposome contains but is not limited to as described in Jeffs etc. 55% cholesterol, 20% Distearoyl Phosphatidylcholine (DSPC), 10%PEG-S-DSG and 15%1, bis- oil base oxygroup of 2-- N, N- dimethylaminopropanecompounds (DODMA).As another example, certain Liposomal formulations can contain but be not limited to such as Heyes Deng 48% cholesterol, 20%DSPC, 2%PEG-c-DMA and 30% cation lipid, wherein cation lipid can be 1,2- distearyl oxygroup-N, N- dimethylaminopropanecompounds (DSDMA), DODMA, DLin-DMA or 1, bis- linolenyl oxygroup of 2-- 3- dimethylaminopropanecompounds (DLenDMA).
In one embodiment, pharmaceutical composition may include liposome, can be formed to deliver codified at least one The mmRNA of immunogene.MmRNA can by it is liposomal encapsulated and/or it may include then can be by liposomal encapsulated aqueous core (referring to international publication number WO2012031046, WO2012031043, WO2012030901 and WO2012006378；The patent It is respectively incorporated herein in its entirety by reference).In another embodiment, the mmRNA of codified immunogene can be prepared In cationic oil in water emulsion, wherein emulsion grain includes that oily core and cation lipid, cation lipid are mutual with mmRNA Effect is to be anchored into emulsion grain (referring to international publication number WO2012006380 for the molecule；It is whole by reference Body is incorporated herein).In another embodiment again, lipid formulations may include at least cation lipid, the rouge of transfection can be enhanced Matter and at least one lipid (international publication number WO2011076807 and U.S. containing the hydrophilic head base for being connected to lipid part Publication No. 20110200582；The patent is respectively incorporated herein in its entirety by reference).In another embodiment, The polynucleotides of encoding immunogens, primary construct and/or mmRNA can be prepared in lipid vesicle, and lipid vesicle can be in function There is crosslinking (referring to U.S. Publication No 20120177724, to be incorporated hereby this between the double-layer of lipoid of change Text).
In one embodiment, polynucleotides, primary construct and/or mmRNA can be prepared in lipid vesicle, lipid Vesica can have crosslinking between functionalized double-layer of lipoid.
In one embodiment, polynucleotides, primary construct and/or mmRNA can be prepared comprising cation lipid Liposome in.The molar ratio (N: P ratio) of the phosphate in nitrogen-atoms and RNA in the cation lipid of liposome can be 1: 1 Between 20: 1, as being incorporated herein in its entirety by reference described in international publication number WO2013006825.Another In a embodiment, N: P ratio of liposome may be greater than 20: 1 or less than 1: 1.
In one embodiment, polynucleotides, primary construct and/or mmRNA can be prepared multiple in lipid-polycation It closes in object.The formation of lipid-polycation complexes can be by as is generally known in the art and/or such as in U.S. Publication No Method described in 20120178702 is completed, and the patent is incorporated herein in its entirety by reference.It is unrestricted as one Property example, polycation may include that cationic peptide or polypeptide are such as, but not limited to polylysine, poly ornithine and/or poly arginine And the cationic peptide described in international publication number WO2012013326；The patent is incorporated hereby this Text.In another embodiment, polynucleotides, primary construct and/or mmRNA can be prepared compound in lipid-polycation In object, lipid-polycation complexes can further comprise neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidyl Ethanol amine (DOPE).
Liposomal formulation can be influenced by (but being not limited to) following factor: the selection of cation lipid component, cation lipid Saturation degree, PEGylated property, all components ratio and biophysical parameters such as size.In (the Semple etc. such as Semple Nature Biotech.2010 28:172-176；It is incorporated herein in its entirety by reference) an example in, liposome Preparation is mainly by 57.1% cation lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3% cholesterol and 1.4%PEG- C-DMA composition.As another example, siRNA more effectively can be delivered to various antigens by the composition for changing cation lipid In delivery cell (the Mol Ther.2011 such as Basha 19:2186-2200；It is incorporated herein in its entirety by reference).
In some embodiments, the PEG ratio in lipidic nanoparticles (LNP) preparation can be increasedd or decreased and/or can The carbon chain lengths of PEG lipid are modified to C18 from C14 to change the pharmacokinetics and/or bio distribution of LNP preparation. As a non-limiting example, compared with cation lipid, DSPC and cholesterol, PEG- of the LNP preparation containing 1%-5% The lipid molar ratios of c-DOMG.In another embodiment, PEG-c-DOMG can be substituted by PEG lipid, the PEG lipid example Such as, but not limited to, PEG-DSG (1,2- distearyl acyl group-sn- glycerol, methoxy poly (ethylene glycol)) or PEG-DPG (1,2- bis- palm Acyl group-sn- glycerol, methoxy poly (ethylene glycol)).Cation lipid can be selected from any lipid as known in the art, such as but not It is limited to DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.
In one embodiment, polynucleotides, primary construct or mmRNA can be prepared in lipidic nanoparticles, such as Described in the international publication number WO2012170930 those, the patent is incorporated herein in its entirety by reference.
In one embodiment, cation lipid may be selected from but not limited to international publication number WO2012040184, WO2011153120、WO2011149733、WO2011090965、WO2011043913、WO2011022460、 WO2012061259, WO2012054365, WO2012044638, WO2010080724, WO201021865 and WO2008103276, U.S. Patent number 7,893,302,7,404,969 and 8,283,333 and U.S. Patent Publication number Cation lipid described in US20100036115 and US20120202871；The patent is respectively whole by reference It is incorporated herein.In another embodiment, cation lipid may be selected from but not limited to international publication number WO2012040184, WO2011153120、WO2011149733、WO2011090965、WO2011043913、WO2011022460、 Formula A described in WO2012061259, WO2012054365 and WO2012044638；The patent is respectively by reference It is integrally incorporated herein.In another embodiment again, cation lipid may be selected from but not limited to international publication number The formula CLI-CLXXIX of WO2008103276, the formula CLI-CLXXIX of U.S. Patent number 7,893,302, U.S. Patent number 7,404, Formulas I-the VI of 969 formula CLI-CLXXXXII and U.S. Patent Publication number US20100036115；The patent is respectively to draw Mode is integrally incorporated herein.As a non-limiting example, cation lipid can be selected from (20Z, 23Z)-N, N- diformazan Base 29-20,23- diene-10- amine, (17Z, 20Z)-N, N- dimethyl 26-17,20- diene-9- amine, (1Z, 19Z)-N5N- dimethyl 25-16,19- diene-8- amine, (13Z, 16Z)-N, N- dimethyl 22-13,16- diene- 5- amine, (12Z, 15Z)-N, N- dimethyl 21-12,15- diene-4- amine, (14Z, 17Z)-N, N- dimethyl 23- 14,17- diene-6- amine, (15Z, 18Z)-N, N- dimethyl 24-15,18- diene-7- amine, (18Z, 21Z)-N, N- diformazan Base 27-18,21- diene-10- amine, (15Z, 18Z)-N, N- dimethyl 24-15,18- diene-5- amine, (14Z, 17Z)-N, N- dimethyl 23-14,17- diene-4- amine, (19Z, 22Z)-N, N- dimethyl 28-19,22- diene- 9- amine, (18Z, 21Z)-N, N- dimethyl 27-18,21- diene-8- amine, (17Z, 20Z)-N, N- dimethyl 26- 17,20- diene-7- amine, (16Z, 19Z)-N, N- dimethyl 25-16,19- diene-6- amine, (22Z, 25Z)-N, N- diformazan Base 31-22,25- diene-10- amine, (21Z, 24Z)-N, N- dimethyl 30-21,24- diene-9- amine, (18Z)-N, N- 27-18- alkene-10- amine of dimethyl, 26-17- alkene-9- amine of (17Z)-N, N- dimethyl, (19Z, 22Z)-N, N- diformazan Base 28-19,22- diene-7- amine, N, 27-10- amine of N- dimethyl, (20Z, 23Z)-N- ethyl-N-methyl 20 Nine-20,23- diene-10- amine, 1- [(11Z, 14Z)-1- nonyl 20-11,14- diene-1- base] pyrrolidines, (20Z)-N, N- 27-20- alkene-10- amine of dimethyl, 27-15- alkene-10- amine of (15Z)-N, N- dimethyl, (14Z)-N, N- dimethyl 29-14- alkene-10- amine, 29-17- alkene-10- amine of (17Z)-N, N- dimethyl, (24Z)-N, N- dimethyl 33- 24- alkene-10- amine, 29-20- alkene-10- amine of (20Z)-N, N- dimethyl, 31-22- alkene of (22Z)-N, N- dimethyl- 10- amine, 25-16- alkene-8- amine of (16Z)-N, N- dimethyl, (12Z, 15Z)-N, N- dimethyl-2- nonyl 21-12, 15- diene-1- amine, (13Z, 16Z)-N, N- dimethyl-3- nonyl 22-13,16- diene-1- amine, N, N- dimethyl-1- [(1S, 2R)-2- octylcyclopropenyl] 17-8- amine, 1- [(1S, 2R)-2- hexyl cyclopropyl]-N, 19-10- of N- dimethyl Amine, N, N- dimethyl-1- [(1S, 2R)-2- octylcyclopropenyl] 19-10- amine, N, [(1S, 2R)-2- is pungent by N- dimethyl-21- Cyclopropyl] 21-10- amine, N, N- dimethyl-1- [(1S, 2S)-2- { [(1R, 2R)-2- pentylcyclopropyl] methyl } ring Propyl] 19-10- amine, N, N- dimethyl-1- [(1S, 2R)-2- octylcyclopropenyl] 16-8- amine, N, N- dimethyl-[(1R, 2S)-2- undecyl cyclopropyl] 14-5- amine, N, N- dimethyl-3- { 7- [(1S, 2R)-2- octylcyclopropenyl] heptyl } ten Two-1- amine, 1- [(1R, 2S)-2- heptyl cyclopropyl]-N, 18-9- amine of N- dimethyl, 1- [(1S, 2R)-2- decyl cyclopropyl Base]-N, 15-6- amine of N- dimethyl, N, N- dimethyl-1- [(1S, 2R)-2- octylcyclopropenyl] 15-8- amine, R-N, N- bis- Methyl-1-[(9Z, 12Z)-ten eight-9,12- diene-1- base oxygroup]-3- (octyl oxygroup) propyl- 2- amine, S-N, N- dimethyl-1- [(9Z, 12Z)-ten eight -9,12- diene -1- base oxygroup] -3- (octyl oxygroup) propyl- 2- amine, 1- 2- [(9Z, 12Z)-ten eight -9, 12- diene -1- base oxygroup] -1- [(octyl oxygroup) methyl] ethyl } pyrrolidines, (2S)-N, N- dimethyl -1- [(9Z, 12Z) - 18-9,12- diene-1- base oxygroup]-3- [(5Z)-octyl- 5- alkene-1- base oxygroup] propyl- 2- amine, 1- { 2- [(9Z, 12Z)-ten Eight -9,12- diene -1- base oxygroup] -1- [(octyl oxygroup) methyl] ethyl } azetidine, (2S) -1- (hexyl oxygroup)-N, N- dimethyl -3- [(9Z, 12Z)-ten eight -9,12- diene -1- base oxygroup] propyl- 2- amine, (2S) -1- (heptyl oxygroup)-N, N- bis- Methyl -3- [(9Z, 12Z)-ten eight -9,12- diene -1- base oxygroup] propyl- 2- amine, N, N- dimethyl -1- (nonyl oxygroup) -3- [(9Z, 12Z)-ten eight -9,12- diene -1- base oxygroup] propyl- 2- amine, N, N- dimethyl -1- [(9Z)-ten eight -9- alkene -1- base oxygen Base] -3- (octyl oxygroup) propyl- 2- amine；(2S)-N, N- dimethyl -1- [(6Z, 9Z, 12Z)-ten eight -6,9,12- triolefin -1- bases Oxygroup] -3- (octyl oxygroup) propyl- 2- amine, (2S) -1- [(11Z, 14Z)-two ten -11,14- diene -1- base oxygroup]-N, N- bis- Methyl -3- (amyl oxygroup) propyl- 2- amine, (2S) -1- (hexyl oxygroup) -3- [(11Z, 14Z)-two ten -11,14- diene -1- base Oxygroup]-N, N- dimethyl propylene -2- amine, 1- [(11Z, 14Z)-two ten -11,14- diene -1- base oxygroup]-N, N- dimethyl -3- (octyl oxygroup) propyl- 2- amine, 1- [(13Z, 16Z)-two 10 two -13,16- diene -1- base oxygroup]-N, N- dimethyl -3- (octyl Oxygroup) propyl- 2- amine, (2S) -1- [(13Z, 16Z)-two 10 two -13,16- diene -1- base oxygroup] -3- (hexyl oxygroup)-N, N- Dimethyl propylene-2- amine, (2S)-1- [(13Z)-two 12-13- alkene-1- base oxygroup]-3- (hexyl oxygroup)-N, N- dimethyl propylene- 2- amine, 1- [(13Z)-two 12-13- alkene-1- base oxygroup]-N, N- dimethyl-3- (octyl oxygroup) propyl- 2- amine, 1- [(9Z)- 16-9- alkene-1- base oxygroups]-N, N- dimethyl-3- (octyl oxygroup) propyl- 2- amine, (2R)-N, N- dimethyl-H (1- formoxyl Octyl) oxygroup] -3- [(9Z, 12Z)-ten eight -9,12- diene -1- base oxygroup] propyl- 2- amine, (2R) -1- [(3,7- dimethyl-octas Base) oxygroup]-N, N- dimethyl -3- [(9Z, 12Z)-ten eight -9,12- diene -1- base oxygroup] propyl- 2- amine, N, N- dimethyl -1- (octyl oxygroup) -3- ({ 8- [(1S, 2S) -2- { [(1R, 2R) -2- pentylcyclopropyl] methyl } cyclopropyl] octyl } oxygroup) propyl- 2- amine, N, N- dimethyl -1- { [8- (2- octylcyclopropenyl) octyl] oxygroup } -3- (octyl oxygroup) propyl- 2- amine and (11E, 20Z, 23Z)-N, 29-11,20,2- triolefin-10- amine of N- dimethyl or its pharmaceutically acceptable salt or stereoisomer.
In one embodiment, lipid can be the lipid of cleavable, such as retouch in international publication number WO2012170889 Those of state, the patent is incorporated herein in its entirety by reference.
In one embodiment, cation lipid can be by as is generally known in the art and/or such as in international publication number WO2012040184、WO2011153120、WO2011149733、WO2011090965、WO2011043913、 WO2011022460, WO2012061259, WO2012054365, WO2012044638, WO2010080724 and WO201021865 Described in method synthesis；The patent is respectively incorporated herein in its entirety by reference.
In one embodiment, the LNP preparation of polynucleotides, primary construct and/or mmRNA can contain 3% lipid PEG-c-DOMG under molar ratio.In another embodiment, the LNP system of polynucleotides, primary construct and/or mmRNA Agent contains the PEG-c-DOMG under 1.5% lipid molar ratios.
In one embodiment, the pharmaceutical composition of polynucleotides, primary construct and/or mmRNA may include in state At least one PEGylated lipid, the patent described in border publication No. 2012099755 are hereby incorporated herein by.
In one embodiment, LNP preparation can contain (1, the 2- bis- myristoyl-sn- glycerol-of PEG-DMG 2000 3- phosphoethanolamine-N- [methoxyl group (polyethylene glycol) -2000).In one embodiment, LNP preparation can contain PEG-DMG 2000, cation lipid as known in the art and at least one other component.In another embodiment, LNP preparation PEG-DMG 2000, cation lipid as known in the art, DSPC and cholesterol can be contained.As a non-limiting reality Example, LNP preparation can contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol.As another non-limiting example, LNP preparation contains PEG-DMG 2000, DLin-DMA, DSPC and cholesterol that molar ratio is 2: 40: 10: 48 (referring to example Such as Geall, Nonviral delivery of self-amplifying RNA vaccines, PNAS 2012；PMID: 22908294；It is incorporated herein in its entirety by reference).As another non-limiting example, modification RNA described herein It can prepare in the nano particle as described in U.S. Publication No 20120207845 to be delivered by parenteral route；It is described Patent is incorporated herein in its entirety by reference.
In one embodiment, LNP preparation can be by international publication number WO2011127255 or WO2008103276 Described in method prepare, the patent is respectively incorporated herein in its entirety by reference.As a non-limiting example, Modification RNA described herein can be encapsulated in the LNP preparation as described in WO2011127255 and/or WO2008103276；Institute Patent is stated respectively to be incorporated herein in its entirety by reference.
In one embodiment, LNP preparation described herein may include polycation composition.It is unrestricted as one Property example, polycation composition can be selected from the formula 1-60 of U.S. Patent Publication number US20050222064；The patent is to quote Mode be integrally incorporated herein.In another embodiment, the LNP preparation comprising polycation composition can be used in vivo And/or modification RNA described herein is delivered in vitro.
In one embodiment, LNP preparation described herein can additionally comprise penetration enhancer molecule.It is unrestricted Penetration enhancer molecule describes in U.S. Patent Publication number US20050222064；The patent is whole by reference simultaneously Enter herein.
In one embodiment, pharmaceutical composition can be prepared in liposome, such as, but not limited to DiLa2 liposome (Marina Biotech, Bothell, WA),(Marina Biotech, Bothell, WA), it is based on The neutral liposome of DOPC (1,2-dioleoyl-sn-glycerol -3- phosphocholine) is (for example, the siRNA for oophoroma is delivered (20065 (12) 1708-1713 of the Cancer Biology & such as Landen Therapy)；It is incorporated hereby Herein) and the liposome of hyaluronic acid coated (Quiet Therapeutics, Israel).
Nanoparticle formulations can be the carbon aquation comprising carbohydrate carrier and modified nucleic acid molecule (for example, mmRNA) Close object nano particle.As a non-limiting example, carbohydrate carrier may include but be not limited to the plant of acid anhydrides modification Plant glycogen β-paste that glycogen or sugared leiomyoma cells, ocentyl succinic plant glycogen, plant glycogen powder-beta-dextrin, acid anhydrides are modified Essence.(see, for example, international publication number WO2012109121；It is incorporated herein in its entirety by reference).
It can be by substituting institute with the Biodegradable cationic lipid of referred to as quick elimination type lipidic nanoparticles (reLNP) Cation lipid is stated to improve lipid nanoparticle preparation.Have been displayed ionizable cation lipid such as, but not limited to, DLinDMA, DLin-KC2-DMA and DLin-MC3-DMA is accumulated in blood plasma and tissue over time and can is potential toxicity source. The tachymetabolism of quick elimination type lipid can be such that lipidic nanoparticles tolerance in rats and therapeutic index improves from 1mg/ Kg dosage to 10mg/kg dosage the order of magnitude.The degradation and metabolism that ester bond including enzymatic degradation can improve cationic components are generally Condition, while still maintaining the activity of reLNP preparation.Ester bond can be located at lipid chain inside or it can be located at lipid chain terminal on. Any carbon in the alternative lipid chain of lactone bond.
In one embodiment, lactone bond can be located on the either side of saturated carbon.The non-limiting example packet of reLNP It includes:
And
It in one embodiment, can may include the lipid nanometer of a nanometer type, polymer and immunogene by delivering Grain causes immune response.(U.S. Publication No 20120189700 and international publication number WO2012099805；It is respectively to draw Mode is integrally incorporated herein).Polymer can encapsulate nanometer type or partly encapsulate nanometer type.Immunogene can be recombination Albumen, modification RNA and/or primary construct described herein.In one embodiment, lipidic nanoparticles can be formulated for Such as, but not limited to support in antiviral vaccine.
Lipidic nanoparticles can be engineered to change the surface nature of particle, so that lipidic nanoparticles can penetrate mucous membrane Barrier.Mucus is located on mucosal tissue, such as, but not limited to oral cavity (for example, oral cavity and esophagus film and tonsil), eye, stomach Intestines (for example, stomach, small intestine, large intestine, colon, rectum), nose, respiratory tract (for example, nose, pharynx, trachea and bronchus film), genitals The mucosal tissue of (for example, vagina, uterine neck and urethra film).It is preferred for higher drug encapsulation efficiency and is capable of providing extensive drug The nano particle greater than 10-200nm of continual delivery has been considered too big and cannot quickly diffuse through mucosal barrier.Mucus connects Continue secretion, outflow, abandon or digest and recycle, so the particle of most of captures can be in several seconds or within a few hours from viscous It is removed on membrane tissue.It has intensively been coated with big polymer nano granules (the diameter 200nm- of low molecular poly (PEG) Mucus (the PNAS such as Lai 2007 only 500nm) are diffused through with 4 to 6 times of the degree lower than identical particle diffusion in water 104 (5): 1482-487；The Adv Drug Deliv Rev.2009 61 such as Lai (2): 158-171；The bibliography is respectively equal It is incorporated herein in its entirety by reference).Transmission rate and/or fluorescence microscopy can be used to determine turning for nano particle Fortune, the technology include but is not limited to fluorescence recovery after photo- bleaching (FRAP) and the more particle trackings (MPT) of high-resolution.It is non-as one Limitative examples, the composition that can penetrate mucosal barrier can be made, the patent according to U.S. Patent number 8 described in 241,670 It is incorporated herein in its entirety by reference.
Engineering may include polymer material (i.e. polymer core) and/or polymerization with the lipidic nanoparticles for penetrating mucus Object-vitamin conjugate and/or triblock copolymer.Polymer material may include but be not limited to polyamine, polyethers, polyamide, gather Ester, polyurethanes, polyureas, polycarbonate, poly- (styrene), polyimides, polysulfones, polyurethane, polyacetylene, polyethylene, Polyethyleneimine, polyisocyanate, polyacrylate, polymethacrylates, polyacrylonitrile and polyarylate.Polymer material It can be biodegradable and/or biocompatible.In addition polymer material can be radiated.As a non-limiting example, Polymer material can be radiated by γ (see, for example, international application no WO201282165, is incorporated hereby this Text).The non-limiting example of specific polymer includes poly- (caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), gathers (lactic acid) (PLA), poly (L-lactic acid) (PLLA), poly- (glycolic) (PGA), poly- (lactic-co-glycolic acid) (PLGA), poly- (L- cream Sour -co- glycolic) (PLLGA), poly(D,L-lactide) (PDLA), poly- (L- lactide) (PLLA), poly- (D, L- lactide- Co- caprolactone), poly- (D, L- lactide-co-caprolactone -co- glycolide), poly- (D, L- lactide-co-PEO- co- D, L- third Lactide), poly- (D, L- lactide-co-PPO- co- D, L- are total), Polyalkylcyanoacrylanano, polyurethane, poly-L-Lysine (PLL), hydroxy propyl methacrylate (HPMA), polyethylene glycol, Poly-L-glutamic acid, poly- (carboxylic acid), polyanhydride, polyorthoester, Poly- (esteramides), polyamide, poly- (ester ether), polycarbonate, polyolefin such as polyethylene and polypropylene, polyalkylene glycol are such as poly- (ethylene glycol) (PEG), polyalkylene oxide (PEO), polyalkylene terephthalates for example poly- (ethylene glycol terephthalate), poly- second Enol (PVA), polyvinylether, polyvinyl ester for example poly- (vinyl acetate), gather at polyvinyl halides for example poly- (vinyl chloride) (PVC) Vinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethane, derivative cellulose such as alkylcellulose, hydroxy alkyl are fine Tie up element, cellulose ether, cellulose esters, NC Nitroncellulose, hydroxypropyl cellulose, carboxymethyl cellulose, acrylic acid polymer such as Poly- ((methyl) methyl acrylate) (PMMA), poly- ((methyl) ethyl acrylate), poly- ((methyl) butyl acrylate), poly- ((first Base) isobutyl acrylate), poly- ((methyl) Hexyl 2-propenoate), poly- ((methyl) isodecyl acrylate), poly- ((methyl) acrylic acid Lauryl), poly- ((methyl) phenyl acrylate), poly- (methacrylic acid), poly- (isopropyl acrylate), poly- (i-butyl Ester), poly- (octadecyl acrylate) and its copolymer and mixture, polydioxanone and its copolymer, poly- hydroxyl alkane Carboxylate, polypropylene glycol fumarate, polyformaldehyde, poloxamer (poloxamer), poly- (ortho acid) ester, poly- (butyric acid), poly- (penta Acid), poly- (lactide-co-caprolactone) and trimethylene carbonate, polyvinylpyrrolidone.Lipidic nanoparticles can coat There is copolymer or associate with it, such as, but not limited to block copolymer is (as described in the international publication number WO2013012476 Branched polyether-polyamide block copolymer, the patent are incorporated herein in its entirety by reference) and (poly(ethylene glycol))-it is (poly- (propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see, for example, the U.S. announce 20120121718 and the U.S. announce 20100003337 and U.S. Patent number 8,263,665；It is respectively incorporated herein in its entirety by reference).Copolymer can To be generally acknowledged that the formation of safe (GRAS) polymer and lipidic nanoparticles can be without the side of new chemical entities generation Formula carries out.For example, lipidic nanoparticles may include the poloxamer for coating PLGA nano particle without forming new chemical entities, It still is able to the quick penetration people mucus (Angew.Chem.Int.Ed.2011 such as Yang 50:2597-2600；It is with reference Mode is integrally incorporated herein).
Polymer-vitamin conjugate vitamin can be vitamin E.The vitamin moieties of conjugate can be by other suitable Component replace, such as, but not limited to VitAVitE, other vitamins, cholesterol, hydrophobic part or other surfaces are living The hydrophobic components (for example, sterol chain, fatty acid, hydrocarbon chain and alkylene oxide chain) of property agent.
Engineering may include surface modification agent such as, but not limited to, mmRNA, anion with the lipidic nanoparticles for penetrating mucus Protein (for example, bovine serum albumin(BSA)), surfactant are (for example, cationic surfactant such as dimethyl double 18 Alkyl bromination ammonium), sugar or sugar derivatives (for example, cyclodextrin), nucleic acid, polymer be (for example, heparin, polyethylene glycol and pool Lip river are husky Nurse), mucolytic agent is (for example, N-acetylcystein, argy wormwood, bromelain, papain, dragon spit pearl (clerodendrum), acetylcysteine, bromhexine (bromhexine), carbocisteine (carbocisteine), according to pula Ketone (eprazinone), mesna (mesna), ambroxol (ambroxol), Sobrerol (sobrerol), dimiodol (domiodol), Letosteine (letosteine), Stepronin (stepronin), Tiopronin (tiopronin), coagulate it is molten Glue protein, extrasin beta 4 Dornase Alfa, Neltenexine (neltenexine), Erdosteine (erdosteine)) and it is various DNA enzymatic includes rhDNA enzyme.Surface modification agent can embed or be embedded in the surface of particle or placement is (for example, by coating, inhale Attached, covalent linkage or other methods) on the surface of lipidic nanoparticles.(20100215580 and beauty are announced see, for example, the U.S. State announces 20080166414；It is incorporated herein in its entirety by reference).
It may include at least one mmRNA described herein that mucus, which penetrates lipidic nanoparticles,.MmRNA can be encapsulated in lipid and receive In rice grain and/or it is placed on the surface of particle.MmRNA can be covalently coupled to lipidic nanoparticles.Mucus penetrates lipid and receives The preparation of rice grain may include multiple nano particles.In addition, preparation can around be glued containing that can interact and change with mucus The structure of liquid and/or adhesive property penetrate lipidic nanoparticles to viscous to reduce the particle of mucosal adhesive, so as to increase mucus Membrane tissue delivering.
In one embodiment, polynucleotides, primary construct or mmRNA are formulated as lipid complex, such as but not It is limited to ATUPLEXTMSystem, DACC system, DBTC system and come from Silence Therapeutics (London, United Kingdom other siRNA- lipid complex technologies)；It comes from(Cambridge, MA's) STEMFECTTMAnd targeting based on polyethyleneimine (PEI) or nucleoprotamine and non-targeted delivery of nucleic acids (Aleku etc., Cancer Res.2008 68:9788-9798；Strumberg etc., Int J Clin Pharmacol Ther 2,012 50: 76-78；2006 13:1222-1234 of Santel etc., Gene Ther；2006 13:1360- of Santel etc., Gene Ther 1370；Gutbier etc., Pulm Pharmacol.Ther.2010 23:334-344；Kaufmann etc., Microvasc Res 2010 80:286-293；Weide etc., J Immunother.2009 32:498-507；Weide etc., J Immunother.2008 31:180-188；Pascolo Expert Opin.Biol.Ther.4:1285-1294；Fotin- Mleczek etc., 2011 J.Immunother.34:1-15；Song etc., Nature Biotechnol.2005,23:709-717； Peer etc., Proc Natl Acad Sci U S A.2007 6；104:4095-4100；deFougerolles Hum Gene Ther.2008 19:125-132；All bibliography are incorporated herein in its entirety by reference).
In one embodiment, such preparation be also configured such that or composition can be changed be so that its it is passive in vivo or Actively it is directed toward different cell types, the cell type includes but is not limited to that liver cell, immunocyte, tumour cell, endothelium are thin Born of the same parents, antigen presenting cell and leucocyte (the Mol Ther.2010 such as Akinc 18:1357-1364；Song etc., Nat Biotechnol.2005 23:709-717；Judge etc., J Clin Invest.2009 119:661-673；Kaufmann etc., 2010 80:286-293 of Microvasc Res；2006 13:1222-1234 of Santel etc., Gene Ther；Santel etc., 2006 13:1360-1370 of Gene Ther；Gutbier etc., Pulm Pharmacol.Ther.2010 23:334-344； Basha etc., Mol.Ther.2011 19:2186-2200；Fenske and Cullis, Expert Opin Drug Deliv.2008 5:25-44；Peer etc., Science.2008 319:627-630；Peer and Lieberman, Gene Ther.2011 18: 1127-1133；All bibliography are incorporated herein in its entirety by reference).Preparation passive target liver cell One example includes the lipid nanoparticle preparation based on DLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA, has been shown Show in combination with apo E and these preparations is promoted to combine and be absorbed into liver cell (Akinc etc., Mol in vivo Ther.2010 18:1357-1364；It is incorporated herein in its entirety by reference).Preparation can also be matched by difference on its surface The expression of body and selectively targeting, such as, but not limited to by folic acid, transferrins, N- acetylgalactosamine (GalNAc) and The method of antibody target illustrates (Kolhatkar etc., Curr Drug Discov Technol.2011 8:197-206； Musacchio and Torchilin, Front Biosci.2011 16:1388-1412；Yu etc., Mol Membr Biol.2010 27:286-298；Patil etc., Crit Rev Ther Drug Carrier Syst.2008 25:1-61；Benoit etc., Biomacromolecules.2011 12:2708-2714；Zhao etc., Expert Opin Drug Deliv.2008 5:309- 319；Akinc etc., Mol Ther.2010 18:1357-1364；Srinivasan etc., Methods Mol Biol.2012 820:105-116；Ben-Arie etc., Methods Mol Biol.2012 757:497-507；Peer 2010 J Control Release.20:63-68；Peer etc., Proc Natl Acad Sci U S A.2007 104:4095-4100；Kim etc., Methods Mol Biol.2011 721:339-353；Subramanya etc., Mol Ther.2010 18:2028-2037； Song etc., Nat Biotechnol.2005 23:709-717；Peer etc., Science.2008 319:627-630；Peer and Lieberman, Gene Ther.2011 18:1127-1133；All bibliography are incorporated hereby Herein).
In one embodiment, polynucleotides, primary construct or mmRNA are formulated as solid lipid nano-particles.Gu Body lipidic nanoparticles (SLN) can be to be spherical, and wherein average diameter is in 10nm between 1000nm.SLN, which possesses, can make lipophilic Molecular melting and solid lipid paralinin that can be stable with surfactant and/or emulsifier.In a further embodiment, Lipidic nanoparticles can for self assembly lipid-polymer nano particle (referring to Zhang etc., ACS Nano, 2008,2 (8), the 1696-1702 pages；It is incorporated herein in its entirety by reference).
Liposome, lipid complex or lipidic nanoparticles can be used to improve polynucleotides, primary construct or mmRNA and draw The effect of protein led generates, because these preparations can increase by polynucleotides, primary construct or mmRNA progress Cell transfecting；And/or increase the translation of the protein of coding.Such a example is related to realizing that polymerization is multiple using lipid encapsulated The effective system for closing object (polyplex) Plasmid DNA delivers (Heyes etc., Mol Ther.2007 15:713-720；It is with reference Mode be integrally incorporated herein).Liposome, lipid complex or lipidic nanoparticles may further be used to increase polynucleotides, primary The stability of construct or mmRNA.
In one embodiment, polynucleotides of the invention, primary construct and/or mmRNA can be formulated for controlling Release and/or targeted delivery.As used herein, " control release " refers to the specific release mould followed for realizing treatment results The pharmaceutical composition or compound of formula discharge overview.In one embodiment, polynucleotides, primary construct or mmRNA can It is encapsulated into the delivery agents for being described herein and/or be used to control as is generally known in the art release and/or targeted delivery.As made herein With term " encapsulating " means to seal, surround or encase.When it is related to the preparation of the compounds of this invention, encapsulating can be for generally , it is complete or partial.Term " generally encapsulate " means at least more than 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.9% or the pharmaceutical composition or change of the invention greater than 99.999% Closing object can seal in delivery agents, surrounds or encase." part encapsulate " mean less than 10%, 10%, 20%, 30%, 40%, 50% or less pharmaceutical composition or compound of the invention can be sealed in delivery agents, surround or be encased.Advantageously, can lead to The evolution that pharmaceutical composition or compound of the invention are measured using fluorescence and/or electron micrograph or activity are crossed to determine packet Envelope.For example, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or pharmaceutical composition of the invention or compound packet greater than 99.99% It is enclosed in delivery agents.
In one embodiment, control delivery formulations may include but be not limited to triblock copolymer.As a non-limit Property example processed, preparation may include two distinct types of triblock copolymer (international publication number WO2012131104 and WO2012131106；It is respectively incorporated herein in its entirety by reference).
In another embodiment, polynucleotides, primary construct or mmRNA can be encapsulated into lipidic nanoparticles or fast In fast elimination type lipidic nanoparticles, and then lipidic nanoparticles or quick elimination type lipidic nanoparticles can be encapsulated into this In text description and/or polymer as known in the art, hydrogel and/or surgical sealants.As a non-limiting example, Polymer, hydrogel or surgical sealants can for PLGA, ethylene vinyl acetate (EVAc), poloxamer, (Nanotherapeutics, Alachua, Inc.FL),(Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrin original copolymer (Ethicon Inc.Cornelia, GA), (Baxter International, Deerfield, Inc, IL), the sealant based on PEG and(Baxter International, Deerfield Inc, IL).
In another embodiment, lipidic nanoparticles can be encapsulated into as known in the art when being injected into subject When can be formed in any polymer of gel.As another non-limiting example, lipidic nanoparticles can be encapsulated into can biology In degradable polymer matrix.
In one embodiment, for control the polynucleotides of release and/or targeted delivery, primary construct or MmRNA preparation may also include at least one control release coating.Control discharges coatingIt is poly- Vinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, hydroxyl Ethyl cellulose, EUDRAGITEUDRAGITAnd cellulose derivative such as Ethylcellulose aqueous dispersion (With)。
In one embodiment, control release and/or targeted delivery preparation may include at least one can containing poly- sun from The degradable polyester of sub- side chain.Degradable polyester including but not limited to poly- (serine ester), poly- (L- lactide-co-L- relies ammonia Acid), poly- (4-hydroxy-L-proline ester) with and combinations thereof.In another embodiment, degradable polyester may include that PEG sews It closes to form PEGylated polymer.
In one embodiment, polynucleotides of the invention, primary construct and/or mmRNA can be encapsulated in therapeutic In nano particle.Therapeutic nano particle can be prepared by being described herein with method as known in the art, such as but unlimited In international publication number WO2010005740, WO2010030763, WO2010005721, WO2010005723, WO2012054923, U.S. Publication No US20110262491, US20100104645, US20100087337, US20100068285, US20110274759, US20100068286 and US20120288541 and U.S. Patent number 8,206,747,8,293,276, 8,318,208 and 8,318,211, respectively it is incorporated herein in its entirety by reference.In another embodiment, it treats Property polymer nano granules can be identified by the method described in U.S. Publication No US20120140790, the patent with The mode of reference is integrally incorporated herein.
In one embodiment, therapeutic nano particle can be formulated for sustained release.As used herein, " continue Release " refers to the pharmaceutical composition or compound that rate of release is followed in special time period.Period may include but be not limited to A few houres, several days, a few weeks, some months and several years.As a non-limiting example, sustained release nano particle may include gathering Object and therapeutic agent polynucleotides such as, but not limited to, of the invention, primary construct and mmRNA are closed (referring to international publication number 2010075072 and U.S. Publication No US20100216804, US20110217377 and US20120201859, respectively to draw Mode is integrally incorporated herein).
In one embodiment, therapeutic nano particle can be formulated as target-specific.As a non-limiting reality Example, therapeutic nano particle may include corticosteroid (referring to international publication number WO2011084518；It is whole by reference Body is incorporated herein).In one embodiment, therapeutic nano particle can be formulated as cancer specific.As a non-limit Property example processed, therapeutic nano particle can prepare be described in international publication number WO2008121949, WO2010005726, WO2010005725, WO2011084521 and U.S. Publication No US20100069426, US20120004293 and In nano particle in US20100104655, the patent is respectively incorporated herein in its entirety by reference.
In one embodiment, nano particle of the invention may include polymer substrate.As a non-limiting reality Example, nano particle may include two or more polymer, such as, but not limited to polyethylene, polycarbonate, polyanhydride, poly- hydroxyl Sour, poly- propyl fumarate, polyamide, polyacetals, polyethers, polyester, poly- (ortho esters), polybutylcyanoacrylate, gathers polycaprolactone Vinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylates, polybutylcyanoacrylate, polyureas, polystyrene, Polyamine, polylysine, poly- (aziridine), poly- (serine ester), poly- (L- lactide-co-L-lysine), poly- (4- hydroxyl-L- Proline ester) or combinations thereof.
In one embodiment, therapeutic nano particle includes diblock copolymer.In one embodiment, two is embedding Section copolymer may include PEG and combination of polymers, and the polymer is such as, but not limited to polyethylene, polycarbonate, polyanhydride, gathers Carboxylic acid, poly- propyl fumarate, polycaprolactone, polyamide, polyacetals, polyethers, polyester, poly- (ortho esters), poly- alpha-cyanoacrylate Ester, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylates, polybutylcyanoacrylate, polyureas, polyphenyl Ethylene, polyamine, polylysine, poly- (aziridine), poly- (serine ester), poly- (L- lactide-co-L-lysine), poly- (4- hydroxyl Base-L-PROLINE ester) or combinations thereof.
As a non-limiting example, therapeutic nano particle includes PLGA-PEG block copolymer (referring to U.S.'s public affairs Cloth US20120004293 and U.S. Patent number 8,236,330, are respectively incorporated herein in its entirety by reference).Another In one non-limiting example, therapeutic nano particle is the hidden of the diblock copolymer comprising PEG and PLA or PEG and PLGA Shape (stealth) nano particle is (respectively equal referring to U.S. Patent number 8,246,968 and international publication number WO2012166923 It is incorporated herein in its entirety by reference).
In one embodiment, therapeutic nano particle may include segmented copolymer (see, for example, U.S. Patent number 8,263,665 and 8,287,910；It is respectively incorporated herein in its entirety by reference).
In one embodiment, block copolymer described herein may include in poly ion complexes, it is described gather from Sub- compound includes non-polymer micella and block copolymer.(see, for example, U.S. Publication No 20120076836；It is with reference Mode be integrally incorporated herein).
In one embodiment, therapeutic nano particle may include at least one acrylate copolymer.Acroleic acid polymerization Object includes but is not limited to acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymer, methyl methacrylate copolymer Object, ethoxyethyl methacrylates, methacrylic acid cyanaoethyl methacrylate, amino alkyl methacrylate copolymer, poly- (propylene Acid), poly- (methacrylic acid), polybutylcyanoacrylate with and combinations thereof.
In one embodiment, therapeutic nano particle may include at least one be described herein and/or this field in The cationic polymer known.
In one embodiment, therapeutic nano particle may include at least one amine-containing polymer, such as, but not limited to Polylysine, polyethyleneimine, poly- (amidoamines) dendritic, poly- (beta-amino ester) are (see, for example, U.S. Patent number 8,287,849；It is incorporated herein in its entirety by reference) with and combinations thereof.
In one embodiment, therapeutic nano particle may include at least one dropping containing polycation side chain Solve polyester.Degradable polyester including but not limited to poly- (serine ester), poly- (L- lactide-co-L-lysine), poly- (4- hydroxyl- L-PROLINE ester) with and combinations thereof.In another embodiment, degradable polyester may include that PEG conjugation is PEGylated to be formed Polymer.
In another embodiment, therapeutic nano particle may include the conjugation of at least one targeting ligand.Targeting is matched Body can be any ligand as known in the art, such as, but not limited to monoclonal antibody.(Kirpotin etc., Cancer Res.2006 66:6732-6740；It is incorporated herein in its entirety by reference).
In one embodiment, therapeutic nano particle can prepare in the aqueous solution that can be used to target on cancer (referring to International publication number WO2011084513 and U.S. Publication No US20110294717, is respectively incorporated hereby Herein).
In one embodiment, polynucleotides, primary construct or mmRNA can be encapsulated in synthesis nano-carrier, even It is connected to the synthesis nano-carrier and/or associates with it.Synthesizing nano-carrier includes but is not limited in international publication number WO2010005740、WO2010030763、WO201213501、WO2012149252、WO2012149255、WO2012149259、 WO2012149265、WO2012149268、WO2012149282、WO2012149301、WO2012149393、 WO2012149405, WO2012149411, WO2012149454 and WO2013019669 and U.S. Publication No Described in US20110262491, US20100104645, US20100087337 and US20120244222 those, the patent Respectively it is incorporated herein in its entirety by reference.It can be used as is generally known in the art and/or method described herein prepare synthesis Nano-carrier.As a non-limiting example, synthesis nano-carrier can by be described in international publication number WO2010005740, WO2010030763 and WO201213501 and U.S. Publication No US20110262491, US20100104645, Method in US20100087337 and US2012024422 is prepared, and the patent is respectively incorporated hereby Herein.In another embodiment, synthesis nano-carrier preparation can by be described in international publication number WO2011072218 and Method in U.S. Patent number 8,211,473 is lyophilized；The patent is respectively incorporated herein in its entirety by reference.
In one embodiment, synthesis nano-carrier can contain release polynucleotides described herein, primary construct And/or mmRNA reactive group (referring to international publication number WO20120952552 and U.S. Publication No US20120171229, It is respectively incorporated herein in its entirety by reference).
In one embodiment, immune response of the synthesis nano-carrier containing enhancing from synthesis nano-carrier delivering Immunostimulant.As a non-limiting example, synthesis nano-carrier may include can enhance immune system based on Th1's The Th1 immunostimulant of response is (each referring to international publication number WO2010123569 and U.S. Publication No US20110223201 From being incorporated herein in its entirety by reference).
In one embodiment, synthesis nano-carrier can be formulated for Targeting delivery.In one embodiment, it synthesizes Nano-carrier be formulated at specified pH and/or discharged after desired time interval polynucleotides, primary construct and/ Or mmRNA.As a non-limiting example, synthesis nano particle can be configured to after 24 hours and/or release under being 4.5 in pH Put polynucleotides, primary construct and/or mmRNA (referring to international publication number WO2010138193 and WO2010138194 and U.S. Publication No US20110020388 and US20110027217, are respectively incorporated herein in its entirety by reference).
In one embodiment, synthesis nano-carrier can be formulated for control release and/or sustained release is described herein Polynucleotides, primary construct and/or mmRNA.As a non-limiting example, it is carried for the synthesis nanometer of sustained release Body can by as is generally known in the art, be described herein and/or such as in international publication number WO2010138192 and U.S. Publication No Method described in 20100303850 is prepared, and the patent is respectively incorporated herein in its entirety by reference.
In one embodiment, synthesis nano-carrier can be prepared as vaccine.In one embodiment, nanometer is synthesized Carrier can encapsulate at least one polynucleotides for encoding at least one antigen, primary construct and/or mmRNA.It is non-as one Limitative examples, synthesis nano-carrier may include at least one antigen and the excipient for vaccine dosage (referring to international publication Number WO2011150264 and U.S. Publication No US20110293723, is respectively incorporated herein in its entirety by reference).Make For another non-limiting example, vaccine dosage may include at least two synthesis nano-carriers and identical or different antigen and Excipient is (referring to international publication number WO2011150249 and U.S. Publication No US20110293701, respectively with the side of reference Formula is integrally incorporated herein).Can by being described herein, as is generally known in the art and/or in international publication number WO2011150258 and beauty Method described in state publication No. US20120027806 selects vaccine dosage, and the patent is respectively whole by reference Body is incorporated herein.
In one embodiment, synthesis nano-carrier may include at least one multicore glycosides for encoding at least one adjuvant Acid, primary construct and/or mmRNA.As non-limiting examples, adjuvant may include GERBU Adjuvant 100, two The double octadecyl ammonium chloride of methyl, dimethyldioctadecylammonium base ammonium phosphate or dimethyldioctadecylammonium guanidine-acetic acid ammonium (DDA) and The nonpolar fraction of the total lipid extract of mycobacteria or the nonpolar fraction part (see, for example, U.S. Patent number 8, 241,610；It is incorporated herein in its entirety by reference).In another embodiment, synthesis nano-carrier may include at least A kind of polynucleotides, primary construct and/or mmRNA and adjuvant.As a non-limiting example, comprising the synthesis of adjuvant Nano-carrier can by the method that is described in international publication number WO2011150240 and U.S. Publication No US20110293700 come It prepares, the patent is respectively incorporated herein in its entirety by reference.
In one embodiment, synthesis nano-carrier can encapsulate at least one peptide, segment or area of the coding from virus Polynucleotides, primary construct and/or mmRNA.As a non-limiting example, synthesis nano-carrier may include but unlimited In being described in international publication number WO2012024621, WO201202629, WO2012024632 and U.S. Publication No Nano-carrier in US20120064110, US20120058153 and US20120058154, the patent is respectively with reference Mode is integrally incorporated herein.
In one embodiment, synthesis nano-carrier, which can be coupled to, can trigger body fluid and/or cytotoxic T The polynucleotides of cell (CTL) response, primary construct or mmRNA (see, for example, international publication number WO2013019669, with The mode of reference is integrally incorporated herein).
In one embodiment, nano particle can optimize for oral administration.Nano particle may include at least one sun Biopolymer, such as, but not limited to chitosan or derivatives thereof.As a non-limiting example, nano particle can lead to The method that is described in U.S. Publication No 20120282343 is crossed to prepare；The patent is incorporated herein in its entirety by reference.
Polymer, Biodegradable nano particle and core-shell nanoparticles
Natural and/or synthesis polymer can be used to prepare for polynucleotides, primary construct and mmRNA of the invention. The non-limiting example for the polymer that can be used for delivering includes but is not limited to come fromBio (Madison, WI) and The DYNAMIC of Roche Madison (Madison, WI)(Arrowhead Reasearch Corp., Pasadena, CA) preparation, PHASERXTMPolymer formulations such as, but not limited to, SMARTT POLYMER TECHNOLOGYTM(Seattle, WA), DMRI/DOPE, poloxamer, from Vical (San Diego, CA)Adjuvant, chitosan, the ring for coming from Calando Pharmaceuticals (Pasadena, CA) Dextrin, dendritic and poly- (lactic-co-glycolic acid) (PLGA) polymer.RONDELTM(RNAi/ oligonucleotide nano Particle delivery) to be total to block poly- for polymer (Arrowhead Research Corporation, Pasadena, CA) and pH responsiveness Close object such as, but not limited to,(Seattle, WA).
One non-limiting example of chitosan preparations include positively charged chitosan core and negatively charged bottom Exterior section (the U.S. Publication No 20120258176 of object；It is incorporated herein in its entirety by reference).Chitosan includes but not Be limited to N- trimethyl chitin, mono- N- carboxymethyl chitosan (MCC), N- palmityl chitosan (NPCS), EDTA- chitosan, Low-molecular weight chitoglycan, chitosan derivatives or combinations thereof.
In one embodiment, processing has been undergone for the polymer in the present invention to be not intended to reduce and/or inhibit Attachment of the substance such as, but not limited to, bacterium to polymer surfaces.Can by as is generally known in the art and/or description and/or in the world Method described in publication No. WO2012150467 carrys out processable polymer, and the patent is incorporated herein in its entirety by reference.
One non-limiting example of PLGA preparation includes but is not limited to PLGA injectable reservoir (for example, by by PLGA It is dissolved in 66%N- N-methyl-2-2-pyrrolidone N (NMP) and residue is aqueous solvent and Leuprorelin (leuprolide) And formedOnce injection, PLGA and Leuprorelin peptide are deposited in subcutaneous space).
Be proved these many polymer process in vivo oligonucleotide delivery into cytoplasm effect (summary in In deFougerolles Hum Gene Ther.2008 19:125-132；The bibliography is whole by reference simultaneously Enter herein).It has generated nucleic acid and (has utilized two kinds of polymerizations of the delivering firm in vivo of siRNA (siRNA)) in this case Object space method is the more conjugates of dynamics (dynamic polyconjugate) and the nano particle based on cyclodextrin.These deliverings The first in method has used the more conjugates of dynamics and has had been displayed and effectively delivered siRNA in mouse and make in liver cell Endogenous said target mrna silencing (Rozema etc., Proc Natl Acad Sci U S A.2007 104:12982-12887；Its It is incorporated herein in its entirety by reference).This specific method is multicomponent polymeric system, and important feature includes nucleic acid (in this case for siRNA) is by the film activity polymer of disulfide bond covalent coupling and wherein PEG (is covered for charge Cover) and N- acetylgalactosamine (for liver cell targeting) group all by key connection (Rozema etc., Proc of pH sensitivity Natl Acad Sci U S A.2007 104:12982-12887；It is incorporated herein in its entirety by reference).It is being integrated to When liver cell and entrance inner body, polymer complex decomposes under low ph conditions, and wherein polymer exposes its positive charge, causes Inner body evolution and siRNA from polymer is discharged into cytoplasm.By substituting N- acetyl galactose amine groups with sweet dew alcohol groups, Having been displayed from the liver cell of expression asialoglycoprotein-receptors can change into sinusoidal endothelial cell and Kupffer is thin for targeting Born of the same parents.Another polymer process is related to the polycation nano particle containing cyclodextrin targeted using transferrins.These nanometers Particle is proved the EWS-FLI1 gene product targeted silent in the Ewing's sarcoma tumour cell for making to express TfR (Hu-Lieskovan etc., Cancer Res.2005 65:8984-8982；It is incorporated herein in its entirety by reference) and Preparation well-tolerated (Heidel etc., Proc Natl in non-human primate body in the siRNA in these nano particles 2007 104:5715-21 of Acad Sci USA；It is incorporated herein in its entirety by reference).These delivery strategies all combine Use the rational method of targeted delivery and inner body evolution mechanism.
Polymer formulations allow the lasting or sustained release of polynucleotides, primary construct or mmRNA (for example, in flesh After interior injection or subcutaneous injection).The release overview of the change of polynucleotides, primary construct or mmRNA, which can lead to, for example to exist The protein of translation coding in the extended period.Polymer formulations may further be used to increase polynucleotides, primary construct or The stability of mmRNA.Biodegradable polymeric has previously been used to protect the nucleic acid in addition to mmRNA not degraded and Display cause in vivo payload sustained release (Rozema etc., Proc Natl Acad Sci U S A.2007 104: 12982-12887；Sullivan etc., Expert Opin Drug Deliv.2010 7:1433-1446；Convertine etc., Biomacromolecules.2010 Oct 1；Chu etc., Acc Chem Res.2012 Jan 13；Manganiello etc., Biomaterials.2012 33:2301-2309；Benoit etc., Biomacromolecules.2011 12:2708-2714； Singha etc., Nucleic Acid Ther.2011 2:133-147；deFougerolles Hum Gene Ther.2008 19:125-132；Schaffert and Wagner, Gene Ther.2008 16:1131-1138；Chaturvedi etc., Expert Opin Drug Deliv.2011 8:1455-1468；Davis, Mol Pharm.2009 6:659-668；Davis, Nature 2010 464:1067-1070；It is respectively incorporated herein in its entirety by reference).
In one embodiment, pharmaceutical composition can be extended release preparation.In a further embodiment, it persistently releases Putting preparation can be used for subcutaneous delivery.Extended release preparation may include but be not limited to PLGA microballoon, ethylene vinyl acetate (EVAc), Poloxamer,(Nanotherapeutics, Inc.Alachua, FL),(Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrin original copolymer (Ethicon Inc.Cornelia, GA)、(Baxter International, Inc Deerfield, IL), the sealant based on PEG and(Baxter International, Inc Deerfield, IL).
As a non-limiting example, there can be adjustable rate of release (for example, several days and several weeks) by preparing PLGA microballoon and the integrality for modification mRNA being encapsulated in PLGA microballoon while being maintained in encapsulation process modification mRNA, come Modification mRNA is prepared in PLGA microballoon.EVAc is that be widely used in the application of preclinical sustained release implants abiotic can Degradation, biocompatible polymer are (for example, extended release product Ocusert, i.e., a kind of pilocarpine for glaucoma (pilocarpine) eye insert or progestasert, i.e., a kind of sustained release progesterone intrauterine device；Transdermal delivery system Unite Testoderm, Duragesic and Selegiline；Conduit).Poloxamer F-407NF is to have at a temperature of less than 5 DEG C The hydrophilic nonionic type surfactant triblock copolymer of the polyoxyethylene-poly-oxypropylene polyoxyethylene of low viscosity, and Solid gel is formed at a temperature of greater than 15 DEG C.Surgical sealants based on PEG include two kinds of synthesis being blended in delivery apparatus PEG component can prepare in one minute, seal in 3 minutes and reuptake in 30 days.The day and Right polymer can at site of administration gelatinizing-in-situ.They have been displayed to treat by ionic interaction and protein and peptide Candidate interacts to provide stabilizing effect.
Polymer formulations can also such as, but not limited to, pass through folic acid, turn iron by the expression selectively targeting of different ligands Albumen and N- acetylgalactosamine (GalNAc) illustrate (Benoit etc., Biomacromolecules.2011 12: 2708-2714；Rozema etc., Proc Natl Acad Sci U S A.2007 104:12982-12887；Davis, Mol Pharm.2009 6:659-668；2010 464:1067-1070 of Davis, Nature；It is respectively whole by reference It is incorporated herein).
Modification of nucleic acids and mmRNA of the invention can be prepared with polymerizable compound or be prepared in polymerizable compound.Polymer May include at least one polymer such as, but not limited to, polyethylene, polyethylene glycol (PEG), poly- (1- lysine) (PLL), be grafted to PEG, cation lipid polymer, Biodegradable cationic lipid polymer, the polyethyleneimine (PEI), cross-linked branched of PLL Poly- (alkylene imine), polyamine derivatives, the poloxamer of modification, biodegradable polymer, the degradable polymerization of elastic biological Object, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable Polyester block copolymer, biodegradable polyesters blocky random copolymers, segmented copolymer, the copolymerization of linear biodegradable Object, it is poly- [α-(4- aminobutyl)-L- glycolic) (PAGA), Biodegradable cross-linked cationic multi-block copolymers, poly- carbonic acid Ester, polyanhydride, polyhydroxy acid, poly- propyl fumarate, polycaprolactone, polyamide, polyacetals, polyethers, polyester, poly- (ortho esters), Polybutylcyanoacrylate, polyvinyl alcohol, polyurethane, polyphosphazene, polyacrylate, polymethacrylates, poly- alpha-cyanoacrylate Ester, polyureas, polystyrene, polyamine, polylysine, poly- (aziridine), poly- (serine ester), poly- (L- lactide-co-L- relies Propylhomoserin), poly- (4-hydroxy-L-proline ester), acrylate copolymer, amine-containing polymer, dextran polymer, dextran polymer Derivative or combinations thereof.
As a non-limiting example, modification of nucleic acids of the invention or mmRNA are available such as U.S. Patent number 6,177,274 Described in prepared with the polymerizable compound of the PEG of PLL grafting；The patent is incorporated herein in its entirety by reference.System Agent can be used for in-vitro transfection cell or for delivering modification of nucleic acids and mmRNA in vivo.In another example, modification of nucleic acids and MmRNA can be suspended in the solution or medium with cationic polymer, is in dry pharmaceutical compositions or be in can be as In the solution being dried described in U.S. Publication No 20090042829 and 20090042825；The patent is respectively to draw Mode is integrally incorporated herein.
As another non-limiting example, polynucleotides of the invention, primary construct or mmRNA can use PLGA-PEG Block copolymer (referring to U.S. Publication No US20120004293 and U.S. Patent number 8,236,330, it is whole by reference Body is incorporated herein) or PLGA-PEG-PLGA block copolymer (referring to U.S. Patent number 6,004,573, it is whole by reference Body is incorporated herein) it prepares.As a non-limiting example, polynucleotides of the invention, primary construct or mmRNA are available The diblock copolymer of PEG and PLA or PEG and PLGA prepare (referring to U.S. Patent number 8,246,968, by reference It is integrally incorporated herein).
Polyamine derivatives can be used to deliver nucleic acid or treatment and/or prevention disease or be included in implantable or injectable device In (U.S. Publication No 20100260817, be incorporated herein in its entirety by reference).As a non-limiting example, medicine Compositions may include modification of nucleic acids and mmRNA and the (institute of the polyamine derivatives described in U.S. Publication No 20100260817 The content for stating patent is incorporated herein in its entirety by reference).As a non-limiting example, polynucleotides of the invention, Polyamide polymer can be used to deliver in primary construct and mmRNA, polyamide (polyaminde) polymer for example but It is not limited to comprising by combining two nitrine monomer of carbohydrate with the diine unit (dilkyne unite) comprising oligomeric amine Polymer (the U.S. Patent number 8,236,280 of prepared 1,3- dipolar addition polymer；It is incorporated hereby Herein).
In one embodiment, polynucleotides of the invention, primary construct or mmRNA can be used and be described in international publication At least one in number WO2011115862, WO2012082574 and WO2012068187 and U.S. Publication No 20120283427 Kind polymer and/or its derivative are prepared, and the patent is respectively incorporated herein in its entirety by reference.In another implementation In scheme, modification of nucleic acids of the invention or mmRNA can use formula Z polymer formulation described in WO2011115862, the patent It is incorporated herein in its entirety by reference.In another embodiment again, modification of nucleic acids or mmRNA can use international publication number Formula Z, Z described in WO2012082574 or WO2012068187 and U.S. Publication No 2012028342 ' or Z " polymer It prepares, the patent is respectively incorporated herein in its entirety by reference.The polymer prepared together with modification RNA of the invention It can be synthesized by the method described in international publication number WO2012082574 or WO2012068187, the patent is respectively equal It is incorporated herein in its entirety by reference.
Polynucleotides, primary construct or mmRNA of the invention can be prepared at least one acrylate copolymer.Acrylic acid Polymer includes but is not limited to that acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymer, methyl methacrylate are total Polymers, ethoxyethyl methacrylates, methacrylic acid cyanaoethyl methacrylate, amino alkyl methacrylate copolymer, poly- (third Olefin(e) acid), poly- (methacrylic acid), polybutylcyanoacrylate with and combinations thereof.
The preparation of polynucleotides of the invention, primary construct or mmRNA may include at least one amine-containing polymer, such as But be not limited to polylysine, polyethyleneimine, poly- (amidoamines) dendritic or combinations thereof.
For example, modification of nucleic acids or mmRNA of the invention can be prepared in medical compounds, the medical compounds includes poly- (alkylene imine), Biodegradable cationic lipid polymer, biodegradable block copolymer, biodegradable polymerization Object or biodegradable random copolymer, biodegradable polyesters block copolymer, biodegradable polyesters polymer, biology Degradable polyester random copolymer, linear biodegradable copolymer, PAGA, Biodegradable cross-linked cationic multiblock Object or combinations thereof.Biodegradable cationic lipid polymer can by as is generally known in the art and/or in U.S. Patent number 6, 696,038, method described in U.S. Application No. 20030073619 and 20040142474 is made, the patent respectively with The mode of reference is integrally incorporated herein.Poly- (alkylene imine) can be used as is generally known in the art and/or such as in U.S. Publication No Method described in 20100004315 is made, and the patent is incorporated herein in its entirety by reference.Biodegradable polymerization Object, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyesters block copolymer, biology can Degradation polyester polymers or biodegradable polyesters random copolymer can be used as is generally known in the art and/or such as in U.S. Patent number Method described in 6,517,869 and 6,267,987 is made, and the patent is respectively incorporated hereby this Text.Linear biodegradable copolymer can be used as is generally known in the art and/or as described in U.S. Patent number 6,652,886 Method is made.PAGA polymer can be used as is generally known in the art and/or such as the side described in U.S. Patent number 6,217,912 Method is made, and the patent is incorporated herein in its entirety by reference.Can by PAGA polymer copolymerization with polymer as but not It is limited to poly-L-Lysine, poly arginine, poly ornithine, histone, avidin, nucleoprotamine, polyactide and poly- (third Lactide-co-glycolide) form copolymer or block copolymer.Biodegradable cross-linked cationic multi-block copolymers can pass through As is generally known in the art and/or as the method described in U.S. Patent number 8,057,821 or U.S. Publication No 2012009145 is come It is made, the patent is respectively incorporated herein in its entirety by reference.For example, segmented copolymer can be used and the poly- second of branch Alkene imines compares L-PEI (LPEI) block with alternative forms to synthesize.In addition, composition or pharmaceutical composition Object can by as is generally known in the art, be described herein or such as in U.S. Publication No 20100004315 or U.S. Patent number 6,267,987 With 6, method described in 217,912 is made, and the patent is respectively incorporated herein in its entirety by reference.
Polynucleotides of the invention, primary construct and mmRNA can with it is at least one containing polycation side chain can Polyester of degrading is prepared.Degradable polyester including but not limited to poly- (serine ester), gathers at poly- (L- lactide-co-L-lysine) (4-hydroxy-L-proline ester) with and combinations thereof.In another embodiment, degradable polyester may include PEG conjugation with shape At PEGylated polymer.
Polynucleotides of the invention, primary construct, mmRNA can be prepared at least one cross-linking polyester.It is cross-linking poly- Ester include be known in the art and described in the U.S. Publication No 20120269761 those, side of the patent to quote Formula is integrally incorporated herein.
In one embodiment, polymer described herein can be conjugated to the PEG of lipid sealing end.It is unrestricted as one Property example, PLGA can be conjugated to lipid sealing end PEG, to form PLGA-DSPE-PEG.As another non-limiting reality Example, the PEG conjugate being used in conjunction with the invention is described in international publication number WO2008103276, and the patent is to quote Mode is integrally incorporated herein.Ligand conjugates can be used such as, but not limited to, to describe in U.S. Patent number 8,273,363 for polymer Conjugate be conjugated, the patent is incorporated herein in its entirety by reference.
In one embodiment, modification RNA described herein can be conjugated with another compound.Conjugate it is unrestricted Property example be described in U.S. Patent number 7,964,578 and 7, in 833,992, the patent respectively it is whole by reference simultaneously Enter herein.In another embodiment, modification RNA of the invention can with such as in U.S. Patent number 7,964,578 and 7,833, The conjugate of formula 1-122 described in 992 is conjugated, and the patent is respectively incorporated herein in its entirety by reference.It retouches herein Polynucleotides, primary construct and/or the mmRNA stated can be conjugated with metal such as, but not limited to, gold.(see, for example, Giljohann Deng Journ.Amer.Chem.Soc.2009 131 (6): 2072-2073；It is incorporated herein in its entirety by reference).Another In one embodiment, polynucleotides, primary construct and/or mmRNA described herein can be conjugated and/or be encapsulated in gold nano In particle.(international publication number WO201216269 and U.S. Publication No 20120302940；It is respectively whole by reference It is incorporated herein).
Such as (patent is incorporated herein in its entirety by reference), base described in U.S. Publication No 20100004313 Because delivering compositions may include nucleotide sequence and poloxamer.For example, modification of nucleic acids and mmRNA of the invention can be used for having In the gene delivery composition of the poloxamer described in U.S. Publication No 20100004313.
In one embodiment, polymer formulations of the invention can by make may include cation carrier polymer system Agent is stablized with the cation lipid polymer contact that can be covalently attached to cholesterol and polyethylene group.Polymer formulations can Using the method and cation lipid polymer contact being described in U.S. Publication No 20090042829, the patent is to quote Mode be integrally incorporated herein.Cation carrier may include but be not limited to polyethyleneimine, poly- (tetrahydroform), poly- (four Methylene imine), polypropyleneimine, aminoglycoside-polyamine, dideoxy-diamino-b- cyclodextrin, spermine, spermidine, poly- first Base acrylic acid (2- dimethylamino) ethyl ester, poly- (lysine), poly- (histidine), poly- (arginine), cationized gelatin, branch Shaped polymer, chitosan, 1,2- dioleoyl -3- trimethylammonium-propane (DOTAP), N- [1- (2,3- dioleoyl oxygroup) third Base]-N, N, N- trimethyl ammonium chloride (DOTMA), 1- [2- (oleoyl oxygroup) ethyl] -2- oil base -3- (2- ethoxy) imidazoles Quinoline chloride (DOTIM), 2,3- dioleoyl oxygroup-N- [2 (sperminecarboxamido) ethyl]-N, N- dimethyl -1- propyl trifluoro Ammonium acetate (DOSPA), 3B- [N- (N ', N '-dimethylamino ethane)-carbamyl] cholesterol hydrochloride (DC-cholesterol HCl), two (heptadecyl) acylamino- glycyl spermidines (DOGS), N, N- distearyl-N, N- ditallowdimethyl ammonium bromide (DDAB), N- (1,2- myristyl oxygroup propyl- 3- yl)-N, N- dimethyl-N-hydroxy ammonium bromide (DMRIE), N, N- bis- Oil base-N, N- alkyl dimethyl ammonium chloride DODAC) with and combinations thereof.
It is multiple that polynucleotides of the invention, primary construct and/or mmRNA can prepare the polymerization in one or more polymer Close (U.S. Publication No 20120237565 and 20120270927 in object；It is respectively incorporated herein in its entirety by reference). In one embodiment, polymer composites include two or more cationic polymers.Cationic polymer may include gathering (aziridine) (PEI), such as linear PEI.
It is nano particle that formulated in combination below, which also can be used, in polynucleotides, primary construct and mmRNA of the invention: poly- Close object, lipid and/or other biodegradable agent such as, but not limited to, calcium phosphate.Component can be combined in core-shell structure copolymer, hybrid and/or Enhance the delivering of polynucleotides, primary construct and mmRNA in laminated construction to allow to fine-tune nano particle (Wang etc., Nat Mater.2006 5:791-796；Fuller etc., Biomaterials.2008 29:1526-1532； DeKoker etc., Adv Drug Deliv Rev.2011 63:748-761；Endres etc., Biomaterials.2011 32: 7721-7731；Su etc., Mol Pharm.2011 Jun 6；8 (3): 774-87；It is incorporated herein in its entirety by reference). As a non-limiting example, nano particle may include multiple polymers such as, but not limited to, hydrophilic-hydrophobic polymer (for example, PEG-PLGA), hydrophobic polymer (for example, PEG) and/or hydrophilic polymer (international publication number WO20120225129；It is to draw Mode is integrally incorporated herein).
It has been displayed and delivers multicore glycosides in vivo with the biodegradable calcium phosphate nanoparticles of lipid and/or combination of polymers Acid, primary construct and mmRNA.In one embodiment, the lipid also containing targeting ligand such as anisamide coats Calcium phosphate nanoparticles can be used to deliver polynucleotides of the invention, primary construct and mmRNA.For example, in order to turn in mouse SiRNA is effectively delivered in shifting property lung model, calcium phosphate nanoparticles (Li et al., the J Contr for having used lipid to coat Rel.2010 142:416-421；Li et al., J Contr Rel.2012 158:108-114；Yang etc., Mol Ther.2012 20:609-615；It is incorporated herein in its entirety by reference).This delivery system is combined in targeted nano particle and enhancing The component calcium phosphate of body evolution is to enhance the delivering of siRNA.
In one embodiment, the calcium phosphate with PEG- polyanion block copolymer can be used to deliver multicore glycosides Acid, primary construct and mmRNA (Kazikawa etc., J Contr Rel.2004 97:345-356；Kazikawa etc., J Contr Rel.2006 111:368-370；It is incorporated herein in its entirety by reference).
In one embodiment, PEG- charge transformation polymer (Pitella etc., Biomaterials.2011 32: It 3106-3114) can be used to form the nano particle for delivering polynucleotides of the invention, primary construct and mmRNA.It is poly- in PEG- Anionic block copolymers by be cracked into polycation at acidic when, PEG- charge transformation polymer can improve, thus Enhance inner body evolution.
The high throughput side of synthesizing cationic crosslinking nano gel core and various shells is in addition concentrated on using core-shell nanoparticles In method (Siegwart etc., Proc Natl Acad Sci U S A.2011 108:12996-13001).It can be by changing nanometer Chemical composition in the core and shell component of particle accurately to control the compound of polymer/nanoparticle, delivering and internalization.For example, core- It is thin that siRNA effectively can be delivered to Mouse Liver after they are by cholesterol covalent attachment to nano particle by core-shell nanoparticles Born of the same parents.
In one embodiment, the hollow lipid core of the external neutral lipid layer comprising intermediate PLGA layers and containing PEG It can be used to deliver polynucleotides of the invention, primary construct and mmRNA.As a non-limiting example, fluorescence is being carried In the mouse of plain expression of enzymes tumour, it has been determined that compared with conventional liposome compound, lipid-polymer-lipid hybridized nanoparticle Significant compacting luciferase expression (Shi etc., Angew Chem Int Ed.2011 50:7027-7031；It is by reference It is integrally incorporated herein).
In one embodiment, lipidic nanoparticles may include core and the polymerization of modified nucleic acid molecule disclosed herein Object shell.Polymer shell can be any polymer described herein and known in the art.In a further embodiment, gather Closing object shell can be used to protect the modification of nucleic acids in core.
It describes the core-shell nanoparticles for being used together with modified nucleic acid molecule of the invention and description can be passed through Method in U.S. Patent number 8,313,777 is formed, and the patent is incorporated herein in its entirety by reference.
In one embodiment, core-shell nanoparticles may include the core of modified nucleic acid molecule disclosed herein and gather Close object shell.Polymer shell can be any polymer described herein and known in the art.In a further embodiment, Polymer shell can be used to protect the modified nucleic acid molecule in core.As a non-limiting example, core-shell nanoparticles can be used to Treat disease of eye or illness (see, for example, U.S. Publication No 20120321719, being incorporated herein in its entirety by reference).
In one embodiment, the polymer being used together with preparation described herein can be for such as in international publication number Modified polymer described in WO2011120053 (such as, but not limited to modification polyacetals), the patent is whole by reference Body is incorporated herein.
Peptide and protein
Polynucleotides, primary construct and mmRNA of the invention can be prepared with peptide and/or protein to increase by multicore The cell transfecting that thuja acid, primary construct or mmRNA are carried out.In one embodiment, peptide such as, but not limited to cell-penetrating Peptide and the protein and peptide for being able to achieve Intracellular delivery can be used to deliver pharmaceutical preparation.It can make together with pharmaceutical preparation of the invention The non-limiting example of cell-penetrating peptides includes that the cell for being attached to polycation for being delivered to intercellular spaces is promoted to wear Saturating peptide sequence, such as tat peptide, cell-penetrating peptide (penetratin), transit peptides (transportan) or the source hCT in the source HIV- Cell-penetrating peptides (see, for example, Caron etc., Mol.Ther.3 (3): 310-8 (2001)；Langel, Cell- Penetrating Peptides:Processes and Applications (CRC Press, Boca Raton FL, 2002)；El-Andaloussi etc., Curr.Pharm.Des.11 (28): 3597-611 (2003)；And Deshayes etc., Cell.Mol.Life Sci.62 (16): 1839-49 (2005), all bibliography are incorporated hereby Herein).Composition can also be prepared to include cell-penetrating agent such as liposome, enhance composition passing to intercellular spaces It send.Polynucleotides of the invention, primary construct and mmRNA compound can such as, but not limited to come to peptide and/or protein The peptide of Aileron Therapeutics (Cambridge, MA) and Permeon Biologics (Cambridge, MA) and/or Protein is to be able to achieve Intracellular delivery (Cronican etc., ACS Chem.Biol.2010 5:747-752；McNaughton Deng 2009 106:6111-6116 of Proc.Natl.Acad.Sci.USA；Sawyer, Chem Biol Drug Des.2009 73:3-6；Verdine and Hilinski, Methods Enzymol.2012；503:3-33；All bibliography are to draw Mode is integrally incorporated herein).
In one embodiment, cell penetrating peptide may include first structure domain and the second structural domain.First structure domain It may include the polypeptide of hypercharge.Second structural domain may include protein binding partner.As used herein, " protein binding partner Body " includes but is not limited to antibody and its function fragment, scaffolding protein or peptide.Cell penetrating peptide can further include for albumen The intracellular binding partner of binding partners.Cell penetrating peptide can be from can wherein introduce polynucleotides, primary building The secretion of the cell of body or mmRNA.
Preparation including peptide or protein matter can be used to increase the cell carried out by polynucleotides, primary construct or mmRNA Transfection changes the bio distribution of polynucleotides, primary construct or mmRNA (for example, by targeting specific organization or cell class Type) and/or increase coding protein translation.(see, for example, international publication number WO2012110636；It is by reference It is integrally incorporated herein).
Cell
Polynucleotides of the invention, primary construct and mmRNA can ex vivo transfection into cell, the cell is then transplanted Into subject.As non-limiting examples, pharmaceutical composition may include that will to modify RNA delivery red to liver and bone marrow cell Cell delivers the virion of modification RNA and such as, but not limited to coming from for delivering modification RNA with virus-like particle (VLP)It (Gaithersburg, MD) and comes fromThe electroporation of cells of (Lyon, France).? The example for describing the payload using red blood cell, virion and electroporation of cells delivering other than mmRNA (Godfrin etc., Expert Opin Biol Ther.2012 12:127-133；Fang etc., Expert Opin Biol Ther.2012 12:385-389；Hu etc., Proc Natl Acad Sci U S A.2011 108:10980-10985；Lund Deng Pharm Res.2010 27:400-420；Huckriede etc., J Liposome Res.2007；17:39-47；Cusi, Hum Vaccin.2006 2:1-7；De Jonge etc., Gene Ther.2006 13:400-411；All bibliography are equal It is incorporated herein in its entirety by reference).
Polynucleotides, primary construct and mmRNA can be by being described in international publication number WO2011085231 and the U.S. The synthesis VLP delivering of method synthesis in publication No. 20110171248, the patent are respectively incorporated hereby Herein.
The preparation based on cell of polynucleotides of the invention, primary construct and mmRNA can be used to ensure that cell transfecting (for example, in cell carrier) changes the bio distribution of polynucleotides, primary construct or mmRNA (for example, by making cell Carrier targets specific organization or cell type) and/or increase coding protein translation.
Various methods be it is as known in the art and suitable for nucleic acid to be introduced into cell, the method includes viruses With the technology of non-viral mediation.The example of the technology of typical non-viral mediation includes but is not limited to electroporation, calcium phosphate mediation Transfer, nuclear transfection, sonoporation, heat shock, magnetic transfection, liposome-mediated transfer, micro-injection, the transfer of micro-bullet mediation The transfer (DEAE- glucan, polyethyleneimine, polyethylene glycol (PEG) etc.) or thin that (nano particle), cationic polymer mediate Born of the same parents' fusion.
The technology of sonoporation or cell ultrasound is to change wearing for cytoplasma membrane using sound (for example, ultrasonic frequency) Permeability.Sonoporation method is well known by persons skilled in the art and is used to delivering nucleic acid (Yoon and Park, Expert in vivo Opin Drug Deliv.2010 7:321-330；Postema and Gilja, Curr Pharm Biotechnol.2007 8: 355-361；Newman and Bettinger, Gene Ther.2007 14:465-475；It is all it is whole by reference simultaneously Enter herein).Sonoporation method is as known in the art and also for example when it is related to bacterium, introduction is public in United States Patent (USP) It is instructed in such as U.S. Patent Publication 20100009424 in cloth 20100196983 and when it is related to other cell types, The patent is respectively incorporated herein in its entirety by reference.
Electroporation technology is also well known in the art and is used in vivo and clinically deliver nucleic acid (Andre etc., Curr Gene Ther.2010 10:267-280；Chiarella etc., Curr Gene Ther.2010 10:281-286；Hojman, Curr Gene Ther.2010 10:128-138；It is all to be incorporated herein in its entirety by reference).In an embodiment In, polynucleotides, primary construct or mmRNA can be by delivering such as the electroporation described in embodiment 8.
Hyaluronidase
The intramuscular or Local subdermal injection liquid of polynucleotides of the invention, primary construct or mmRNA may include that catalysis is saturating The bright acid-hydrolyzed hyaluronidase of matter.By being catalyzed the hydrolysis of the component transparent matter acid of interstitial barrier, hyaluronidase is reduced The viscosity of hyaluronic acid, to increase penetration into tissue (Frost, Expert Opin.Drug Deliv. (2007) 4:427- 440；It is incorporated herein in its entirety by reference).Accelerate the dispersion and system of the coding protein generated by the cell transfected Distribution is useful.Alternatively, hyaluronidase can be used to increase be exposed to intramuscular or subcutaneous administration polynucleotides of the invention, The cell number of primary construct or mmRNA.
Nano particle analogies
Polynucleotides, primary construct or mmRNA of the invention can be encapsulated in nano particle analogies and/or be absorbed into Nano particle analogies.Nano particle analogies analog organism or particle such as, but not limited to pathogen, virus, bacterium, Fungi, helminth, prion and cell delivery functions.As a non-limiting example, polynucleotides of the invention, just (referring to international publication number in the non-viral body particle for the delivery functions that grade construct or mmRNA can be encapsulated in analog virus WO2012006376 is incorporated herein in its entirety by reference).
Nanotube
Polynucleotides, primary construct or mmRNA of the invention can adhere to or be integrated in another way at least one Nanotube, such as, but not limited to rosiness nanotube, rosiness nanotube, carbon nanotube and/or list with double alkali yl connector Wall carbon nano tube.Polynucleotides, primary construct or mmRNA can pass through power such as, but not limited to non-coplanar force, ionic forces, covalent force And/or other power are integrated to nanotube.
In one embodiment, one or more polynucleotides, primary construct or mmRNA can be discharged by nanotube In cell.Size and/or the surface texture of at least one nanotube can be changed to adjust the phase interaction of the intracorporal nanotube of body With and/or attachment or be integrated to polynucleotides disclosed herein, primary construct or mmRNA.In one embodiment, can change The functional group of the structural unit of structure changes unit and/or at least one nanotube of attachment so as to adjust nanotube size and/or Characteristic.As a non-limiting example, the length of nanotube can be changed to hinder nanotube to pass through the hole in normal blood vessels wall But still the sufficiently small biggish hole in blood vessel to pass through tumor tissues.
In one embodiment, at least one nanotube can also be coated with delivering enhancing compound comprising polymer Such as, but not limited to, polyethylene glycol.In another embodiment, at least one nanotube and/or polynucleotides, primary construct Or mmRNA can be mixed with pharmaceutically acceptable excipient and/or delivery vehicle.
In one embodiment, polynucleotides, primary construct or mmRNA adhere to and/or combine in another way To at least one rosiness nanotube.Rosiness nanotube can be by method as known in the art and/or by international public Method described in cloth WO2012094304 is formed, and the patent is incorporated herein in its entirety by reference.At least one Polynucleotides, primary construct and/or mmRNA can be adhered to by such as the method described in international publication number WO2012094304 And/or it is integrated at least one rosiness nanotube in another way, the patent is incorporated herein in its entirety by reference, Wherein it can cause at least one polynucleotides, primary construct or mmRNA attachment or be integrated to rosiness in another way Under conditions of nanotube, by the module and at least one polynucleotides, primary of rosiness nanotube or formation rosiness nanotube Construct and/or mmRNA mixing are in an aqueous medium.
In one embodiment, polynucleotides, primary construct or mmRNA can adhere to and/or tie in another way Close at least one carbon nanotube.As a non-limiting example, polynucleotides, primary construct or mmRNA can be coupled to Bridging agent and bridging agent can be coupled to carbon nanotube (see, for example, U.S. Patent number 8,246,995；It is whole by reference Body is incorporated herein).Carbon nanotube can be single-walled nanotube (see, for example, U.S. Patent number 8,246,995；It is by reference It is integrally incorporated herein).
Conjugate
Polynucleotides, primary construct and mmRNA of the invention includes conjugate, is such as covalently attached to carrier or targeting base The polynucleotides of group, primary construct or mmRNA, or two including generating fusion protein jointly code areas are (for example, have target To group and therapeutic protein or peptide).
Conjugate of the invention includes naturally occurring material, if protein is (for example, human serum albumins (HSA), low close Spend lipoprotein (LDL), high-density lipoprotein (HDL) or globulin)；Carbohydrate is (for example, glucan, amylopectin, shell are more Sugar, chitosan, inulin, cyclodextrin or hyaluronic acid)；Or lipid.Ligand can be also recombination or synthetic molecules, such as synthesized polymer Object, such as synthesis polyaminoacid, oligonucleotides (for example, aptamer).The example of polyaminoacid includes that polyaminoacid is polylysine (PLL), poly- L-Aspartic acid, L-glutamic acid, styrene-maleic anhydride copolymer, poly- (L- lactide-co-glycolide) are total Polymers, divinyl ether-copolymer-maleic anhydride, N- (2- hydroxypropyl) methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly- (2- ethylacrylic acid), n-isopropyl acrylamide polymer or poly-phosphine piperazine.It is poly- The example of amine includes: that polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamine, false peptide-polyamine, peptide mimics are poly- Amine, dendritic polyamine, arginine, amidine, nucleoprotamine, cation lipid, cationic porphyrin, the quaternary salt of polyamine or α spiral shell Revolve peptide.
The representative United States Patent (USP) for instructing the preparation of polynucleotides conjugate (specially RNA) includes but is not limited to that the U.S. is special Benefit number 4,828,979；4,948,882；5,218,105；5,525,465；5,541,313；5,545,730；5,552,538；5, 578,717；5,580,731；5,591,584；5,109,124；5,118,802；5,138,045；5,414,077；5,486, 603；5,512,439；5,578,718；5,608,046；4,587,044；4,605,735；4,667,025；4,762,779；4, 789,737；4,824,941；4,835,263；4,876,335；4,904,582；4,958,013；5,082,830；5,112, 963；5,214,136；5,082,830；5,112,963；5,214,136；5,245,022；5,254,469；5,258,506；5, 262,536；5,272,250；5,292,873；5,317,098；5,371,241；5,391,723；5,416,203；5,451, 463；5,510,475；5,512,667；5,514,785；5,565,552；5,567,810；5,574,142；5,585,481；5, 587,371；5,595,726；5,597,696；5,599,923；5,599,928 and 5,688,941；6,294,664；6,320, 017；6,576,752；6,783,931；6,900,297；7,037,646；It is respectively incorporated herein in its entirety by reference.
In one embodiment, conjugate of the invention can be used as the load for modification of nucleic acids and mmRNA of the invention Body.Conjugate may include cationic polymer such as, but not limited to, polyamine, polylysine, polyalkyleneimine and can be grafted to poly- The polyethyleneimine of (ethylene glycol).As a non-limiting example, conjugate can be similar to polymeric conjugates and synthesize poly- The method for closing conjugate is described in U.S. Patent number 6,586,524, and the patent is incorporated herein in its entirety by reference.
Conjugate may also include targeting group, such as cell or tissue targeting agent, for example, agglutinin, glycoprotein, lipid or Protein, such as it is integrated to the antibody of designated cell type such as nephrocyte.Target group can for thyroid-stimulating hormone, melanotropin, Agglutinin, glycoprotein, surfactant protein A, mucoprotein carbohydrate, multivalence lactose, multivalence galactolipin, N- acetyl group-half Lactose amine, multivalence fucose, glycosylated polyaminoacid, multivalence galactolipin, turns iron at n-acetyl-glucosamine multivalence mannose Albumen, diphosphonate, polyglutamate, polyaspartic acid salts, lipid, cholesterol, steroids, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptide analogies or aptamer.
Targeting group can be protein, such as glycoprotein or peptide, such as have the molecule of pathoklisis to total ligand, Or antibody, such as it is integrated to the designated cell type such as antibody of cancer cell, endothelial cell or osteocyte.Targeting group may also include Hormone and hormone receptor.They may also include non-peptide type, such as lipid, agglutinin, carbohydrate, vitamin, co-factor, more Valence lactose, multivalence galactolipin, GalNAc, n-acetyl-glucosamine multivalence mannose, multivalence fucose or suitable Body.Ligand may be, for example, the activator of lipopolysaccharides or p38 map kinase.
Targeting group can be any ligand that can target special receptor.Example includes but is not limited to folic acid, GalNAc, half Lactose, mannose, mannose -6P, aptamer, integrin receptor ligand, chemokine receptor ligands, transferrins, biotin, 5-hydroxytryptamine receptor ligand, PSMA, Endothelin, GCPII, Somat, LDL and HDL ligand.In specific embodiment party In case, targeting group is aptamer.Aptamer can be unmodified or with modification disclosed herein any combination.
In one embodiment, pharmaceutical composition of the invention may include that chemical modification is such as, but not limited to, similar to lock core The modification of acid.
Introduction lock nucleic acid (LNA) such as preparation from those of Santaris representative United States Patent (USP) include but is not limited to Below: U.S. Patent number 6,268,490；6,670,461；6,794,499；6,998,484；7,053,207；7,084,125 with And 7,399,845, respectively it is incorporated herein in its entirety by reference.
The representative United States Patent (USP) for instructing the preparation of PNA compound includes but is not limited to U.S. Patent number 5,539,082；5, 714,331 and 5,719,262, respectively it is hereby incorporated herein by.Other religious doctrines of PNA compound are found in for example Nielsen etc., Science, in 1991,254,1497-1500.
Some embodiments distinctive in the present invention include the polynucleotides with phosphorothioate backbone, primary structure The few nucleosides of body or mmRNA and the main chain with other modifications, and U.S. Patent number 5 specially cited above are built, 489,677 -- CH2--NH--CH2--、--CH2--N(CH3)--O--CH2-- [referred to as methylene (methyl-imino) or MMI master Chain], -- CH2--O--N(CH3)--CH2--、--CH2--N(CH3)--N(CH3)--CH2-- and -- N (CH3)--CH2--CH2-- [wherein natural phosphodiester backbone is expressed as -- O-P (O)2--O--CH2--] and U.S. Patent number cited above 5, 602,240 amide backbone.In some embodiments, distinctive polynucleotides have the U.S. cited above herein The morpholino backbone structure of the patent No. 5,034,506.
Modification on 2 ' positions may also aid in delivering.Preferably, the modification on 2 ' positions is not located at the sequence of coding polypeptide In, i.e., not can be in translated region.Modification on 2 ' positions can be located at 5 ' UTR, 3 ' UTR and/or tailing area (tailing Region in).Modification on 2 ' positions may include the following one on 2 ' positions: H (that is, 2 '-deoxidations)；F；O-, S- or N- alkane Base；O-, S- or N- alkenyl；O-, S- or N- alkynyl；Or O- alkyl-O- alkyl, wherein alkyl, alkenyl and alkynyl can for replace or Unsubstituted C1To C10Alkyl or C2To C10Alkenyl and alkynyl.Illustrative suitable modification includes O [(CH2)nO]mCH3、O (CH2)nOCH3、O(CH2)nNH2、O(CH2)nCH3、O(CH2)nONH2And O (CH2)nON[(C2)nCH3]2, wherein n and m be 1 to About 10.In other embodiments, polynucleotides, primary construct or mmRNA include the following one on 2 ' positions: C1To C10 Low alkyl group, substituted low alkyl group, alkaryl, aralkyl, O- alkaryl or O- aralkyl, SH, SCH3、OCN、Cl、Br、 CN、CF3、OCF3、SOCH3、SO2CH3、ONO2、NO2、N3、NH2, Heterocyclylalkyl, heteroalkylaryl, aminoalkyl amino, more alkyl Amino, substituted silicyl, RNA cracking group, reporter group, intercalator, for improving pharmacokinetic profile Group or the group for improving pharmacodynamic properties and other substituent groups with similarity.In some embodiments, Modification includes 2 '-methoxy ethoxy (2 '-O-CH2CH2OCH3, also known as 2 '-O- (2- methoxy ethyl) or 2 '-MOE) (Martin etc., Helv.Chim.Acta, 1995,78:486-504), i.e. alkoxy-alkoxy.Another is exemplary to be modified to 2 '-dimethylamino oxygroup ethyoxyls, i.e. O (CH2)2ON(CH3)2Group, also known as 2 '-DMAOE, such as this paper following embodiment Described in and 2 '-dimethylamino ethoxy ethyoxyls (in the art be also known as 2 '-O- dimethylamino ethoxy ethyls Or 2 '-DMAEOE), i.e., the also 2 '-O--CH described in this paper following embodiment2--O--CH2--N(CH2)2.Other modification packets Include 2 '-methoxyl group (2 '-OCH3), 2 '-amino propoxyl group (2 '-OCH2CH2CH2NH2) and 2 '-fluorine (2 '-F).Similar modification is also It can be carried out in other positions, 3 ' positions of the sugar in specially 3 ' terminal nucleotides or in the dsRNA of 2 ' -5 ' connection and 5 ' ends 5 ' positions of terminal nucleotide.Polynucleotides of the invention can also have sugared analogies such as cyclobutyl moiety to substitute furan pentose base Sugar.The representative United States Patent (USP) for instructing the preparation of the sugared structure of such modification includes but is not limited to U.S. Patent number 4,981,957； 5,118,800；5,319,080；5,359,044；5,393,878；5,446,137；5,466,786；5,514,785；5,519, 134；5,567,811；5,576,427；5,591,722；5,597,909；5,610,300；5,627,053；5,639,873；5, 646,265；5,658,873；5,670,633 and 5,700,920, and the patent is respectively herein incorporated by reference this Text.
In other embodiments, polynucleotides, primary construct or mmRNA are covalently conjugated to cell penetrating peptide. Cell-penetrating peptides may also include signal sequence.Conjugate of the invention can design have increased stability；Increased cell Transfection；And/or the bio distribution (for example, the specific tissue of targeting or cell type) changed.
In one embodiment, polynucleotides, primary construct or mmRNA can be conjugated to reagent to enhance delivering.Make For a non-limiting example, the reagent can be monomer or polymer, such as target monomer or have such as in international publication number The polymer of block is targeted described in WO2011062965, the patent is incorporated herein in its entirety by reference.Another In a non-limiting example, medicament can be the transport agents for being covalently coupled to polynucleotides of the invention, primary construct or mmRNA (see, for example, U.S. Patent number 6,835.393 and 7,374,778, be respectively incorporated herein in its entirety by reference).? Again in another non-limiting example, reagent can be envelope barrier transport enhancer such as in U.S. Patent number 7,737,108 and 8, Described in 003,129 those, the patent is respectively incorporated herein in its entirety by reference.
In another embodiment, polynucleotides, primary construct or mmRNA can be conjugated to SMARTT POLYMER(Inc.Seattle, WA).
Self-assembling nanoparticles
Nucleic acid self-assembling nanoparticles
Self-assembling nanoparticles have clearly defined size, accurately control as that can be easy the nucleic acid reprogramed Chain.For example, the optimal granularity of the nanometer delivery vector for cancer targeting is 20-100nm, because the diameter greater than 20nm avoids Kidney is removed and is enhanced by the penetrability and reserve effects of enhancing to the delivering of certain tumours.It is received using self assembly nucleic acid Rice grain is, it can be achieved that the single uniform group of size and shape with the spatial orientation accurately controlled and for enhancing delivering Cancer targeting ligand density.As a non-limiting example, using the programmable of short dna segment and therapeutic siRNA Self assembly prepares oligonucleotide nano particle.These nano particles on molecule with controllable granularity and targeting ligand position And density is identical.DNA fragmentation and siRNA are self-assembled in single step reaction to generate the DNA/ for being used for internal targeted delivery SiRNA tetrahedron nano particle.(2012 7:389-393 of Lee etc., Nature Nanotechnology；It is with the side of reference Formula is integrally incorporated herein).
In one embodiment, polynucleotides disclosed herein, primary construct and/or mmRNA can be formulated as from group Accommodate rice grain.As a non-limiting example, nucleic acid can be used to be made polynucleotides for use in the present invention, primary building Nano particle in the delivery system of body and/or mmRNA is (see, for example, international publication number WO2012125987；It is with reference Mode is integrally incorporated herein).
In one embodiment, nucleic acid self-assembling nanoparticles may include polynucleotides disclosed herein, primary building The core and polymer shell of body or mmRNA.Polymer shell can be any polymer described herein and known in the art. In a further embodiment, polymer shell can be used to protect polynucleotides, primary construct and mmRNA in core.
Self-assembling nanoparticles based on polymer
Polymer can be used to form the thin slice self-assembled in nano particle.These nano particles can be used to deliver the present invention Polynucleotides, primary construct and mmRNA.In one embodiment, these self-assembling nanoparticles can be for by RNA hair clip Long polymer formed microsponge, the long polymer of the RNA hair clip is formed before self-assembling to microsponge crystallize ' beat Pleat ' thin slice.These microsponges are the cavernous transformation particle of intensive hematocrit, can be used as effective carrier and can pass cargo It send to cell.The diameter of microsponge can be 1um to 300nm.Microsponge can be compound with shape with other reagents as known in the art The microsponge of Cheng Geng great.As a non-limiting example, microsponge can be compound to form promotion cell absorption with a kind of reagent Outer layer such as polycation polyethyleneimine (PEI).This compound may be formed under high temperature (150 DEG C) be able to maintain it is stable Particle (Grabow and Jaegar, the Nature Materials 2012,11:269-269 of 250-nm diameter；It is with the side of reference Formula is integrally incorporated herein).In addition, these microsponges, which can show, is protected from the extraordinary of ribonuclease degradation Degree.
In another embodiment, the such as, but not limited to, microsponge of the self-assembling nanoparticles based on polymer can be complete Programmable nano particle.Geometry, size and the Chemical Measurement of nano particle are accurately controlled to generate for delivering goods The best nano particle of object such as, but not limited to, polynucleotides, primary construct and/or mmRNA.
In one embodiment, the nano particle based on polymer may include polynucleotides disclosed herein, primary structure Build body and/or the core and polymer shell of mmRNA.Polymer shell can be for any polymer described herein and be this field institute It is known.In a further embodiment, polymer shell can be used to protect polynucleotides in core, primary construct and/or mmRNA。
In another embodiment again, the nano particle based on polymer may include non-core acid polymer, and it includes more A heterobifunctional monomer as described in the international publication number WO2013009736 those, the patent is incorporated hereby Herein.
Inorganic nanoparticles
Polynucleotides, primary construct and/or mmRNA of the invention can prepare in inorganic nanoparticles (United States Patent (USP) Numbers 8,257,745, be incorporated herein in its entirety by reference).Inorganic nanoparticles may include but be not limited to water-swellable Clay material.As a non-limiting example, inorganic nanoparticles may include synthesizing montmorillonite made from simple silicate Clay (see, for example, U.S. Patent number 5,585,108 and 8,257,745, respectively it is incorporated hereby this Text).
In one embodiment, inorganic nanoparticles may include the core and polymer of modification of nucleic acids disclosed herein Shell.Polymer shell can be any polymer described herein and known in the art.In a further embodiment, it polymerize Object shell can be used to protect the modification of nucleic acids in core.
Semiconductive nano particle and metal nanoparticle
Polynucleotides, primary construct and/or mmRNA of the invention can be prepared comprising semiconductive material or metal material Water dispersible nanoparticles in (U.S. Publication No 20120228565；It is incorporated herein in its entirety by reference) or in magnetic Property nano particle in formed (U.S. Publication No 20120265001 and 20120283503；It is respectively whole by reference It is incorporated herein).Water dispersible nanoparticles can be hydrophobic nanoparticles or hydrophilic nano.
In one embodiment, semiconductive nano particle and/or metal nanoparticle may include multicore disclosed herein The core and polymer shell of thuja acid, primary construct and/or mmRNA.Polymer shell can be for any polymer described herein simultaneously And it is known in the art.In a further embodiment, polymer shell can be used to protect polynucleotides in core, primary building Body and/or mmRNA.
Gel and hydrogel
In one embodiment, polynucleotides disclosed herein, primary construct and/or mmRNA can be encapsulated into ability In domain in known any hydrogel, when being injected into subject, the hydrogel can form gel.Hydrogel is hydrophilic poly- The network of object chain is closed, and is existed sometimes by the colloidal gel form of decentralized medium of wherein water.Hydrogel is highly absorbent (they can contain have more than 99% water) natural or synthetic polymer.Hydrogel is also possessed very due to its a large amount of water content Similar to the pliability of natural tissues.Hydrogel described herein can be used to encapsulate bio-compatible, biodegradable and/or porous Lipidic nanoparticles.
As a non-limiting example, hydrogel can be the hydrogel of aptamer functionalization.The hydrogel of aptamer functionalization Nucleic acid hybridization can be used to program to discharge one or more polynucleotides, primary construct and/or mmRNA.(Battig etc., J.Am.Chem.Society.2012 134:12410-12413；It is incorporated herein in its entirety by reference).
As another non-limiting example, hydrogel be can shape as inverted opal.Opal hydrogel is shown Higher swelling ratio and the equally also fast an order of magnitude of Swelling Dynamics.Generate the method and opal of opal hydrogel The description of hydrogel describes in international publication number WO2012148684, and the patent is incorporated herein in its entirety by reference.
In another non-limiting example again, hydrogel can be antibacterium hydrogel.Antibacterium hydrogel may include medicine Acceptable salt or organic material on, such as, but not limited to pharmaceutical grade and/or medical grade silver salt and aloe gel or extract. (international publication number WO2012151438, be incorporated herein in its entirety by reference).
In one embodiment, modification mRNA can be encapsulated in lipidic nanoparticles and then lipidic nanoparticles can It is encapsulated into hydrogel.
In one embodiment, polynucleotides disclosed herein, primary construct and/or mmRNA can be encapsulated into ability In domain in known any gel.As a non-limiting example, gel can be for fluorouracil injectable gel or containing originally The fluorouracil injectable gel of known chemical compound and/or drug in field.As another example, polynucleotides, Primary construct and/or mmRNA can be encapsulated in containing in adrenergic fluorouracil gel (see, for example, Smith etc., Cancer Chemotherapty and Pharmacology, 1,999 44 (4): 267-274；It is whole by reference simultaneously Enter herein).
In one embodiment, polynucleotides disclosed herein, primary construct and/or mmRNA can be encapsulated into fiber In protein gel, fibrin hydrogel or Fibrin Glue.In another embodiment, polynucleotides, primary construct And/or mmRNA can be prepared before being encapsulated into fibrin gel, fibrin hydrogel or Fibrin Glue and be received in lipid In rice grain or quick elimination type lipidic nanoparticles.In another embodiment again, polynucleotides, primary construct and/ Or mmRNA can be formulated as lipid complex before being encapsulated into fibrin gel, hydrogel or Fibrin Glue.Fiber egg White gel, hydrogel and glue include two kinds of components, i.e., fibrinogen solution and rich in calcium thrombin solution (see, for example, Spicer and Mikos, Journal of Controlled Release 2010.148:49-55；The Journal such as Kidd of Controlled Release 2012.157:80-85；It is respectively incorporated herein in its entirety by reference).Changeable fibre The concentration of the component of fibrillarin gel, hydrogel and/or glue is big to change the feature of gel, hydrogel and/or glue, network sieve pore Small and/or degradation feature, such as, but not limited to the release overview of change fibrin gel, hydrogel and/or glue.(referring to example Such as, Spicer and Mikos, Journal of Controlled Release 2010.148:49-55；Kidd etc., Journal Of Controlled Release 2012.157:80-85；Catelas etc., Tissue Engineering 2008.14: 119-128；It is respectively incorporated herein in its entirety by reference).This feature can be used to deliver modification disclosed herein It is advantageous when mRNA.(see, for example, Kidd etc., Journal of Controlled Release 2012.157:80-85； Catelas etc., Tissue Engineering 2008.14:119-128；It is respectively incorporated hereby this Text).
Cation and anion
The preparation of polynucleotides disclosed herein, primary construct and/or mmRNA may include cation or anion.? In one embodiment, preparation include metal cation such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ with and combinations thereof.As One non-limiting example, preparation may include polymer and the polynucleotides compound with metal cation, primary construct and/or MmRNA (see, for example, U.S. Patent number 6,265,389 and 6,555,525, respectively it is incorporated hereby this Text).
The nano particle and particle of molding
Polynucleotides, primary construct and/or mmRNA disclosed herein can be prepared in nano particle and/or particle.This A little nano particles and/or particle may be molded as any size, shape and chemical property.As an example, LIQUIDA can be used(Morrisville, NC's)Technology be made nano particle and/or particle (referring to Such as international publication number WO2007024323；It is incorporated herein in its entirety by reference).
In one embodiment, the nano particle of molding may include polynucleotides disclosed herein, primary construct and/ Or the core and polymer shell of mmRNA.Polymer shell can be any polymer described herein and known in the art.? In other embodiments, polymer shell can be used to protect polynucleotides, primary construct and/or mmRNA in core.
Nanometer jacket (NanoJacket) and nano liposomes
Polynucleotides, primary construct and/or mmRNA disclosed herein can be prepared in Keystone Nano (State College, PA) nanometer jacket and nano liposomes in.Nanometer jacket by the compound being naturally present in body include calcium, Phosphate is made, and may also include a small amount of silicate.The size of nanometer jacket can be within the scope of 5nm to 50nm, and can be used To deliver hydrophilic and hydrophobic compound such as, but not limited to, polynucleotides, primary construct and/or mmRNA.
Nano liposomes are made of lipid, the lipid being such as, but not limited to naturally present in body.Nano liposomes Size can be within the scope of 60nm to 80nm, and can be used to deliver hydrophilic and hydrophobic compound such as, but not limited to, polynucleotides, just Grade construct and/or mmRNA.In an aspect, polynucleotides disclosed herein, primary construct and/or mmRNA are prepared In nano liposomes such as, but not limited to, ceramide nano liposome.
Excipient
Pharmaceutical preparation can additionally comprise pharmaceutically acceptable excipient, and as used herein excipient includes being suitable for Any and all solvents, decentralized medium, diluent or the other liquid vehicles of desired specific dosage form, dispersion or suspend helps Agent, surfactant, isotonic agent, thickener or emulsifier, preservative, solid binder, lubricant etc..The The of Remington Science and Practice of Pharmacy, the 21st edition, A.R.Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006；It is incorporated herein in its entirety by reference) it discloses for compounding pharmaceutical composition Various excipient and for its preparation known technology.In addition to such as by generate any undesirable biological effect or in addition with Any other component of harmful mode and pharmaceutical composition interaction and with substance or derivatives thereof it is incompatible it is any often It advises other than excipient medium, the use of the excipient is included within the scope of the invention.
In some embodiments, pharmaceutically acceptable excipient be at least 95%, at least 96%, at least 97%, extremely Few 98%, at least 99% or 100% are pure.In some embodiments, excipient is approved for people and uses for animal doctor. In some embodiments, excipient is ratified by Food and Drug Adminstration of the US.In some embodiments, excipient is medicinal Grade.In some embodiments, excipient meets United States Pharmacopeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia and/or international medicine The standard of allusion quotation.
The pharmaceutically acceptable excipient used in the manufacture of pharmaceutical composition include but is not limited to inert diluent, Dispersing agent and/or granulating agent, surfactant and/or emulsifier, disintegrating agent, adhesive, preservative, buffer, lubricant and/ Or oil.Such excipient can be optionally included in pharmaceutical composition.
Exemplary thinning agents include but is not limited to calcium carbonate, sodium carbonate, calcium phosphate, Dicalcium Phosphate, calcium sulfate, phosphoric acid hydrogen Calcium, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbierite, inositol, sodium chloride, Gan Dian Powder, cornstarch, Icing Sugar etc. and/or combination thereof.
Exemplary granulating agent and/or dispersing agent include but is not limited to potato starch, cornstarch, tapioca, glycolic acid Sodium starch, clay, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and Wood products, natural sponge, cation Exchanger resin, calcium carbonate, silicate, sodium carbonate, crosslinking polyvinylpyrrolidone) (Crospovidone), sodium carboxymethyl starch (hydroxyl Amylcose acetate sodium), hydroxymethyl cellulose, croscarmellose sodium (Croscarmellose), methylcellulose, pregelatinization Starch (starch 1500), Microcrystalline Starch, the insoluble starch of water, calcium carboxymethylcellulose, aluminium-magnesium silicateMonth Osmanthus sodium sulphate, quaternary ammonium compound etc. and/or combination thereof.
Exemplary surfactants and/or emulsifier include but is not limited to naturally occurring emulsifying agent (such as gum arabic, fine jade Rouge, alginic acid, sodium alginate, bassora gum, chondrin, cholesterol, xanthan gum, pectin, gelatin, yolk, casein, lanolin, Cholesterol, wax and lecithin), colloidal clays (such as bentonite [alumina silicate] and[aluminium-magnesium silicate]), long-chain Amino acid derivativges, high molecular weight alcohol (such as stearyl alcohol, cetanol, oleyl alcohol, monostearate glyceryl triacetate, distearyl acid Glycol ester, glycerin monostearate and propylene glycolmonostearate, polyvinyl alcohol), carbomer (carbomer) (such as carboxyl Polymethylene, polyacrylic acid, acrylate copolymer and carboxy vinyl polymer), carrageenan, cellulose derivative (such as Sodium carboxymethylcellulose, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, Methyl cellulose Element), sorbitan fatty acid esters (such as Tween 20It is poly- Ethylene oxide sorbitanPolysorbate 80It is de- Water sorbitan monopalmitateSorbitan monostearateSorbitan Alcohol tristearateGlyceryl monooleate, dehydrated sorbitol mono-fatty acid ester), polyoxyethylene Ester (such as polyoxyl 40 stearateCrodaret, polyoxyethylenated castor oil, polyoxy Methylene stearate and), sucrose fatty ester, cithrol (such as), polyoxyethylene ether (such as polyoxyethylene lauryl ether), polyvinylpyrrolidone), Diethylene glycol monolaurate, Emulphor FM, enuatrol, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, laurel Sodium sulphate,68、188, cetyl trimethylammonium bromide, hexadecyl Pyridine, benzalkonium chloride, docusate sodium etc. and/or combination thereof.
Exemplary adhesive includes but is not limited to starch (such as cornstarch and gelatinized corn starch)；Gelatin；Sugar (such as sucrose, Glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol)；Natural and synthesis natural gum (such as gum arabic, Sodium alginate, chondrus extract, panwar natural gum, ghatti gum, the mucilaginous substance of isapol skin, carboxymethyl cellulose, methyl Cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, acetic acid Cellulose, polyvinylpyrrolidone), aluminium-magnesium silicateAnd larch arabinogalactan)；Alginate； Polyethylene oxide；Polyethylene glycol；Inorganic calcium salt；Silicic acid；Polymethacrylates；Wax；Water；Alcohol etc.；With and combinations thereof.
Exemplary preservative may include but be not limited to antioxidant, chelating agent, anti-microbial preservative, antimycotic anti-corrosion Agent, alcohol preservative, acidic preservative and/or other preservatives.Exemplary antioxidants include but is not limited to alpha tocopherol, anti-bad Hematic acid, ascorbyl palmitate, Butylated Hydroxyanisole, Butylated Hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, gallic acid third Ester, sodium ascorbate, sodium hydrogensulfite, sodium pyrosulfite and/or sodium sulfite.Exemplary chelators include ethylenediamine tetra-acetic acid (EDTA), citric acid monohydrate, natrium adetate, EDTAP dipotassium ethylene diamine tetraacetate, edetic acid(EDTA), fumaric acid, malic acid, phosphoric acid, edetic acid(EDTA) Sodium, tartaric acid and/or edetate trisodium.Exemplary anti-microbial preservative includes but is not limited to benzalkonium chloride, benzethonium chloride, benzene Methanol, bronopol, cetrimonium bromide, cetylpyridinium chloride, Chlorhexidine, methaform, chloreresol, chloroxylenol, first Phenol, ethyl alcohol, glycerol, Hexetidine, miaow urea, phenol, Phenoxyethanol, benzyl carbinol, phenylmercuric nitrate, propylene glycol and/or thimerosal.Show Example property antifungal preservative includes but is not limited to butyl p-hydroxybenzoate, methyl p-hydroxybenzoate, P-hydroxybenzoic acid second Ester, propylparaben, benzoic acid, hydroxybenzoic acid, Potassium Benzoate, potassium sorbate, sodium benzoate, sodium propionate and/or Sorbic acid.Exemplary alcohols preservative includes but is not limited to ethyl alcohol, polyethylene glycol, phenol, phenolic compound, bis-phenol, methaform, hydroxyl Yl benzoic acid ester and/or benzyl carbinol.Illustrative acid preservative includes but is not limited to vitamin A, vitamin C, vitamin E, β- Carrotene, citric acid, acetic acid, dehydroactic acid, ascorbic acid, sorbic acid and/or phytic acid.Other preservatives include but is not limited to Tocopherol, tocopherol acetate, deferoxamine mesylate, cetrimonium bromide, Butylated Hydroxyanisole (BHA), Butylated Hydroxytoluene (BHT), ethylenediamine, the moon Osmanthus base sodium sulphate (SLS), Sodium Lauryl Ether Sulphate (SLES), sodium hydrogensulfite, sodium pyrosulfite, potassium sulfite, burnt sulfurous Sour potassium, GLYDANT Methyl p-hydroxybenzoate,115、II、NEOLONETM、KATHONTMAnd/or
Examples of buffers includes but is not limited to that citrate buffer solution, acetate buffer solution, phosphate-buffered are molten Liquid, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, neo-calglucon, Calcium Glucoheptonate, calcium gluconate, D- gluconic acid, glycerol Calcium phosphate, calcium lactate, propionic acid, calcium levulinate, valeric acid, calcium monohydrogen phosphate, phosphoric acid, tricalcium phosphate, calcium hydroxy phosphate, potassium acetate, Potassium chloride, potassium gluconate, potassium mixture, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, potassium phosphate mixture, sodium acetate, sodium bicarbonate, Sodium chloride, sodium citrate, sodium lactate, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium phosphate mixture, tromethamine, hydroxide Magnesium, aluminium hydroxide, alginic acid, apirogen water, isotonic saline solution, ringer's solution (Ringer ' s solution), ethyl alcohol etc. and/or A combination thereof.
Exemplary lubricants include but is not limited to magnesium stearate, calcium stearate, stearic acid, silica, talcum, malt, Compritol 888 ATO, hydrogenated vegetable oil, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, lauryl magnesium sulfate, NaLS etc. with and combinations thereof.
It is exemplary oil include but is not limited to almond, almond, avocado, Babassu coconut, bergamot, ' Heijialun ' seed, Common Borage, Needle juniper, chamomile, mustard, Caraway, palm wax, castor-oil plant, cortex cinnamomi, cocoa butter, coconut, fish liver, coffee, corn, cottonseed, Er Emu, eucalyptus, oenothera biennis, fish, linseed, citronellol, cucurbit, grape pip, fibert, hyssop, isopropyl myristate, George Simond Wood, Hawaii drupe, miscellaneous lavender, lavender, lemon, the fruit of a cubeb litsea tree, Queensland nut, high mallow, mango seed, Bai Manghua seed, mink, Nutmeg, olive, citrus, orange connect fin salmon, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin, rapeseed, rice bran, fan Repeatedly perfume, safflower, sandalwood, camellia, tower flower, sea-buckthorn, sesame, sher butter, silicone, soybean, sunflower, tea tree, Ji, Chinese toon, Cus-cus, English walnut and wheat-germ oil.Exemplary oil including but not limited to butyl stearate, Trivent OCG, capric acid is sweet Oily three esters, cyclomethicone, diethyl sebacate, dimethyl silicone polymer 360, isopropyl myristate, mineral oil, octyl 12 Alkanol, oleyl alcohol, silicone oil and/or combination thereof.
According to the judgement of makers-up, excipient such as cocoa butter and suppository wax, colorant, coating agent, sweetener, flavoring agent And/or aromatic may be present in composition.
Delivering
The disclosure covers by considering that any appropriate approach of drug delivery science may be promoted for any therapeutic agent, medicine The delivering of object, diagnosticum or the polynucleotides of imaging, primary construct or mmRNA.Delivering can be exposed or preparation.
Exposed delivering
Polynucleotides, primary construct or mmRNA of the invention exposed can be delivered to cell.As used herein, " exposed " refer to polynucleotides, primary construct or mmRNA of the delivering without the reagent for promoting transfection.For example, being delivered to the more of cell Nucleotide, primary construct or mmRNA, which can be free of, modification.Can be used as is generally known in the art will with administration method described herein Exposed polynucleotides, primary construct or mmRNA is delivered to cell.
The delivering of preparation
Usable method described herein prepares polynucleotides of the invention, primary construct or mmRNA.Preparation can contain It can modify and/or unmodified polynucleotides, primary construct or mmRNA.Preparation can further comprise but be not limited to cell-penetrating Agent, pharmaceutically acceptable carrier, delivery agents, Bio-erodable or biocompatible polymer, solvent and sustained release are passed Send reservoir.Can be used as is generally known in the art with administration method described herein by the polynucleotides of preparation, primary construct or MmRNA is delivered to cell.
Any mode that composition can be also formulated in a manner of several in this field is directly delivered to organ or tissue, The mode includes but is not limited to the fabric or biodegradable material that composition is such as coated or impregnated with by using matrix, It directly impregnates or immerses by conduit by gel, powder, ointment, creme, gel, lotion and/or drops.
Application
Can by generate treat effective result any approach apply polynucleotides of the invention, primary construct or mmRNA.These include but is not limited to enteral, stomach and intestine, Epidural cavity, oral, transdermal, Epidural cavity (epidural) (Epidural cavity (peridural)), intracerebral (enter brain), (entering the ventricles of the brain) in the ventricles of the brain, skin outer (coating is on the skin), intradermal (enter skin Itself), subcutaneous (under the skin), nose application (passing through nose), intravenous (entering vein), intra-arterial (entering artery), intramuscular Infusion (entering marrow) in (entering heart) in (enter muscle), heart, bone, intrathecal (entering canalis spinalis), (infusion or note in peritonaeum Be mapped in peritonaeum), intravesical infusion, (passing through eyes), intracavernous injection (entering penis bottom), intravaginal are applied in vitreum With, apply outside intrauterine, amnion, transdermal (diffusing through intact skin for system distribution), transmucosal (diffuse through viscous Film), be blown into and (smell suction), under sublingual, lip, bowel lavage, eye drops (drop on conjunctiva) or use auristilla.In specific embodiment In, composition can permit it and apply across the mode of blood-brain barrier, vascular barrier or other epithelial barriers.For of the invention The non-limiting approach of the application of polynucleotides, primary construct or mmRNA is described below.
Parenteral and injection application
Liquid dosage form for parenteral administration includes but is not limited to pharmaceutically acceptable emulsion, microemulsion, solution, mixes Suspension, syrup and/or elixir.Besides the active ingredients, liquid dosage form may include inertia usually used in the art Diluent, such as water or other solvents, lytic agent and emulsifier such as ethyl alcohol, isopropanol, ethyl carbonate, ethyl acetate, benzene first Alcohol, Ergol, propylene glycol, 1,3-BDO, dimethylformamide, oil are (specifically for cottonseed oil, peanut oil, corn oil, embryo Bud oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan aliphatic ester with And its mixture.Besides inert diluents, oral composition may include adjuvant such as wetting agent, emulsifier and suspending agent, sweet tea Taste agent, flavoring agent and/or aromatic.In certain embodiments for parenteral administration, composition and lytic agent are such asAlcohol, oil, modified oil, glycols, polysorbate, cyclodextrin, polymer and/or combination thereof mixing.
Injectable formulation, such as sterile injection is aqueous or oil-based suspension can use suitable dispersion according to known technology Agent, wetting agent and/or suspending agent are prepared.Sterile injectable preparation can be the acceptable diluent of non-toxic parenteral and/or molten Sterile injectable solution, suspension and/or emulsion in agent, the solution such as in 1,3-BDO.It is adoptable acceptable Medium and solvent are water, ringer's solution (U.S.P.) and isotonic sodium chlorrde solution.Sterile expressed oi be conventionally used as solvent or Suspension media.Any mild expressed oi, monoglyceride or diglyceride including synthesis can be used for this purpose.Fat Acid such as oleic acid can be used in the preparation of injectable agent.
Injectable formulation can be for example by filtration bacteria-retaining filter and/or by can dissolve or disperse before the use Bactericidal agent is added in aseptic solid composite in sterile water or other sterile injectable mediums to sterilize.
In order to extend the effect of active constituent, it is often desirable to slow down to the active constituent from subcutaneous injection or intramuscular injection Absorption.This can be realized by using the crystallization of poorly water-soluble or the liquid suspension of non-setting material.The absorption speed of drug Rate then depends on its rate of dissolution, and rate of dissolution may depend on crystal size and crystalline form.Alternatively, by by drug dissolution or It is suspended in oily medium and completes the delay absorption of the medicament forms of parenteral administration.Injectable reservoir type passes through in biology The microcapsule matrix of drug is formed in degradable polymer such as polylactide-polyglycolide to be made.Depending on drug with polymerize The property of the ratio of object and used specific polymer can control the rate of drug release.Other biodegradable polymers Example include poly- (ortho esters) and poly- (acid anhydrides).Reservoir injectable formulation is compatible with bodily tissue by being embedded in drug It is prepared in liposome or microemulsion.
Rectum and vaginal application
Composition for rectum or vaginal application is usually suppository, can by by composition be at ambient temperature Solid but under body temperature for liquid and therefore in rectum or vaginal canal fusing and discharge active component it is suitable non-stimulated Property excipient such as cocoa butter, polyethylene glycol or suppository wax mixing to prepare.
Oral administration
Liquid dosage form for oral administration includes but is not limited to pharmaceutically acceptable emulsion, microemulsion, solution, suspension Liquid, syrup and/or elixir.Besides the active ingredients, liquid dosage form may include that inertia usually used in the art is dilute Release agent, such as water or other solvents, lytic agent and emulsifier for example ethyl alcohol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, Ergol, propylene glycol, 1,3-BDO, dimethylformamide, oil are (specifically for cottonseed oil, peanut oil, corn oil, plumule Oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan aliphatic ester and Its mixture.Besides inert diluents, oral composition may include adjuvant such as wetting agent, emulsifier and suspending agent, sweet taste Agent, flavoring agent and/or aromatic.In certain embodiments for parenteral administration, composition and lytic agent are such asAlcohol, oil, modified oil, glycols, polysorbate, cyclodextrin, polymer and/or combination thereof mixing.
Solid dosage forms for oral administration includes capsule, tablet, pill, powder and granule.In such solid dosage forms In, active constituent and at least one pharmaceutically acceptable excipient of inertia such as sodium citrate or Dicalcium Phosphate and/or filler Or incremental agent (such as starch, lactose, sucrose, glucose, mannitol and silicic acid), adhesive (such as carboxymethyl cellulose, sea Alginates, gelatin, polyvinylpyrrolidone, sucrose and gum arabic), moisturizer (such as glycerol), disintegrating agent (such as fine jade Rouge, calcium carbonate, potato or tapioca, alginic acid, certain silicates and sodium carbonate), dissolution delayer (such as paraffin), inhale Receive promotor (such as quarternary ammonium salt compound), wetting agent (such as cetanol and glycerin monostearate), absorbent (such as kaolinite Soil and POLARGEL NF) and lubricant (such as talcum, calcium stearate, magnesium stearate, solid polyethylene glycol, lauryl sulfate Sodium) and the mixing of its mixture.In the case where capsule, tablet and pill, dosage form may include buffer.
Part or transdermal administration
As described herein, the composition containing polynucleotides of the invention, primary construct or mmRNA can be formulated for office Portion's application.Skin can be the ideal target area of delivering, because it is easily entered.It not only can be (potential to skin confine expression of genes Ground avoids non-specific toxicity), and the gene expression of certain layer and cell type in skin is limited.
The position of the skin expression of institute's delivering compositions will depend on the approach of delivery of nucleic acids.Usually consider that three approach will Polynucleotides, primary construct or mmRNA are delivered to skin: (i) local coating (such as part/region treatment and/or change Cosmetic coating)；(ii) intramuscular injection (such as part/region treatment and/or makeup application)；And (iii) system is passed Send (such as treating the skin disease for influencing skin and skin exterior domain).Several not Tongfangs as known in the art can be passed through Polynucleotides, primary construct or mmRNA are delivered to skin by method.Have been displayed most of local delivery methods can DNA delivery, example Such as, but not limited to, local coating non-cationic lipid body-DNA compound, cationic-liposome-DNA compound, particle mediate (particle gun) punctures the gene transfection and Viral delivery method mediated.After delivering nucleic acid, in many different skin cells Detect gene product in type, including but not limited to basic keratinocyte, sebocyte cell, skin fibroblasts with And skin macrophage.
In one embodiment, the present invention is provided to conveniently and/or effectively carry out the various of method of the invention to apply Expect (for example, wound dressing) or bandage (for example, adhesive bandage).Usual dressing or bandage may include the described herein of sufficient amount Pharmaceutical composition and/or polynucleotides, primary construct or mmRNA are to allow user to carry out the multiple treatment to subject.
In one embodiment, the present invention is provided to the polynucleotides of more than one injected delivery, primary construct Or mmRNA composition.
In one embodiment, before part and/or transdermal administration, at least one tissue regions such as skin can be by The device and/or solution effects of penetrability can be increased.In one embodiment, tissue can be worn device effect to increase The penetrability (referring to U.S. Patent Publication number 20080275468, being incorporated herein in its entirety by reference) of skin.Another In a embodiment, tissue can be acted on by ultrasonic enhancement device.Ultrasonic enhancement device may include but be not limited to announce in the U.S. Numbers 20040236268 and U.S. Patent number 6,491,657 and 6,234,990 described in device；The patent is respectively to draw Mode is integrally incorporated herein.The method of enhancing penetration into tissue is described in 20040171980 He of U.S. Publication No 20040236268 and U.S. Patent number 6,190,315 in；The patent is respectively incorporated herein in its entirety by reference.
In one embodiment, device can be used to increase tissue before the preparation for delivering modification mRNA described herein Penetrability.The penetrability of skin can be by as is generally known in the art and/or the method described in U.S. Patent number 6,190,315 It measures, the patent is incorporated herein in its entirety by reference.As a non-limiting example, modification mRNA preparation can lead to The delivery method that is described in U.S. Patent number 6,190,315 is crossed to deliver, the patent is whole by reference simultaneously Enter herein.
It, can be before tissue can be acted on by the device that can increase penetrability, period in another non-limiting example And/or matter (EMLA) creme is fused altogether with local anesthetic later and handles tissue.(the Anesth Analg (2004) such as Katz；98: 371-76；It is incorporated herein in its entirety by reference) it confirms that EMLA creme is applied in combination with low energy, ultrasonic with low energy Surface skin analgesia is seen within 5 minutes after pretreatment soon to start.
In one embodiment, can organize it is processed come before, during and/or after increasing penetrability by reinforcing agent It is applied to tissue.Reinforcing agent includes but is not limited to transport enhancer, physical enhancers and cavitation-enhanced agent.Reinforcing agent it is unrestricted Property example is described in U.S. Patent number 6, and in 190,315, the patent is incorporated herein in its entirety by reference.
In one embodiment, device can be used to increase tissue before the preparation for delivering modification mRNA described herein Penetrability, the preparation can be further containing causing the substance of immune response.In another non-limiting example, containing drawing The preparation for playing the substance of immune response can pass through the method described in U.S. Publication No 20040171980 and 20040236268 To deliver；The patent is respectively incorporated herein in its entirety by reference.
Dosage form for part and/or transdermal administration composition may include ointment, paste, creme, lotion, gel, powder, Solution, spray, inhalant and/or patch.In general, aseptically by active constituent and pharmaceutically acceptable carrier And/or any desired preservative and/or the buffer that may be needed mix.
In addition, the present invention covers the use of transdermal patch, usually there is the transdermal patch offer to be controlled compound The additional advantage for being delivered to body.Such dosage form can be for example by dissolving compound and/or being scattered in medium appropriate To prepare.Alternatively, or in addition, can be by providing rate controlling membranes and/or by dispersing polymer substrate for compound And/or carry out speed control in gel.
Preparation suitable for local application includes but is not limited to liquid and/or semi-liquid preparations such as liniment, lotion, oil-in-water And/or water-in-oil emulsion such as creme, ointment and/or paste, and/or solution and/or suspension.
Can the preparation of local application can be for example comprising about 0.1% to about 10% (w/w) active constituent, although active constituent Concentration can be as so high such as the solubility limit of active constituent in a solvent.Formulations for topical administration can further include herein One or more other ingredients of description.
Reservoir application
As described herein, in some embodiments, composition is prepared in the reservoir for extended release.In general, special Determine organ or tissue's (" target tissue ") to be targeted for applying.
In some aspects of the invention, polynucleotides, primary construct or mmRNA are spatially remained in target tissue Or neighbouring target tissue.It provides by making composition (the specially nucleic acid component of composition) generally be retained in target tissue In, it is meant that at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or the composition greater than 99.99% be retained in target tissue under conditions of make target Tissue (it contains one or more target cells) contacts to the target tissue of mammalian subject with composition and provides composition Method.Advantageously, it is protected by measuring the amount being present in into the nucleic acid in the composition of one or more target cells to determine It stays.For example, following period of time after administration, at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99% or applying greater than 99.99% It is present in into the cell with the nucleic acid to subject.For example, using the water-based composition of qiagen rnase and transfection reagent carry out to Mammalian subject intramuscular injection, and composition is determined by measuring the amount of ribonucleic acid being present in myocyte Retain.
Aspect of the invention is related to by making target tissue under conditions of being retained in composition generally in target tissue (containing one or more target cells), which is contacted with composition to the target tissue of mammalian subject, provides the method for composition. Composition contains a effective amount of polynucleotides, primary construct or mmRNA so that target polypeptides are at least one target cell It generates.Composition usually contains cell-penetrating agent, although being also covered by " exposed " nucleic acid (as not having cell-penetrating agent or other The nucleic acid of reagent) and pharmaceutically acceptable carrier.
In some cases, the amount of the protein generated by the cell in tissue desirably increases.Preferably, protein This increase generated is spatially confined to the cell in target tissue.It thus provides increasing the group of mammalian subject The method for knitting the generation of middle target protein.Composition containing polynucleotides, primary construct or mmRNA, feature are provided It is to have determined that the composition of unit quantity produces in the cell of the significant percentage in the target tissue for being contained in predetermined volume Raw target polypeptides.
In some embodiments, composition includes a variety of different polynucleotides, primary construct or mmRNA, wherein A kind of or more than one polynucleotides, primary construct or mmRNA encoding target polypeptide.Optionally, composition also contains cell Penetrating agent is to help the Intracellular delivery of composition.To include predefine volume target tissue in significant percentage it is thin The dosage that composition required for target polypeptides is generated in born of the same parents is determined (in general, close to preparatory scheduled volume or in target Organize the significant generation for not inducing target polypeptides in the tissue of distal end).After determining herein, determining dosage is introduced directly into In the tissue of mammalian subject.
In one embodiment, the present invention is provided to inject more than once or by the more of fractionated dose injected delivery Nucleotide, primary construct or mmRNA.
In one embodiment, the present invention can be used small disposable drug reservoir, patch pump or osmotic pumps and retain Near target tissue.The non-limiting example of patch pump include by(Franklin Lakes, NJ), Insulet Corporation (Bedford, MA), SteadyMed Therapeutics (San Francisco, CA), Medtronic (Minneapolis, MN) (for example, MiniMed), UniLife (York, PA), Valeritas (Bridgewater, NJ) and Those of SpringLeaf Therapeutics (Boston, MA) manufacture and/or sale.One non-limiting reality of osmotic pumps Example include by(Cupertino, CA) (for example,WithThose of) manufacture.
Lung application
Pharmaceutical composition can be adapted for dosage form preparation, packaging and/or sale that lung application is carried out by oral cavity.This Kind preparation may include dry particl, and it includes active constituent and it is in about 0.5nm to about 7nm or about 1nm to about 6nm range Interior diameter.Such composition suitably exists with dry powder form to be administered, and it includes that can instruct to push away that the dry powder, which uses, The device of the dry powder reservoir of its dispersion powders is flowed to and/or using self-propelled solvent/powder-dispensing container such as comprising molten into agent The device of the active constituent in the low boiling propellant in sealing container is solved and/or is suspended in apply.Such powder includes Grain, the wherein at least particle of 98 weight % have with the particle of diameter and at least 95% number greater than 0.5nm is less than 7nm Diameter.It is less than alternatively, the particle of at least 95 weight % has with the particle of diameter and at least 90% number greater than 1nm The diameter of 6nm.Dry powder composition be may include solid fine powder diluent such as sugar and eligibly be provided with unit dosage forms.
Low boiling propellant generally includes the liquid propellant that boiling point at atmosheric pressure is lower than 65 °F.In general, propellant 50% to 99.9% (w/w) of composition can be accounted for, and active constituent can account for 0.1% to 20% (w/w) of composition.Propellant It can further include other ingredient, such as liquid non-ionic and/or solid anionic surfactant and/or solid diluent (its granularity can be identical as the order of magnitude of the particle comprising active constituent).
As a non-limiting example, polynucleotides described herein, primary construct and/or mmRNA can prepare use In pass through the method described in U.S. Patent number 8,257,685 carry out pulmonary delivery；The patent is whole by reference It is incorporated herein.
Be formulated for pulmonary delivery pharmaceutical composition can with the drops of solution and/or suspension provide activity at Point.Such preparation can be in aqueous and/or dilution alcoholic solution and/or suspension formation system optionally sterile, comprising active constituent Standby, packaging and/or sale, and can eligibly be applied using any spraying and/or atomising device.Such preparation can be wrapped further Containing one or more other ingredients, including but not limited to flavoring agent for example saccharin sodium, volatile oil, buffer, surfactant and/or Preservative such as methyl hydroxybenzoate.The drop provided by this administration method can have within the scope of about 0.1nm to about 200nm Average diameter.
Intranasally, nose and oral administration
It can be used for the intranasal delivery of pharmaceutical composition as the preparation described herein that can be used for pulmonary delivery.Suitable for nose Another preparation of interior application is the corase meal that comprising active constituent and average grain is about 0.2 μm to 500 μm.This preparation It is applied in a manner of using snuffing, i.e., by quickly sucking from the powder container being placed near nose across nasal passage.
Suitable for nose application preparation can for example comprising as little as 0.1% (w/w) and the activity of up to 100% (w/w) at Point, and may include one or more other ingredients described herein.Pharmaceutical composition can be adapted for the preparation of oral administration Form preparation, packaging and/or sale.Such preparation can be for example in the form of using tablet made from conventional method and/or pastille In the presence of, and can be for example comprising 0.1% to 20% (w/w) active constituent, remaining includes oral dissolvable and/or degradable group Close object and optionally one or more other ingredients described herein.Alternatively, be suitable for oral administration preparation may include containing The powder of active constituent and/or the solution and/or suspension of aerosolization and/or atomization.Such powdery, aerosolization and/or aerosol The preparation of change can have average grain and/or droplet size within the scope of about 0.1nm to about 200nm in dispersion, and can Further include one or more any other ingredients described herein.
Eye application
Pharmaceutical composition can be adapted for dosage form preparation, packaging and/or the sale of eye application.Such preparation can be such as Exist in the form of eye drops, 0.1%/1.0% (w/w) including such as active constituent in aqueous or oil-based liquid excipient Solution and/or suspension.Such dropping liquid can further include buffer, salt and/or other described herein one or more What other ingredient.The preparation that useful other eyes can be applied includes with microcrystalline form and/or to include existing for Liposomal formulation Those of active constituent.Auristilla and/or eye drops are covered within the scope of the invention.Plural layers device can be prepared as containing For delivery to eyes and/or the pharmaceutical composition of surrounding tissue.
Payload application: detectable agent and therapeutic agent
Polynucleotides, primary construct or mmRNA described herein can be used in many different scenes, wherein it is desirable to will Substance (" payload ") is delivered to biological target, such as delivering for detecting the detectable substance or delivering therapeutic agent of target. Detection method may include but be not limited to be imaged in vitro and in-vivo imaging method, such as immunohistochemistry, biodiversity resources (BLI), magnetic resonance imaging (MRI), positron emission tomography (PET), electron microscopy, X-ray computed tomography are taken the photograph Shadow, optical coherence tomography, absorbs imaging, thermal imaging, fluorescent reflection imaging, fluorescence microscopy, fluorescent molecule at Raman image Tomographic imaging, Magnetic resonance imaging, X-ray imaging, ultrasonic imaging, photoacoustic imaging, experimental determination are needing label/dye Color/imaging is in any case.
Polynucleotides, primary construct or mmRNA can design connector and payload including being in any useful orientation.Example Such as, the connector held there are two having can be used to for one end to be attached to payload and the other end be attached to nucleobase, such as de- On the position C-7 or C-8 of nitrogen adenosine or denitrogenation guanosine or to the position N-3 or C-5 of cytimidine or uracil.Multicore of the invention Thuja acid may include more than one payload (for example, marker and transcription inhibitor) and a kind of cracking joint.At one In embodiment, modified nucleoside acid is triphosphoric acid 7- denitrogenation-adenosine of modification, and wherein it is de- to be attached to 7- for one end of cracking joint The position nitrogen-adenosine C7, the other end of connector are attached to inhibitor (for example, the position C5 for the nucleobase being attached on cytidine), And marker (for example, Cy5) is attached to center (Fig. 5 and the 9th He see, for example, U.S. Patent number 7,994,304 of connector Nothing in 10 column emits the compound 1 of A*pCp C5 Parg, and the patent is hereby incorporated herein by).In three will modified When phosphoric acid 7- denitrogenation-adenosine is incorporated to code area, the obtained polynucleotides with cracking joint be attached to marker and Inhibitor (for example, polymerase inhibitors).In connector cracking (for example, reduction has two sulphur of cleavable under the reducing conditions The connector of key section), release mark object and inhibitor.This document describes other connector and payload (for example, therapeutic agent, Detectable marker and cell-penetrating payload).
Scheme 12 below depicts illustrative modified nucleoside acid, and wherein nucleobase adenine is in 7- denitrogenation adenine Connector is attached on C-7 carbon.In addition, scheme 12 is depicted with the connector and payload (example on the 3 ' ends for being incorporated into mRNA Such as detectable agent) modified nucleoside acid.Disulfide bond cracking and 1, the 2- addition of the mercapto on alkynes propyl ester release detectable agent. Remaining structure (being for example portrayed as pApC5Parg in scheme 12) is inhibitor.The principle of structure for modified nucleoside acid is The inhibitor tied spatially interferes polymerase to be incorporated to dibasic ability.It is therefore essential that tethers is to be enough to influence this The length and inhibitor of kind function are in inhibition or prevention second and succeeding nucleotide enters in the polynucleotide chain of growth In stereochemical orientation.
Scheme 12
For example, polynucleotides described herein, primary construct or mmRNA can be used for reprograming induced multipotency and do In cell (iPS cell), its cell that can directly track transfection compared with the total cell of cluster.It in another example, can be through Cross connector be attached to polynucleotides, primary construct or mmRNA and can the drug of fluorescent marker can be used in vivo for example thin Tracking drug intracellular.Other examples include but is not limited to be delivered in cell in reversible pharmacological using polynucleotides, primary building Body or mmRNA.
Polynucleotides, primary construct or mmRNA described herein can be used for for example detectable agent of payload or treatment Agent is in the intracellular targeting of specific cells device.Illustrative intracellular target may include but be not limited to process for advanced mRNA Nuclear location or be connected to the nuclear localization sequence (NLS) of the mRNA containing inhibitor.
In addition, polynucleotides described herein, primary construct or mmRNA can be used to for therapeutic agent being delivered to such as activity The intracorporal cell or tissue of object.For example, polynucleotides described herein, primary construct or mmRNA can be used to deliver height pole The chemotherapeutant of property is to kill cancer cell.It can by polynucleotides, primary construct or mmRNA that connector is attached to therapeutic agent Promote to allow therapeutic agent to advance in cell to reach the film of intracellular target and penetrate.
In an example, connector be attached on 2 '-positions of ribose ring and/or polynucleotides, primary construct or It (see, for example, international publication number WO2012030683, is incorporated hereby on the 3 ' of mmRNA and/or 5 ' positions Herein).Connector can be disclosed herein, it is any as is generally known in the art and/or disclosed in international publication number WO2012030683 Connector, the patent are incorporated herein in its entirety by reference.
In another example, polynucleotides, primary construct or mmRNA can be attached to multicore glycosides by cracking joint Acid, primary construct or mmRNA HIV suppression peptide (VIP).Cracking joint can be such that VIP and dyestuff is discharged into cell.Another In one example, polynucleotides, primary construct or mmRNA can be attached to ADP- ribosyl compound (ADP- by connector Ribosylate), it is responsible for the effect of some bacteriotoxins such as cholera toxin, diphtheria toxin and pertussis toxin.These toxin Albumen is the ADP- ribosyltransferase for changing the target protein in people's cell.For example, Cholera Toxin A DP- ribosyl compound G-protein By causing a large amount of fluid secretions for the diarrhea for leading to life-threatening to change people's cell from small intestine internal layer.
In some embodiments, payload can be therapeutic agent such as cytotoxin, isotopic ion, chemotherapeutant Or other therapeutic agents.Cytotoxin or cytotoxic agent include can any medicament harmful to cell.Example includes but is not limited to Taxol, Gramicidin D, ethidium bromide, ipecine, mitomycin, Etoposide (etoposide), replaces cytochalasin B Buddhist nun moors glycosides (teniposide), vincristine, vincaleukoblastinum, colchicin, adriamycin, daunorubicin, chinizarin (dihydroxyanthracinedione), mitoxantrone, mithramycin, actinomycin D, 1- dehydrogenation testosterone, glucocorticoid, Procaine (procaine), totokaine (tetracaine), lidocaine (lidocaine), Propranolol (propranolol), puromycin, maytansinoid (maytansinoid) such as Ansamitocin Po (maytansinol) (referring to U.S. Patent number 5,208,020, be integrally incorporated herein), draw miramycin (rachelmycin) (CC-1065, referring to beauty State's patent No. 5,475,092,5,585,499 and 5,846,545, all patents are hereby incorporated herein by) with And the like or homologue.Isotopic ion include but is not limited to iodine (for example, I125 or iodine 131), strontium 89, phosphorus, palladium, caesium, Iridium, phosphate, cobalt, Y90, samarium 153 and praseodymium.Other therapeutic agents include but is not limited to antimetabolite (for example, methotrexate (MTX), 6- Purinethol, 6-thioguanine, cytarabine, 5 FU 5 fluorouracil, dacarbazine), alkylating agent is (for example, mustargen, plug replace Group, Chlorambucil, draw miramycin (CC-1065), melphalan (melphalan), Carmustine (carmustine) (BSNU), Lomustine (lomustine) (CCNU), cyclophosphamide, busulfan (busulfan), dibromannitol, streptozotocin, mitogen Mycin C and cis- dichlorodiamine platinum (II) (DDP), cis-platinum), anthracycline (anthracyclines) is (for example, daunorubicin (being formerly referred to as daunomycin) and adriamycin), antibiotic is (for example, dactinomycin D (dactinomycin) (is formerly referred to as actinomyces Element), bleomycin, mithramycin and Anthramycin (anthramycin) (AMC)) and antimitotic agent (for example, Changchun New alkali, vincaleukoblastinum, taxol and maytansinoid).
In some embodiments, payload can be detectable agent, such as various small organic molecules, inorganic compound, Nano particle, enzyme or zymolyte, fluorescent material, luminescent material (for example, luminol (luminal)), bioluminescent material (example Such as, luciferase, fluorescein and aequorin), chemiluminescent material, radioactive material (for example,18F、67Ga、81mKr、82Rb 、111In、123I、133Xe、201Tl、125I、35S、14C、3H or99mTc is (for example, such as pertechnetate (pertechnetate (VII), TcO4 -)) And contrast agent is (for example, golden (for example, gold nano grain), gadolinium (for example, Gd of chelating), iron oxide are (for example, superparamagnetism oxygen Change iron (SPIO), monocrystaline iron oxide nanoparticles nano particle (MION) and Ultrasmall superparamagnetic iron oxide (USPIO)), magnesium chelate (example Such as, Mn-DPDP), barium sulfate, iodide contrast medium (Iohexol), microvesicle or perfluocarbon).Such detectable label of optics Object includes for example being not limited to: 4- 4 '-isothiocyanato of acetylaminohydroxyphenylarsonic acid talan -2,2 ' disulfonic acid；Acridine and derivative (for example, acridine and acridine isocyanates)；5- (2 '-amino-ethyl) amino naphthalenes -1- sulfonic acid (EDANS)；4- amino-N- [3- second Alkenylsufonyl) phenyl] naphthalimide -3,5 disulfonate；N- (4- anilino- -1- naphthalene) maleimide；O-amino benzoyl Amide；BODIPY；It is bright orange；Cumarin and derivative are (for example, cumarin, 7- amino -4- methylcoumarin (AMC, cumarin 120) and 7- amino -4- trifluoromethyl cumarin (coumarin 1 51))；Cyanine dye；Phloxin；4 ', 6- diamidino -2- benzene Base indoles (DAPI)；5 ' 5 "-dibromo pyrogallol-sulfonephthalein (bromopyrogallol red)；7- diethylamino -3- (4 '-isothiocyanic acids Root closes phenyl) -4- methylcoumarin；Diethylene-triamine pentaacetic acid ester；4,4 '-diisothiocyanic acid roots close dihydro-talan- 2,2 '-disulfonic acid；4,4 '-diisothiocyanic acid roots close 2,2 '-disulfonic acid of talan-；5- [dimethylamino]-naphthalene -1- sulphonyl Chlorine (DNS, dansyl chloride)；4- dimethyl aminophenylazo phenyl -4 '-isothiocyanates (DABITC)；Eosin and derivative (example Such as, eosin and eosin isothiocyanates)；Algae red and derivative (for example, algae red B and algae red isothiocyanates)；Second ingot；Fluorescein With derivative (for example, 5-carboxyfluorescein (FAM), 5- (4,6- dichlorotriazine -2- base) Aminofluorescein (DTAF), 2 ', 7 ' - The chloro- 6- Fluoresceincarboxylic acid of dimethoxy-4 ' ' 5 '-two, fluorescein, fluorescein isothiocynate, X- rhodamine -5- (and -6)-different sulphur Cyanate (QFITC or XRITC) and fluorescamine)；2- [2- [3- [[1,3- dihydro -1,1- dimethyl -3- (3- sulfopropyl) - 2H- benzo [e] indoles -2- subunit] ethylidene] -2- [4- (ethoxy carbonyl) -1- piperazinyl] -1- cyclopentene -1- base] ethylene Base] -1,1- dimethyl -3- (3- sulfopropyl) -1H- benzo [e] indoles hydroxide, inner salt, with n, n- diethyl ethanamine is compound (1:1)(IR144)；The chloro- 2- of 5- [2- [3- [(chloro- 3- ethyl -2 (3H)-benzothiazole-subunit of 5-) ethylidene] -2- (diphenyl Amino) the amyl- 1- yl of -1- ring] vinyl] -3- ethyl-benzothiazole perchlorate (IR140)；Malachite green isothiocyanate；4- Methyl umbelliferone o-cresolphthalein；Nitrotyrosine；Pararosaniline；It is phenol red；B- phycoerythrin；Phthalic aldehyde；Pyrene and derivative (for example, pyrene, pyrene butyrate and succinimido 1- pyrene)；Butyrate quantum dot；Reactive Red 4 (CIBACRONTMAzarin 3B- A)；Rhodamine and derivative (for example, 6- carboxy-X-rhodamine (ROX), 6- carboxyrhodamine (R6G), Sulforhodamine B, Sulfonic acid chloride rhodamine (Rhod), rhodamine B, Rhodamine 123, rhodamine X isothiocyanates, Sulforhodamine B, Sulforhodamine 101, sulfonyl chloride derivatives (texas Red), the N, N, N of Sulforhodamine 101 ', N ' tetramethyl -6- carboxyrhodamine (TAMRA) tetramethylrhodamine and tetramethylrhodamine isothiocyanates (TRITC))；Riboflavin；Rosolic acid；Terbium chelate Derivative；Cyanine -3 (Cy3)；Cyanine -5 (Cy5)；Cyanine -5.5 (Cy5.5), cyanine -7 (Cy7)；IRD 700；IRD 800； Alexa 647；La Jolta Blue；Phthalocyanine and naphthalene phthalocyanine.
In some embodiments, detectable agent can be become in activation detectable non-detectable precursor (for example, The tetrazine fluorogen construct to fluoresce is (for example, tetrazine-BODIPY FL, tetrazine-Ao Legang green 488 or tetrazine-BODIPY TMR-X) or enzyme can activate the agent that fluoresces (for example,(VisEn Medical))).Enzyme label can be used The external test of composition include but is not limited to enzyme linked immunosorbent assay (ELISA) (ELISA), immune precipitation determination, immunofluorescence technique, Enzyme immunoassay (EIA) (EIA), radioimmunoassay (RIA) and western blot analysis.
Combination
Polynucleotides, primary construct or mmRNA can with one or more other therapeutic agents, prophylactic, diagnosticum or at As agent is applied in combination." combination " is not intended to imply that medicament must be administered simultaneously and/or be formulated for be delivered together, although these Delivering method is within the scope of this disclosure.Composition can one or more other desired therapeutic agents or medical procedures simultaneously, Before or after apply.In general, every kind of medicament will be under doses and/or according to the time top application determined for the medicament With.In some embodiments, the disclosure covers and can improve its bioavailability, reduction and/or change its metabolism, inhibit it Drain and/or change the pharmaceutical agent combinations delivering pharmaceutical composition that it is distributed in body, prevention composition, diagnosis composition or at As composition.As a non-limiting example, nucleic acid or mmRNA can with for treating cancer or control excessive proliferated cell Drug agent is applied in combination.In U.S. Patent number 7,964,571 (it is incorporated herein in its entirety by reference), describes and be used for The combination treatment of primary or metastatic solid tumors is treated, the DNA plasmid and lipid for including encoding Interleukin -12 are used At least one anticancer agent of the pharmaceutical composition of polymer and also delivering or chemotherapeutant.In addition, coding antiproliferative molecule Nucleic acid and mmRNA of the invention can be present in pharmaceutical composition (see, for example, U.S. Publication No together with lipid polymer 20110218231, it is incorporated herein in its entirety by reference, it is desirable that DNA plasmid and rouge comprising encoding antiproliferative molecule The pharmaceutical composition of matter polymer), described pharmaceutical composition can be applied together at least one chemotherapeutant or anticancer agent.
It will be further understood that the therapeutically active agent being applied in combination, prophylactic activity agent, diagnosis activating agent or imaging active Agent can be applied together or with different components separate administration with single composition.Usually, it is contemplated that the medicament being applied in combination is not surpassing It crosses under the level that they are used alone and uses.In some embodiments, the level being applied in combination will be less than that being used alone It is a little horizontal.In one embodiment, the combination, respectively application or one can be applied according to divided doses scheme described herein Play application.
Administration
The present invention provides include the egg that modification mRNA and its coding according to the present invention are applied to subject in need The method of white matter or compound.Can be used effective for prevent, treat, diagnose or be imaged disease, illness and/or symptom (for example, Disease relevant to working memory deficit, illness and/or symptom) any amount and any administration method to subject apply core Acid, protein or compound or its pharmaceutical composition, image forming composition, diagnosis composition or prevention composition.Required essence Really amount may depend on type, age and the general status of subject, the seriousness of disease, concrete composition, its method of application, its Mode of action etc. is different between subject and subject.Composition according to the present invention is typically formulated as convenient for application and dosage Uniform dosage unit form.It will be understood, however, that total every consumption per day of composition of the invention can closed by attending physician It manages in medical judgment scope and determines.The dosage level that the specific treatment of any specific patient is effective, prevention is effective or is suitably imaged Various factors will be depended on, the factor includes the seriousness of treated illness and illness；Used particular compound Activity；Used concrete composition；Patient age, weight, general health, gender and diet；Used materialization Close administration time, administration method and the discharge rate of object；Duration for the treatment of；It is combined with used particular compound or simultaneously The drug used；And well known similar factor in medical domain.
In certain embodiments, composition according to the present invention can deliver about 0.0001mg/kg to about daily enough 100mg/kg, about 0.001mg/kg are to about 0.05mg/kg, about 0.005mg/kg to about 0.05mg/kg, about 0.001mg/kg to about 0.005mg/kg, about 0.05mg/kg are to about 0.5mg/kg, about 0.01mg/kg to about 50mg/kg, about 0.1mg/kg to about 40mg/ Kg, about 0.5mg/kg are to about 30mg/kg, about 0.01mg/kg to about 10mg/kg, about 0.1mg/kg to about 10mg/kg or about 1mg/ It is applied once or several times a day under kg to the about dosage level of 25mg/kg subject's weight, to obtain desired treatment, examine Disconnected, prevention or imaging effect.It can three times a day, twice a day, once a day, every other day, every three days, weekly, every two weeks, often Three weeks or the desired dosage of every four weeks delivering.In certain embodiments, can be used multiple applications (for example, twice, three times, Four times, five times, six times, seven times, eight times, nine times, ten times, it is ten primary, 12 times, ten three times, 14 times or more time applications) pass Send desired dosage.When using multiple applications, divided doses scheme can be used, as described herein those.
In accordance with the present invention it has been found that generating more Gao Shui in mammalian subjects with divide dosage schedule application mmRNA Flat protein.As used herein, " divided dose " is to be divided into single unit dose or total daily dosage two or more times Dosage, such as the application two or more times of single unit dose.As used herein, " single unit dose " is with primary agent The dosage of any therapeutic agent of amount/primary/single approach/single contact point application, that is, single administration event.As made herein With " total daily dosage " is to give or defined amount in 24 hour period.Total daily dosage can be used as single unit dose Application.In one embodiment, mmRNA of the invention is applied to subject with divided dose.MmRNA can only be prepared in buffer In or prepare in preparation described herein.
Dosage form
Pharmaceutical composition described herein can be configured to dosage form described herein, it is such as part, intranasal, endotracheal or Injectable (for example, it is intravenous, intraocular, vitreum is interior, in intramuscular, heart, in peritonaeum, subcutaneously).
Liquid dosage form
Liquid dosage form for parenteral administration includes but is not limited to pharmaceutically acceptable emulsion, microemulsion, solution, mixes Suspension, syrup and elixir.Besides the active ingredients, liquid dosage form may include that inertia usually used in the art is dilute Release agent, including but not limited to water or other solvents, lytic agent and emulsifier for example ethyl alcohol, isopropanol, ethyl carbonate, ethyl acetate, Benzyl alcohol, Ergol, propylene glycol, 1,3-BDO, dimethylformamide, oil are (specifically for cottonseed oil, peanut oil, corn Oil, embryo oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and sorbitan fatty acid Ester and its mixture.In certain embodiments for parenteral administration, composition can be with lytic agent such asAlcohol, oil, modified oil, glycols, polysorbate, cyclodextrin, polymer and/or combination thereof mixing.
Injectable agent
Injectable formulation, such as sterile injection is aqueous or oil-based suspension can be prepared according to known technology and may include Suitable dispersing agent, wetting agent and/or suspending agent.Sterile injectable preparation can be the acceptable diluent of non-toxic parenteral And/or sterile injectable solution, suspension and/or emulsion in solvent, such as the solution in 1,3-BDO.It is adoptable Acceptable medium and solvent include but is not limited to water, ringer's solution (U.S.P.) and isotonic sodium chlorrde solution.It is sterile not Volatile oil is conventionally used as solvent or suspension media.Any mild expressed oi can be used for this purpose, including the sweet of synthesis Oily monoesters or diglyceride.Fatty acid such as oleic acid can be used in the preparation of injectable agent.
Injectable formulation can be for example by filtration bacteria-retaining filter and/or by can dissolve or disperse before the use Bactericidal agent is added in aseptic solid composite in sterile water or other sterile injectable mediums to sterilize.
In order to extend the effect of active constituent, it may be desirable to slow down to the active constituent from subcutaneous injection or intramuscular injection It absorbs.This can be realized by using the crystallization of poorly water-soluble or the liquid suspension of non-setting material.Polynucleotides, primary structure The absorption rate for building body or mmRNA then depends on its rate of dissolution, and rate of dissolution may depend on crystal size and crystalline form.Or Person, the delay of the polynucleotides of parenteral administration, primary construct or mmRNA absorbs can be by constructing polynucleotides, primary Body or mmRNA dissolution are suspended in oily media to complete.Injectable reservoir type in the poly- second of such as polylactide-by handing over The microcapsule matrix of polynucleotides, primary construct or mmRNA is formed in the biodegradable polymer of ester to be made.It depends on The property of the ratio and used specific polymer of polynucleotides, primary construct or mmRNA and polymer, can control multicore The rate of thuja acid, primary construct or mmRNA release.The example of other biodegradable polymers is including but not limited to poly- (former Acid esters) and it is poly- (acid anhydrides).Reservoir injectable formulation can be by by polynucleotides, primary construct or mmRNA is embedded in and body It is prepared in the liposome or microemulsion of tissue compatible.
Lung
It can also be used in the intranasal delivery of pharmaceutical composition as the preparation described herein that can be used for pulmonary delivery.It is suitable for Another preparation of intranasal administration can be the corase meal that comprising active constituent and average grain is about 0.2 μm to 500 μm.It is this Preparation can be applied by the way of snuffing, i.e., by quickly sucking from the powder container being placed near nose across nasal passage.
Suitable for nose application preparation can for example comprising as little as 0.1% (w/w) and the activity of up to 100% (w/w) at Point, and may include one or more other ingredients described herein.Pharmaceutical composition can be adapted for the preparation of oral administration Form preparation, packaging and/or sale.Such preparation can be for example in the form of using tablet made from conventional method and/or pastille In the presence of, and can be for example containing about 0.1% to 20% (w/w) active constituent, wherein remaining may include oral dissolvable and/or can The composition of degradation and optionally one or more other ingredients described herein.Alternatively, the preparation for being suitable for oral administration can Solution and/or suspension comprising the powder containing active constituent and/or aerosolization and/or atomization.Such powdery, aerosolization And/or the preparation of aerosolization can have average grain within the scope of about 0.1nm to about 200nm and/or drop big in dispersion It is small, and can further include one or more any other ingredients described herein.
General Consideration in terms of preparing and/or manufacturing medicament is found in such as Remington:The Science And Practice of Pharmacy the 21st edition, Lippincott Williams & Wilkins, 2005 (it is with reference Mode is integrally incorporated herein) in.
Coating or shell
Tablet, dragee, glue can be prepared with well known enteric coating and other coatings in coating and shell such as pharmaceutical-formulating art The solid dosage forms of capsule, pill and granule.They optionally comprising opacifier and can have them only or preferably in intestines The optionally composition of discharge active component in a delayed fashion in certain a part in road.The example packet of workable embedding composition Include polymeric material and wax.Excipient and high-molecular-weight poly second such as lactose or toffee can be used in the solid composite of similar type Glycol etc. is come the filler that is used as in soft and hard filling gelatine capsule.
The characteristic of pharmaceutical composition
Pharmaceutical composition described herein can be by one or more of bioavailability, therapeutic window and/or volume of distribution To characterize.
Bioavailability
When being configured to composition with delivery agents as described herein, polynucleotides, primary construct or mmRNA can be shown The increase of bioavailability compared with the composition for lacking delivery agents as described herein.As used herein, term " biological utilisation Rate " is directed to the system availability of the polynucleotides of the specified rate of mammal application, primary construct or mmRNA.It can pass through Measurement compound is applied to the area under the curve (AUC) that the compound of form is had not been changed after mammal or maximum serum or Plasma concentration (Cmax) evaluate bioavailability.AUC is the serum or plasma concentration pair along the compound of ordinate (Y-axis) Along the determination for the area under the curve that the time of abscissa (X-axis) maps.In general, can be used known to those of ordinary skill in the art And such as G.S.Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, Volume 72, method described in Marcel Dekker, New York, Inc., 1996 calculates the AUC of particular compound, described Bibliography is incorporated herein in its entirety by reference.
CmaxValue is the chemical combination reached in the serum or blood plasma of mammal after compound to be applied to mammal The maximum concentration of object.Method known to persons of ordinary skill in the art can be used to measure the C of particular compoundmaxValue.As herein Used phrase " increasing bioavailability " or " improving pharmacokinetics " mean total with delivery agents as described herein It is used as AUC, C in mammals when with applicationmaxOr CminFirst polynucleotides of measurement, primary construct or mmRNA are Utilization rate ratio of uniting is bigger when there is no this co-administration.In some embodiments, polynucleotides, primary construct or The bioavailability of mmRNA can increase at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, At least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, At least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, At least about 95% or about 100%.
Therapeutic window
When being configured to composition with delivery agents as described herein, polynucleotides, primary construct or mmRNA can be shown Polynucleotides, primary construct or the therapeutic window of mmRNA composition applied and being applied for shortage delivery agents as described herein The therapeutic window of polynucleotides, primary construct or mmRNA composition, which is compared, increased." treatment as used herein Window " refers to the horizontal extent of the therapeutic active substance at the range or site of action of plasma concentration, treats and makees with high initiation A possibility that using.In some embodiments, polynucleotides, primary construct when being co-administered with delivery agents as described herein Or the therapeutic window of mmRNA can increase at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, extremely Few about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, extremely Few about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, extremely Few about 95% or about 100%.
Volume of distribution
When being configured to composition with delivery agents as described herein, polynucleotides, primary construct or mmRNA can be shown Relative to the composition volume of distribution (V for lacking delivery agents as described hereindist) improve to some extent, such as reduce or target.Distribution Volume (Vdist) connects the amount of the intracorporal drug of body and the concentration of the drug in blood or blood plasma.As used herein, Term " volume of distribution " refer to under the same concentrations in blood or blood plasma in body accommodate drug total amount required for stream Body volume: Vdist is equal to the drug concentration in the intracorporal medication amount/blood of body or blood plasma.For example, for 10mg dosage and The plasma concentration of 10mg/L, volume of distribution will be 1 liter.Volume of distribution reflects that drug is present in the degree in extravascular tissue. Big volume of distribution reflects the tendency of the compound combination structural constituent compared with plasma protein combines.In clinical setting, Vdist may be used to determine the load doses for realizing Css.In some embodiments, total with delivery agents as described herein With polynucleotides, primary construct or mmRNA when application volume of distribution can be reduced by least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%.
Biological effect
In one embodiment, the biological effect for being delivered to the modification mRNA of animal can be by the albumen in analyzing animal Matter is expressed to classify.Protein expression can be by analyzing the biology collected from the mammal for applying modification mRNA of the invention Sample determines.In one embodiment, the expression of the modification mRNA coding by being administered to mammal of at least 50pg/ml Protein can be preferred.For example, the egg of the 50-200pg/ml by the protein for the modification mRNA coding for being delivered to mammal White matter expresses the protein that can regard therapeutically effective amount in the mammalian body as.
Modification of nucleic acids is detected by mass spectrography
Mass spectrography (MS) is that structure and molecular mass about molecule/concentration letter can be provided after molecule is converted into ion The analytical technology of breath.First ionized molecule with obtain positive charge or negative electrical charge and then they travel across mass analyzer with Different zones according to its matter/lotus (m/z) than reaching detector.
Carry out mass spectrography using mass spectrograph, the mass spectrograph include for ionize be classified isolated sample and generation for into The ion source of the charged molecule of one step analysis.Such as the ionization of sample can pass through electrospray ionisation (ESI), atmospheric pressure chemical electricity From (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid phase secondary ionization (LSIMS), ground substance assistant laser Desorption/ionization (MALDI), field-ionization, field desorption, thermojet/plasma jet ionization and particle beams ionization are to carry out. Skilled artisan will appreciate that can based on analyte to be measured, sample type, detector type, positive negative mode selection etc. really Determine the selection of ionization method.
After sample is ionized, resulting positively charged or negatively charged ion can be analyzed to determine mass-to-charge ratio (that is, m/z).For determining that the suitable analyzer of mass-to-charge ratio includes quadrupole analyzer, ion trap analyzer and flight time point Parser.Several detection pattern detection ions can be used.For example, detectable (that is, using selected ion monitoring mode (SIM)) inspection The ion of selection is surveyed, or optionally, scan pattern such as multiple reaction monitoring (MRM) or Selective reaction monitoring (SRM) can be used To detect ion.
It has been displayed and is monitored with liquid chromatography-multiple reaction of the peptide reference substance dilution of stable isotope labelling coupling It (LC-MS/MRM) is the effective ways for protein verifying (for example, Keshishian etc., Mol Cell Proteomics 2009 8:2339-2349；2009 55:1108-1117 of Kuhn etc., Clin Chem；Lopez etc., Clin Chem 2010 56:281-290；It is respectively incorporated herein in its entirety by reference).Unlike being frequently used in biomarker discovery research The mass spectrography not targeted, the MS method of targeting be collect Instrumental whole analysis ability it is tens of to several in complex mixture The MS mode based on peptide sequence on the peptide of hundred kinds of selections.It is limited only to by will test and be classified separation from target protein Those of matter peptide, sensitivity and reproducibility significantly improve compared with discovery mode MS method.Multiple reaction based on mass spectrography This method for monitoring (MRM) quantitative protein can be drawn by quick, targeting, the multiplication protein expression profile of clinical sample The discovery of biomarker is influenced significantly and is quantified.
In one embodiment, it can be analyzed by the method for MRM-MS containing by least one modification of the invention The biological sample of at least one protein of mRNA coding.Biological sample quantitatively can further comprise but be not limited to as internal standard The peptide or protein matter of the isotope labelling of object.
According to the present invention, biological sample is once obtain from subject can be by enzymic digestion.As used herein, term " digestion " means to be fragmented into shorter peptide.As used herein, phrase " processing sample is with digesting protein " means in order to broken The mode of protein in bad sample manipulates sample.These enzymes include but is not limited to trypsase, endo protease Glu-C and pancreas Chrymotrypsin.In one embodiment, enzyme can be used to digest to contain and be compiled by least one modification mRNA of the invention The biological sample of at least one protein of code.
In one embodiment, it can be used electrospray ionisation analysis containing the egg by modification mRNA coding of the invention Protein in the biological sample of white matter.Electrospray ionisation (ESI) mass spectrography (ESIMS) helps ion passing through using electric energy Mass spectrography from solution is transferred to gas phase before being analyzed.It can be used method analysis sample as known in the art (for example, Ho Deng Clin Biochem Rev.2003 24 (1): 3-12；It is incorporated herein in its entirety by reference).Dispersion electricity can be passed through The fine spray of lotus drop evaporates solvent and projects ion to generate the thin of altitudinal belt charge drop from charged droplets Mist will be transferred in gas phase comprising ionic species in the solution.At least one, at least two, at least three or at least can be used 4 mass analyzers such as, but not limited to, four-electrode quality analyzer analyzes the mist of altitudinal belt charge drop.In addition, mass spectrometry method It may include purification step.As a non-limiting example, settable first quadrupole is to select single m/z ratio, it can be filtered in this way Fall other molecular ions with different m/z ratios, this can eliminate the Sample Purification on Single process of complicated and time consumption before MS analysis.
In one embodiment, analysis can contain by of the invention in series connection ESIMS system (for example, MS/MS) Modify the protein in the biological sample of the protein of mRNA coding.As non-limiting examples, can be used Product scan (or son Ion scan), precursor scan (precursor scans), neutral loss or multiple reaction monitoring come analysis of the droplet.
In one embodiment, substance assistant laser desorpted/ionization (MALDI) mass spectrography (MALDIMS) point can be used Biological sample of the analysis containing the protein by modification mRNA coding of the invention.MALDI provides macromolecular and small molecule such as egg The non-destructive of white matter vaporizes and ionization.In MALDI analysis, make the matrix compounds of analyte Yu a large amount of molar excess first Cocrystallization, the matrix compounds may also include but are not limited to absorb the weak organic acid of ultraviolet light.For the matrix in MALDI Non-limiting example be alpha-cyano -4- hydroxycinnamic acid, 3,5- dimethoxy-4 '-hydroxycinnamic acid and 2,5- dihydroxy benzenes first Acid.The laser emission of analyte-substrate mixture can cause the vaporization of matrix and analyte.The desorption of induced with laser provides complete The macroion yield of analyte and allow accurately measure compound.Method analysis sample as known in the art can be used (for example, Lewis, Wei and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894； It is incorporated herein in its entirety by reference).As non-limiting examples, it can be wrapped for the mass analyzer in MALDI analysis Include linear flight time (TOF), TOF reflector or Fourier transformation mass analyzer.
In one embodiment, dry droplet method can be used to form analyte-substrate mixture.By biological sample with Matrix is mixed to generate the saturation matrix solution that matrix and sample ratio are about 5000: 1.Then allow to be saturated matrix solution Aliquot (about 0.5-2.0uL) is dry to form analyte-substrate mixture.
In one embodiment, thin layer method can be used to form analyte-substrate mixture.It is initially formed the even film of matrix And then sample application, and analyte-substrate mixture can be formed by matrix absorption.
In one embodiment, thick bed method can be used to form analyte-substrate mixture.With NC Nitroncellulose matrix Additive forms the even film of matrix.Once obtaining uniform NC Nitroncellulose hypothallus, just sample application and it is absorbed into matrix To form analyte-substrate mixture.
In one embodiment, sandwich method can be used to form analyte-substrate mixture.Such as in thin layer method that The thin layer of sample host crystal then adds the drop of aqueous trifluoroacetic acid, sample and matrix.Then it is absorbed into sample in matrix To form analyte-substrate mixture.
V. polynucleotides of the invention, primary construct and mmRNA purposes
In preferred embodiments, polynucleotides of the invention, primary construct and mmRNA are designed to avoid or return Keep away harmful biological response such as immune response and/or degradation pathway, overcome expression threshold value and/or improve protein generate ability, The egg of improved expression rate or translation efficiency, improved drug or protein half life and/or protein concentration, optimization is provided White matter positioning, improve it is one of following or a variety of: stability and/or removing, acceptor absorbance and/or dynamics in tissue, The cell of composition enters, with the shape of being connected of machine translator, secernment efficiency (where applicable), the accessibility recycled and/or cell State, function and/or active adjusting.
Therapeutic agent
Therapeutic agent
Polynucleotides of the invention, primary construct or mmRNA such as modification of nucleic acids and modification RNA and from described herein The protein of the polynucleotides, primary construct or mmRNA translation can be used as therapeutic agent or prophylactic.They can be used for medicine In.For example, polynucleotides described herein, primary construct or mmRNA can be administered to subject, wherein polynucleotides, primary Construct or mmRNA are translated in vivo to generate therapeutic or preventative polypeptide in subject's body.It provides for diagnosing, controlling Treat or prevent composition, method, kit and the reagent of people and other diseases or symptom in the mammalian body.Work of the invention Property therapeutic agent include polynucleotides, primary construct or mmRNA, containing polynucleotides, primary construct or mmRNA cell or The polypeptide translated from polynucleotides, primary construct or mmRNA.
In certain embodiments, there is provided herein contain one or more polynucleotides, primary construct or mmRNA Agent is treated in combination, the polynucleotides, primary construct or mmRNA contain the immunity of coding promotion mammalian subject One or more protein, together with induction of antibodies dependent cells toxicity protein can translated region.For example, there is provided herein contain There is controlling for one or more nucleic acid of coding Herceptin (trastuzumab) and G-CSF (G-CSF) Treat agent.Specifically, such combined therapy agent has the Her2+ patients with mastocarcinoma for developing induction of resistance to Herceptin.(ginseng See, such as Albrecht, Immunotherapy.2 (6): 795-8 (2010)).
There is provided herein use polynucleotides, primary construct or mmRNA described herein to induce recombination in cell mass The method of the translation of polypeptide.Such translation can be in vivo, in vitro, in culture or in vitro.Contain cell mass with a effective amount of Have nucleic acid composition contact, the nucleic acid have at least one nucleosides modification and encoding recombinant polypeptide can translated region.Make Nucleic acid navigate in one or more cells of cell mass and in cell from translated nucleic acid recombinant polypeptide under conditions of connect Touch group.
The physical features of target tissue, target cell type, method of application, nucleic acid are based at least partially on (for example, size and repairing Adorn nucleosides degree) and other determinants provide " effective quantity " composition.In general, a effective amount of composition is in cell Effective protein is provided to generate, it is preferably more more effective than the composition containing corresponding unmodified nucleic acid.Cell transfecting can be passed through (that is, with percentage of the cell of nucleic acid transfection) increases, increases from the protein translation of nucleic acid, nucleolysis reduce it is (such as logical Cross from the duration of the protein translation of modification of nucleic acids increase confirmed) or host cell innate immune response reduce Confirm that efficiency increases.
Aspect of the invention is related to the side of the translation of recombinant polypeptide in inductor in mammalian subject in need Method.Wherein, a effective amount of composition containing nucleic acid, the nucleic acid tool are applied to subject using delivering method described herein Have at least one structure or chemical modification and an encoding recombinant polypeptide can translated region.So that nucleic acid navigates to the cell of subject In and in the cell under the amount of translated nucleic acid recombinant polypeptide and it is other under the conditions of nucleic acid is provided.It can be with a wheel or more than a wheel Nucleic acid apply to target the cell or there are the tissues of cell that nucleic acid is positioned.
In certain embodiments, the polynucleotides of application, primary construct or mmRNA guide one or more recombinations more Peptide generates, and it is living that the recombinant polypeptide provides the function being generally not present in the cell, tissue or organism for translating recombinant polypeptide Property.For example, the functional activity of missing substantially can be enzymatic, structure or gene regulation.In the relevant embodiments, it applies Polynucleotides, primary construct or mmRNA guide one or more recombinant polypeptides to generate, and the recombinant polypeptide increases (example Such as, synergistically it) translates and exists but generally insufficient functional activity in the cell of recombinant polypeptide.
In other embodiments, the polynucleotides of application, primary construct or mmRNA guide one or more recombinations more Peptide generates, a kind of polypeptide for being generally not present in the cell of recombinant polypeptide substitution translation recombinant polypeptide (or it is a variety of more Peptide).It is such that there is no can be caused by the gene mutation of encoding gene or its regulatory pathway.In some embodiments, it recombinates more Peptide makes the level of the endogenous protein in cell increase to desired level；This increase can make the level of endogenous protein Reach normal level from lower than normal level or reach hypernormal level from normal level.
Alternatively, recombinant polypeptide plays the endogenous egg that antagonism is present in cell, secretes on the surface of cell or from cell The active effect of white matter.In general, the activity of the endogenous protein is to subject's nocuousness；Such as due to endogenous protein Mutation cause activity or positioning change.In addition, directly or indirectly antagonism is present in cell, in the table of cell recombinant polypeptide The activity for the biological moieties secreted on face or from cell.Example by the biological moieties of antagonism include lipid (for example, cholesterol), Lipoprotein (for example, low-density lipoprotein), nucleic acid, carbohydrate, proteotoxin such as shiga toxin and tetanus toxin or small Molecule toxin such as botulin toxin, cholera toxin and diphtheria toxin.In addition, can be to show not wishing by the biomolecule of antagonism The activity of the prestige such as endogenous protein of cytotoxicity or cell inhibitory activity.
Recombinant protein described herein can be engineered for positioning in the cell, potentially in specific compartment such as nucleus It is interior, or engineering is for from cell secretion or transposition to the plasma membrane of cell.
In some embodiments, the polypeptide of modification mRNA and its coding according to the present invention can be used for treating any various Disease, illness and/or symptom, it is including but not limited to below one or more: autoimmune disorder (such as diabetes, lupus, Multiple sclerosis, psoriasis, rheumatoid arthritis)；Inflammatory conditions (such as arthritis, inflammatory pelvic disease)；It is infectious Disease (such as virus infection (for example, HIV, HCV, RSV), bacterium infection, fungal infection, pyemia)；Neuropathic conditions (such as Alzheimer's, Heng Yandunshi disease；Autism；Duchenne muscular dystrophy)；Cardiovascular disease (such as artery congee Sample hardening, hypercholesterolemia, thrombosis, coagulation disorders, angiogenesis illness such as macular degeneration)；Proliferative disorders (such as Cancer, benign tumour)；Respiratory disorder (such as chronic obstructive pulmonary disease)；Digest illness (such as inflammatory bowel disease, burst Ulcer)；Musculoskeletal disorders (such as fibromyoma, arthritis)；(such as diabetes, sclerotin are dredged for endocrine, metabolism and nutrition illness Loose disease)；Disease in the urological system (such as nephrosis)；Psychological disorders (such as depression, schizophrenia)；Skin disorder (such as hurt Mouth, eczema)；Blood and lymph illness (such as anaemia, hemophilia) etc..
Be characterized in that dysfunction or the active disease of abnormal protein include cystic fibrosis, it is sickle cell anemia, big Blister epidermidolysis, amyotrophic lateral sclerosis and glucose-6-phosphate dehydrogenase lack.The present invention provides for passing through It introduces tested containing polynucleotides provided herein, the nucleic acid of primary construct or mmRNA or therapeutic agent treatment based on cell The method of the intracorporal such symptom of person or disease, wherein polynucleotides, primary construct or mmRNA encode antagonism or with other Mode overcomes the active protein of the abnormal protein being present in subject cell.The specific example of dysfunction protein For the missense mutation variant of cystic fibrosis transmembrane conductance regulator (CFTR) gene, generation causes cystic fibrosis The dysfunction protein variant of CFTR albumen.
It is characterized in that missing (or being generally reduced so that suitable (or normal) physiologic proteins function does not occur) albumen The active disease of matter includes cystic fibrosis, c-type Niman-Peek's disease, β major thalaseemia, pseudohypertrophic myotrophy Bad, hurler syndrome, Hunter syndrome and haemophilia A.This proteinoid may not be present or there is no function. The present invention provides for by introduce containing polynucleotides provided herein, primary construct or mmRNA nucleic acid or be based on The method of the therapeutic agent intracorporal such symptom for the treatment of subject or disease of cell, wherein polynucleotides, primary construct or The protein for the protein active that mmRNA coding substitution subject target cell is lacked.The specific reality of dysfunction protein Example is the nonsense mutation variant of cystic fibrosis transmembrane conductance regulator (CFTR) gene, and generation causes cystic fibrosis The non-functional protein variant of CFTR albumen.
It thus provides by making under conditions of being present in a effective amount of CTFR polypeptide in cell, subject's is thin Born of the same parents with encoding function CFTR polypeptide can polynucleotides, primary construct or the mmRNA of translated region contact and treats the food in one's mouth The method of the cystic fibrosis of newborn animal subjects.Preferred target cell is epithelial cell, endothelial cell and mesothelial cell, such as Lung, and method of administration is determined in view of target tissue；It is directed to pulmonary delivery, RNA molecule is formulated for applying by sucking.
In another embodiment, the present invention provides for (passing through genome at present by that will encode Sortilin Study characterization protein) modification mRNA molecule be introduced into the hyperlipidemia in the cell mass of subject so as to improve subject Come treat subject hyperlipidemia method.SORT1 gene coding is known as the trans-Golgi network (TGN) of Sortilin Transmembrane protein.The individual that genetic research has been displayed 1/5th has single nucleotide in the 1p13 locus of SORT1 gene Polymorphism rs12740374 makes them tend to have low-level low-density lipoprotein (LDL) and very low density lipoprotein (VLDL).The each copy for the small allele being present in about 30% crowd makes LDL cholesterol change 8mg/dL, and is present in Two copies of the small allele in about 5% group reduce LDL cholesterol by 16mg/dL.Small allele also has been displayed Carrier has 40% reduced risk of myocardial infarction.In vivo functionality research in mouse describes in mouse liver tissue SORT1 overexpression causes to significantly reduce LDL- cholesterol levels, such as reduces as many as 80%, and silencing SORT1 keeps LDL gallbladder solid (Musunuru K etc., From noncoding variant to phenotype via the SORT1 at of alcohol increase about 200% the 1p13 cholesterol locus.Nature 2010；466:714-721).
In another embodiment, the present invention provides for treating hematopoietic disorders, cardiovascular disease, oncology, sugar Urinate disease, cystic fibrosis, neurogenic disease, inborn metabolism errors, skin and systemic disorder and blindness method.It has retouched Stated treat these disease specifics molecular target identity (Templeton writes, Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, the 3rd edition, Bota Raton, FL:CRC Press；It is with reference Mode be integrally incorporated herein).
There is provided herein prevention infection and/or pyemias in the subject in development infection and/or pyemia risk Method, method include to have it is such prevention need subject application enough prevention infection and/or pyemic amount combination Object, the composition include the more of coding antimicrobial polypeptide (for example, antibacterial polypeptide) or part thereof or form processing completely Nucleotide, primary construct or mmRNA precursor.In certain embodiments, in development infection and/or pyemia risk by Examination person can be cancer patient.In certain embodiments, cancer patient can undergo pretreating scheme (conditioning regimen).In some embodiments, pretreating scheme may include but be not limited to chemotherapy, radiotherapy or the two. As a non-limiting example, polynucleotides, primary construct or mmRNA codified PROTEIN C, its proenzyme or preceding former albumen, The activation form (APC) of PROTEIN C or the variant of PROTEIN C as known in the art.Polynucleotides, primary construct or mmRNA can It is modified by sulphation and is delivered to cell.It can be in the non-limiting example packet of the polypeptide of chemical modification mRNA interior coding of the invention Those of introduction in United States Patent (USP) 7,226,999,7,498,305,6,630,138 is included, the patent is respectively with reference Mode is integrally incorporated herein.These patents teach PROTEIN C sample molecule, variant and derivative, any PROTEIN C sample molecule, Variant and derivative can be in the molecule interior codings of chemical modification of the invention.
The infection and/or pyemic method for the treatment of subject further provided herein, method include to there is such control Subject's application composition for the treatment of infection and/or pyemic amount enough of needs is treated, the composition resists micro- comprising coding Biological polypeptide (for example, antibacterial polypeptide), antimicrobial polypeptide for example described herein or part thereof or completely form processing Polynucleotides, primary construct or mmRNA precursor.In certain embodiments, the subject for having treatment to need is cancer patient. In certain embodiments, cancer patient undergoes pretreating scheme.In some embodiments, pretreating scheme may include but not It is limited to chemotherapy, radiotherapy or the two.
In certain embodiments, subject can show acute or chronic microorganism infection (for example, bacterium infection).? In certain embodiments, subject can receive or can receive a kind of therapy.In certain embodiments, the therapy It may include but be not limited to radiotherapy, chemotherapy, steroids, ultraviolet radioactive or combinations thereof.In certain embodiments, patient It can be by microvascular disorder.In some embodiments, microvascular disorder can be diabetes.In certain embodiments, patient There can be wound.In some embodiments, wound can be ulcer.In a specific embodiment, wound can be diabetes Property foot ulcers.In certain embodiments, subject can have one or more burn wounds.In certain embodiments, Application can be part or system.In certain embodiments, application can be subcutaneous.In certain embodiments, application can It is intravenous.In certain embodiments, application can be oral.In certain embodiments, application can be local.? In certain embodiments, application can be to pass through sucking.In certain embodiments, application can be rectum.In certain embodiment party In case, application can be vagina.
The other aspects of the disclosure be related to will the cell transplantation containing polynucleotides, primary construct or mmRNA to lactation Animal subjects.To mammalian subject dosed cells be it is known to persons of ordinary skill in the art, and including but it is unlimited In implant region (for example, part or subcutaneous administration), organ delivering or systemic injection (for example, intravenous injection or sucking) and The pharmaceutically cell preparation in acceptable carrier.Such composition containing polynucleotides, primary construct or mmRNA can Be formulated for it is intramuscular, through in artery, peritonaeum, intravenous, intranasal, subcutaneous, endoscope, transdermal or intrathecal application.In some implementations In scheme, composition can be formulated for extended release.
It can be subject to disease, illness or deleterious condition to its subject for applying therapeutic agent or can be at developing disease, disease In the risk of disease or deleterious condition.The method for the subject that identifies, diagnoses and classify, the method are provided based on these bases It may include clinical diagnosis, biomarker level, genome-wide association study (GWAS) and other sides as known in the art Method.
Rare liver diseases or illness
Cholestasis (PFIC) in progressive familial gallbladder
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat Cholestasis (PFIC) in progressive familial gallbladder.As used herein term " progressive familial gallbladder Interior Cholestasis " or " PFIC " refer to the liver disorders that can lead to liver failure.PFIC is characterized in that bile is formed and liver is thin The cholestatic autosomal recessive defect of born of the same parents.As used herein, " (hepatocellular) of liver cell " is for retouching Stating influences or is related to the term (also known as liver cell (hepatocyte)) of some cases of liver cell.As used herein, Term " cholestasia ", which refers to, is characterized in that bile from the symptom that liver flowing is slow or interrupts.As used herein, term " gallbladder Juice " refer to by liver generate comprising water, bile salt, mucus, fat, inorganic salts and cholesterol liquid substance, help meals Eat fat emulsification and digestion.
There are the PFIC of three kinds of known types (PFIC-1, PFIC-2 and PFIC-3), and they have traced back to and have been related to liver The mutation of cell transport system and chologenetic protein coding gene.
PFIC-1 and PFIC-2 is usually diagnosed in infancy, but can be paid a home visit during period before birth or non-neonate It is disconnected.As used herein, in the period of term " before birth " refers to that generation is in antenatal organism life.As made herein With term " neonatal period " refers to one period occurred in organism life after birth.For the mankind, neonatal period, can Including from birth to postnatal about 1 month, to about 3 about or to about 6 months one periods.As used herein, term " infancy " refer to occur birth and childhood between organism life in one period.For the mankind, infancy can Including from birth to about 1 one full year of life, to about 2 one full year of life, to about 3 one full year of life or to the period of about 4 one full year of life.
PFIC-3 can be diagnosed during period before birth, during neonatal period or during infancy.In some feelings Under condition, PFIC-3 can escape diagnosis until childhood or the adolescence.As used herein, term " childhood " refer to generation In the period of in the organism life after infancy and before the adolescence.For the mankind, childhood may include from about 2 one full year of life to About 10 one full year of life, about 3 one full year of life to about 11 one full year of life, about 4 one full year of life to about 12 one full year of life or about 5 one full year of life to about 13 one full year of life period.As herein Used, term " adolescence " refer to occur childhood and the manhood between organism life in the period of.For the mankind, Adolescence may include from about 10 one full year of life to about 16 one full year of life, from about 11 one full year of life to about 17 one full year of life, from about 12 one full year of life to about 18 one full year of life, from About 13 one full year of life are to about 19 one full year of life and from about 14 one full year of life to the period of about 20 one full year of life.
The clinical manifestation of PFIC includes but is not limited to itch, cholestasia and jaundice.As used herein, term " itch " Refer to cause to scratch grab or rubdown affected part part impulsion uncomfortable feeling.As used herein, term " jaundice " refers to spy Sign is the symptom of skin caused by being increased by bilirubin, eyes and/or mucous membrane jaundice.
Most patients with PFIC develop fibrosis before the manhood and by liver failures.It can be faced by observation Bed performance, cholangiography, liver ultrasonic examination, liver histological and heredity test are to diagnose the individual with PFIC.It can be into Childhood that the other test of row is to exclude to cause cholestatic other illnesss.
The dysfunction of one of three kinds of different cell trafficking proteins can cause each in 1 type, 2 types and 3 type PFIC.Often Kind all refers to lipid transfer and every kind plays an important role to bile from hepatic secretion.1 type PFIC is by leading to I class 8B type member 1 The gene mutation of ATP enzyme aminophospholipids transport protein (ATP8B1) dysfunction cause.ATP8B1 is played phosphatidyl silk ammonia The effect of acid and phosphatidyl-ethanolamine from a lateral translocation of phospholipid bilayer to the other side.PFIC-2 and PFIC-3 is by influence ATP- Cause in conjunction with the mutation of the function of box (ABC) transport protein.(ABCB11) transport protein of ABC subfamily B member 11 is by by ox Sulphur cholic acid and other cholic acid related compounds are transported in bile to support bile to generate from liver cell.Defective ABCB11 Function leads to PFIC-2.Phosphatide, preferably phosphatidyl choline transport are passed through liver plasma membrane by ABC subfamily B member 4 (ABCB4) For being incorporated into bile.The gene mutation for leading to ABCB4 dysfunction is the reason of causing PFIC-3.(Davit-Spraul, A. etc., Progressive familial intrahepatic cholestasis.Orphanet J Rare Dis.2009 Jan 8；4:1；Degiorgio, D. etc., Molecular characterization and structural implications of 25 new ABCB4 mutations in progressive familial intrahepatic cholestasis type 3(PFIC3).Eur J Hum Genet.2007 Dec；15 (12): 1230-8；It is respectively to draw Mode is integrally incorporated herein).
In one embodiment, the rare liver disease of at least one of the invention can be included to patient's application with PFIC The composition of disease or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness polynucleotides, primary structure Build body or mmRNA codified peptide, protein or its segment, such as, but not limited to ATP-binding cassette subfamily (MDR/TAP) member 4 (ABCB4), the ATP enzyme amino phosphorus of ATP-binding cassette subfamily (MDR/TAP) member 11 (ABCB11) and I class 8B type member 1 Rouge transport protein (ATP8B1).
In one embodiment, PFIC-1 can be treated by applying composition of the invention, the composition includes Encode the peptide of ATP8B1, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary construct Or mmRNA.In another embodiment, PFIC-1, the composition packet can be treated by applying composition of the invention The rare liver disease of at least one of peptide, protein or its segment containing coding comprising SEQ ID NO:855 or SEQ ID NO:856 Disease or illness polynucleotides, primary construct or mmRNA.
In one embodiment, PFIC-2 can be treated by applying composition of the invention, the composition includes Encode the peptide of ABCB11, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary construct Or mmRNA.In another embodiment, PFIC-2, the composition packet can be treated by applying composition of the invention The rare liver diseases of at least one or illness multicore glycosides of peptide, protein or its segment containing coding comprising SEQ ID NO:865 Acid, primary construct or mmRNA.
In one embodiment, PFIC-3 can be treated by applying composition of the invention, the composition includes Encode the peptide of ABCB4, protein or its segment the rare liver diseases of at least one or illness polynucleotides, primary construct or mmRNA.In another embodiment, PFIC-3 can be treated by applying composition of the invention, the composition includes The rare liver diseases of at least one or illness multicore of peptide of the coding comprising SEQ ID NO:866-871, protein or its segment Thuja acid, primary construct or mmRNA.
Familial hypercholesterolemia
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat familial hypercholesterolemia (FH).As used herein, term " familial hypercholesterolemia " or " FH " Refer to the relevant raised inherited disorder of cholesterol levels of the low-density lipoprotein being characterized in that in blood plasma (LDL).With FH's Patient or subject can have increased risk of cardiovascular diseases when young.In some embodiments, the blood of these individuals High-level LDL in liquid can be the result of the gene mutation of coding ldl receptor.It is believed that (but it is not intended that limitation), ldl receptor exists In circulation combine LDL and promote LDL endocytosis to express on it to receptor cell in.When this receptor is dysfunction When, LDL level keeps increasing and promoting the development of atherosclerosis in the circulating cycle.Individual with FH is for FH- correlation Gene mutation can for heterozygosis or homozygosis.Symptom in homozygous individual can be even more serious.Childhood or the adolescence during lead to Crossing method as known in the art includes but is not limited to appear the physical examination of vitiligoidea (fatty skin growth) to be able to achieve diagnosis.It is early The diagnosis of phase FH can be carried out by analysis family history and science of heredity.(Sjouke, B. etc., Familial Hypercholesterolemia:present and future management.Curr Cardiol Rep.2011 Dec； 13 (6): 527-36；Avis, H.J. etc., A systematic review and meta-analysis of statin therapy in children with familial hypercholesterolemia.Arterioscler Thromb Vasc Biol.2007 Aug；27 (8): 1803-10；It is respectively incorporated herein in its entirety by reference).
In one embodiment, the rare liver diseases of at least one of the invention can be included to patient's application with FH Or the composition of illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness polynucleotides, primary building Body or mmRNA codified peptide, protein or its segment, such as, but not limited to LDL receptor (LDLR), apolipoprotein B (APOB) and proprotein convertases subtilin/9 type science-stars (kexin) (PCSK9).
In one embodiment, FH can be treated by applying composition of the invention, the composition includes coding The peptide of LDLR, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary construct or mmRNA.In another embodiment, FH can be treated by applying composition of the invention, the composition includes coding The rare liver diseases of at least one or illness multicore glycosides of peptide, protein or its segment comprising SEQ ID NO:1145-1151 Acid, primary construct or mmRNA.
In one embodiment, FH can be treated by applying composition of the invention, the composition includes coding The peptide of APOB, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary construct or mmRNA.In another embodiment, FH can be treated by applying composition of the invention, the composition includes coding The rare liver diseases of at least one or illness multicore glycosides of peptide, protein or its segment comprising SEQ ID NO:819 or 819 Acid, primary construct or mmRNA.
In one embodiment, FH can be treated by applying composition of the invention, the composition includes coding The peptide of PCSK9, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary construct or mmRNA.In another embodiment, FH can be treated by applying composition of the invention, the composition includes coding The rare liver diseases of at least one or illness multicore glycosides of peptide, protein or its segment comprising SEQ ID NO:1241-1243 Acid, primary construct or mmRNA.
Ornithine carbamoyltransferase deficiency disease
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat Ornithine carbamoyltransferase deficiency disease (OTCD).As used herein, " ornithine turns term Shifting enzyme deficiency disease " or " OTCD " refer to the urea as caused by the gene mutation of codase ornithine transcarbamylase (OTC) The inherited disorder of synthesis.These mutation can lead to hyperammonemia, nerve problem and the death rate and increase.OTC is urea cycle Important component, catalysis form citrulling from carbamyl phosphate and ornithine.This process is for removing excess of ammonia from body It is crucial.Gene for OTC is carried in X chromosome, so that OTCD be made to become the chain illness of X.As most of X are chain Illness is the same, and OTCD mainly influences male, and women is carrier.The seriousness of OTCD depends on the property of gene mutation.It leads The gene mutation in the individual of nonfunctional OTC is caused to typically result in those individuals dead in the first month of life.Bosom can be passed through It doubts by the genetic analysis in the individual of OTCD and finds such mutation.With the gene mutation for leading to different degrees of enzyme function Can survive longer time and response of individual in treatment reduce the influence of disease.It can observe symptom and detected in urine The diagnosis of OTCD is made after to high-level ammonia and orotic acid.(Brunetti-Pierri, N. etc., Phenotypic correction of ornithine transcarbamylase deficiency using low dose helper- dependent adenoviral vectors.J Gene Med.2008 Aug；10 (8): 890-6；Wilmslow, U.K., Ornithine transcarbamylase deficiency:a urea cycle defect.Eur J Paediatr Neurol.2003；7 (3): 115-21；It is respectively incorporated herein in its entirety by reference).
In one embodiment, the rare liver disease of at least one of the invention can be included to patient's application with OTC The composition of disease or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness polynucleotides, primary structure Build body or mmRNA codified peptide, protein or its segment, such as, but not limited to ornithine transcarbamylase (OTC).
In one embodiment, OTCD can be treated by applying composition of the invention, the composition includes to compile The code peptide of OTC, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary construct or mmRNA.In another embodiment, OTCD can be treated by applying composition of the invention, the composition includes to compile Peptide of the code comprising SEQ ID NO:1192, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, Primary construct or mmRNA.
Crigler-Najjar syndrome (Crigler-Najjar Syndrome)
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat crigler-Najjar syndrome.As used herein, term " crigler-Najjar syndrome ", which refers to, influences enzyme bilirubin- The birth defects of the function of the 1A1 isoform (UGT1A1) of uridine diphosphate glucoside acyltransferase.UGT1A1 is hydrophobic It is crucial in the detoxication of bilirubin (toxic by-products of hemachrome degradation).It is believed that UGT1A1 is red by making hydrophobic gallbladder Element is connected to glucuronic acid and is worked with being excreted in bile.Non- knot can be undergone by the individual of crigler-Najjar syndrome Mould assembly hyperbilirubinemia (or hydrophobic bilirubin is excessive in circulation), leads to nerve damage and needs as patient age becomes More carry out about inefficient conventional light therapy.The diagnosis of crigler-Najjar syndrome normally starts from and observes jaundice in child, and so After can pass through enzymatic determination as known in the art and liver and measure and evaluate enzyme function.(Lysy, P.A. etc., Liver cell Transplantation for Crigler-Najjar syndrome type I:update and perspectives.World J Gastroenterol.2008 Jun 14；14 (22): 3464-70；Sugatani, J., Function, genetic polymorphism, and transcriptional regulation of human UDP- Glucuronosyltransferase (UGT) 1A1.Drug Metab Pharmacokinet.2012 Oct 23. [is printing Electronic publication before]；It is respectively incorporated herein in its entirety by reference).
It in one embodiment, can include of the invention at least one to patient's application with crigler-Najjar syndrome The composition of the rare liver diseases of kind or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness multicore Thuja acid, primary construct or mmRNA codified peptide, protein or its segment, such as, but not limited to UGT1A1.
In one embodiment, crigler-Najjar syndrome can be treated by applying composition of the invention, described group Closing object includes the coding peptide of UGT1A1, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, just Grade construct or mmRNA.In another embodiment, Ke-can be treated by applying composition of the invention, and to receive Er Shi comprehensive Simulator sickness, the composition include at least one of peptide of the coding comprising SEQ ID NO:1357 or 1358, protein or its segment Rare liver diseases or illness polynucleotides, primary construct or mmRNA.
Ceruloplasmin deficiency disease
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat ceruloplasmin deficiency disease.As used herein, term " ceruloplasmin deficiency disease " refers to plasma copper Azurin (CP) can due to the gene mutation in CP gene defective and/or dysfunction disease.It is believed that CP is responsible for By iron from cell traffic to capillary in transport protein.Once iron is in combination with ferritin and enters blood in capillary Stream.In the case where functionality CP is not present, iron accumulates in the cell and serum ferritin level is got higher.(Ogimoto, M. Deng Criteria for early identification of aceruloplasminemia.Intern Med.2011；50 (13): 1415-8；Miyajima, H., Aceruloplasminemia.GeneReviews [the Internet] .Seattle (WA): University of Washington, Seattle；2003 Aug 12 [updating on 2 17th, 2011]；It is respectively equal It is incorporated herein in its entirety by reference).
Iron accumulation can be especially serious in liver and in eyes, brain and pancreas.Drawn by the genetic defect in CP gene The ceruloplasmin deficiency disease risen is autosomal recessive illness.As used herein, term " autosomal recessive " refers to It can influence the disease to those of responsible gene pure people, illness, character or phenotype.The symptom of disease is usually in 25 one full year of life and 60 Show between one full year of life.It can be analyzed by the serum analysis of ceruloplasmin and iron level or by Typical AVM to check that iron accumulates To be considered the diagnosis of the individual by disease.
In one embodiment, can to ceruloplasmin deficiency disease patient application comprising it is of the invention at least A kind of composition of rare liver diseases or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness are more Nucleotide, primary construct or mmRNA codified peptide, protein or its segment, such as, but not limited to CP.
In one embodiment, ceruloplasmin deficiency disease, institute can be treated by applying composition of the invention Stating composition includes the coding peptide of CP, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, just Grade construct or mmRNA.In another embodiment, ceruloplasmin can be treated by applying composition of the invention Deficiency disease, the composition include the rare liver of at least one of peptide of the coding comprising SEQ ID NO:891, protein or its segment Dirty disease or illness polynucleotides, primary construct or mmRNA.
α-mannosidosis
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat α-mannosidosis.As used herein, term " α-mannosidosis " refers to by leading to lyase Illness caused by defective and/or the dysfunction α of body thesaurismosis-D-MANNOSE glycosides enzyme enzymatic activity.It (is compiled by MAN2B1 gene Code α-D-MANNOSE glycosides enzyme) in genetic defect caused by α-mannosidosis be autosomal recessive illness.By α- The feature of those of mannosidosis people includes but is not limited to immune deficiency, face and skeletal abnormality, impaired hearing and intelligence Power is impaired.It includes but is not limited to that analysis phenotypic characteristic and liver and Dyssplenism come that method as known in the art, which can be used, Diagnosis is made during child's early stage.By childhood and adolescence, the disease compared with mild forms can keep not diagnosing.It is usually logical α-D-MANNOSE glycosides enzyme the enzymatic activity crossed in measurement whole blood cells (while blood cell) confirms the diagnosis of disease. (Malm, D. etc., Alpha-mannosidosis.Orphanet J Rare Dis.2008 Jul 23；3:21；It is with reference Mode is integrally incorporated herein).
In one embodiment, of the invention at least one can be included to α-mannosidosis patient application The composition of the rare liver diseases of kind or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness multicore Thuja acid, primary construct or mmRNA codified peptide, protein or its segment, such as, but not limited to MAN2B1.
In one embodiment, α-mannosidosis can be treated by applying composition of the invention, it is described Peptide of the composition comprising coding MAN2B1, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, Primary construct or mmRNA.In another embodiment, α-mannoside can be treated by applying composition of the invention Excessive disease, the composition include at least one of peptide of the coding comprising SEQ ID NO:1156-1158, protein or its segment Rare liver diseases or illness polynucleotides, primary construct or mmRNA.
Tyrosinemia
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat tyrosinemia.As used herein, term " tyrosinemia " refers to the tyrosine being characterized in that in blood And/or the raised illness of level and/or symptom of tyrosine by-product.In some embodiments, tyrosinemia is by causing The gene mutation of defective necessary to tyrosine is degraded and/or dysfunction enzyme causes.Symptom includes but is not limited to develop Stagnation diarrhea, vomiting, jaundice, the increase of nose is bleeding trend, microcephaly, is trembled, incoordination, autotomy, fine movement association Adjust obstacle, language defect, spasm and/or cabbage sample smell.(Nakamura, K. etc., Animal models of tyrosinemia.J Nutr.2007 Jun；137 (6 supplementary issues 1): 1556S-1560S；discussion 1573S-1575S； Bergeron, A. etc., Hereditary tyrosinemia:an endoplasmic reticulum stress Disorder? Med Sci (Paris) .2003 Oct；19 (10): 976-80；Mehere, P. etc., Tyrosine Aminotransferase:biochemical and structural properties and molecular dynamics simulations.Protein Cell.2010 Nov；1 (11): 1023-32；It is respectively incorporated hereby Herein).
1 type tyrosinemia refers to the illness form as caused by shortage fumarylacetoacetate hydrolase (FAH) activity. FAH activity shortage in 1 type tyrosinemia causes metabolin fumaryl acetoacetate ester to accumulate, and triggering cellular activity is for example thin Born of the same parents' apoptosis, mutation occur, aneuploid induces (aneugenesis) and mitosis occurs.
2 type tyrosinemias refer to the illness form as caused by shortage tyrosine transaminase (TAT) activity.TAT is to cause The reason of tyrosine and other aromatic amino acids include but is not limited to the reversible ammonia of p-hydroxybenzene pyruvic acid (HPP) (pHPP).
It in one embodiment, can be rare comprising at least one of the invention to patient's application with tyrosinemia The composition of liver diseases or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness polynucleotides, Primary construct or mmRNA codified peptide, protein or its segment, such as, but not limited to FAH or TAT.
In one embodiment, 1 type tyrosinemia, the combination can be treated by applying composition of the invention Object includes the peptide of coding FAH, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary building Body or mmRNA.In another embodiment, 1 type tyrosinemia can be treated by applying composition of the invention, it is described Composition includes the rare liver diseases of at least one of peptide of the coding comprising SEQ ID NO:985-987, protein or its segment Or illness polynucleotides, primary construct or mmRNA.
In one embodiment, 2 type tyrosinemias, the combination can be treated by applying composition of the invention Object includes the peptide of coding TAT, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary building Body or mmRNA.In another embodiment, 2 type tyrosinemias can be treated by applying composition of the invention, it is described Composition includes peptide of the coding comprising SEQ ID NO:1356, the rare liver diseases of at least one of protein or its segment or disease Disease polynucleotides, primary construct or mmRNA.
Hemochromatosis
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat hemochromatosis.As used herein, term " hemochromatosis ", which refers to, is characterized in that be lacked by heredity The illness or symptom of the overload of iron caused by falling into.The symptom of hemochromatosis include but is not limited to cirrhosis, hyperpigmentation, Pituitary function decline, diabetes and/or arthritis.Genetic defect relevant to hemochromatosis is used to characterize the shape of disease Formula.1 type hemochromatosis is caused by the gene mutation in HFE gene.2A type and 2B type are respectively in HFE2 and HAMP gene Mutation result.Although the symptom of 1 type hemochromatosis usually until the manhood just exist, 2A type and 2B type color The symptom of plain hemachromatosis usually childhood during there is.(Papanikolaou, G. etc., Hepcidin in iron overload disorders.Blood.2005 May 15；105 (10): 4103-5；Nandar, W. etc., HFE gene variants affect iron in the brain.J Nutr.2011 Apr 1；141 (4): 729S-739S；Wallace, D.F. etc., Non-HFE haemochromatosis.World J Gastroenterol.2007 Sep 21；13 (35): 4690-8；It is respectively incorporated herein in its entirety by reference).
It in one embodiment, can be rare comprising at least one of the invention to patient's application with hemochromatosis See the composition of liver diseases or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness multicore glycosides Acid, primary construct or mmRNA codified peptide, protein or its segment, such as, but not limited to HFE2, HAMP, Solute Carrier man 40 member 1 (SLC40A1) of race and transferrin receptor 2 (TFR2).
In one embodiment, 1 type hemochromatosis can be treated by applying composition of the invention, described group Close the peptide that object includes coding HFE, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary structure Build body or mmRNA.In another embodiment, 1 type hemochromatosis can be treated by applying composition of the invention, The composition includes the rare liver of at least one of peptide of the coding comprising SEQ ID NO:1038-1050, protein or its segment Dirty disease or illness polynucleotides, primary construct or mmRNA.
In one embodiment, 2 type hemochromatosis can be treated by applying composition of the invention, described group Close the peptide that object includes coding HFE2 or HAMP, the rare liver diseases of at least one of protein or its segment or illness multicore glycosides Acid, primary construct or mmRNA.In another embodiment, 2 type colors can be treated by applying composition of the invention Plain hemachromatosis, the composition include coding comprising the peptide of SEQ ID NO:1051-1057,1067 or 1068, protein or its The rare liver diseases of at least one or illness polynucleotides of segment, primary construct or mmRNA.
In one embodiment, hemochromatosis, the combination can be treated by applying composition of the invention Object includes the rare liver diseases of at least one or illness multicore glycosides of the peptide of coding TFR2 or SLC40A1, protein or its segment Acid, primary construct or mmRNA.In another embodiment, hemochrome can be treated by applying composition of the invention Hemachromatosis, the composition include peptide, protein or its piece that coding includes SEQ ID NO:1290-1296 or 1323-1326 The rare liver diseases of at least one or illness polynucleotides of section, primary construct or mmRNA.
Glycogen storage disease
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat glycogen storage disease IV.As used herein, term " glycogen storage disease " or " GSD ", which refer to, is characterized in that glycogen Illness in defective synthesis and/or the organism decomposed.In some embodiments, GSD includes but is not limited to also known as to pacify The 4 type GSD that the gloomy disease of moral, brancher deficiency, amylopectinosis and glycogen branching enzyme lack.IV type GSD is encoded by GBE1 gene The shortage of glycogen branching enzyme (GBE) cause, cause to be formed for the abnormal glycogen of cytotoxicity.Although due to tissue specificity The expression of isoform and there is variation, but the patient with classics IV type liver GSD look like at birth it is healthy；However The hardening of liver occurs usually to lead to liver failure before 5 one full year of life in life early stage.(Ozen, H., Glycogen Storage diseases:new perspectives.World J Gastroenterol.2007 May 14；13 (18): 2541-53；It is incorporated herein in its entirety by reference).
In one embodiment, the rare liver disease of at least one of the invention can be included to patient's application with GSD The composition of disease or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness polynucleotides, primary structure Build body or mmRNA codified peptide, protein or its segment, such as, but not limited to glucan (Isosorbide-5-Nitrae-α -) branching enzyme 1 (GBE1).
In one embodiment, IV type GSD can be treated by applying composition of the invention, the composition includes Encode the peptide of GBE1, protein or its segment the rare liver diseases of at least one or illness polynucleotides, primary construct or mmRNA.In another embodiment, GSD IV can be treated by applying composition of the invention, the composition includes Peptide of the coding comprising SEQ ID NO:1010-1012, the rare liver diseases of at least one of protein or its segment or illness are more Nucleotide, primary construct or mmRNA.
Fanconi cystinosis (Fanconi Cystinosis)
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat Fanconi cystinosis.As used herein, term " cystinosis ", which refers to, is characterized in that Guang ammonia in lysosome The disease of sour abnormal accumulation.The most commonly encountered diseases of Fanconi syndrome are because being to prevent metabolin from reuptaking to make its substitution in blood flow The kidney trouble that ground passes through in urine.Cystine passes through the disulfide bond formation between two cysteine residues.Lysosome is water The site of protein digestibility and the intracorporal cystine accumulation of lyase are solved dependent on the carrier mediated transhipment for its release.By The cystinosis albumen (cystinosin) of CTNS gene coding is that cystine and and other oroteins are combined in lysosome Cooperatively facilitate its seven-transmembrane albumen removed from lysosome.Individual on cystinosis protein function with major defect exists Meeting is impacted between 6 monthly ages and 12 monthly ages and has body fluid and electrolyte loss and other kidney complication and metabolism simultaneously Disease is sent out to exist.Kidney failure usually by 10 years old when occur.Current treatment is using can crack cystine molecule, to allow it The drug cysteamine removed from lysosome.(Kalatzis, V. etc., Cystinosis:from gene to disease.Nephrol Dial Transplant.2002 Nov；17 (11): 1883-6；It is incorporated hereby Herein).
In one embodiment, the patient with hemochromatosis can apply rare comprising at least one of the invention The composition of liver diseases or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness polynucleotides, Primary construct or mmRNA codified peptide, protein or its segment, such as, but not limited to cystinosis albumen, half Guang of lysosome Propylhomoserin transport protein (CTNS).
In one embodiment, Fanconi cystinosis can be treated by applying composition of the invention, described group Close the peptide that object includes coding CTNS, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, primary Construct or mmRNA.In another embodiment, Fanconi cystine can be treated by applying composition of the invention Disease, the composition include the rare liver of at least one of peptide of the coding comprising SEQ ID NO:938-941, protein or its segment Dirty disease or illness polynucleotides, primary construct or mmRNA.
Farber lipogranulomatosis (Farber Lipogranulomatosis)
In one embodiment, rare liver diseases of the invention or illness polynucleotides, primary construct or mmRNA It can be used to treat farber lipogranulomatosis.As used herein, term " farber lipogranulomatosis ", " method Bai Shi fat meat The swollen disease of bud " or " farber's disease " refer to the lysosomal storage disease identified in nineteen fifty-seven by Sidney Farber.Disease is by N- acyl group mind The shortage of enzyme acid ceramidase through 1 (ASAH1) gene of sphingol hydroamidase (acid ceramidase) coding is drawn It rises.The shortage of this enzyme prevents intracellular ceramide to be normally decomposed into sphingosine and fatty acid and causes to lead to disease The ceramide abnormal accumulation of disease symptoms.Symptom is typically found in child's early stage, but can also occur in later life.In disease In classical field formalism, symptom occurs within former weeks of life and may include joint deformity, there are subcutaneous nodules and hoarseness. Patient can also often result in infancy death by nerve damage.Patient with slight nerve damage can survive to its four Ten years old.Currently, there is no the treatments for being available for farber lipogranulomatosis.(Ekici, B. etc., Farber disease:A Clinical diagnosis.J Pediatr Neurosci.2012 May；7 (2): 154-5；Farber, S. etc., Lipogranulomatosis；a new lipo-glycoprotein storage disease.J Mt Sinai Hosp N Y.1957 November in year-December；24 (6): 816-37；It is respectively incorporated herein in its entirety by reference).
It in one embodiment, can be rare comprising at least one of the invention to patient's application with hemochromatosis See the composition of liver diseases or illness polynucleotides, primary construct or mmRNA.Rare liver diseases or illness multicore glycosides Acid, primary construct or mmRNA codified peptide, protein or its segment, such as, but not limited to N- acyl sphingosine amide Hydrolase (acid ceramidase) 1 (ASAH1).
In one embodiment, farber lipogranulomatosis can be treated by applying composition of the invention, it is described Composition includes the coding peptide of ASAH1, the rare liver diseases of at least one of protein or its segment or illness polynucleotides, just Grade construct or mmRNA.In another embodiment, Fanconi cystine can be treated by applying composition of the invention Disease, the composition include that at least one of peptide of the coding comprising SEQ ID NO:1171-1175, protein or its segment is rare Liver diseases or illness polynucleotides, primary construct or mmRNA.
Treatment of wounds
Polynucleotides, primary construct or mmRNA of the invention can be used for Wound healing and bone regeneration, such as be cured for showing delay The wound of conjunction.There is provided herein include the method for applying polynucleotides, primary construct or mmRNA to manage Wound healing and bone regeneration. Methods herein can further comprise the step carried out prior to, concurrently with, or after application polynucleotides, primary construct or mmRNA Suddenly.Example, it may require cleaning and preparing wound bed to promote wound healing and it is desirable that obtain wound closure.It can be used several Strategy is to promote wound healing and to realize wound closure, including but not limited to: (i) debridement, optionally repeats, quick debridement Art (tissue that operation removal is die or infected from wound), optionally includes chemical debridement system agent such as enzyme to remove necrotic tissue； (ii) wound dressing, for providing the environment of damp warm for wound and promoting tissue repair and healing.
The example of material for preparing wound dressing includes but is not limited to: hydrogel (for example,), hydrocolloid (for example, ), foam (for example,) and alginate (for example, )；(iii) other growth factor, for stimulating cell differentiation and proliferation and promoting wound healing, such as Becaplermin (becaplermin)(REGRANEX), i.e., it is a kind of to recombinate blood for treating the people of nerve foot ulcers by FDA approval Platelet source property growth factor；(iv) soft tissue wound covering, it is a kind of to obtain skin required for clean non-healing of wound Skin graft.The example that can be used for the skin graft of soft tissue covering includes but is not limited to: auto-skin grafting Object, cadaver skin graft, Bioengineered skin substitutes (for example,)。
In certain embodiments, polynucleotides of the invention, primary construct or mmRNA can be into one Step include hydrogel (for example,), hydrocolloid (for example,), foam (for example, ) and/or Alginate (for example,).In certain embodiments, multicore glycosides of the invention Acid, primary construct or mmRNA can be used together with skin graft, and the skin graft includes but is not limited to autologous skin Graft, cadaver skin graft or Bioengineered skin substitutes (for example, ).In some embodiments, polynucleotides, primary construct or mmRNA can be with wound dressing systems Agent and/or skin graft apply together or they can separate and apply, but method is such as, but not limited to, impregnated or sprayed.
It in some embodiments, may include coding antimicrobial polypeptide (for example, anti-thin for the composition for the treatment of of wounds Bacterium polypeptide) and/or antiviral polypeptide polynucleotides, primary construct or mmRNA.The precursor of codified antimicrobial polypeptide or Partially or completely form processing.Composition can be formulated for applying using bandage (for example, cohesive bandage).Antimicrobial polypeptide And/or antiviral polypeptide can be mutually mixed with dressing composition or for example can separate coating by impregnating or spraying.
The generation of antibody
In one embodiment of the invention, polynucleotides, primary construct or mmRNA codified antibody and such anti- The segment of body.These can be generated by either method described herein.Antibody can have any different subclass or isotype Immunoglobulin, such as, but not limited to, IgA, IgG or IgM or any other subclass.It can exemplary antibodies prepared in accordance with the present invention Molecule and segment include but is not limited to immunoglobulin molecules, generally complete immunoglobulin molecules and can contain paratope Those of immunoglobulin molecules part.Such part of antibody containing paratope includes but is not limited to Fab, Fab ', F (ab’)2, F (v) and those parts as known in the art.
Polynucleotides codified variant antibodies polypeptide of the invention, can with reference polypeptide sequence have certain identity or There is similar or dissimilar binding characteristic to reference polypeptide sequence.
The antibody obtained by means of the present invention can be chimeric antibody, and it includes the non-human antibodies from immune animal Source variable region sequences and human antibody source constant-region sequences.In addition, they can be also humanized antibody, it includes derive to exempt from The complementary determining region (CDR) of the non-human antibody of epidemic disease animal and framework region (FR) and constant region from human antibody.Another In a embodiment, method provided herein can have for enhancing the antibody protein products collection efficiency in cell cultivation process.
Infection management
In one embodiment, it provides for polynucleotides, the primary structure by application coding antimicrobial polypeptide Body or mmRNA is built to treat or prevent microorganism infection (for example, bacterium infection) in subject and/or feel with microorganism or virus Contaminate relevant disease, illness or symptom or the method for its symptom.The application can with antimicrobial (for example, antibacterial agent), Such as antimicrobial polypeptide or small molecule Antimicrobe compound described herein combine.Antimicrobial includes but is not limited to anti- Bacteriocin, antivirotic, antifungal agent, antiprotozoal, antiparasitic and anti-prion agent.
The medicament (delivering while for example, providing two kinds of medicaments) for example can be administered simultaneously with combined unit dose. It can also be spaced lower application medicament, the such as, but not limited to interval of a few minutes, hours, days or weeks at the appointed time.It is logical Often, medicament can be biological utilisation simultaneously in subject's body, such as detectable.It in some embodiments, can be substantially Medicament is administered simultaneously, such as applies the combination unit dose of two unit doses or two kinds of medicaments under same time.Other In embodiment, the unit dose that can be separated delivers medicament.It can be in any order or as including two or more medicaments One or more preparations apply medicament.It in preferred embodiments, can be in the number of another medicament (for example, second medicament) One of the medicament (example is carried out in minute, one hour, two hours, three hours or four hours or in even one day or two days Such as, the first medicament) application at least once.In some embodiments, combining can be achieved synergistic results, be greater than adduction knot Fruit, such as at least 25%, 50%, 75%, 100%, 200%, 300%, 400% or 500% greatly than adduction result.
Symptom relevant to bacterium infection
Can disease relevant to bacterium infection, illness or symptom include but is not limited to below one or more: abscess is put Line bacterium disease, acute prostatitis, Aeromonas hydrophila, annual ryegrass toxicity, anthrax, bacilus purpura, bacteremia, bacterium Property enterogastritis, meningitis, bacterial pneumonia, bacterial vaginitis, the relevant skin symptom of bacterium, bartonellosis (bartonellosis), BCG-oma, staphylococosis, botulismus, brazilian purpuric fever, Brodie tumour, brucellosis, Bu Luli ulcer, campylobacteriosis, saprodontia, carrion's disease, cat scratch disease, cellulitis, choamydiae infection, cholera, chronic bacterial Property prostatitis, the more stove osteomyelitis of chronic recurrent, clostridium necrotic enteritis, periodontic endodontic lesion, ox infectiousness pleura Pneumonia, diphtheria, diphtheritic stomatitis, Ehrlichiosis (ehrlichiosis), erysipelas, epiglottiditis (piglottitis), erysipelas, Fitz-Hugh-Curtis syndrome (Fitz-Hugh-Curtis syndrome), the propagated spotted fever of flea, foot rot (infectiousness Foot dermatitis) plus Lei Shi sclerosing osteomyelitis, stranguria syndrome, granuloma inguinale, human granular leukocyte anaplasmosis, the thermophilic monocyte angstrom of people In wish body disease, pertussis, impetigo, late congenital syphilis eye disease, legionellosis, Le meter Ai syndrome (Lemierre ' s Syndrome), leprosy (hansen's disease), leptospirosis, listerellosis, Lyme disease, lymphnoditis, glander-like disease, brain Meningococcus disease, Meningococcal septicaemia, methicillin-resistant staphylococcus aureus (MRSA) infection, mycobacterium avium-intracellulare (MAI), Eaton agent pneumonia, necrotizing fasciitis, nocardiasis, stomatonoma (subcutaneous ulcer of galloping along on horseback or stomatonecrosis), navel Inflammation, orbital cell ulitis, osteomyelitis, the splenectomy postoperative infection (OPSI) that can not be resisted, sheep brucella, pasteurellosis, Eye socket surrounding cells knit scorching, pertussis (pertussis) (pertussis (whooping cough)), the plague, pneumococcus lung Inflammation, Bo Teshi disease, rectitis, pseudomonas infection, psittacosis, pyemia, purulent myositis, Q heat, relapsing fever (relapsing Fever) (relapsing fever (typhinia)), rheumatic fever, American spotted fever (RMSF), rickettsiosis, salmonellosis, orangutan Red heat, sepsis, Serratieae infection, shigellosis, southern tick correlation exanthema, staphylococcus scald skin syndrome, chain Coccus pharyngitis, swimming pool granuloma, traum's disease, syphilis, syph-ilitic aortitis, tetanus, toxic shock are comprehensive Levy (TSS), trachoma, trench fever, Malabar ulcer, tuberculosis, tularemia, typhoid fever, typhus, urogenital tuberculosis, urine Road infection, vancomycin resistance infection of staphylococcus aureus, fertile-two Cotard (Waterhouse-Friderichsen of fluorine Syndrome), pseudotuberculosis (Yersinia ruckeri) disease and Yersiniosis.Other diseases relevant to bacterium infection, illness And/or symptom may include such as Alzheimer's disease, anorexia nervosa, asthma, atherosclerosis, pay attention to lacking and move more Disease, autism, autoimmune disease, bipolarity mental disease, cancer (for example, colorectal cancer, gallbladder cancer, lung cancer, cancer of pancreas with And gastric cancer), chronic fatigue syndrome, chronic obstructive pulmonary disease, Crohn's disease, coronary heart disease, dementia, depression, Ji-bars of synthesis Levy (Guillain-Barr é syndrome), metabolic syndrome, multiple sclerosis, myocardial infarction, obesity, obsessive compulsive Disease, paranoid fears, psoriasis, rheumatoid arthritis, sarcoidosis, schizophrenia, apoplexy, thromboangiitis obliterans (Burger Family name's disease) and tourette's syndrome.
Bacterial pathogens
Bacterium described herein can be gram-positive bacterium or gramnegative bacterium.Bacterial pathogens include but unlimited In Baume acinetobacter calcoaceticus (Acinetobacter baumannii), Bacillus anthracis (Bacillus anthracis), withered grass Bacillus (Bacillus subtilis), Bordetella pertussis (Bordetella pertussis), Bollinger body dredge spiral Body (Borrelia burgdorferi), Bacillus abortus (Brucella abortus), Brucellacanis (Brucella Canis), Bacterium melitense (Brucella melitensis), Brucella suis (Brucella suis), jejunum campylobacter Bacillus (Campylobacter jejuni), chlamydia pneumoniae (Chlamydia pneumoniae), chlamydia trachomatis (Chlamydia trachomatis), Chlamydophila psittaci (Chlamydophila psittaci), meat poisoning fusiform brood cell's bar Bacterium (Clostridium botulinum), clostridium difficile (Clostridium difficile), Clostridium perfringens brood cell's bar Bacterium (Clostridium perfringens), clostridium tetani (Clostridium tetani), coagulase-negative staphylococci (coagulase Negative Staphylococcus), Bacterium diphtheriae (Corynebacterium diphtheria), Enterococcus faecalis (Enterococcus faecalis), enterococcus faecium (Enterococcus faecium), Escherichia coli (Escherichia coli), enterotoxigenic E.Coli (enterotoxigenic Escherichia coli) (ETEC), Enteropathogenic E.Coli (enteropathogenic E.coli), Escherichia coli O 157: H7 (E.coli O157:H7), intestines Bacillus (Enterobacter sp.), francisella tularensis (Francisella tularensis), influenza are bloodthirsty Bacillus (Haemophilus influenzae), helicobacter pylori (Helicobacter pylori), klebsiella pneumoniae (Klebsiella pneumoniae), legionella pneumophilia (Legionella pneumophila), leptospria interrogans (Leptospira interrogans), Listeria Monocytogenes (Listeria monocytogenes), catarrh Catarrhalis (Moraxella catarralis), Mycobacterium leprae (Mycobacterium leprae), tuberculosis branch bar Bacterium (Mycobacterium tuberculosis), mycoplasma pneumoniae (Mycoplasma pneumoniae), Neisseria gonorrhoeae (Neisseria gonorrhoeae), meningococcus (Neisseria meningitides), proteus mirabilis (Preteus mirabilis), Proteus (Proteus sps.), Pseudomonas aeruginosa (Pseudomonas Aeruginosa), Rickettsia rickettsii (Rickettsia rickettsii), salmonella typhi (Salmonella Typhi), salmonella typhimurium (Salmonella typhimurium), serratia marcescens (Serratia Marcesens), Fu Shi Shigella (Shigella flexneri), Shigella (Shigella sonnei), golden yellow in pine Color staphylococcus (Staphylococcus aureus), staphylococcus epidermis (Staphylococcus epidermidis), corruption Raw staphylococcus (Staphylococcus saprophyticus), Streptococcusagalactiae (Streptococcus Agalactiae), Streptococcus mutans (Streptococcus mutans), streptococcus pneumonia (Streptococcus Pneumoniae), streptococcus pyogenes (Streptococcus pyogenes), Tyreponema pallidum (Treponema Pallidum), comma bacillus (Vibrio cholerae) and Yersinia pestis (Yersinia pestis).Bacterial disease Substance may also include the bacterium for causing drug resistant bacterial infections, such as the clostridium difficile of resistance to clindamycin, the difficult shuttle of resistance to fluoquinolone Bacterium, methicillin-resistant staphylococcus aureus (MRSA), multi-drug resistant object enterococcus faecalis, multi-drug resistant object enterococcus faecium, multi-drug resistant object Pseudomonas aeruginosa, multi-drug resistant object wave U.S. acinetobacter calcoaceticus and vancomycin resistance staphylococcus aureus (VRSA).
Antibiotic combinations
In one embodiment, modification mRNA of the invention can be applied together with one or more antibiotic.These Including but not limited to Aknilox, Ambisome (Ambisome), Amoxicillin (Amoxycillin), ammonia It must XiLin (Ampicillin), Augmentin (Augmentin), Moxifloxacin (Avelox), azithromycin (Azithromycin), mupirocin (Bactroban), povidone iodine (Betadine), betamethasone valerate (Betnovate), sulfacetamide prednisolone ocular fluid (Blephamide), Cefaclor (Cefaclor), cefadroxil (Cefadroxil), Cefdinir (Cefdinir), Cefepime (Cefepime), Cefixime (Cefix), Cefixime (Cefixime), Cefoxitin (Cefoxitin), Cefpodoxime (Cefpodoxime), Cefprozil (Cefprozil), cephalo Cefuroxime (Cefuroxime), Cefzil (Cefzil), cefalexin (Cephalexin), cephazoline (Cephazolin), Cefotaxime (Ceptaz), chloramphenicol (Chloramphenicol), Chlorhexidine (Chlorhexidine), chloramphenicol (Chloromycetin), Chlorsig, Ciprofloxacin (Ciprofloxacin), clarithromycin (Clarithromycin), chlorine Lincomycin phosphate (Clindagel), clindamycin (Clindamycin), Clindatech, cloxacillin (Cloxacillin), colistin (Colistin), Compound New Nomin (Co-trimoxazole), demeclocycline (Demeclocycline), dicloxacillin (Diclocil), dicloxacillin (Dicloxacillin), fortimicin (Doxycycline), cefadroxil (Duricef), erythromycin (Erythromycin), flamazine (Flamazine), Floxin, actiline (Framycetin), fucidin (Fucidin), furantoin (Furadantin), fusidinic acid (Fusidic), gatifloxacin (Gatifloxacin), gemifloxacin (Gemifloxacin), gemifloxacin (Gemifloxacin), Erythromycin Estolate (Ilosone), iodine, Levaquin (Levaquin), lavo-ofloxacin (Levofloxacin), Lomefloxacin (Lomefloxacin), Maxaquin (Maxaquin), cefoxitin mefoxin (Mefoxin), Meropenem (Meronem), minocycline (Minocycline), Moxifloxacin (Moxifloxacin), ethamine Butanol (Myambutol), nystatin (Mycostatin), neosporin (Neosporin), Netilmicin (Netromycin), furantoin (Nitrofurantoin), Norfloxacin (Norfloxacin), Norilet, Ofloxacin (Ofloxacin), Cefdinir piece (Omnicef), Amoxicillin trihydrate (Ospamox), oxytetracycline (Oxytetracycline), chloramphenicol (Paraxin), penicillin (Penicillin), Pnu-Imune 23 (Pneumovax), Polymyxin B (Polyfax), povidone (Povidone), R-76-1 (Rifadin), rifampin (Rifampin), rifaximin (Rifaximin), Rifinah (Rifinah), Rimactane (Rimactane), Ceftriaxone (Rocephin), roxithromycin (Roxithromycin), seromycin (Seromycin), neomycin (Soframycin), Sparfloxacin (Sparfloxacin), flucloxacillin sodium capsule (Staphlex), Targocid (Targocid), tetracycline (Tetracycline), Doxycycline (Tetradox), Armyl (Tetralysal), tobramycin (tobramycin), tobramycin (Tobramycin), 2-ethylisonicotinthionamide (Trecator), tigecycline (Tygacil), through the ages Mycin, fortimicin (Vibramycin), rifaximin (Xifaxan), spreads total nurse at Cefradine (Velosef) (Zagam), Zitrotek, Zoderm, Zymar and this fertile (Zyvox).
Antibacterial agent
Exemplary antibacterial agent includes but is not limited to aminoglycoside (for example, amikacin (amikacin)Gentamicin (gentamicin)Kanamycins (kanamycin)Neomycin (neomycin)Netilmicin (netilmicin)TobramycinParomomycin (Paromomycin) ), ansamycin (ansamycin) (for example, geldanamycin (geldanamycin), herbimycin (herbimycin)), carbon Cephem (carbacephem) is (for example, Loracarbef (loracarbef)Carbapenem (Carbapenem) (for example, ertapenem (ertapenem)Doripenem (doripenem)Imipenem (imipenem)/cilastatin (cilastatin)Metro training Southern (meropenem)Cephalosporin (cephalosporin) (first generation) is (for example, cefadroxilCephazoline (cefazolin)Cefoxitin (cefalotin) or cefoxitin (cefalothin)Cefalexin (cefalexin)Cephalosporin (second generation) (example Such as, CefaclorCefamandole (cefamandole)CefoxitinCefprozilCefuroxime), cephalosporin (third generation) is (for example, CefiximeCefdinirCephalo It holds in the palm logical sequence (cefditoren)Cefoperazone (cefoperazone)Cephalo Thiophene oxime (cefotaxime)CefpodoximeCefotaximeHead Spore cloth alkene (ceftibuten)Ceftizoxime (ceftizoxime)Ceftriaxone (ceftriaxone)), cephalosporin (forth generation) is (for example, Cefepime)、 Cephalosporin (the 5th generation) is (for example, Ceftobiprole (ceftobiprole)), glycopeptide is (for example, impersonate drawing Rather (teicoplanin)VancomycinTe Lawan star (telavancin)), lincosamide (lincosamide) is (for example, clindamycinLincomycin), lipopeptid is (for example, Daptomycin (daptomycin)), macrolide (for example, Ah Miramycin ( ), clarithromycin Dirithromycin (dirithromycin)Erythromycin Roxithromycin, troleandomycin (troleandomycin)Ketek (telithromycin)Spectinomycin (spectinomycin)), monobactam is (for example, aztreonam (aztreonam)), nitrofuran is (for example, furazolidoneFurantoin), penicillin is (for example, Amoxicillin AminobenzylpenicillinAzlocillin (azlocillin), Carbenicillin (carbenicillin)Cloxacillin (cloxacillin)DicloxacillinFlucloxacillin (flucloxacillin)Mezlocillin (mezlocillin)MethicillinNaphthlazole (nafcillin)Benzene azoles XiLin (oxacillin)Benzyl penicillinOspenPiperacillin (piperacillin)Temocillin (temocillin)Ticarcillin (ticarcillin)), penicillin combination (for example, Amoxicillin/gram Clavulanic acid saltAminobenzylpenicillin/Sulbactam (sulbactam)Piperacillin/tri- Zababatin (tazobactam)Ticarcillin/Clavulanate), polypeptide (for example, Bacitracin, colistinPolymyxin B, quinolone are (for example, Ciprofloxacin Enoxacin (enoxacin) GatifloxacinLavo-ofloxacinLomefloxacinMoses Sha XingAcidum nalidixicumNorfloxacinOfloxacinTrovafloxacin (trovafloxacin)Grepafloxacin (grepafloxacin)SparfloxacinTemafloxacin (temafloxacin)), sulfanilamide (SN) is (for example, mafenide (mafenide)Sulfanilamide (SN) chrysoidine (sulfonamidochrysoidine)Sulfacetamide (sulfacetamide)Sulphadiazine (sulfadiazine)Sulphur Amic metadiazine silverSulfamethizole (sulfamethizole) (THIOSULFIL), sulfanilamide (SN) First oxazole (sulfamethoxazole)Sulfanilamide (SN) Sulfafurazole (sulfanilamide), willow nitrogen sulphur Pyridine (sulfasalazine)Bacteresulf (sulfisoxazole)Trimethoprim (trimethoprim) First Oxygen benzyl pyridine-sulfamethoxazole (Compound New Nomin) (TMP-SMX)), tetracycline (for example, demeclocyclineDoxycyclineMinocyclineOxytetracyclineTetracycline ), resist mycobacteria drug (for example, Clofazimine (clofazimine)Dapsone (dapsone)Capreomycin (capreomycin)SeromycinEthambutol2-ethylisonicotinthionamideIsoniazid (isoniazid) PyrazinamideRifampinMycobutin (rifabutin)Rifapentine (rifapentine)Streptomysin) and its Its antibacterial agent is (for example, arsphenamine (arsphenamine)ChloramphenicolPhosphonomycin (fosfomycin)Fusidinic acidLinezolid (linezolid)Flagyl (metronidazole)MupirocinPlate mycin (platensimycin), Quinupristin (quinupristin)/Dalfopristin (dalfopristin)Rifaximin Thiamphenicol (thiamphenicol), tigecyclineTinidazole)。
Symptom relevant to virus infection
In another embodiment, provide for by with antivirotic (for example, antiviral polypeptide described herein Or small molecule antiviral) be administered in combination encoding antiviral polypeptide (such as antiviral polypeptide described herein) polynucleotides, Primary construct or mmRNA to treat or prevent virus infection and/or disease relevant to virus infection, illness in subject Or the method for symptom or its symptom.
Disease relevant to virus infection, illness or symptom include but is not limited to acute febrile pharyngitis, pharyngo-conjunctival fever, prevalence Property keratoconjunctivitis sicca, infantile gastroenteritis, Coxsackie virus infection (Coxsackie infection), infectious mononucleosis Disease, Burkitt lymphoma (Burkitt lymphoma), oxyhepatitis, chronic hepatitis, cirrhosis, hepatocellular carcinoma, primary HSV-1 infection (for example, children's gingivostomatitis, adult tonsillitis and pharyngitis, keratoconjunctivitis), latency HSV-1 infect (example Such as, herpes labialis and cold sore), primary HSV-2 infection, latency HSV-2 infection, aseptic meningitis, infectiousness monokaryon it is thin Born of the same parents' increase disease, cytomegalic inclusion disease, Kaposi sarcoma (Kaposi sarcoma), multicenter type Ka Siman disease (multicentric Castleman disease), lymphoma primary effusion, AIDS, influenza, Reye's syndrome (Reye syndrome), morbilli, postinfectious encephalomyelitis, parotitis, epithelial proliferation venereal disease become (for example, verruca vulgaris, verruca plana, Plantar wart and anogenital wart, papilloma of larynx, epidermodysplasia verruciformis), cervical carcinoma, squamous cell carcinoma, croup (croup), pneumonia, bronchiolitis, common cold, polio, rabies, bronchiolitis, pneumonia, influenza sample Syndrome, severe bronchiolitis are with pneumonia, German measles (German measle), congenital rubella, varicella and band-like Bleb.
Viral pathogen
Viral pathogen includes but is not limited to adenovirus, Coxsackie virus, dengue fever virus, encephalitis viruses, Ai-bars of Er Shi Virus, hepatitis A virus, hepatitis type B virus, Hepatitis C Virus, herpes simplex virus type 1, herpes simplex virus type 2, Cytomegalovirus, human herpesvirus 8,hhv 8, human immunodeficiency virus, influenza virus, measles virus, mumps virus, human milk head Tumor virus, parainfluenza virus, poliovirus, hydrophobin, Respiratory Syncytial Virus(RSV), rubella virus, varicella Herpesviral, west nile virus and flavivirus.Viral pathogen may also include the virus for causing drug-resistant viral infection.
Antivirotic
Exemplary antivirotic includes but is not limited to Abacavir (abacavir)Abacavir/drawing Meter Fu Ding (lamivudine)/Zidovudine (zidovudine)Acyclovir (aciclovir) or acyclovir (acyclovir) Adefovirdipivoxil (adefovir)AmantadineAmprenavir (amprenavir)Pacify Puli nearly (ampligen), arbidol (arbidol), atazanavir (atazanavir)Boceprevir (boceprevir), cidofovir (cidofovir), Prezista (darunavir)Delavirdine (delavirdine)DidanosineTwo DodecanolEdoxudine (edoxudine), efavirenz (efavirenz) Emtricitabine (emtricitabine)Emtricitabine/tenofovir (tenofovir)/efavirenzEn Fuwei (enfuvirtide)Entecavir (entecavir)Famciclovir (famciclovir)Fomivirsen (fomivirsen)Fosamprenavir (fosamprenavir)Foscanet (foscarnet)Phosphorus acetate (fosfonet), Ganciclovir (ganciclovir)GS 9137Imiquimod (imiquimod) Indinavir (indinavir)Inosine, isoprinosine (inosine pranobex)I type interferon, II type interferon, type iii interferon, kutapressinLamivudine Lamivudine/Qi Duofu It is fixedLopinavir (lopinavir), Loviride (loviride), Maraviroc (maraviroc)Metisazone (methisazone), MK-2048, moroxydine, Na Feina Wei (nelfinavir)Nevirapine (nevirapine)Oseltamivir (oseltamivir)Peg-IFN alpha-2b α -2aPenciclovir (penciclovir)Peramivir (peramivir), pleconaril (pleconaril), PodophyllotoxinIt draws for Wei (raltegravir) is drawnLi Ba Wei Lin (ribavirin)WithRimantadineRitonavir (ritonavir)It is phonetic Pyridine, inverase (saquinavir) Stavudine (stavudine), tea Set oily (thousand layers of oil (melaleuca oil)), tenofovirTenofovir/emtricitabineTipranavir (tipranavir)TrifluridineIt is bent AmantadineValaciclovir (valaciclovir)Valganciclovir (valganciclovir)Wei Liweiluo (vicriviroc), arabinosy ladenosine (vidarabine), Wei La Miaow determines (viramidine), zalcitabine (zalcitabine), zanamivir (zanamivir)And Zidovudine (retrovir (AZT),)。
Symptom relevant to fungal infection
Disease relevant to fungal infection, illness or symptom include but is not limited to aspergillosis, blastomycosis, Candida Disease, coccidioidomycosis, cryptococcosis, histoplasmosis, madura diseasemadura foot, paracoccidioidomycosis and epidermophytosis interdigitale.In addition, tool There is the people of immune deficiency to be especially susceptible to infection by fungi such as aspergillus (Aspergillus), Mycotoruloides (Candida), hidden Coccus (Cryptoccocus), Histoplasma (Histoplasma) and Pneumocystis category (Pneumocystis) cause Disease.Other fungies can encroach on eye, nail, hair, and especially (so-called dermatophyte and thermophilic keratin are true for skin Bacterium), and cause various symptom, wherein relatively conventional with ringworm such as athlete's foot (athlete ' s foot).Fungal spore is also The main reason for allergy, and some allergic reactions can be excited from the wide scope fungi of different classifications group.
Fungal pathogens
Fungal pathogens include but is not limited to Ascomycota (Ascomycota) (for example, sharp born of the same parents sickle-like bacteria (Fusarium Oxysporum), Pneumocystis carinii (Pneumocystis jirovecii), Eurotium (Aspergillus spp.), thick ball Pityrosporion ovale (Coccidioides immitis)/coccidioides immitis (posadasii), Candida albicans (Candida Albicans)), Basidiomycota (Basidiomycota) is (for example, filobasidiella neoformans (Filobasidiella Neoformans), trichosporon bacteria (Trichosporon)), Microsporida (Microsporidia) is (for example, rabbit brain original intracellular Worm (Encephalitozoon cuniculi), diarrhea protozoa (Enterocytozoon bieneusi)) and mucor subphylum (Mucoromycotina) (for example, volume branch Mucor (Mucor circinelloides), Rhizopus oryzae (Rhizopus oryzae), Absidia corymbifera (Lichtheimia corymbifera)).
Antifungal agent
Exemplary antifungal agent includes but is not limited to polyene antifungal agent (for example, natamycin (natamycin), cracking kill Rhzomorph (rimocidin), filipin (filipin), nystatin, amphotericin B, candicin (candicin), Kazakhstan are mould Plain (hamycin)), imidazole antifungal agents are (for example, Miconazole (miconazole)Ketoconazole (ketoconazole) Clotrimazole (clotrimazole) Econazole (econazole), Omoconazole (omoconazole), Bifonazole (bifonazole), butoconazole (butoconazole), Fenticonazole (fenticonazole), Isoconazole (isoconazole), Oxiconazole (oxiconazole), Sertaconazole (sertaconazole)Sulphur Health azoles (sulconazole), tioconazole (tioconazole)), triazole anti-fungal agents are (for example, Ah 's health azoles (albaconazole), Fluconazole (fluconazole), Itraconazole (itraconazole), Chinese mugwort Saperconazole (isavuconazole), ravuconazole (ravuconazole), posaconazole (posaconazole), voriconazole (voriconazole), terconazole (terconazole)), thiazole antifungal agent (for example, Abafungin (abafungin)), alkene Propylamine is (for example, Terbinafine (terbinafine)Naftifine (naftifine)Cloth For naphthalene fragrant (butenafine) (Ultra)), echinocandin (echinocandin) is (for example, anidulafungin (anidulafungin), Caspofungin (caspofungin), mikafen (micafungin)) and other antifungal agents (for example, polygodial (polygodial), benzoic acid, Ciclopirox, TolnaftateUndecenoic acid, Flucytosine or 5-flurocytosine, ash Flavomycoin (griseofulvin), Haloprogin (haloprogin), sodium bicarbonate, allicin).
Symptom relevant to protozoal infections
Disease relevant to protozoal infections, illness or symptom include but is not limited to amcbiasis, Giardiasis, hair drop Parasitosis, lethargus, U.S.'s difussa, leishmaniasis (kala-azar (Kala-Azar)), balantidiasis, toxoplasmosis, malaria Disease, Acanthamoeba Keratitis and babesiasis.
Protozoal pathogens
Protozoal pathogens include but is not limited to entamoeba historlytica (Entamoeba histolytica), merchant's whip Caterpillar category (Giardia lambila), trichomonas vaginalis (Trichomonas vaginalis), trypanosoma bocagei Gambia (Trypanosoma brucei), schizotrypanum cruzi (T.cruzi), Leishmania donovani (Leishmania donovani), bowel bag Eimeria (Balantidium coli), toxoplasma gondii (Toxoplasma gondii), Plasmodium (Plasmodium spp.) And Babesiamicrofti (Babesia microti).
Antiprotozoal
The lively agent of Exemplary antigens includes but is not limited to Eflornithine (eflornithine), furazolidoneMelarsoprol (melarsoprol), metronidazoleOrnidazole (ornidazole), paromomycin sulfate (paromomycin sulfate)Pentamidine (pentamidine), pyrimethamine (pyrimethamine)With And Tinidazole (tinidazole)
Symptom relevant to parasitic infection
Disease relevant to parasitic infection, illness or symptom include but is not limited to Acanthamoeba Keratitis, amcbiasis, Roundworm disease, babesiasis, balantidiasis, shellfish roundworm disease (baylisascariasis), chagas disease, clonorchiasis, cone fly Disease (cochliomyia), Cryptosporidiosis, bothrio-cephaliasis, drachunculiasis, echinococcosis, elephantiasis, enterobiasis, piece Fluke disease, fasciolopsiasis, filariasis, giardiasis, gnathostomiasis, hymenolepiasis, isosporiasis, oncomelania Heat, leishmaniasis, Lyme disease, malaria, metagonimiasis, fly-blown, onchocercosis, pediculosis, scabies, snail fever, lethargic sleep Disease, strongyloidiasis, taeniasis, toxocarasis, toxoplasmosis, trichinosis and trichuriasis.
Parasitic agent
Parasitic agent include but is not limited to Acanthamoeba (Acanthamoeba), Anisakid nematode (Anisakis), Ascaris lumbricoides (Ascaris lumbricoides), horse botfly (botfly), Balantidium, bedbug, Cestoda (Cestoda), trombiculid, screwfly (Cochliomyia hominivorax), entamoeba historlytica, Fasciola hepatica (Fasciola Hepatica), giardia lamblia (Giardia lamblia), hookworm, Leishmania (Leishmania), zigzag ligulate Worm (Linguatula serrata), liver fluke, Loa loa (Loa loa), Paragonimus (Paragonimus), pinworm, Plasmodium falciparum (Plasmodium falciparum), Schistosoma (Schistosoma), Strongyloides intestinalis (Strongyloides stercoralis), mite, tapeworm, toxoplasma gondii (Toxoplasma gondii), Trypanosomonas (Trypanosoma), whipworm, wuchereria bancrofti (Wuchereria bancrofti).
Antiparasitic
Exemplary antiparasitic includes but is not limited to anti-nematode agent (for example, mebendazole, Pyrantel Pamoate, thiophene Parbendazole, diethylcarbamazine, ivermectin (ivermectin)), anti-tapeworm agent is (for example, niclosamidum (niclosamide), pyrrole quinoline Ketone (praziquantel), albendazole (albendazole)), anti-fluke agent (for example, praziquantel), resistance to deformation worm agent (example Such as, rifampin, amphotericin B) and antiprotozoal (for example, melarsoprol, Eflornithine, metronidazole, Tinidazole).
To prion-infected relevant symptom
It include but is not limited to Creutzfeldt-Jakob disease to prion-infected relevant disease, illness or symptom (CJD), medicine origin Creutzfeldt-Jakob disease (iCJD), variant Creutzfeldt-Jakob disease (vCJD), Familial Creutzfeldt-Jakob disease (fCJD), sporadic Creutzfeldt-Jakob disease (sCJD), lattice- It is Shi-sand syndrome (GSS), fatal familial insomnia (FFI), kuru, scrapie, bovine spongiform encephalopathy (BSE), crazy Cattle disease, infectious mink encephalopathy (TME), chronic wasting disease (CWD), cat spongiform encephalopathy (FSE), exotic ungulate encephalopathy (EUE) and spongiform encephalopathy.
Anti-prion agent
Exemplary anti-prion agent includes but is not limited to Flupirtine (flupirtine), the more sulfate of pentosan (pentosan polysuphate), quinacrine (quinacrine) and tetracyclic compound.
The adjusting of immune response
Immune response avoids
As described herein, the useful feature of polynucleotides of the invention, primary construct or mmRNA is can to reduce, escape Keep away or avoid the innate immune response of cell.On the one hand, there is provided herein the polynucleotides of encoding target polypeptide, primary building Body or mmRNA cause the immune response from host compared with the response triggered by reference compound when it is delivered to cell Reduced, the reference compound for example, corresponds to the unmodified of polynucleotides of the invention, primary construct or mmRNA Polynucleotides or different polynucleotides of the invention, primary construct or mmRNA.As used herein, " reference compound " is Cause any point of the innate immune response with the immunostimulation of known degree, level or amount when applying to mammal Son or substance.Reference compound is not necessarily nucleic acid molecules and it is not necessarily any polynucleotides of the invention, primary construct Or mmRNA.Therefore, polynucleotides, just can be indicated relative to any compound or substance of such a response of known triggering Grade construct or mmRNA are avoided, are escaped or cannot trigger the measurement of immune response.
Term " innate immune response " includes answering the cell of external source single-chain nucleic acid (being typically derived from virus or bacterium) It answers, is related to inducing cytokine expression and release (specially interferon) and cell death.As used herein, congenital Immune response or interferon response are causing cytokine-expressing, cytokine release, the full inhibition of protein synthesis, cell RNA Full destruction, the up-regulation of ajor histocompatibility molecule and/or apoptotic death induction, the genetic transcription for the gene for being related to apoptosis lure It leads, operate under antibiosis is long and congenital and adaptive immunity cell-stimulating individual cell level.By some of I type IFN induction Gene include PKR, ADAR (adenosine deaminase for acting on RNA), OAS (2 ', 5 '-oligoadenylate synthetase), RNA enzyme L and Mx albumen.PKR and ADAR causes to inhibit translation initiation and rna editing respectively.OAS is activator RNA restriction endonuclease RNA enzyme L drop Solve the dsRNA- dependence synzyme of ssRNA.
In some embodiments, innate immune response includes I type or II type interferon expression, and I type or II type Interferon expression is the same as from not yet compared with the reference of the cell of polynucleotides of the invention, primary construct or mmRNA contact Do not increase more than twice.
In some embodiments, innate immune response includes the expression of one or more IFN signature genes, and its The expression of middle one or more IFN signature gene with from not yet being connect with polynucleotides of the invention, primary construct or mmRNA The reference of the cell of touching, which is compared, is more than three times without increasing.
And in some cases, the innate immune response eliminated in cell can be to be advantageous, and the present invention provides applying Used time causes immune response (including interferon signal transduction) generally to reduce (significantly smaller) but does not completely eliminate this answer Polynucleotides, primary construct and the mmRNA answered.
In some embodiments, compared with the immune response induced by reference compound, immune response reduction by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9% or be greater than 99.9%.Immune response Itself can be by the expression of determining 1 type interferon or activity level or interferon regulation gene such as toll sample receptor (for example, TLR7 And TLR8) expression measure.The water of the measurement cell death reduction after one or many applications to cell mass can also be passed through It puts down to measure the reduction of innate immune response；Such as cell death is compared for cell death observed by reference compound Frequency lacks 10%, 25%, 50%, 75%, 85%, 90%, 95% or more than 95%.In addition, cell death can influence to be less than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or less than 0.01% with polynucleotides, primary structure Build the cell of body or mmRNA contact.
In another embodiment, the immunogenicity of polynucleotides of the invention, primary construct or mmRNA is significantly small In with mutually homotactic unmodified vitro synthesized RNA molecule polynucleotides or primary construct or reference compound.Such as this Text is used, and " immunogenicity significantly less than " refers to the detectable reduction of immunogenicity.In another embodiment, term is Refer to that the multiple of immunogenicity reduces.In another embodiment, term refer to so that can apply a effective amount of polynucleotides, just Grade construct or mmRNA but the reduction without triggering detectable immune response.In another embodiment, term is to instigate Application polynucleotides, primary construct or mmRNA must be repeated but do not cause to be enough detectably to reduce recombinant protein expression Immune response reduction.In another embodiment, reduction be so that repeatable application polynucleotides, primary construct or MmRNA but the immune response for not causing to be enough to eliminate detectable recombinant protein expression.
In another embodiment, the immunogenicity of polynucleotides, primary construct or mmRNA pair more unmodified than its Answer object or reference compound 2 times small.In another embodiment, immunogenicity reduces 3 times.In another embodiment, Immunogenicity reduces 5 times.In another embodiment, immunogenicity reduces 7 times.In another embodiment, immunogene Property reduce 10 times.In another embodiment, immunogenicity reduces 15 times.In another embodiment, immunogenicity subtracts Small certain multiple.In another embodiment, immunogenicity reduces 50 times.In another embodiment, immunogenicity subtracts It is 100 times small.In another embodiment, immunogenicity reduces 200 times.In another embodiment, immunogenicity reduces 500 times.In another embodiment, immunogenicity reduces 1000 times.In another embodiment, immunogenicity reduces 2000 times.In another embodiment, it is poor to reduce another multiple for immunogenicity.
The method for determining immunogenicity is well known in the art, and including for example measuring cell factor (such as IL- 12, IFN α, TNF-α, RANTES, MIP-1 α or MIP-1 β, IL-6, IFN-β or IL-8) secretion, measurement DC activation marker object The ability of the adjuvant for adaptive immune response is served as in the expression or measurement of (such as CD83, HLA-DR, CD80 and CD86).
Polynucleotides of the invention, primary construct or mmRNA (including the modification combination instructed herein) can have and make it It is more suitable for the advantageous characteristic of therapeutic modality.
It is relevant to the modification therapeutical uses of mRNA to have determined that " all or none " model in this field is not enough to describe very much Biological phenomenon.Inventor has determined that in order to improve protein and generate, people are contemplated that modification or modify combined property, modification hundred Divide than and investigate the effect of more than one cell factor or measurement are to determine specific modification mRNA and risk profile.
In one aspect of the invention, compared with unmodified determine modification mRNA validity method be related to measurement and One or more cell factors are analyzed, the expression of the cell factor is triggered by applying exogenous nucleic acid of the invention.By this Applications or gauge such as cytokine response, PolyIC, R-848 of a little values with unmodified nucleic acid or it is as known in the art its Its reference substance compares.
One example of the gauge developed herein is the coding polypeptide that measurement generates in cell, tissue or organism The level of (protein) is measured and triggers its expression in cell, tissue or organism due to apply or contacting modification of nucleic acids One or more (or one group) levels of cell factor or the ratio of amount.Such ratio is referred to herein as protein: cell because Son ratio or " PC " ratio.PC ratio is higher, and modification of nucleic acids (polynucleotides of the measured protein of coding) effect is bigger.Of the invention Preferred PC ratio in terms of cell factor may be greater than 1, be greater than 10, be greater than 100, be greater than 1000, be greater than 10,000 or more.Have The modification of nucleic acids of PC ratio more higher than the modification of nucleic acids of different or unmodified construct is preferred.
PC ratio can be further limited by the modification percentage being present in polynucleotides.For example, being normalized to 100% Modification of nucleic acids, it may be determined that as the protein of cell factor (or risk) or the function of cytokine profile generate.
In one embodiment, the present invention provides for by comparing modification of nucleic acids (polynucleotides, primary construct Or mmRNA) PC ratio and any specific modification multicore glycosides is determined by chemical property, cell factor or modification percentage The method of the relative potency of acid, primary construct or mmRNA.
It can produce the mmRNA containing different nucleobase substitution levels, Protein requirement is generated to increase and be dived with immunostimulation Power reduces.The relative percentage of any modified nucleoside acid and its naturally occurring nucleotide counterpart can be in IVT reaction process Change (for example, using 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.1%, 0.01%5 methylcytidine and cytidine； Use 100%, 50%, 25%, 10%, 5%, 2.5%, 1%, 0.1%, 0.01% pseudouridine or N1- methyl-pseudouridine and urine Glycosides).Also the mmRNA using different ratios can be made (for example, not using 2 or more different nucleotide to identical base With pseudouridine and N1- methyl-pseudouridine of ratio).It can also be made on more than one " base " position while there is blending ratio MmRNA, such as 5 methylcytidines/cytidine and pseudouridine/N1- methyl-pseudouridine/uridine ratio.Use the modification with change The modification mRNA of nucleotide ratio can be beneficial to reduce the potential exposure to chemically modified nucleoside acid.Finally, regulatory protein matter produces It is also possible that the position that raw or immunostimulatory potential or the modified nucleoside acid of the two enter mmRNA, which introduces,.It external (can use The measurement of PBMC measuring method as described herein) ability that the mmRNA shows these improvement characteristics is evaluated, and can also be internal It is generated by the protein of measurement mmRNA coding with the mediators such as cell factor of congenital immunity identification and is evaluated.
In another embodiment, by determining the unmodified nucleotide or reference compound degree that cause with specified rate The amount of polynucleotides required for one of identical above response, primary construct or mmRNA determines polynucleotides, primary structure Build the comparative immunogenicity of body or mmRNA and its unmodified counterpart.For example, if identical response is caused to need twice Polynucleotides, primary construct or mmRNA, then the immunogenicity of polynucleotides, primary construct or mmRNA is than unmodified nucleosides Acid or reference compound are twice small.
In another embodiment, it is responded by determining relative to same amount of unmodified nucleotide or reference compound The cell factor secreted by the application of polynucleotides, primary construct or mmRNA (such as IL-12, IFN α, TNF-α, RANTES, MIP-1 α or MIP-1 β, IL-6, IFN-β or IL-8) amount come determine polynucleotides, primary construct or mmRNA with And its comparative immunogenicity of unmodified counterpart.For example, if secretion half cell factor, polynucleotides, primary structure The immunogenicity for building body or mmRNA is twice smaller than unmodified nucleotide.In another embodiment, in calculating above method Immunogenicity before deduct stimulation background level.
It is also provided herein the titration for carrying out the immune response in a kind of cell or a group cell, reduces or eliminates Method.In some embodiments, cell is contacted simultaneously with identical polynucleotides, primary construct or the mmRNA for changing dosage And evaluation dose response.In some embodiments, make cell under identical or different dosage many different polynucleotides, just Grade construct or mmRNA contact are used to generate to wish that the optimal of effect forms to determine.About immune response, it is desirable to effect can For the immune response for avoiding, escaping or reduce cell.The efficiency that desired effect can also generate for change protein.
Polynucleotides, primary construct and/or mmRNA of the invention is may be used in international publication number WO2013003475 The method of description is used to reduce immune response, and the patent is incorporated herein in its entirety by reference.
The activation of immune response: vaccine
In addition, certain modified nucleosides or its group when being introduced into polynucleotides of the invention, primary construct or mmRNA Conjunction will activate innate immune response.When with polypeptide and/or other vaccine combinations, such activating molecules can be used as adjuvant.? In certain embodiments, activating molecules contain coding be used as vaccine polypeptide sequence can translated region, thus provide as from help The ability of agent.
In one embodiment, polynucleotides of the invention, primary construct and/or mmRNA codified immunogene.It compiles The delivering of the polynucleotides of code immunogene, primary construct and/or mmRNA can activate immune response.It is non-limiting as one Example, the polynucleotides of encoding immunogens, primary construct and/or mmRNA can be delivered to cell to trigger multiple congenital answer Approach is answered (referring to international publication number WO2012006377；It is incorporated herein in its entirety by reference).As another non-limit Property example processed, the polynucleotides of the invention of encoding immunogens, primary construct and mmRNA can be sufficiently large to vertebrate The dose delivery of tool immunogenicity is to vertebrate (referring to international publication number WO2012006372 and WO2012006369；It is each From being incorporated herein in its entirety by reference).
Polynucleotides of the invention, primary construct or mmRNA codified vaccine polypeptide sequence and can further wrap Containing inhibitor.Inhibitor can damage antigen submission and/or inhibit various approach as known in the art.It is non-limiting as one Example, polynucleotides of the invention, primary construct or mmRNA can be combined with the inhibitor that can damage antigen submission and be used for vaccine (referring to international publication number WO2012089225 and WO2012089338；It is respectively incorporated herein in its entirety by reference).
In one embodiment, polynucleotides of the invention, primary construct or mmRNA can be the RNA of self-replacation. The RNA molecule of self-replacation can enhance the expression of the efficiency of RNA delivery and the gene product of encapsulating.In one embodiment, Polynucleotides, primary construct or mmRNA may include being described herein and/or at least one modification as known in the art.One In a embodiment, the RNA of self-replacation may be designed so that self-replacation RNA will not inducing infectious virion production It is raw.It as a non-limiting example, can be by U.S. Publication No US20110300205 and international publication number Method described in WO2011005799 designs the RNA of self-replacation, and the patent is respectively incorporated hereby Herein.
In one embodiment, the polynucleotides of self-replacation of the invention, primary construct or mmRNA codified can Cause the protein of immune response.As a non-limiting example, polynucleotides, primary construct or mmRNA can be that can compile The mRNA of the self-replacation of at least one antigen of code is (referring to U.S. Publication No US20110300205 and international publication number WO2011005799, WO2013006838 and WO2013006842；It is respectively incorporated herein in its entirety by reference).
In one embodiment, this can be used in the polynucleotides of self-replacation of the invention, primary construct or mmRNA Text description or method as known in the art are prepared.As a non-limiting example, the RNA of self-replacation can pass through Geall Method (Nonviral delivery of self-amplifying RNA vaccines, the PNAS 2012 of equal descriptions； PMID:22908294 it) is formulated for delivering.
In one embodiment, polynucleotides of the invention, primary construct or mmRNA codified peptide amphiphile and/ Or immunogenicity peptide amphiphile.
In one embodiment, the preparation of polynucleotides of the invention, primary construct or mmRNA can further include Peptide amphiphile and/or immunogenicity peptide amphiphile.It as a non-limiting example, include peptide amphiphile and/or immunogenicity The polynucleotides of peptide amphiphile, primary construct or mmRNA can be such as U.S. Publication No US20110250237 and international publication numbers It is prepared described in WO2010009277 and WO2010009065；The patent is respectively incorporated herein in its entirety by reference.
In one embodiment, polynucleotides of the invention, primary construct or mmRNA can be immunostimulating. As a non-limiting example, polynucleotides, primary construct or mmRNA codified positive-sense strand or antisense strand RNA virus base It is all or part of (respectively equal referring to international publication number WO2012092569 and U.S. Publication No US20120177701 because of group It is incorporated herein in its entirety by reference).In another non-limiting example, immunostimulating polynucleotides of the invention, Primary construct or mmRNA can be used the excipient as described herein and/or as known in the art for application (referring to state Border publication No. WO2012068295 and U.S. Publication No US20120213812 are respectively incorporated hereby this Text).
In one embodiment, can be enhanced with inductive treatment effect by being described herein by adding various compounds Method prepare vaccine response.As a non-limiting example, vaccine preparation may include MHC II binding peptide or have The peptide of the sequence similar with MHC II binding peptide is (public referring to international publication number WO2012027365, WO2011031298 and the U.S. Cloth US20120070493, US20110110965, are respectively incorporated herein in its entirety by reference).As another Example, vaccine preparation may include the nicotinic compounds that the modification of antibody response can be generated to the intracorporal nicotine residue of subject It is (respectively whole by reference referring to international publication number WO2012061717 and U.S. Publication No US20120114677 It is incorporated herein).
Naturally occurring mutant
In another embodiment, it can be used polynucleotides, primary construct and/or mmRNA naturally occurring to express Protein variant, the variant have improved disease modifying activity, bioactivity, improved patient including enhancing turn Return or defencive function etc..Many such modifiers (Nadeau, Current Opinion in is described in mammals 2003 13:290-295 of Genetics & Development；Hamilton and Yu, PLoS Genet.2012；8: e1002644；1994 7:180-184 of Corder etc., Nature Genetics；All bibliography are whole by reference Body is incorporated herein).Example for the mankind includes Apo E2 albumen, Apo A-I misfolded proteins (Apo A-I Milano, Apo A-I Paris), high activity IX factor protein (IX factor Padua Arg338Lys), transthyretin mutant (TTR Thr119Met).Have been displayed the expression of ApoE2 (cys112, cys158) by reduce to Alzheimer's disease and possibly its The easy acceptabilily of its symptom such as cardiovascular disease comes relative to other ApoE isoforms (ApoE3 (cys112, arg158) and ApoE4 (arg112, arg158)) it gives and protects (1994 7:180-184 of Corder etc., Nature Genetics；Seripa etc., Rejuvenation Res.2011 14:491-500；Liu et al., Nat Rev Neurol.2013 9:106-118；All references Document is incorporated herein in its entirety by reference).The expression of Apo A-I variant it is related with cholesterol reduction (deGoma and Rader, 2011 Nature Rev Cardiol 8:266-271；Nissen etc., 2003 JAMA 290:2292-2300；It is all Bibliography is incorporated herein in its entirety by reference).The amino acid sequence of ApoA-I changes into Apo in certain groups (Arg 151 changes for cysteine (Arg 173 changes into Cys) in A-I Milano and the cysteine in Apo A-I Paris Become Cys).IX element mutation (FIX Padua) on the R338L of position generates the IX factor protein that activity increases about 10 times (Simioni etc., N Engl J Med.2009 361:1671-1675；Finn etc., Blood.2012 120:4521-4523； Cantore etc., Blood.2012 120:4517-20；All bibliography are incorporated herein in its entirety by reference).It has shown The mutation (Arg104 His, Thr119Met) for showing the transthyretin on position 104 or 119 is also to carry to cause The patient of the disease of Val30Met mutation provides protection (Saraiva, Hum Mutat.2001 17:493-503；DATA BASE ON TRANSTHYRETIN MUTATIONS http://www.ibmc.up.pt/mjsaraiva/ttrmut.html；All ginsengs It examines document to be incorporated herein in its entirety by reference).Observe respectively carry Met 119 and His104 mutation Portugal and The difference of clinical manifestation and symptom severity between Japanese 30 patient of Met is obvious with being generated by avirulence mutant Protective effect (Coelho etc., 1996 Neuromuscular Disorders (Suppl) 6:S20；Terazaki etc., 1999.Biochem Biophys Res Commun 264:365-370；All bibliography it is whole by reference simultaneously Enter herein), the avirulence mutant gives molecule more multistability.Encode the modification of these protectiveness TTR allele MRNA can be expressed in the patient of TTR amyloidosis, to reduce the effect of causative mutation TTR albumen.
Major groove acts on gametophyte
As described herein, phrase " major groove effect gametophyte " refers to through the phase interaction with nucleotide or the big groove face of nucleic acid It is detected with (such as in conjunction with) and response is in the RNA identification receptor of RNA ligand.Equally, such as comprising modified nucleoside acid or nucleic acid The RNA ligand of polynucleotides described herein, primary construct or mmRNA reduces the interaction with major groove binding partners And therefore reduce innate immune response.
Exemplary major groove effect (as combined) gametophyte includes but is not limited to following nuclease and unwindase.In film, TLR (TOll- sampleReceptor) 3,7 and 8 can response in single-stranded and double-stranded RNA.In cytoplasm, DEX (D/H) unwindase and ATP enzyme it is super The member of 2 class of family can sense RNA to originate antiviral response.These unwindases include RIG-I (Retinoic acid-Induced gene I) and MDA5 (Melanoma Differentiation-It is relatedGene 5).Other examples includeScience of heredityWithPhysiology Experiment roomAlbumen 2 (LGP2) contains There are the protein of HIN-200 structural domain or the protein containing helicase domain.
The targeting of Pathogenic organisms or diseased cells
There is provided herein for using Codocyte to inhibit the polynucleotides of polypeptide or cytotoxic polypeptide, primary construct Or mmRNA is come the method that targets pathogenic microorganism such as bacterium, yeast, protozoan, worm etc. or diseased cells such as cancer cell.It is excellent Selection of land, introduced mRNA contain special or translate preferably in target Pathogenic organisms to reduce the possible effect of missing the target of therapeutic agent The modified nucleoside answered or the modification of other nucleic acid sequences.Such method, which has, is present in any life for removing Pathogenic organisms or killing Object material includes that blood, sperm, ovum and graft materials include diseased cells in embryo, tissue and organ.
Biological treatment
Method provided herein can have for enhancing the protein product yield in cell cultivation process.Containing multiple places In the cell culture of chief cell, polynucleotides, primary construct or the mmRNA for being incorporated herein description cause relative to correspondence not The protein generation efficiency of modification of nucleic acids increases.Can for example it be increased by display cell transfecting, from polynucleotides, primary construct Or the protein translation of mmRNA increases, nucleolysis is reduced and/or the innate immune response of host cell is reduced to prove State the increase of protein generation efficiency.Protein can be measured by enzyme linked immunosorbent assay (ELISA) (ELISA) to generate, and this can be passed through Known various functional examinations measure protein active in field.It can be sent out during mammal that is continuous or being fed in batches Raw egg white matter generates.
In addition, optimization potential target cell line or a series of particular polypeptide in cell lines expression be it is useful, it is described Polypeptide is specially the protein variant that target polypeptides such as have the reference protein matter of known activity.In one embodiment, it mentions It has supplied by the multiple target cell types of offer and independently to make each in multiple target cell types and encoding target polypeptide Polynucleotides, primary construct or mmRNA contact optimize the method for the expression of the target polypeptides in target cell.Cell can be same When or successively transfected with two or more polynucleotides, primary construct or mmRNA.
In certain embodiments, more wheel method described hereins can be used to obtain with increased one or more mesh Mark the cell of the expression of nucleic acid or protein.For example, one or more polynucleotides of available code target nucleic acid or protein, Primary construct or mmRNA transfect cell.Before separating again, by the one or more encoding target cores of other a few wheels Before other nucleic acid transfections of acid or protein, cell can be separated according to the method described in this article.The method can have for generating The cell of expression with increased following substance: the compound of protein, the nucleic acid in identical or relevant biological approach Or protein, upstream each other or downstream effects nucleic acid or protein, each other have adjust, activation or pressing function nucleic acid Or protein, depending on function each other or the nucleic acid or protein of active nucleic acid or protein or shared homology.
In addition, changeable condition of culture is to increase protein generation efficiency.Then, detection and/or quantitative multiple target cells The presence and/or level of target polypeptides in type, to allow by selecting effective target cell and relative cell Condition of culture optimizes the expression of polypeptide.Such method polypeptide contain one or more posttranslational modifications or have a large amount of three Level structure and the case where usually make effective protein generate complication when it is particularly useful.
In one embodiment, it can cultivate for the cell in method of the invention.Can suspend culture or adherent training Feeding form culture cell.Cell, including but not limited to bioreactor, cell culture bags can be cultivated in various containers (cell bag), bag (wave bag), culture plate, culture bottle and other containers well known within the skill of those ordinarily skilled are shaken.Carefully Born of the same parents can include but is not limited to that chemistry is fixed in IMDM (Invitrogen, catalog number (Cat.No.) 12440-53) or any other suitable culture medium It is cultivated in the culture based formulation of justice.The environmental condition such as temperature and Atmospheric composition for being applicable to cell culture are those skilled in the art Known to member.Method of the invention is used in combination with any cell generated suitable for protein.
The present invention provides modification of nucleic acids is repeatedly introduced (for example, transfection) for example in vitro, in vitro, in situ or in vivo Into target cell group.For example, contact same cell group may be repeated one or more times (as twice, three times, four times, five times or be more than Five times).In some embodiments, to make the number for reaching predetermined protein translation efficiency in cell mass enough Carry out the step of repetitive cell group contacts with polynucleotides, primary construct or mmRNA.In view of the target cell provided by nucleic acid modification The cytotoxicity of group usually reduces, can be real in a series of different cell types and in various tissues as herein provided It now repeats to transfect.
In one embodiment, bioremediation of the invention can be used to generate antibody or its function fragment.Function Segment may include Fab, Fab ', F (ab ')2, Fv structural domain, scFv or double antibody.They can include complementary determining region in any area It (CDR) is variable in.In one embodiment, there are complete diversity in the area CDR3.In another embodiment, In addition in the area CDR3, antibody is substantially conservative.
The antibody in conjunction with any biomolecule or to associate, the biomolecule either people source, pathogenicity can be made Or inhuman source.Pathogen may be present in non-human mammal, in clinical sample or from commercial product such as cosmetics or drug Material.They may also be combined with any sample or sample, including clinical sample or tissue sample from any organism.
In some embodiments, selected from by 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 72 hours, 84 Under the frequency of the group of the composition of hour, 96 hours and 108 hours and it is being less than 20nM, is being less than 50nM, less than 80nM or less than 100nM Concentration under be repeated as many times contact procedure.Can also be less than 1mM, less than 5mM, less than 10mM, less than 100mM or be less than Composition is applied under 500mM.
In some embodiments, with 50 molecule/cells, 100 molecule/cells, 200 molecule/cells, 300 Molecule/cell, 400 molecule/cells, 500 molecule/cells, 600 molecule/cells, 700 molecule/cells, 800 points Son/cell, 900 molecule/cells, 1000 molecule/cells, 2000 molecule/cells or 5000 molecule/cell amounts add Add nucleotide, primary construct or mmRNA.
In other embodiments, to add polynucleotides, primary construct under the concentration selected from the group being made up of Or mmRNA:0.01fmol/106 cell, 0.1fmol/106 cell, 0.5fmol/106 cell, 0.75fmol/106 Cell, 1fmol/106 cell, 2fmol/106 cell, 5fmol/106 cell, 10fmol/106 cell, 20fmol/ It is 106 cells, 30fmol/106 cell, 40fmol/106 cell, 50fmol/106 cell, 60fmol/106 thin Born of the same parents, 100fmol/106 cell, 200fmol/106 cell, 300fmol/106 cell, 400fmol/106 cell, 500fmol/106 cell, 700fmol/106 cell, 800fmol/106 cell, 900fmol/106 cell and 1pmol/106 cell.
In some embodiments, biologic is detected by the one or more measurable bioprocess parameters of monitoring It generates, the measurable bioprocess parameter is, for example, to be selected from the parameter for the group being made up of: cell density, pH, oxygen water Flat, glucose level, lactate level, temperature and protein generate.Protein generation can measure as specific production rate (SP) (in solution Product such as heterogenous expression polypeptide concentration) and can be expressed as mg/L or g/L；In an alternate embodiment, specific production rate It can be expressed as pg/ cell/day.SP increases the absolute or relative increase for the production concentration that can refer to generate under conditions of defining for two groups (for example, when compared with no control handled with modification mRNA).
Cell
In one embodiment, cell is selected from the group being made up of: mammalian cell, bacterial cell, plant are thin Born of the same parents, microbial cell, alga cells and fungal cell.In some embodiments, cell is mammalian cell, such as but not It is limited to people, mouse, rat, goat, horse, rabbit, hamster or milk cow cell.In a further embodiment, cell may be from built Vertical cell line, including but not limited to HeLa, NS0, SP2/0, KEK 293T, Vero, Caco, Caco-2, MDCK, COS-1, COS-7、K562、Jurkat、CHO-K1、DG44、CHOK1SV、CHO-S、Huvec、CV-1、Huh-7、NIH3T3、HEK293、 293, A549, HepG2, IMR-90, MCF-7, U-20S, Per.C6, SF9, SF21 or Chinese hamster ovary (CHO) cell.
In certain embodiments, cell is fungal cell, such as, but not limited to Chrysosporium (Chrysosporium) Cell, aspergillus (Aspergillus) cell, trichoderma (Trichoderma) cell, dictyostelium (Dictyostelium) cell, Mycotoruloides (Candida) cell, saccharomyces (Saccharomyces) cell, fission yeast Belong to (Schizosaccharomyces) cell and Penicillium (Penicillium) cell.
In certain embodiments, cell is bacterial cell, such as, but not limited to Bacillus coli cells, bacillus subtilis (B.subtilis) cell or BL21 cell.Primary cell and passage (secondary) to transfect by means of the present invention Cell can be obtained from various tissues, and include but is not limited to all cell types that can be maintained in culture.For example, can The primary cell and passage cell transfected by means of the present invention include but is not limited to fibroblast, keratinocyte, Epithelial cell (for example, galactophore epithelial cell, enterocyte), endothelial cell, Deiter's cells, nerve cell, blood shape At the precursor of ingredient (for example, lymphocyte, bone marrow cell), myocyte and these cell somatic types.Primary cell can also be from The donor of identical type or from another type (for example, mouse, rat, rabbit, cat, dog, pig, milk cow, bird, sheep, goat, horse) It obtains.
Purifying and separation
Those of ordinary skill in the art should be able to determine that method is used to that target egg is purified or separated from the cell of culture White matter.In general, this by using compatibility in conjunction with or the catching method that purifies of non-compatibility complete.If the cell of culture is not Target protein is secreted, then should carry out the cracking of culture cell before purifying or separation.Can be used in the present invention containing Target protein, together with cell culture medium component and cell culture additive such as defoaming compounds and other nutrients and benefit Fill the unclarified cell culture fluids of agent, cell, cell fragment, host cell proteins matter, DNA, virus etc..It can be biological anti- It answers and carries out the method in device itself.Fluid can be pre-processed to desired stimulation such as pH, temperature or other stimulation characteristics or can Treatment fluid or polymer can be added in carrier liquid when adding polymer, the carrier liquid is properly handled to poly- Close the required parameter that object is dissolved in required incentive condition in fluid.Polymer is allowed sufficiently to recycle with fluid and then Stimulation (pH, temperature, salinity variation etc.) can be applied and desired protein and polymer can be made to be settled out from solution Come.Polymer and desired protein can be isolated from remaining fluid and optionally washed once or repeatedly with removal Any supplementary set or loosely bound pollutant.Then desired protein can be recycled from polymer for example, by elution etc.. Preferably, polymer can made to keep its precipitation form and retaining it in the selection elution process of desired protein Any impurity a series of conditions under eluted.Polymer and protein and any impurity may be dissolved in new fluid such as water Or in buffer solution, and can have by such as compatibility, ion exchange, hydrophobicity or to protein comparison polymer or impurity Some other types of chromatographic means of preferable and selectivity recycle protein.Then the protein of elution can be recycled simultaneously And if applicable, other procedure of processing can be carried out to it, such as sample step or continue to flow through step in batches.
In another embodiment, optimize the expression of potential target cell line or a series of particular polypeptide in cell lines Can be to be useful, the polypeptide is specially the protein variant that target polypeptides such as have the reference protein matter of known activity.One In a embodiment, provide by providing multiple target cell types and independently making each in multiple target cell types It is contacted with the modification mRNA of coding polypeptide to optimize the method for the expression of the target polypeptides in target cell.In addition, changeable culture Condition is to increase protein generation efficiency.Then, detection and/or the presence of the target polypeptides in quantitative multiple target cell types And/or it is horizontal, to allow by selecting effective target cell and relative cell culture condition come optimization aim polypeptide Expression.Such method can target polypeptides contain one or more posttranslational modifications or have a large amount of tertiary structure and usually Making effective protein is useful when generating complication.
Protein Recovery
Target protein is preferably used as secrete polypeptide to recycle from culture medium, or if expressing no secretion signal, It can be recycled from host cell lysats.From other recombinations in a manner of obtaining the generally preparation of the target protein of homogeneous Protein of interest matter can be required in albumen and host cell proteins matter.Cell can be removed from culture medium or lysate And/or particulate cell debris.It then can be for example by the classification separation of immunoaffinity or ion exchange column, ethanol precipitation, anti- Phase HPLC (RP-HPLC),The chromatography of chromatography, silica or cation exchange resin such as DEAE are come The purification of target product from the soluble protein, polypeptide and nucleic acid of pollution.It purifies by the protein of host cell heterogenous expression Method is well known in the art.
Method described herein and composition can be used to generate can cut down or block endogenous agonist biological response and/ Or the protein of the receptor or signal transduction molecule in antagonism mammalian subjects.For example, IL-12 and IL-23 receptor letter Number conduction can be in chronic autoimmune disorder such as multiple sclerosis and diseases associated with inflammation such as rheumatoid arthritis, psoriasis, erythema Enhanced (Kikly K, Liu L, Na S, Sedgwich JD (2006) in lupus, ankylosing spondylitis and Crohn's disease Cur.Opin.Immunol.18 (6): 670-5).In another embodiment, the antagonism of nucleic acid encode chemokine receptors Agent.Chemokine receptors CXCR-4 and CCR-5 be HIV enter required for host cell (Arenzana-Seisdedos F etc., (1996)Nature.Oct 3；383 (6599): 400).
Gene silencing
Polynucleotides, primary construct and mmRNA described herein have for making one of cell mass or a variety of target bases The expression silencing (that is, prevent or generally reduce) of cause.Coding is capable of to the mesh of boot sequence specific histone H3 methylation Mark polynucleotides, primary construct or the mmRNA of polypeptide the polypeptide be translated and methylated by histone H 3 and after Continuous heterochromatin forms under conditions of reducing the genetic transcription of target gene, to be introduced into the cell of cell mass.In some realities It applies in scheme, Silencing Mechanisms is carried out to the cell mass being present in mammalian subject.It is useful by non-limiting example Target gene is -2 family member of Janus kinases of mutation, wherein mammalian subject expression mutation target gene, by by exception Myeloproliferative disease caused by kinase activity.
The co-administration of polynucleotides, primary construct and mmRNA and RNAi agent is also provided herein.
The adjusting of biological approach
Rapid translation polynucleotides, primary construct and the mmRNA being introduced into cell are provided and are adjusted target biological approach Hope mechanism.Such antagonism or agonism adjusted including given approach.In one embodiment, use is provided In by enable polynucleotides, primary construct and mmRNA navigate in cell and target polypeptides in cell from Make cell and a effective amount of multicore comprising encoding target polypeptide under conditions of polynucleotides, primary construct and mmRNA translation The composition contact of thuja acid, primary construct or mmRNA carrys out the method for the biological approach in antagonism cell, wherein the polypeptide presses down The activity of the polypeptide to work in biological approach is made.Exemplary bio approach is that autoimmunity or inflammatory conditions are for example multiple In property hardening, rheumatoid arthritis, psoriasis, lupus erythematosus, ankylosing spondylitis or Crohn's disease it is defective those；Tool For body, the antagonism of IL-12 and IL-23 signal transduction path has particular utility.(referring to Kikly K, Liu L, Na S, Sedgwick JD (2006) Curr.Opin.Immunol.18 (6): 670-5).
Further it is provided that the polynucleotides of the antagonist of coding chemokine receptors, primary construct or mmRNA；Chemotactic Factor acceptor CXCR-4 and CCR-5 be such as required for entering in host cell HIV (Arenzana-Seisdedos F, (1996)Nature.Oct 3；383 (6599): 400).
Alternatively, provide by enable nucleic acid navigate in cell and recombinant polypeptide in cell from nucleic acid Contact cell with the polynucleotides of a effective amount of encoding recombinant polypeptide, primary construct or mmRNA to swash The method of biological approach in dynamic cell, and recombinant polypeptide is induction of the activity of the polypeptide to work in biological approach.Show The biological approach of example property excitement includes the approach for adjusting cell fate and determining.Such excitement is reversible or irreversible.
The expression of ligand or receptor on cell surface
In some respects and in the embodiment of aspects described herein, polynucleotides described herein, primary construct Or mmRNA can be used to ligand or ligand receptor (for example, part of going back to the nest) on expression cell surface.It is attached to matching for cell surface Body or ligand receptor, which may be allowed cell, has desired biological interaction with tissue or medicament in vivo.Ligand can for antibody, Antibody fragment, aptamer, peptide, vitamin, carbohydrate, protein or polypeptide, receptor such as cell surface receptor, adherency point Son, glycoprotein, saccharide residue, therapeutic agent, drug, glycosaminoglycan or any combination thereof.For example, ligand can be special for identification cancer cell Property antigen antibody, so that presenting can preferably interact with tumour cell to allow the tumour-specific of modified cells fixed The cell of position.Ligand can give the ability that cell composition accumulates in tissue to be treated, because preferred ligand can It interacts with the target molecule on the outer surface of tissue to be treated.Has the ligand of conditional cross reactivity with other tissues It is usually preferred.
In some cases, ligand may act as part of going back to the nest, permissive cell target specific organization or with particular ligand phase Interaction.Such part of going back to the nest may include but be not limited to specific binding to, antibody, monoclonal antibody or derivatives thereof or similar Any member of object comprising but be not limited to: Fv segment, scFv (scFv) segment, Fab ' segment, 2 segment of F (ab '), unijunction Structure domain antibodies, camelised antibodies and antibody fragment, humanized antibody and antibody fragment and above-mentioned multivalent forms；Multivalence combines Reagent, including but not limited to: Mono-specific antibodies or bispecific antibody, as the stable Fv segment of disulfide bond, scFv connect ((SCFV) 2 segment), double antibody, three antibody or four antibody are usually covalently attached or stable (that is, bright in another way Propylhomoserin zipper or spiral are stable) scFv segment；And other parts of going back to the nest, including such as aptamer, receptor and fusion protein.
In some embodiments, going back to the nest partially to be surface bound antibody, may be allowed adjustment cell-targeting specificity. This is particularly useful, because the antibody of high degree of specificity can be generated for the target epitope for desired targeting moiety.One In a embodiment, Multiple Antibodies are expressed on the surface of cell, and every kind of antibody can have difference to desired target Specificity.Such method can increase the affinity and specificity of interaction of going back to the nest.
Those of skill in the art can desired positioning based on cell or function select any part of going back to the nest, such as estrogen by Body ligand, as tamoxifen (tamoxifen) can have cell-targeting estrogen dependent breast cancer cell on cell surface There is the estrogen receptor for increasing number.Other non-limiting examples of ligand/receptor interaction include CCRI (for example, being used for Treat the inflammation joint tissue or brain in rheumatoid arthritis and/or multiple sclerosis), CCR7, CCR8 (for example, targeting lymph Nodal tissue), CCR6, CCR9, CCR10 (for example, targeting intestinal tissue), CCR4, CCR10 (such as targeting skin), CXCR4 (for example, for general enhancing migrating), HCELL (such as treating inflammation and inflammatory conditions, marrow), 4 β 7 of α (such as Targeted for intestinal mucosa), VLA-4/VCAM-1 (such as targeting endothelium).In general, being related to targeting any of (for example, cancer metastasis) Receptor can be used in method described herein and composition.
The adjusting of cell lineage
Provide the method that inducing cell destiny changes in target mammalian cell.Target mammalian cell can be precursor Cell and changes to can be related to drive and be divided into a kind of pedigree or block such differentiation.Alternatively, target mammalian cell can be point The cell of change, and cell fate change include drive dedifferente for versatility precursor or block it is such dedifferente, such as make Cancer cell dedifferentes as cancer stem cell.In the case where wishing cell fate variation, in the item for changing inducing cell destiny The mRNA of a effective amount of Codocyte destiny inducing polypeptide is introduced into target cell under part.In some embodiments, it modifies MRNA has for cell subsets to be reprogrammed into the second phenotype from the first phenotype.This reprogram can be temporary or permanent 's.
Optionally, inducing target cell is reprogramed using intermediate phenotype.
In addition, method of the invention is transfected the ability of cell due to high transfection efficiency, again and generated in target cell The amount of recombinant polypeptide can retentivity (tenability) and be particularly useful in generate induction multipotent stem cells (iPS is thin Born of the same parents).Additionally, it is contemplated that the generation that there is reduced teratoma to be formed for the use of the iPS cell generated using method described herein Rate.
Additionally provide the method for reducing the cell differentiation in target cell group.For example, before making polypeptide be translated and reduce Under conditions of somatic differentiation, makes the target cell group containing one or more precursor cell types and have a effective amount of coding more The composition contact of the polynucleotides of peptide, primary construct and mmRNA.In a not limiting embodiment, target cell group by Containing the tissue haveing damage in the mammalian subject or tissue that surgical procedure influences.Precursor is that such as matrix precursor is thin Born of the same parents, neural precursor or mesenchymal precursor cells.
In a particular embodiment, the multicore for encoding one or more differentiation factor Gata4, Mef2c and Tbx4 is provided Thuja acid, primary construct or mmRNA.These mRNA factor generated is introduced into fibroblast and is driven and is reprogramed For cardiac muscle cell.This reprogram can be in vivo by making the patch containing mRNA or other materials contact impaired heart group It knits and is carried out with promoting cardiac regeneration.This method promotes the myogenesis opposite with fibre modification.
The mediation of cell death
In one embodiment, polynucleotides, primary construct or mmRNA composition can be used to make it is dead by The expression increase of body, death receptor ligand or combinations thereof carrys out inducing cell (for example, cancer cell) apoptosis.This method can be used to lure Lead the cell death of any desired cell and particularly useful to the cancer for the treatment of cells escape natural apoptosis signal.
Can be by multiple independent signaling pathways come apoptosis-induced, the independent signaling pathways are concentrated on by belonging to A variety of meridian genomics between several " death receptors " and its ligand of tumor necrosis factor (TNF) receptor/ligand superfamily At final effect handset system.The death receptor most sufficiently characterized is CD95 (" Fas "), TNFRI (p55), 3 (DR3 of death receptor Or Apo3/TRAMO), DR4 and DR5 (apo2-TRAIL-R2).The final effect handset system of apoptosis can be referred to as caspase (caspase) a series of activation of protease.The activation of these caspases leads to a series of cracking of living cells albumen And cell death.The molecular mechanism of death receptor/ligand induction apoptosis is well known in the art.For example, by with lower section Formula come induce Fas/FasL- mediate apoptosis: by the end C- death domain (DD) come the three of zygotic induction Fas receptor trimerization A FasL molecule transfers to recruit adaptin FADD (the Fas GAP-associated protein GAP with death domain) and caspase -8. The oligomeric of this three molecular complexes Fas/FAIDD/ caspase -8 leads to -8 proteolytic cleavage of proenzyme caspase For active caspase -8, transfer through the other downstream caspases of Proteolytic activation (including caspase-3 mRNA) To start apoptotic process.In general, death ligand is apoptosis when becoming tripolymer or more advanced structure.As monomer, they It can be by serving as anti-apoptotic agent with for combining the tripolymer of death receptor to compete.
In one embodiment, polynucleotides, primary construct or mmRNA composition coding death receptor (for example, Fas, TRAIL, TRAMO, TNFR, TLR etc.).Expressed by the transfection of polynucleotides, primary construct and mmRNA it is dead by The cell of body becomes easy the death being subjected to by activating the ligand induction of the receptor.Similarly, such as on the surface thereof it expresses The cell of death ligand by when the cell of transfection contacts target cell with the death of receptor-inducible cell.In another embodiment In, polynucleotides, primary construct and mmRNA composition coding death receptor ligand (for example, FasL, TNF etc.).At another In embodiment, polynucleotides, primary construct and mmRNA composition coding caspase are (for example, Caspase-3, half Guang aspartase 8, caspase 9 etc.).The increasing that cannot be suitably divided into non-proliferative or control is often exhibited in cancer cell In the case where growing form, in another embodiment, the polynucleotides of synthesis, primary construct and mmRNA composition coding Death receptor and its activation ligand appropriate.In another embodiment, the polynucleotides of synthesis, primary construct and By Cell differentiation inducing activity at avirulence or non-self refresh table when mmRNA composition coding is expressed in cancer cell such as cancer stem cell Type (for example, cell growth rate reduces, cell division is reduced etc.) or inducing cell enter the dormant cells phase (for example, G0Stop Phase) differentiation factor.
It will be apparent to those skilled in the art that polynucleotides, primary construct or mmRNA can be required using apoptosis induction technology Target such as tumour cell suitably to prevent undesirable extensive cell death.Therefore, passing for identification cancer antigen can be used Mechanism (for example, ligand or antibody, the liposome of targeting etc. of attachment) is sent, so that polynucleotides, primary construct or mmRNA Only expressed in cancer cell.
Cosmetic applications
In one embodiment, polynucleotides, primary construct and/or mmRNA can be used for cosmetology symptom treatment, Improve or prevents.Such symptom includes acne, rosacea, spot formation, wrinkle, eczema, shingles zoster, psoriasis, old age Spot, birthmark, dry skin, callus, fash (for example, diaper, heat), scabies, nettle rash, wart, insect bites, leucoderma, scalp Bits, freckle and aging sign.
VI. kit and device
Kit
The present invention provides various for carrying out conveniently and/or effectively the kit of method of the invention.Usual kit will Multiple experiments are treated and/or carried out to component including sufficient amount and/or number to allow user to carry out the multiple of subject.
On the one hand, the present invention provides the reagent comprising molecule (polynucleotides, primary construct or mmRNA) of the invention Box.In one embodiment, kit includes one or more functional antibodies or its function fragment.
The kit can be used for protein generation, it includes include can the first polynucleotides of translated region, primary building Body or mmRNA.Kit can further include packaging and specification and/or form the delivery agents of preparation compositions.Delivery agents can Include salt water, buffer solution, lipoids or any delivery agents disclosed herein.
In one embodiment, buffer solution may include sodium chloride, calcium chloride, phosphate and/or EDTA.At another In embodiment, buffer solution may include but be not limited to salt water, the salt water with 2mM calcium, 5% sucrose, 5% with 2mM calcium Sucrose, 5% mannitol, 5% mannitol with 2mM calcium, ringer lactate, sodium chloride, the sodium chloride with 2mM calcium and Mannose is (see, for example, U.S. Publication No 20120258046；It is incorporated herein in its entirety by reference).In other implementation In scheme, buffer solution can be precipitating or it can be freeze-drying.It is consistent, reproducible to realize that the amount of every kind of component can be changed Higher concentration salt water or Sample Buffer preparation.Component can be changed also to increase through after a period of time and/or in various items Stability of the RNA in buffer solution is modified under part.On the one hand, the present invention is provided to the kit of protein production, packets Contain: including can translated region polynucleotides, primary construct or mmRNA, effective in the generation when being introduced into target cell Desired amount by can translated region coding protein amount provide；The second polynucleotides comprising inhibition nucleic acid, to have The amount imitated in the innate immune response for generally inhibiting cell provides；And packaging and specification.
On the one hand, the present invention is provided to protein generate kit, it includes: including can translated region multicore glycosides Acid, primary construct or mmRNA, wherein polynucleotides show to degrade as caused by cellular nucleic acid enzyme and reduce；And packaging and Specification.
On the one hand, the present invention is provided to protein generate kit, it includes: including can translated region multicore glycosides Acid, primary construct or mmRNA, wherein polynucleotides show to degrade as caused by cellular nucleic acid enzyme and reduce；And it is suitable for Translate the first nucleic acid can translated region mammalian cell.
In one embodiment, the level of PROTEIN C can be measured by immunoassays.Measurement is purchased from and from any number Supplier obtain, including BioMerieux Inc. (Durham, NC), Abbott Laboratories (Abbott Park, IL), Siemens Medical Solutions USA, Inc. (Malvern, PA), Diagnostics A/S (Gentofte, Denmark),Life Science Inc. (Houston, TX) or Roche Diagnostic Corporation (Indianapolis, IN).In this embodiment, measurement can be used to evaluate with modification The level of the application of mRNA molecule or the PROTEIN C or its activation form or variant applied in response to modification mRNA molecule.
Device
The present invention provides the device of the polynucleotides that may be incorporated into encoding target polypeptide, primary construct or mmRNA.These dresses It sets comprising the reagent in stabilization formulations, the reagent is immediately delivered to subject in need such as people for synthesizing and suffers from The polynucleotides of the dosage form of person.The non-limiting example of this target polypeptides includes the growth factor for wound healing And/or angiogenesis stimulant, the peptide antibiotic of promotion infection control and rapid stimulation answer the immune of virus newly identified The antigen answered.
Device can be also employed in conjunction with the invention.In one embodiment, device is used to evaluate to modify the shape of mRNA The level of the protein of formula application.Device may include blood, urine or the test of other biofluids.It can be large enough to include automatic Central laboratory's platform or small-sized dispersion desktop apparatus.It can be point-of-care or handheld device.In this embodiment, example Such as, PROTEIN C or APC can before with the modification mRNA of coding PROTEIN C (its proenzyme), APC or its any variant treatment, among or It is quantitative later.PROTEIN C, also known as autoprothrombin IIA and plasma thromboplastin antecedent V are to Coagulation modulation and internal plasmin Solve the proenzyme or precursor of the active serine protease for generating and playing an important role.It is closed in liver in the form of single chain polypeptide At, but undergo post translational processing and generate two chain intermediates.Amino-through 12- residue peptide from the amino terminals of heavy chain to molecule The cracking of the thrombin-mediated of end makes the intermediate forms of PROTEIN C be converted into the active shape of referred to as " activated protein c " (APC) Formula.Device can be used as Duplicate diagnostic test relevant to PROTEIN C in drug discovery research or as pyemia or serious purulence The APC of toxication is treated.In early stage research, it has been suggested that APC has the ability for reducing the death rate in severe sepsis.At this After work, clinical research causes FDA to ratify a kind of compound, and the buckling of activation can net α (drotrecogin alfa) (recombination PROTEIN C).However, removing drug in all sales market according to the result that PROWESS-SHOCK is studied the second half year in 2011 Return, the statistically significant reductions of All-cause death rate in 28 days not being able to satisfy researches show that it in patients with septic shock it is main Endpoint.Present invention offer can be used for diagnosing and treating pyemia, severe sepsis and septicemic cemia modification mRNA molecule, overcome Existing project relevant to the increase of protein expression efficiency in the mammalian body or problem.
In some embodiments, device be it is self-contained and optionally can wireless remote access to obtain for closing At and/or the specification of polynucleotides, primary construct or mmRNA that generates of analysis.Device can flexibly synthesize at least one Kind polynucleotides, primary construct or mmRNA, and the different polynucleotides of preferably unrestricted number, primary building Body or mmRNA.In certain embodiments, device can be transported by one or several individuals.In other embodiments, it scales Device is to be mounted on workbench or table.In other embodiments, device for zooming is to be installed to suitcase, knapsack or similar to big In small object.In another embodiment, device can be point-of-care or hand-held device.In a further embodiment, Device for zooming is to be installed in vehicle such as car, truck or ambulance or military vehicle such as tank or personnel carrier.It generates Information necessary to the modification mRNA of encoding target polypeptide is present in the computer-readable medium for including in device.
In one embodiment, device can be used to evaluate and be applied in the form of polynucleotides, primary construct or mmRNA Protein level.Device may include blood, urine or the test of other biofluids.
In some embodiments, device can be with the database communication (for example, wireless communication) of nucleic acid and polypeptide sequence. Device contains at least one sample blocks (sample block) for being inserted into one or more sample containers.Such sample container It can receive any number of material such as template DNA, nucleotide, enzyme, buffer and the other reagents in liquid or other forms. Sample container can also be heated and cool down by contacting with sample blocks.Sample blocks are usually and at least one sample The device pedestal of one or more electronic control units of block is connected to.Sample blocks preferably contain heating module, this heated mould Block can heat and/or cool sample container and its content to the temperature about between -20C and+100C.Device pedestal and electricity Pressure supply such as battery or external voltage supply connection.Device also contains the work for storing and distributing the material for RNA synthesis Have (means).
Optionally, sample blocks contain the module of the nucleic acid for separating synthesis.Alternatively, device, which contains, is optionally connected to sample The separation module of block.Preferably, device contains the tool of the nucleic acid for analyzing synthesis.Such analysis includes sequence identity (as by hybridization prove), there is no undesirable sequence, measurement synthesis mRNA integrality (such as by with spectrophotometry Combined microfluid viscosimetry) and modification RNA concentration and/or effect (such as passing through spectrophotometry).
In certain embodiments, device with for detecting the pathogen being present in from the biomaterial that subject obtains Tool combinations, the tool be, for example, be used for microorganism identification IBIS PLEX-ID system (Abbott, Abbott Park, IL)。
Suitable device for delivering intradermal drug composition described herein includes hour hand device, such as in United States Patent (USP) 4,886,499；5,190,521；5,328,483；5,527,288；4,270,537；5,015,235；5,141,496 and 5,417, Described in 662 those；The patent is respectively incorporated herein in its entirety by reference.Intradermal composition can be by limiting needle It is applied into the device of effective penetration length of skin, as those are (described specially described in the PCT Publication WO 99/34850 Benefit is incorporated herein in its entirety by reference) and its functional equivalent.By liquid jet injector and/or pass through piercing stratum The layer and jet injection device that liquid composition is delivered to corium is suitable by the needle for generating the jet flow for reaching corium.Injection note Injection device is for example described in United States Patent (USP) 5,480,381；5,599,302；5,334,144；5,993,412；5,649,912；5, 569,189；5,704,911；5,383,851；5,893,397；5,466,220；5,339,163；5,312,335；5,503, 627；5,064,413；5,520,639；4,596,556；4,790,824；4,941,880；4,940,460；And PCT Publication WO In 97/37705 and WO 97/13537；The patent is respectively incorporated herein in its entirety by reference.Come using compressed gas It is suitable for accelerating the outer layer that existing vaccine passes through skin in powder form to reach the trajectory powder/granule delivery apparatus of corium 's.Alternatively, or in addition, conventional syringe can be used in classical awns Tu Shi (mantoux) method of intradermal administration.
In some embodiments, device can for pump or including for across blood-brain barrier apply the compound of the present invention or The conduit of composition.Such device includes but is not limited to pressurize smell delivery apparatus, iontophoresis device, multilayer microfluidic device Deng.Such device can be portable or fixed.Their implantable or outsides are tied to body or combinations thereof.
Can be used the device for application with according to instruct herein single-dose scheme, multiple dosage regimen or by several times to Prescription case delivers polynucleotides of the invention, primary construct or mmRNA.The following describe such devices.
It is susceptible to as known in the art for public to cell, the method and apparatus of organ and tissue multiple applications and this paper The method and composition opened is used in combination as embodiment of the present invention.These include for example with those of multiple needles method With device, the hybrid devices using such as lumen or conduit and the device using heat, electric current or radiation-driven mechanism.
According to the present invention, these multiple applications devices can be used deliver the single dose expected herein, multidose or Divided dose.
Method for therapeutic agent to be delivered to solid tissue has been described by Bahrami etc. and for example in United States Patent (USP) public affairs It is instructed in cloth 20110230839, disclosure is incorporated herein in its entirety by reference.According to Bahrami, by needle battle array Column are incorporated into the dress that the generally fluid of equivalent is delivered on any position along the length of each needle in the solid tissue In setting.
Device for passing through biological tissue's delivering biomaterial has been described by Kodgule etc. and for example in United States Patent (USP) It announces and is instructed in 20110172610, disclosure is incorporated herein in its entirety by reference.It, will be by according to Kodgule One or more metals are made and multiple skies with about 200 microns to about 350 microns of outer diameter and at least 100 micrometer lengths Heart microneedle be incorporated into delivering peptide, protein, carbohydrate, nucleic acid molecules, lipid and other pharmaceutical active ingredients or its In combined device.
Delivering probe for therapeutic agent to be delivered to tissue has been described by Gunday etc. and for example in United States Patent (USP) public affairs It is instructed in cloth 20110270184, disclosure is incorporated herein in its entirety by reference.It, will be multiple according to Gunday Needle is incorporated into the capsule of the mobile attachment between starting position and inactive position to force medicament to come out from capsule across needle Device in.
More injection Medical Devices are described and are for example instructed in U.S. Patent Publication 20110218497, institute by Assaf The content for stating patent is incorporated herein in its entirety by reference.According to Assaf, multiple needles are incorporated into have be connected to one or In the chamber of multiple needles and the device of the tool for continuously refilling chamber with medical fluid after per injection.
In one embodiment, polynucleotides, primary construct or mmRNA are through at least three needle simultaneously or at 60 minutes Period is subendothelial or intramuscular administration to three different, optionally adjacent positions (for example, simultaneously or in 60 minutes sections It is applied to 4,5,6,7,8,9 or 10 positions).It can be used and be described in 20110230839 He of U.S. Patent Publication number Device in 20110218497 applies divided dose to adjacent tissue simultaneously, the patent respectively it is whole by reference simultaneously Enter herein.
For injecting substances at least partly implantable system (the specially telotism stimulation system in patient body System) it is described by Forsell and is for example instructed in U.S. Patent Publication 20110196198, disclosure is to draw Mode is integrally incorporated herein.According to Forsell, multiple needles are incorporated into together with one or more shells adjacent to patient's In the device of left and right cavernous body implantation.Also implantation reservoir and pump is to supply drug by needle.
The method of iron for transdermal delivery therapeutically effective amount has been described by Berenson and for example in United States Patent (USP) public affairs It is instructed in cloth 20100130910, disclosure is incorporated herein in its entirety by reference.According to Berenson, can make Multiple microchannels are formed in cuticula with multiple needles to enhance the transdermal delivery of the ionic iron on iontophoretic patch.
Method for passing through biological tissue's delivering biomaterial has been described by Kodgule etc. and for example in United States Patent (USP) It announces and is instructed in 20110196308, disclosure is incorporated herein in its entirety by reference.According to Kodgule, will contain There are multiple biodegradable microneedles of therapeutic activity ingredient to be incorporated to delivering protein, carbohydrate, nucleic acid molecules, lipid In the device of other pharmaceutical active ingredients or combinations thereof.
Percutaneous plaster comprising botulinum toxin composition has been described by Donovan and for example in U.S. Patent Publication It is instructed in 20080220020, disclosure is incorporated herein in its entirety by reference.According to Donovan, by multiple needles It is incorporated into the patch that botulin toxin is delivered to below cuticula by the needle, the needle projects through the angle of skin Matter layer is without making angiorrhoxis.
The small-sized disposable drug reservoir or patch pump that about 0.2mL can be accommodated to 15mL liquid preparation can be placed in skin Above and use required aperture (for example, 26 to No. 34) continuous subcutaneous delivery preparation.As non-limiting examples, patch pump 76mm can be multiplied for the 50mm with 30 to No. 34 needles and multiply the spring-loaded (BD of 20mmTMMicroinfuser, Franklin Lakes NJ), the 41mm with the 2mL reservoir for drug delivery such as insulin multiply 62mm multiply 17mm ( Insulet Corporation Bedford, MA) or 43-60mm diameter with 0.5mL to 10mL reservoir, 10mm it is thick (SteadyMed Therapeutics, San Francisco, CA).In addition, patch pump can be battery It is power supply and/or rechargeable.
Cold probe for from the position administering active agents to low-temperature treatment has been described by Toubia and for example in the U.S. It is instructed in patent disclosure 20080140061, disclosure is incorporated herein in its entirety by reference.According to Toubia, Multiple needles, which are incorporated into, to be received activating agent in chamber and will be in the probe of pharmacy application to tissue.
For treat or prevent inflammation or promote healthy joint method by Stock etc. describe and for example in the U.S. it is special Benefit is announced instructs in 20090155186, and disclosure is incorporated herein in its entirety by reference.It, will be more according to Stock A needle is incorporated into the device of composition of the application containing signal transduction modulators compound.
Multiple location injecting systems are described by Kimmell etc. and are for example taught in U.S. Patent Publication 20100256594 It leads, disclosure is incorporated herein in its entirety by reference.According to Kimmell, multiple needles are incorporated into will by needle Drug delivery is in the device into cuticula.
Method for interferon to be delivered to intradermal compartment has been described by Dekker etc. and for example in United States Patent (USP) public affairs It is instructed in cloth 20050181033, disclosure is incorporated herein in its entirety by reference.According to Dekker, will have Between 0mm and 1mm multiple needles of the outlet of exposure height be incorporated by between 0.3mm and 2mm depth delivered substance change Into in the device of pharmacokinetics and bioavailability.
It has been described by Desai and for gene, enzyme and biological agent to be delivered to histiocytic method for example in the U.S. It is instructed in patent disclosure 20030073908, disclosure is incorporated herein in its entirety by reference.It, will according to Desai Multiple needles are incorporated into insertion body and are delivered in the device of medicament fluid by the needle.
Method for using treating cardiac arrhythmias with fibroblast cells has been described by Lee etc. and for example in United States Patent (USP) public affairs It is instructed in cloth 20040005295, disclosure is incorporated herein in its entirety by reference.According to Lee, simultaneously by multiple needles Enter in the device into the regional area that fibroblast is delivered to tissue.
Method using the pump of magnetic control for treating brain tumor has been described by Shachar etc. and for example in United States Patent (USP) Introduction, disclosure are incorporated herein in its entirety by reference in 7799012 (methods) and 7799016 (devices).Root According to Shachar, multiple needles are incorporated into and push medicament to pass through in the pump of needle with the rate of control.
The method for treating the intracorporal bladder function venereal disease disease of female has been described by Versi etc. and for example in beauty It is instructed in state's patent 8,029,496, disclosure is incorporated herein in its entirety by reference.It, will be miniature according to Versi Needle array is incorporated into the device that therapeutic agent is delivered directly in trigonumvesicae by needle.
Microneedle transdermal delivery device is described and is for example instructed in United States Patent (USP) 7,364,568, institute by Angel etc. The content for stating patent is incorporated herein in its entirety by reference.According to Angel, multiple needles are incorporated by inserting from different directions The needle entered into surface delivers the substance into the device in body surface.Microneedle transdermal delivery device can be solid microneedle System or hollow miniature needle system.As a non-limiting example, solid miniature needle system, which can have, up to holds to 0.5mg Amount, wherein about 150-700 μm high of 300-1500 every square centimeter solid microneedles are by drug coat.Microneedle penetrates cutin Layer and keep the shorter duration (for example, 20 seconds to 15 minutes) in skin.In another example, hollow microneedle System has up to 3mL capacity to use about 950 μm high of 15-20 microneedle every square centimeter to deliver liquid preparation. Microneedle penetrates skin to allow liquid preparation to be flowed into skin from device.Hollow miniature needle system may depend on volumes of formulation With viscosity and it is 1 to 30 minute lasting.
Device for h inf is described by Dalton etc. and is for example instructed in United States Patent (USP) 7,150,726, Disclosure is incorporated herein in its entirety by reference.According to Dalton, multiple needles are incorporated into fluid through needle It is delivered in the device in subcutaneous tissue.
For by it is micro- intubation Intradermal delivery vaccine and gene therapeutic agents device and method by descriptions such as Mikszta simultaneously And for example instructed in United States Patent (USP) 7,473,247, disclosure is incorporated herein in its entirety by reference.According to At least one hollow microneedle is incorporated between 0.025mm and 2mm by vaccine delivery to subjects skin by Mitszta In the device of depth.
The method for delivering insulin is described and is for example instructed in United States Patent (USP) 7,722,595, institute by Pettis etc. The content for stating patent is incorporated herein in its entirety by reference.According to Pettis, two needles are incorporated into device, wherein this two A needle is substantially simultaneously inserted into skin, and insulin is delivered to intradermal compartment by the depth that first needle is in less than 2.5mm And insulin is delivered to subcutaneous compartment by the depth that second needle is in greater than 2.5mm and less than 5.0mm.
Percutaneous injection delivering under suction is described by Kochamba etc. and is for example taught in United States Patent (USP) 6,896,666 It leads, disclosure is incorporated herein in its entirety by reference.According to Kochamba, the multiple needles that will be relatively adjacent each other It is incorporated into and injects a fluid into the device below cortex.
For being extracted by skin or the device of delivered substance has been described by Down etc. and for example in United States Patent (USP) 6, It is instructed in 607,513, disclosure is incorporated herein in its entirety by reference.According to Down, it is incorporated into device Multiple skin penetration components have about 100 microns to about 2000 microns of length and are about 30 to No. 50.
Device for substance to be delivered to skin has been described by Palmer etc. and for example in United States Patent (USP) 6,537,242 Middle introduction, disclosure are incorporated herein in its entirety by reference.According to Palmer, microneedle array, which is incorporated into, to be made With stretching assembly to enhance contact of the needle with skin and provide in the device more evenly delivered of substance.
Device for casting for localized drug delivery has been described by Zamoyski and for example in United States Patent (USP) 6,468,247 Middle introduction, disclosure are incorporated herein in its entirety by reference.According to Zamoyski, simultaneously by multiple hypodermic needles Enter in retracting the device that the content of hypodermic needle is injected into tissue when the hypodermic needle.
For passing through tissue by improving Interaction enhanced drug between microneedle and application on human skin and biomolecule The method of conveying is described and is for example instructed in United States Patent (USP) 6,743,211, disclosure by Prausnitz etc. It is incorporated herein in its entirety by reference.According to Prausnitz, multiple microneedles are incorporated into and are capable of providing application microneedle It is harder and be unlikely to deform in the device on surface.
Device for applying medicament in organ is described by Ting etc. and is for example taught in United States Patent (USP) 6,077,251 It leads, disclosure is incorporated herein in its entirety by reference.According to Ting, will there is the side for being used to enhance application to open Multiple needles of mouth, which are incorporated by stretching out the needle from needle cavity room and the needle being retracted into chamber, forces medicament from reservoir It is injected into the device in target organ into the needle and by the medicament.
Spininess retainer and subcutaneous multichannel infusion orifice have been described by Brown and for example in United States Patent (USP) 4,695,273 Middle introduction, disclosure are incorporated herein in its entirety by reference.According to Brown, by multiple needles on needle holder Be inserted through the diaphragm of infusion orifice and with the chamber separated in the infusion orifice.
Double syringes are described by Horn and are for example instructed in United States Patent (USP) 3,552,394, the patent Content is incorporated herein in its entirety by reference.According to Horn, two needle intervals being incorporated into device are less than 68mm and can With different patterns and length, therefore realize the injection for carrying out different depth.
Syringe with multiple needles and multiple fluid compartments has been described by Hershberg and for example in United States Patent (USP) 3, It is instructed in 572,336, disclosure is incorporated herein in its entirety by reference.According to Hershberg, simultaneously by multiple needles Enter to the syringe that cannot be mixed for the incompatible drug of one injection with multiple fluid compartments and capable of being administered simultaneously In.
Surgical instrument for intracutaneous injection fluid has been described by Eliscu etc. and for example in United States Patent (USP) 2,588,623 Middle introduction, disclosure are incorporated herein in its entirety by reference.According to Eliscu, multiple needles are incorporated into wider In the utensil of general dispersion intracutaneous injection fluid.
It has been described by Hung for the equipment to multiple breast milk pipes while delivered substance and for example in EP 1818017 Introduction, disclosure are incorporated herein in its entirety by reference.According to Hung, multiple lumens are incorporated into and are inserted through The aperture of conduit network simultaneously delivers a fluid in the device of conduit network.
Conduit for medicament to be introduced to the tissue of heart or other organs is described by Tkebuchava and is for example existed It is instructed in WO2006138109, disclosure is incorporated herein in its entirety by reference.According to Tkebuchava, it is incorporated to Enter two loopers of organ walls with flat track.
Device for delivering medicament is described by Mckay etc. and is for example instructed in WO2006118804, described special The content of benefit is incorporated herein in its entirety by reference.According to Mckay, multiple needles on each needle with multiple apertures are incorporated to Promote the regional delivery to the tissue such as inside dish gap of interverbebral disc into device.
Method for being delivered directly to immune regulator in the intradennal space in mammal skin by Pettis is described and is for example instructed in WO2004020014, and disclosure is incorporated hereby this Text.According to Pettis, multiple needles is incorporated into, substance is delivered between 0.3mm and 2mm in the device of depth by needle.
It is absorbed and improved drug metabolism at least two compartments for substance to be administered in skin with being used for system Dynamic (dynamical) method and apparatus are described and are for example instructed in WO2003094995, disclosure by Pettis etc. It is incorporated herein in its entirety by reference.According to Pettis, multiple needles with about 300 μm of length between about 5mm are incorporated to Into the device delivered simultaneously to intradermal tissue compartment and subcutaneous tissue compartments.
Drug delivery device with needle and roller is described by Zimmerman etc. and is for example taught in WO2012006259 It leads, disclosure is incorporated herein in its entirety by reference.According to Zimmerman, it will be positioned in multiple skies in roller Heart needle is incorporated into the device in roller rotation through the content in needle delivery reservoirs.
Drug delivery device such as bracket is as known in the art and for example in U.S. Patent number 8,333,799, the U.S. It is instructed in publication No. US20060020329, US20040172127 and US20100161032；The respective content of patent with The mode of reference is integrally incorporated herein.Stent delivery polynucleotides described herein, primary construct, the system of mmRNA can be used Agent.In addition, bracket used herein can deliver a variety of polynucleotides, primary construct with identical or variation delivery rate And/or mmRNA and/or preparation.The non-limiting example of bracket manufacturer includes(Miami, FL)Boston Scientific Corporation (Natick, MA)Medtronic (Minneapolis, MN)And Abbott (Abbott Park, IL)
Use the method and apparatus of conduit and/or lumen
It can be used and apply the present invention according to single, multiple or divided doses scheme using the method and apparatus of conduit and lumen MmRNA.The following describe such method and apparatus.
Skeletal myoblast cell to damaged heart cardiac muscle delivering catheter-based by Jacoby etc. describe and for example It is instructed in U.S. Patent Publication 20060263338, disclosure is incorporated herein in its entirety by reference.According to Multiple needles are incorporated into device by Jacoby, and described device is at least partially inserted into blood vessel and passes through needle for groups of cells Object is closed to be delivered in the regional area of subject's heart.
Equipment for using neurotoxin treatment asthma has been described by Deem etc. and for example in U.S. Patent Publication It is instructed in 20060225742, disclosure is incorporated herein in its entirety by reference.According to Deem, simultaneously by multiple needles Enter into the device that neurotoxin is delivered in bronchial tissue by needle.
Method for applying multicomponent therapy is described by Nayak and is for example taught in United States Patent (USP) 7,699,803 It leads, disclosure is incorporated herein in its entirety by reference.According to Nayak, multiple injection cannulas can be incorporated into dress In setting, wherein may include the depth slit for controlling the depth for delivering therapeutic substance within the organization.
For ablation channel and by least one therapeutic agent be delivered to the operation device in the desired area of tissue by McIntyre etc. is described and is for example instructed in United States Patent (USP) 8,012,096, and disclosure is whole by reference Body is incorporated herein.According to McIntyre, multiple needles is incorporated into, therapeutic agent is assigned in the tissue regions for surrounding channel simultaneously spy Not suitable for the device of the revascularization procedures of saturating cardiac muscle.
The method for treating the intracorporal bladder function venereal disease disease of female has been described by Versi etc. and for example in beauty It is instructed in state's patent 8,029,496, disclosure is incorporated herein in its entirety by reference.It, will be miniature according to Versi Needle array is incorporated into the device that therapeutic agent is delivered directly in trigonumvesicae by needle.
For by fluid delivery to the device and method in flexible biological barrier by Yeshurun etc. describe and for example Introduction, disclosure are whole by reference in United States Patent (USP) 7,998,119 (device) and 8,007,466 (method) Body is incorporated herein.According to Yeshurun, the microneedle on device is penetrated and is extended in flexible biological barrier and by hollow The hole injecting fluid of microneedle.
Used in by substance from epicardial injections to the tissue regions for the heart being placed in trunk with epicardial surface Method by Bonner etc. describe and for example instructed in United States Patent (USP) 7,628,780, disclosure is to quote Mode be integrally incorporated herein.According to Bonner, device has for driving needle to enter tissue and inject a medicament by needle Elongated shaft and distal injection head in tissue.
Device for sealing puncturing hole is described by Nielsen etc. and is for example taught in United States Patent (USP) 7,972,358 It leads, disclosure is incorporated herein in its entirety by reference.According to Nielsen, multiple needles are incorporated into sealer It is delivered in the device in the tissue for surrounding puncture path.
It is generated for flesh and the method for angiogenesis has been described by Chiu etc. and for example in United States Patent (USP) 6,551,338 Introduction, disclosure are incorporated herein in its entirety by reference.According to Chiu, by maximum gauge be at least 1.25mm simultaneously 5 to 15 needles with the length effective in offer 6mm to 20mm paracentesis depth are incorporated into device, and described device is inserted into the heart Flesh nearby and by the pipeline at least some needles to the cardiac muscle supplies exogenous angiogenesis factor or life The flesh factor.
Method for treating prostata tissue has been described by Bolmsj etc. and for example in United States Patent (USP) 6,524,270 Introduction, disclosure are incorporated herein in its entirety by reference.According to Bolmsj, including the conduit being inserted by urethra Device have at least one project over the hollow tip in prostata tissue around.By the tip by astringent and only Pain medicine is delivered in the prostata tissue.
It has been described by Findlay etc. and for fluid to be infused to the method at position in bone for example in United States Patent (USP) 6, It is instructed in 761,726, disclosure is incorporated herein in its entirety by reference.According to Findlay, multiple needles are incorporated to To can penetrate the hard encasing material covered by one layer of soft material and will fluid delivery it is predetermined to hard encasing material lower section In device at distance.
It has been described by Vigil etc. and for injecting a medicament into the device in vascular wall for example in United States Patent (USP) 5,713, It is instructed in 863, disclosure is incorporated herein in its entirety by reference.According to Vigil, multiple syringes are mounted on On each flexible pipe in device, medicament fluid is introduced into the flexible pipe by multi lumen catheter and from institute by described device Syringe is stated to come out for being infused into vascular wall.
Conduit for therapeutic agent and/or diagnosticum to be delivered to the tissue of encirclement body passage has been described by Faxon etc. And such as instructed in United States Patent (USP) 5,464,395, disclosure is incorporated herein in its entirety by reference.According to At least one needle cannula is incorporated by being projected into the needle on the outside of conduit for desired drug delivery to tissue by Faxon Conduit in.
Foley's tube for delivering therapeutic agent has been described and has for example been instructed in WO2010024871, institute by Orr etc. The content for stating patent is incorporated herein in its entirety by reference.According to Orr, multiple needles is incorporated into, therapeutic agent is delivered to tissue In the device of interior different depth.On the other hand, medicament elution sacculus can be used to deliver preparation described herein.Medicament elution ball Capsule can be used for target damage application in, such as, but not limited to in-stent restenosis, treatment tortuous vessels in damage, bifurcated lesions, It femur/popliteal injury of muscle and damages at one's knees.
For therapeutic agent (for example, polynucleotides, primary construct or mmRNA) to be delivered to the tissue being placed in around lumen Device by Perry etc. describe and for example instructed in U.S. Patent Publication US20100125239, disclosure It is incorporated herein in its entirety by reference.According to Perry, conduit has can be by describing as is generally known in the art and in Perry Method be coated with therapeutic agent sacculus.As the balloons are inflated, therapeutic agent will contact surrounding tissue.In addition device can have heat source To change the temperature of coating on sacculus, to discharge therapeutic agent to tissue.
Use the method and apparatus of electric current
Can be used method and apparatus using electric current according to instruct herein single-dose scheme, multiple dosage regimen or point Secondary dosage regimen delivers mmRNA of the invention.The following describe such method and apparatus.
The collagen-induced therapy device of electricity has been described by Marquez and for example in U.S. Patent Publication 20090137945 Introduction, disclosure are incorporated herein in its entirety by reference.According to Marquez, multiple needles are incorporated into repetition and are worn Peronium skin simultaneously takes out in the device of a part of substance applied for the first time to skin in skin.
Power driven system is described and is for example instructed in U.S. Patent Publication 20070185432, institute by Etheredge etc. The content for stating patent is incorporated herein in its entirety by reference.According to Etheredge, microneedle is incorporated into, medicine is driven by electric current Agent passes through needle and enters in the device in targeted therapentic part.
Iontophoresis device is described by Matsumura etc. and is for example instructed in United States Patent (USP) 7,437,189, described The content of patent is incorporated herein in its entirety by reference.According to Matsumura, multiple needles are incorporated into can be with higher speed Degree or with higher efficiency by ionizable drug delivery in device into living body.
It has been described by Hoffmann etc. by Needleless injection and electroporation Intradermal delivery bioactivator and for example in beauty It is instructed in state's patent 7,171,264, disclosure is incorporated herein in its entirety by reference.It, will according to Hoffmann One or more needleless injectors are incorporated into electroporation device and the combination of Needleless injection and electroporation is enough to draw medicament Enter into the cell in skin, muscle or mucous membrane.
The method of Intracellular delivery for the penetrating mediation of electricity has been described by Lundkvist etc. and for example in United States Patent (USP) It is instructed in 6,625,486, disclosure is incorporated herein in its entirety by reference.According to Lundkvist, by a pair of of needle Electrode is incorporated into conduit.The conduit is placed in body cavity, then stretches out the pin electrode to penetrate and surround the lumen Tissue in.Then device by least one described pin electrode introduce medicament and by the pin electrode to apply electric field with Allow the medicament to pass through cell membrane to enter in the cell for the treatment of site.
Delivery system for transdermal immune is described and is for example instructed in WO2006003659, institute by Levin etc. The content for stating patent is incorporated herein in its entirety by reference.According to Levin, multiple electrodes are incorporated into and are applied between the electrodes Electric energy to generate microchannel to promote in the device of transdermal delivery in skin.
For method of the RF transmissibility into skin have been described by Schomacker and for example in WO2011163264 Middle introduction, disclosure are incorporated herein in its entirety by reference.According to Schomacker, multiple needles are incorporated into and are applied Add vacuum so that skin is sucked with plate and contacts so that needle is inserted into skin by the hole on plate and is delivered in the device of RF energy.
VII. it defines
The substituent group in each place in the present specification, the compound of the disclosure is disclosed with group or with range.Definitely It is intended to every kind and each independent sub-portfolio of the member that the disclosure includes such group and range.For example, term " C1-6Alkyl " Definitely it is intended to be separately disclosed methyl, ethyl, C3Alkyl, C4Alkyl, C5Alkyl and C6Alkyl.
About: as used herein, term " about " means +/- the 10% of cited value.
Be administered in combination: as used herein, term " combined administration " or " combined application " mean while or making pair There are apply two or more reagents to subject in the period of the effect overlapping of every kind of medicament by patient.In some embodiment party In case, them are applied in each other about 60,30,15,10,5 or 1 minutes.In some embodiments, make the application of medicament enough Closely interval is so that realize combination (for example, collaboration) effect.
Animal: as used herein, term " animal " refers to any member of the animal kingdom.In some embodiments, " dynamic Object " refers to the people under any stage of development.In some embodiments, " animal " refers to inhuman under any stage of development Animal.In certain embodiments, non-human animal be mammal (for example, rodent, mouse, rat, rabbit, monkey, dog, cat, Sheep, ox, primate or pig).In some embodiments, animal include but is not limited to mammal, birds, creep it is dynamic Object, amphibian, fish and worm.In some embodiments, animal be transgenic animals, genetically engineered animal or gram It is grand.
Target antigen or desired antigen: as used herein, term " target antigen " or " desired antigen " include this Those of text offer protein and other biomolecule, by antibody described herein and its segment, mutant, variant and change Immunologic specificity combines.The example of target antigen includes but is not limited to insulin, insulin-like growth factor, hGH, tPA, cell The factor such as interleukins (IL), for example, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, interferon (IFN) α, IFN β, IFN γ, IFN ω Or IFN τ, tumor necrosis factor (TNF) such as TNF α and TNF β, TNF γ, TRAIL；G-CSF, GM-CSF, M-CSF, MCP-1 and VEGF。
About: it is as used herein, when being applied to one or more target values, term " about " or " about " refer to similar In the value of illustrated reference value.In certain embodiments, term " about " or " about " refer in illustrated reference value (being more than or less than) 25% in either direction, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or more a small range value range, unless in addition saying It is bright or apparent (in addition to such number will be more than the 100% of probable value) from context in another way.
Association: it is as used herein, about two or more parts in use, term " association ", " conjugation ", " company Connect ", " attachment " and " tying " mean the part directly or served as the other parts of one or more of bridging agent each other Physical association or connection are to form sufficiently stable structure, so that under the condition such as physiological condition using the structure The part keeps physical association." association ", which needs not be, strictly to be combined by direct covalent chemical.It may further indicate ionic bond It closes or hydrogen bonding or connection based on hybridization is sufficiently stable so that " association " entity keeps physical association.
Difunctional: as used herein, term " difunctional " is refer to or maintain at least two functions any Substance, molecule or part.The function can realize identical result or Different Results.It can be identical or not for generating the structure of function With.For example, the modification RNA codified cytotoxic peptide (the first function) of bi-functional of the invention, while including coding RNA Those of nucleosides be in product in themselves and made from them cytotoxicity (the second function).In this example, double Delivering of functional modification RNA to cancer cell, which will be generated not only, can improve or the peptide or protein matter molecule for the treatment of cancer, but also In case of the translation for degrading rather than modifying RNA, the nucleosides of cytotoxicity payload is also delivered to cell.
Bio-compatible: as used herein, term " bio-compatible " means and living cells, tissue, organ or System compatible, Cause little or no damage, toxicity or the risk repelled by immune system.
Biodegradable: as used herein, term " biodegradable " means to resolve by the effect of being Harmless products.
Bioactivity: as used herein, phrase " bioactivity " refers to be had in biosystem and/or organism The feature of active any substance.For example, having the substance quilt of biological effect to the organism when applying to organism Think bioactivity.In a particular embodiment, if polynucleotides even of the invention, primary construct or mmRNA A part is that the relevant activity of biology is thought in bioactivity or simulation, then it is believed that polynucleotides, primary construct or mmRNA are Bioactivity.
The technical terms of chemistry: the following provide the definition of the various technical terms of chemistry of " acyl group " to " mercapto ".
As used herein term " acyl group ", which is represented, is connected to parent molecular group by carbonyl as herein defined Hydrogen or alkyl as herein defined (for example, halogenated alkyl) and by formoxyl (i.e. carboxy aldehyde group), acetyl group, propionyl Base, bytyry etc. illustrate.Exemplary unsubstituted acyl group includes 1 to 7,1 to 11 or 1 to 21 carbon.In some embodiment party In case, alkyl is further replaced with 1,2,3 or 4 substituent group as described herein.
As used herein term " acylamino- ", which is represented, is connected to parent molecule base by amino as herein defined The acyl group as herein defined of group is (that is,-N (RN1)-C (O)-R, wherein R is the H or C optionally replaced1-6、C1-10Or C1-20Alkane Base and RN1As defined herein).Exemplary unsubstituted acylamino- include 1 to 41 carbon (for example, 1 to 7,1 to 13,1 to 21,2 to 7,2 to 13,2 to 21 or 2 to 41 carbon).In some embodiments, alkyl further use it is as described herein 1,2, 3 or 4 substituent groups replace and/or amino is-NH2Or-NHRN1, wherein RN1It independently is OH, NO2、NH2、NRN2 2、SO2ORN2、 SO2RN2、SORN2, alkyl or aryl, and each RN2It can be H, alkyl or aryl.
As used herein term " acyloxy " represent by oxygen atom be connected to parent molecular group such as this paper institute (that is,-O-C (O)-R, wherein R is the H or C optionally replaced to the acyl group of definition1-6、C1-10Or C1-20Alkyl).It is exemplary unsubstituted Acyloxy includes 1 to 21 carbon (for example, 1 to 7 or 1 to 11 carbon).In some embodiments, alkyl is further used as herein 1,2,3 or 4 substituent group replaces and/or amino is-NH2Or-NHRN1, wherein RN1It independently is OH, NO2、NH2、 NRN2 2、SO2ORN2、SO2RN2、SORN2, alkyl or aryl, and each RN2It can be H, alkyl or aryl.
As used herein term " alkaryl ", which is represented, is connected to parent molecule by alkylidene as herein defined The aryl as herein defined of group.Exemplary unsubstituted alkaryl is 7 to 30 carbon (for example, 7 to 16 or 7 to 20 Carbon, such as C1-6Alkyl-C6-10Aryl, C1-10Alkyl-C6-10Aryl or C1-20Alkyl-C6-10Aryl).In some embodiments, sub- Alkyl and aryl respectively can further be replaced with for corresponding group 1,2,3 or 4 substituent group as herein defined.With phase Other groups plus prefix " alkane-(alk-) " are defined with mode, wherein " alkane " refers to C1-6Alkylidene, unless otherwise noted, and And the chemical structure of connection is as defined herein.
Term " alkyl-cycloalkyl " is represented through alkylidene as herein defined (for example, having 1 to 4,1 to 6,1 to 10 Or the alkylidene of 1 to 20 carbon) it is connected to the naphthenic base as herein defined of parent molecular group.In some embodiments In, alkylidene and naphthenic base respectively can further be taken with for corresponding group 1,2,3 or 4 substituent group as herein defined Generation.
As used herein term " alkenyl " represent unless otherwise specified otherwise have 2 to 20 carbon (for example, 2 to 6 or 2 to 10 carbon) the monovalent straight chain or branched group containing one or more carbon-to-carbon double bonds, and by vinyl, 1- propylene Base, 2- acrylic, 2- methyl-1-propylene base, 1- cyclobutenyl, 2- cyclobutenyl etc. illustrate.Alkenyl include cis-isomer and Transisomer.Alkenyl optionally use 1,2,3 or 4 as herein defined independently selected from amino, aryl, naphthenic base or The substituent group of heterocycle (for example, heteroaryl) or any exemplary alkyl substituent group described herein replace.
The chemical substituents of term " alkenyloxy group " representative formula-OR, wherein R is C2-20Alkenyl is (for example, C2-6Or C2-10Alkenyl), Unless otherwise specified.Exemplary alkenyloxy group includes ethyleneoxy, propenyloxy group etc..In some embodiments, alkenyl can be into one Step is replaced with 1,2,3 or 4 substituent group (for example, hydroxyl) as herein defined.
Term " miscellaneous alkyl aryl " refer to by alkylidene as herein defined be connected to parent molecular group as this Heteroaryl defined in text.Exemplary unsubstituted miscellaneous alkyl aryl is 2 to 32 carbon (for example, 2 to 22,2 to 18,2 to 17,2 To the carbon of 16,3 to 15,2 to 14,2 to 13 or 2 to 12, such as C1-6Alkyl-C1-12Heteroaryl, C1-10Alkyl-C1-12Heteroaryl or C1-20Alkyl-C1-12Heteroaryl).In some embodiments, alkylidene and heteroaryl respectively can be further with for corresponding bases Group's 1,2,3 or 4 substituent group as herein defined replaces.Miscellaneous alkyl aryl is the subset of alkyl heterocyclic.
Term " alkyl heterocyclic " represent by alkylidene as herein defined be connected to parent molecular group as Heterocycle defined in text.Exemplary unsubstituted alkyl heterocyclic is 2 to 32 carbon (for example, 2 to 22,2 to 18,2 to 17,2 To the carbon of 16,3 to 15,2 to 14,2 to 13 or 2 to 12, such as C1-6Alkyl-C1-12Heterocycle, C1-10Alkyl-C1-12Heterocycle or C1-20Alkyl-C1-12Heterocycle).In some embodiments, alkylidene and heterocycle respectively can be further with for corresponding bases Group's 1,2,3 or 4 substituent group as herein defined replaces.
The chemical substituents of term " alkoxy " representative formula-OR, wherein R is C1-20Alkyl is (for example, C1-6Or C1-10Alkyl), Unless otherwise specified.Exemplary alkoxy radicals include methoxyl group, ethyoxyl, propoxyl group (for example, positive propoxy and isopropoxy), uncle Butoxy etc..In some embodiments, alkyl can further with 1,2,3 or 4 substituent group as herein defined (for example, Hydroxyl or alkoxy) replace.
The alkoxy that term " alkyloxy-alkoxy " representative is replaced with alkoxy.Exemplary unsubstituted alkyloxy-alkoxy Including the carbon between 2 to 40 (for example, 2 to 12 or 2 to 20 carbon, such as C1-6Alkoxy -C1-6Alkoxy, C1-10Alkoxy- C1-10Alkoxy or C1-20Alkoxy -C1-20Alkoxy).In some embodiments, each alkoxy can be further with such as this 1 defined in text, 2,3 or 4 substituent groups replace.
The alkyl that term " alkoxyalkyl " representative is replaced with alkoxy.Exemplary unsubstituted alkoxyalkyl includes 2 Carbon between to 40 is (for example, 2 to 12 or 2 to 20 carbon, such as C1-6Alkoxy -C1-6Alkyl, C1-10Alkoxy -C1-10Alkyl or C1-20Alkoxy -C1-20Alkyl).In some embodiments, alkyl and alkoxy respectively can be further with for corresponding groups 1,2,3 or 4 substituent group as herein defined replaces.
As used herein term " alkoxy carbonyl " represent by carbonyl atom be connected to parent molecular group as (for example,-C (O)-OR, wherein R is the H or C optionally replaced to alkoxy as defined herein1-6、C1-10Or C1-20Alkyl).Example Property unsubstituted alkoxy carbonyl include 1 to 21 carbon (for example, 1 to 11 or 1 to 7 carbon).In some embodiments, alcoxyl Base is further replaced with 1,2,3 or 4 substituent group as described herein.
As used herein term " Alkoxycarbonylalkoxy " representative is taken with alkoxy carbonyl as herein defined (for example,-O- alkyl-C (O)-OR, wherein R is the C optionally replaced to the alkoxy as herein defined in generation1-6、C1-10Or C1-20 Alkyl).Exemplary unsubstituted Alkoxycarbonylalkoxy include 3 to 41 carbon (for example, 3 to 10,3 to 13,3 to 17,3 to 21 or 3 to 31 carbon, such as C1-6Alkoxy carbonyl-C1-6Alkoxy, C1-10Alkoxy carbonyl-C1-10Alkoxy or C1-20Alkoxy Carbonyl-C1-20Alkoxy).In some embodiments, each alkoxy is further independently with as described herein 1,2,3 Or 4 substituent groups (for example, hydroxyl) replace.
As used herein term " alkoxy carbonyl alkyl " is represented to be replaced with alkoxy carbonyl as herein defined Alkyl as herein defined (for example,-alkyl-C (O)-OR, wherein R is the C optionally replaced1-20、C1-10Or C1-6Alkyl). Exemplary unsubstituted alkoxy carbonyl alkyl includes 3 to 41 carbon (for example, 3 to 10,3 to 13,3 to 17,3 to 21 or 3 to 31 A carbon, such as C1-6Alkoxy carbonyl-C1-6Alkyl, C1-10Alkoxy carbonyl-C1-10Alkyl or C1-20Alkoxy carbonyl-C1-20Alkane Base).In some embodiments, each alkyl and alkoxy are further independently taken with 1,2,3 or 4 as described herein Dai Ji (for example, hydroxyl) replaces.
As used herein term " alkyl " includes that the straight chain of 1 to 20 carbon (for example, 1 to 10 or 1 to 6) and branch are satisfied And group, unless otherwise specified.Alkyl is by methyl, ethyl, n-propyl and isopropyl, normal-butyl, sec-butyl, isobutyl group and tertiary fourth Base, neopentyl etc. are for example, and optionally with one, two, three or (in the alkyl with two or more carbon In the case where) four substituent groups replace, the substituent group is independently selected from the group being made up of: (1) C1-6Alkoxy；(2) C1-6Alkyl sulphinyl；(3) amino as herein defined is (for example, unsubstituted amino is (that is,-NH2) or replace amino (that is,-N (RN1)2, wherein RN1As defined for amino)；(4)C6-10Aryl-C1-6Alkoxy；(5) azido；(6) halogenated Base；(7)(C2-9Heterocycle) oxygroup；(8) hydroxyl；(9) nitro；(10) oxo base (for example, carboxyl aldehyde or acyl group)；(11)C1-7Spiral shell Ring group；(12) thio alkoxy；(13) mercapto；(14)-CO2RA’, wherein RA’Selected from the group being made up of: (a) C1-20Alkane Base is (for example, C1-6Alkyl), (b) C2-20Alkenyl is (for example, C2-6Alkenyl), (c) C6-10Aryl, (d) hydrogen, (e) C1-6Alkyl-C6-10Virtue Base, (f) amino-C1-20Aryl, (g)-(CH2)s2(OCH2CH2)s1(CH2)s3The polyethylene glycol of OR ', wherein s1 be 1 to 10 it is whole Number (for example, 1 to 6 or 1 to 4), s2 and s3 respectively independently 0 to 10 integer (for example, 0 to 4,0 to 6,1 to 4,1 to 6 Or 1 to 10), and R ' is H or C1-20Alkyl, and (h)-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1The poly- second of amino- Glycol, wherein s1 is integer of 1 to 10 (for example, 1 to 6 or 1 to 4), the respective integer (example independently 0 to 10 of s2 and s3 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and each R such as,N1The C for independently being hydrogen or optionally replacing1-6Alkyl；(15)-C (O)NRB’RC’, wherein RB’And RC’Respectively independently selected from the group being made up of: (a) hydrogen, (b) C1-6Alkyl, (c) C6-10Virtue Base and (d) C1-6Alkyl-C6-10Aryl；(16)-SO2RD’, wherein RD’Selected from the group being made up of: (a) C1-6Alkyl, (b) C6-10Aryl, (c) C1-6Alkyl-C6-10Aryl and (d) hydroxyl；(17)-SO2NRE’RF’, wherein RE’And RF’Respectively independently Selected from the group being made up of: (a) hydrogen, (b) C1-6Alkyl, (c) C6-10Aryl and (d) C1-6Alkyl-C6-10Aryl；(18)-C (O)RG’, wherein RG’Selected from the group being made up of: (a) C1-20Alkyl is (for example, C1-6Alkyl), (b) C2-20Alkenyl is (for example, C2-6 Alkenyl), (c) C6-10Aryl, (d) hydrogen, (e) C1-6Alkyl-C6-10Aryl, (f) amino-C1-20Alkyl, (g)-(CH2)s2 (OCH2CH2)s1(CH2)s3The polyethylene glycol of OR ', wherein s1 is integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are respectively Integer (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10) independently 0 to 10, and R ' is H or C1-20Alkyl with And (h)-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1Amino-polyethylene glycol, wherein s1 be integer of 1 to 10 (for example, 1 To 6 or 1 to 4), the respective integer (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10) independently 0 to 10 of s2 and s3, And each RN1The C for independently being hydrogen or optionally replacing1-6Alkyl；(19)-NRH’C(O)RI’, wherein RH’Selected from what is be made up of Group: (a1) hydrogen and (b1) C1-6Alkyl, and RI’Selected from the group being made up of: (a2) C1-20Alkyl is (for example, C1-6Alkyl), (b2)C2-20Alkenyl is (for example, C2-6Alkenyl), (c2) C6-10Aryl, (d2) hydrogen, (e2) C1-6Alkyl-C6-10Aryl, (f2) amino- C1-20Alkyl, (g2)-(CH2)s2(OCH2CH2)s1(CH2)s3The polyethylene glycol of OR ', wherein s1 be integer of 1 to 10 (for example, 1 to 6 or 1 to 4), the respective integer (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10) independently 0 to 10 of s2 and s3, and And R ' is H or C1-20Alkyl, and (h2)-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1Amino-polyethylene glycol, wherein s1 For integer of 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 respectively independently 0 to 10 integer (for example, 0 to 4,0 to 1 to 4,1 to 6 or 1 to 10), and each R 6,N1The C for independently being hydrogen or optionally replacing1-6Alkyl；(20)-NRJ’C(O)ORK’, Middle RJ’Selected from the group being made up of: (a1) hydrogen and (b1) C1-6Alkyl, and RK’Selected from the group being made up of: (a2) C1-20 Alkyl is (for example, C1-6Alkyl), (b2) C2-20Alkenyl is (for example, C2-6Alkenyl), (c2) C6-10Aryl, (d2) hydrogen, (e2) C1-6Alkyl- C6-10Aryl, (f2) amino-C1-20Alkyl, (g2)-(CH2)s2(OCH2CH2)s1(CH2)s3The polyethylene glycol of OR ', wherein s1 is 1 To 10 integer (for example, 1 to 6 or 1 to 4), s2 and s3 respectively independently 0 to 10 integer (for example, 0 to 4,0 to 6,1 To 4,1 to 6 or 1 to 10), and R ' is H or C1-20Alkyl, and (h2)-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1's Amino-polyethylene glycol, wherein s1 is integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are respectively independently 0 to 10 Integer (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and each RN1The C for independently being hydrogen or optionally replacing1-6Alkane Base；And (21) amidine.In some embodiments, these groups respectively can be further substituted as described herein.For example, C1The alkylidene of alkaryl can further be replaced to obtain corresponding aroyl substituent group with oxo base.
As used herein term " alkylidene " and prefix " alkyl-" are represented through two hydrogen atoms of removal derived from straight The saturated divalent hydrocarbon radical of chain or branched-chain saturated hydrocarbon, and illustrated by methylene, ethylidene, isopropylidene etc..Term " Cx-y Alkylidene " and prefix " Cx-yAlkyl-" represents the alkylidene with the carbon between x and y.For x example values be 1,2,3, 4,5 and 6, and be 2,3,4,5,6,7,8,9,10,12,14,16,18 or 20 (for example, C for the example values of y1-6、C1-10、 C2-20、C2-6、C2-10Or C2-20Alkylidene).In some embodiments, alkylidene can further such as be determined with for alkyl herein 1,2,3 or 4 substituent group of justice replaces.
As used herein term " alkyl sulphinyl ", which is represented, is connected to parent molecular group by-S (O)-group Alkyl.Exemplary unsubstituted alkyl sulphinyl is 1 to 6,1 to 10 or 1 to 20 carbon.In some embodiments, alkane Base can further be replaced with 1,2,3 or 4 substituent group as herein defined.
As used herein term " alkylsulfinylalkyl " represent by alkyl sulphinyl replace such as this paper institute The alkyl of definition.Exemplary unsubstituted alkylsulfinylalkyl is 2 to 12,2 to 20 or 2 to 40 carbon.In some implementations In scheme, each alkyl can further be replaced with 1,2,3 or 4 substituent group as herein defined.
As used herein term " alkynyl " represents 2 to 20 carbon atoms containing carbon-carbon triple bond (for example, 2 to 4,2 To 6 or 2 to 10 carbon) monovalent straight chain or branched group and by acetenyl, 1- propinyl etc. illustrate.Alkynyl can be optional Ground 1,2,3 or 4 substitution independently selected from aryl, naphthenic base or heterocycle (for example, heteroaryl) as herein defined Base or any exemplary alkyl substituent group described herein replace.
The chemical substituents of term " alkynyloxy group " representative formula-OR, wherein R is C2-20Alkynyl is (for example, C2-6Or C2-10Alkynyl), Unless otherwise specified.Exemplary alkynyloxy group includes acetylene oxygroup, propargyl alcoholate etc..In some embodiments, alkynyl can be into one Step is replaced with 1,2,3 or 4 substituent group (for example, hydroxyl) as herein defined.
As used herein term " amidine " representative-C (=NH) NH2Group.
As used herein term " amino " representative-N (RN1)2, wherein each RN1It independently is H, OH, NO2、N(RN2)2、 SO2ORN2、SO2RN2、SORN2, N-protected base, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, naphthenic base, alkyl-cycloalk Base, carboxyalkyl, sulfoalkyl, heterocycle (for example, heteroaryl) or alkyl heterocyclic (for example, miscellaneous alkyl aryl), wherein these The R enumeratedN1Group respectively can optionally replace as defined herein for each group；Or two RN1Combination forms heterocycle Base or N-protected base and wherein each RN2It independently is H, alkyl or aryl.Amino of the invention can be unsubstituted amino (that is,-NH2) or replace amino (that is,-N (RN1)2).In preferred embodiments, amino is-NH2Or-NHRN1, wherein RN1Solely It is on the spot OH, NO2、NH2、NRN2 2、SO2ORN2、SO2RN2、SORN2, alkyl, carboxyalkyl, sulfoalkyl or aryl, and each RN2It can For H, C1-20Alkyl is (for example, C1-6Alkyl) or C6-10Aryl.
Term " amino acid " as described herein refers to side chain, amino and acid groups (for example,-CO2The carboxyl of H or- SO3The sulfo group of H) molecule, wherein amino acid is connected to parent molecule base by side chain, amino or acid groups (for example, side chain) Group.In some embodiments, amino acid is connected to parent molecular group by carbonyl, and wherein side chain or amino are connected to carbonyl Base.Exemplary side chain includes the alkyl optionally replaced, aryl, heterocycle, alkaryl, alkyl heterocyclic, aminoalkyl, amino first Acyl and carboxyalkyl.Exemplary amino acid includes alanine, arginine, asparagine, aspartic acid, half Guang ammonia Acid, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, first sulphur ammonia Acid, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, Soviet Union's ammonia Acid, tryptophan, tyrosine and valine.Amino acid group optionally use one, two, three or (tool there are two or In the case where the amino acid group of more carbon) four substituent groups replace, and the substituent group is independently selected from being made up of Group: (1) C1-6Alkoxy；(2)C1-6Alkyl sulphinyl；(3) amino as herein defined is (for example, unsubstituted amino (that is,-NH2) or replace amino (that is,-N (RN1)2, wherein RN1As defined for amino)；(4)C6-10Aryl-C1-6Alcoxyl Base；(5) azido；(6) halogeno-group；(7)(C2-9Heterocycle) oxygroup；(8) hydroxyl；(9) nitro；(10) oxo base is (for example, carboxylic Base aldehyde or acyl group)；(11)C1-7Loop coil base；(12) thio alkoxy；(13) mercapto；(14)-CO2RA’, wherein RA’Selected from by with The group of lower composition: (a) C1-20Alkyl is (for example, C1-6Alkyl), (b) C2-20Alkenyl is (for example, C2-6Alkenyl), (c) C6-10Aryl, (d) Hydrogen, (e) C1-6Alkyl-C6-10Aryl, (f) amino-C1-20Aryl, (g)-(CH2)s2(OCH2CH2)s1(CH2)s3The poly- second two of OR ' Alcohol, wherein s1 is integer of 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 respectively independently 0 to 10 integer (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and R ' is H or C1-20Alkyl, and (h)-NRN1(CH2)s2(CH2CH2O)s1 (CH2)s3NRN1Amino-polyethylene glycol, wherein s1 is integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are respectively solely On the spot it is integer of 0 to 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and each RN1It independently is hydrogen or optionally takes The C in generation1-6Alkyl；(15)-C(O)NRB’RC’, wherein RB’And RC’Respectively independently selected from the group being made up of: (a) hydrogen, (b)C1-6Alkyl, (c) C6-10Aryl and (d) C1-6Alkyl-C6-10Aryl；(16)-SO2RD’, wherein RD’Selected from being made up of Group: (a) C1-6Alkyl, (b) C6-10Aryl, (c) C1-6Alkyl-C6-10Aryl and (d) hydroxyl；(17)-SO2NRE’RF’, wherein RE’And RF’Respectively independently selected from the group being made up of: (a) hydrogen, (b) C1-6Alkyl, (c) C6-10Aryl and (d) C1-6Alkane Base-C6-10Aryl；(18)-C(O)RG’, wherein RG’Selected from the group being made up of: (a) C1-20Alkyl is (for example, C1-6Alkyl), (b)C2-20Alkenyl is (for example, C2-6Alkenyl), (c) C6-10Aryl, (d) hydrogen, (e) C1-6Alkyl-C6-10Aryl, (f) amino-C1-20Alkane Base, (g)-(CH2)s2(OCH2CH2)s1(CH2)s3The polyethylene glycol of OR ', wherein s1 be integer of 1 to 10 (for example, 1 to 6 or 1 to 4), the respective integer (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10) independently 0 to 10 of s2 and s3, and R ' is H Or C1-20Alkyl and (h)-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1Amino-polyethylene glycol, wherein s1 is 1 to 10 Integer (for example, 1 to 6 or 1 to 4), s2 and s3 respectively independently 0 to 10 integer (for example, 0 to 4,0 to 6,1 to 4,1 to , and each R 6 or 1 to 10)N1The C for independently being hydrogen or optionally replacing1-6Alkyl；(19)-NRH’C(O)RI’, wherein RH’Selected from by Group consisting of: (a1) hydrogen and (b1) C1-6Alkyl, and RI’Selected from the group being made up of: (a2) C1-20Alkyl (for example, C1-6Alkyl), (b2) C2-20Alkenyl is (for example, C2-6Alkenyl), (c2) C6-10Aryl, (d2) hydrogen, (e2) C1-6Alkyl-C6-10Aryl, (f2) amino-C1-20Alkyl, (g2)-(CH2)s2(OCH2CH2)s1(CH2)s3The polyethylene glycol of OR ', wherein s1 is integer of 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 respectively independently 0 to 10 integer (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 To 10), and R ' is H or C1-20Alkyl, and (h2)-NRN1(CH2)s2(CH2CH2O)s1(CH2)s3NRN1The poly- second two of amino- Alcohol, wherein s1 is integer of 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 respectively independently 0 to 10 integer (for example, , and each R 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10)N1The C for independently being hydrogen or optionally replacing1-6Alkyl；(20)-NRJ’C (O)ORK’, wherein RJ’Selected from the group being made up of: (a1) hydrogen and (b1) C1-6Alkyl, and RK’Selected from what is be made up of Group: (a2) C1-20Alkyl is (for example, C1-6Alkyl), (b2) C2-20Alkenyl is (for example, C2-6Alkenyl), (c2) C6-10Aryl, (d2) hydrogen, (e2)C1-6Alkyl-C6-10Aryl, (f2) amino-C1-20Alkyl, (g2)-(CH2)s2(OCH2CH2)s1(CH2)s3The poly- second two of OR ' Alcohol, wherein s1 is integer of 1 to 10 (for example, 1 to 6 or 1 to 4), s2 and s3 respectively independently 0 to 10 integer (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and R ' is H or C1-20Alkyl, and (h2)-NRN1(CH2)s2(CH2CH2O)s1 (CH2)s3NRN1Amino-polyethylene glycol, wherein s1 is integer of 1 to 10 (for example, 1 to 6 or 1 to 4), and s2 and s3 are respectively solely On the spot it is integer of 0 to 10 (for example, 0 to 4,0 to 6,1 to 4,1 to 6 or 1 to 10), and each RN1It independently is hydrogen or optionally takes The C in generation1-6Alkyl；And (21) amidine.In some embodiments, these groups respectively can further be taken as described herein Generation.
As used herein term " aminoalkoxy " represent by amino as herein defined replace such as this paper institute The alkoxy of definition.Alkyl and amino respectively can be further with for 1,2,3 or 4 substitutions as described herein of corresponding group Base replaces (for example, CO2RA’, wherein RA’Selected from the group being made up of: (a) C1-6Alkyl, (b) C6-10Aryl, (c) hydrogen and (d) C1-6Alkyl-C6-10Aryl, such as carboxyl).
As used herein term " aminoalkyl " represent by amino as herein defined replace as this paper determines The alkyl of justice.Alkyl and amino respectively can further be taken with for corresponding group 1,2,3 or 4 substituent group as described herein In generation, is (for example, CO2RA’, wherein RA’Selected from the group being made up of: (a) C1-6Alkyl, (b) C6-10Aryl, (c) hydrogen and (d) C1-6Alkane Base-C6-10Aryl, such as carboxyl).
As used herein term " aryl " represents the monocycle with one or two aromatic ring, bicyclic or polycyclic carbocyclic ring system It unites and by phenyl, naphthalene, 1,2- ihydro naphthyl, 1,2,3,4- tetralyls, anthryl, phenanthryl, fluorenyl, indanyl, indenyl etc. For example, and optionally replaced with 1,2,3,4 or 5 substituent group independently selected from the group being made up of: (1) C1-7Acyl group (for example, carboxyl aldehyde)；(2)C1-20Alkyl is (for example, C1-6Alkyl, C1-6Alkoxy -C1-6Alkyl, C1-6Alkyl sulfenyl Base-C1-6Alkyl, amino-C1-6Alkyl, azido-C1-6Alkyl, (carboxyl aldehyde)-C1-6Alkyl, halogenated-C1-6Alkyl is (for example, complete Fluoroalkyl), hydroxyl-C1-6Alkyl, nitro-C1-6Alkyl or C1-6Thio alkoxy-C1-6Alkyl)；(3)C1-20Alkoxy (for example, C1-6Alkoxy, such as perfluoro alkoxy)；(4)C1-6Alkyl sulphinyl；(5)C6-10Aryl；(6) amino；(7)C1-6Alkyl-C6-10 Aryl；(8) azido；(9)C3-8Naphthenic base；(10)C1-6Alkyl-C3-8Naphthenic base；(11) halogeno-group；(12)C1-12Heterocycle (for example, C1-12Heteroaryl)；(13)(C1-12Heterocycle) oxygroup；(14) hydroxyl；(15) nitro；(16)C1-20Thio alkoxy (example Such as, C1-6Thio alkoxy)；(17)-(CH2)qCO2RA’, the integer that wherein q is 0 to 4, and RA’Selected from what is be made up of Group: (a) C1-6Alkyl, (b) C6-10Aryl, (c) hydrogen and (d) C1-6Alkyl-C6-10Aryl；(18)-(CH2)qCONRB’RC’, wherein q Integer and wherein R for 0 to 4B’And RC’Independently selected from the group being made up of: (a) hydrogen, (b) C1-6Alkyl, (c) C6-10Virtue Base and (d) C1-6Alkyl-C6-10Aryl；(19)-(CH2)qSO2RD’, integer and wherein R that wherein q is 0 to 4D’Selected from by following The group of composition: (a) alkyl, (b) C6-10Aryl and (c) alkyl-C6-10Aryl；(20)-(CH2)qSO2NRE’RF’, wherein q is 0 to 4 Integer and wherein RE’And RF’Respectively independently selected from the group being made up of: (a) hydrogen, (b) C1-6Alkyl, (c) C6-10Virtue Base and (d) C1-6Alkyl-C6-10Aryl；(21) mercapto；(22)C6-10Aryloxy group；(23)C3-8Cycloalkyloxy；(24)C6-10Virtue Base-C1-6Alkoxy；(25)C1-6Alkyl-C1-12Heterocycle (such as C1-6Alkyl-C1-12Heteroaryl)；(26)C2-20Alkenyl；And (27)C2-20Alkynyl.In some embodiments, these groups respectively can be further substituted as described herein.For example, C1- Alkaryl or C1The alkylidene of alkyl heterocyclic can further be replaced to obtain corresponding aroyl and (heterocycle) acyl with oxo base Base substituent group.
As used herein term " alkoxy aryl " represent by oxygen atom be connected to parent molecular group as Alkaryl defined in text.Exemplary unsubstituted alkoxyalkyl include 7 to 30 carbon (for example, 7 to 16 or 7 to 20 carbon, Such as C6-10Aryl-C1-6Alkoxy, C6-10Aryl-C1-10Alkoxy or C6-10Aryl-C1-20Alkoxy).In some embodiments In, alkoxy aryl can be used 1,2,3 or 4 substituent group as herein defined to replace.
The chemical substituents of term " aryloxy group " representative formula-OR ', wherein R ' is the aryl of 6 to 18 carbon, unless in addition referring to It is bright.In some embodiments, aryl can be used 1,2,3 or 4 substituent group as herein defined to replace.
As used herein term " aroyl " represent by carbonyl be connected to parent molecular group as this paper determines The aryl of justice.Exemplary unsubstituted aroyl has 7 to 11 carbon.In some embodiments, aryl is available such as this paper institute 1,2,3 or 4 substituent group of definition replaces.
Term " azido " representative-N3Group is also denoted as-N=N=N.
As used herein term " bicyclic " refers to that tool can be aromatic or non-aromatic there are two the structure of ring 's.Twin nuclei includes two rings of loop coil base as herein defined and shared one or more bridgings, wherein the bridging It may include an atom or the chain including two, three or more atom.Exemplary bicyclic group includes bicyclic carbocyclic group, In the first ring and the second ring be carbocylic radical as herein defined；Bicyclic aryl, wherein the first ring and the second ring are such as this paper institute The aryl of definition；Bicyclic heterocyclic radical, wherein the first ring is heterocycle and the second ring is carbocylic radical (for example, aryl) or heterocycle (for example, heteroaryl)；And bicyclic heteroaryl, wherein the first ring is heteroaryl and the second ring is carbocylic radical (for example, aryl) Or heterocycle (for example, heteroaryl).In some embodiments, bicyclic radicals are available is directed to naphthenic base, heterocycle and aryl such as 1,2,3 or 4 substituent group as defined herein replaces.
As used herein term " carbocyclic ring " and " carbocylic radical " refer to wherein can be aromatic or non-aromatic ring The C optionally replaced formed by carbon atom3-12Monocyclic, bicyclic or tricyclic structure.Carbocyclic ring structure includes naphthenic base, cycloalkenyl and virtue Base.
As used herein term " carbamoyl " representative-C (O)-N (RN1)2, wherein each RN1The meaning see herein In " amino " definition provided.
As used herein term " carbamoylalkyl " representative is replaced by carbamoyl as herein defined Alkyl as herein defined.Alkyl can further be replaced with 1,2,3 or 4 substituent group as described herein.
As used herein term " carbamoyl " refers to structure-NRN1C (=O) OR or-OC (=O) N (RN1)2Carbamate groups, wherein each RN1The meaning see " amino " provided herein definition in, and R be as herein Defined alkyl, naphthenic base, alkyl-cycloalkyl, aryl, alkaryl, heterocycle (for example, heteroaryl) or alkane heterocycle (example Such as, alkane heteroaryl).
As used herein term " carbonyl " represents C (O) group, is also denoted as C=O.
Term " carboxyl aldehyde " represents the acyl group with structure-CHO.
As used herein term " carboxyl " means-CO2H。
As used herein term " Carboxyalkoxy " represent by carboxyl as herein defined replace such as this paper institute The alkoxy of definition.Alkoxy can further be replaced with for alkyl 1,2,3 or 4 substituent group as described herein.
As used herein term " carboxyalkyl " represent by carboxyl as herein defined replace as this paper determines The alkyl of justice.Alkyl can further be replaced with 1,2,3 or 4 substituent group as described herein.
As used herein term " cyano " representative-CN group.
The chemical substituents of term " cycloalkyloxy " representative formula-OR, wherein R is C as herein defined3-8Naphthenic base is removed It is non-otherwise indicated.Naphthenic base can further be replaced with 1,2,3 or 4 substituent group as described herein.Exemplary unsubstituted ring Alkoxy is 3 to 8 carbon.In some embodiments, naphthenic base can further be replaced with 1,2,3 or 4 as described herein Base replaces.
As used herein term " naphthenic base " represents the unit price saturation or unsaturation non-aromatic ring of three to eight carbon Alkyl, unless otherwise specified, and by cyclopropyl, cyclobutyl, cyclopenta, cyclohexyl, suberyl, bicyclic [2.2.1.] heptyl etc. It illustrates.When naphthenic base includes a carbon-to-carbon double bond, naphthenic base can be described as " cycloalkenyl ".Exemplary cycloalkenyl groups include ring Pentenyl, cyclohexenyl group etc..Naphthenic base of the invention can be optionally with following substitution: (1) C1-7Acyl group (for example, carboxyl aldehyde)；(2) C1-20Alkyl is (for example, C1-6Alkyl, C1-6Alkoxy -C1-6Alkyl, C1-6Alkyl sulphinyl-C1-6Alkyl, amino-C1-6Alkyl, Azido-C1-6Alkyl, (carboxyl aldehyde)-C1-6Alkyl, halogenated-C1-6Alkyl (for example, perfluoroalkyl), hydroxyl-C1-6Alkyl, nitre Base-C1-6Alkyl or C1-6Thio alkoxy-C1-6Alkyl)；(3)C1-20Alkoxy is (for example, C1-6Alkoxy, such as perfluoroalkoxy Base)；(4)C1-6Alkyl sulphinyl；(5)C6-10Aryl；(6) amino；(7)C1-6Alkyl-C6-10Aryl；(8) azido；(9) C3-8Naphthenic base；(10)C1-6Alkyl-C3-8Naphthenic base；(11) halogeno-group；(12)C1-12Heterocycle is (for example, C1-12Heteroaryl)； (13)(C1-12Heterocycle) oxygroup；(14) hydroxyl；(15) nitro；(16)C1-20Thio alkoxy is (for example, C1-6Thio alkoxy)； (17)-(CH2)qCO2RA’, the integer that wherein q is 0 to 4, and RA’Selected from the group being made up of: (a) C1-6Alkyl, (b) C6-10 Aryl, (c) hydrogen and (d) C1-6Alkyl-C6-10Aryl；(18)-(CH2)qCONRB’RC’, integer and wherein that wherein q is 0 to 4 RB’And RC’Independently selected from the group being made up of: (a) hydrogen, (b) C1-6Alkyl, (c) C6-10Aryl and (d) C1-6Alkyl-C6-10 Aryl；(19)-(CH2)qSO2RD’, integer and wherein R that wherein q is 0 to 4D’Selected from the group being made up of: (a) C6-10Alkane Base, (b) C6-10Aryl and (c) C1-6Alkyl-C6-10Aryl；(20)-(CH2)qSO2NRE’RF’, wherein q be 0 to 4 integer and Wherein RE’And RF’Respectively independently selected from the group being made up of: (a) hydrogen, (b) C6-10Alkyl, (c) C6-10Aryl and (d) C1-6Alkyl-C6-10Aryl；(21) mercapto；(22)C6-10Aryloxy group；(23)C3-8Cycloalkyloxy；(24)C6-10Aryl-C1-6Alkane Oxygroup；(25)C1-6Alkyl-C1-12Heterocycle (such as C1-6Alkyl-C1-12Heteroaryl)；(26) oxo base；(27)C2-20Alkenyl；With And (28) C2-20Alkynyl.In some embodiments, these groups respectively can be further substituted as described herein.For example, C1Alkaryl or C1The alkylidene of alkyl heterocyclic can further be replaced to obtain corresponding aroyl and (heterocycle) with oxo base Acyl substituent.
As used herein term " diastereoisomer " means each other not for mirror image and non-superimposable vertical each other Body isomers.
The medicament of as used herein term " effective quantity " is to be enough to realize beneficial or desired as a result, for example clinical tie The amount of fruit, and therefore " effective quantity " depends on applying its background.For example, under the background of the medicament of application treating cancer, A effective amount of medicament is for example to be enough to realize the as herein defined of cancer compared with no application medicament response obtained The amount for the treatment of.
Term " enantiomter " as used herein means at least 80% (that is, a kind of at least 90% mapping is different Structure body and at most 10% another enantiomter), preferably at least 90% and more preferably at least 98% optical purity or Every kind of independent optical activity of the compounds of this invention of enantiomter excessive (as determined by the method for this field Plays) Form.
As used herein term " halogeno-group " represents the halogen selected from bromine, chlorine, iodine or fluorine.
As used herein term " halogenated alkoxy " represent by halogen group (that is, F, Cl, Br or I) replaces as Alkoxy defined in text.Halogenated alkoxy can be with one, two, three or (in the alkyl feelings with two or more carbon Under condition) four halogens replace.Halogenated alkoxy includes perfluoro alkoxy (for example,-OCF3)、-OCHF2、-OCH2F、-OCCl3、- OCH2CH2Br、-OCH2CH(CH2CH2Br)CH3And-OCHICH3.In some embodiments, halogenated alkoxy can be further Replaced with for alkyl 1,2,3 or 4 substituent group as described herein.
As used herein term " halogenated alkyl " represent by halogen group (that is, F, Cl, Br or I) replaces such as this paper Defined alkyl.Halogenated alkyl can with one, two, three or (in the case that with two or more carbon alkyl) four A halogen replaces.Halogenated alkyl includes perfluoroalkyl (for example,-CF3)、-CHF2、-CH2F、-CCl3、-CH2CH2Br、-CH2CH (CH2CH2Br)CH3And-CHICH3.In some embodiments, halogenated alkyl can be further with as described herein for alkyl 1,2,3 or 4 substituent group replace.
As used herein term " miscellaneous alkylidene " refers to wherein one or two composition carbon atom respectively by nitrogen, oxygen Or the alkylidene as herein defined of sulphur substitution.In some embodiments, miscellaneous alkylidene can be further with for alkylidene 1,2,3 or 4 substituent group as described herein replaces.
As used herein term " heteroaryl " represents the subset of aromatic heterocycle as herein defined: that is, They contain 4n+2 pi-electron in monocycle or polycyclic loop system.Exemplary unsubstituted heteroaryl have 1 to 12 (for example, 1 to 11,1 to 10,1 to 9,2 to 12,2 to 11,2 to 10 or 2 to 9) a carbon.In some embodiments, heteroaryl is used for miscellaneous 1 defined in ring group, 2,3 or 4 substituent groups replace.
As used herein term " heterocycle ", which represents, contains one, two, three or four heteroatomic 5 member ring, 6 Member ring or 7 member rings (unless otherwise specified), the hetero atom is independently selected from the group being made of nitrogen, oxygen and sulphur.5 member rings have 0 To 2 double bonds, and 6 member rings and 7 member rings have 0 to 3 double bond.Exemplary unsubstituted heterocycle is with 1 to 12 (for example, 1 To 11,1 to 10,1 to 9,2 to 12,2 to 11,2 to 10 or 2 to 9) a carbon.Term " heterocycle " also represents polycyclic with bridging Two non-adjacent members of the heterocyclic compound of structure, wherein one or more carbon and/or heteroatom bridges receipts or other documents in duplicate ring, such as quinine Ring group.Term " heterocycle " includes bicyclic radicals, three cyclic groups and four cyclic groups, more than any of them heterocyclic fused to one, Two or three carbocyclic rings, such as aromatic ring, cyclohexane ring, cyclohexene ring, pentamethylene ring, cyclopentene ring or another monocyclic heterocycles, Such as indyl, quinolyl, isoquinolyl, tetrahydric quinoline group, benzofuranyl, benzothienyl.The example packet of annelated heterocycles Include tropane and 1,2,3,5,8,8a- hexahydro indolizines.Heterocycle includes pyrrole radicals, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazoles Quinoline base, pyrazolidinyl, imidazole radicals, imidazolinyl, imidazolidinyl, pyridyl group, piperidyl, homopiperidinyl, pyrazinyl, piperazinyl, Pyrimidine radicals, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidinyl, morpholinyl, thio-morpholinyl, thiazolyl, thiophene Oxazolidinyl, isothiazolyl, isothiazole alkyl, indyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, dihydro-quinoxaline Base, quinazolyl, cinnoline base, phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, diazosulfide base, furans Base, thienyl, thiazolidinyl, isothiazolyl, triazolyl, tetrazole radical, oxadiazoles base (for example, 1,2,3-oxadiazoles base), purine Base, thiadiazolyl group (for example, 1,2,3- thiadiazolyl group), tetrahydrofuran base, dihydrofuryl, tetrahydro-thienyl, dihydrothiophene, Indolinyl, dihydroquinoline base, tetrahydric quinoline group, tetrahydro isoquinolyl, dihydro-isoquinoline base, pyranose, dihydro pyranyl, Dithiazole base, benzofuranyl, isobenzofuran-base, benzothienyl etc., including its dihydro-form and four hydrogen forms, wherein one A or multiple double bonds are reduced and are substituted with hydrogen.Other examples heterocycle includes: 2,3,4,5- tetrahydro -2- oxos-oxazole Base；2,3- dihydro-2-oxo -1H- imidazole radicals；2,3,4,5- tetrahydro -5- oxo -1H- pyrazolyls are (for example, 2,3,4,5- tetrahydros - 2- phenyl -5- oxo -1H- pyrazolyl)；2,3,4,5- tetrahydro -2,4- dioxo -1H- imidazole radicals are (for example, 2,3,4,5- tetrahydros - 2,4- dioxo -5- methyl -5- phenyl -1H- imidazole radicals)；2,3- dihydro -2- thio -1,3,4- oxadiazoles bases are (for example, 2,3- Thio -5- the phenyl -1,3 of dihydro -2-, 4- oxadiazoles base)；4,5- dihydro -5- oxo -1H- triazolyls are (for example, 4,5- dihydro -3- Methyl -4- amino 5- oxo -1H- triazolyl)；1,2,3,4- tetrahydro -2,4- dioxo pyridyl group is (for example, 1,2,3,4- tetrahydro - 2,4- dioxo -3,3- parvoline bases)；2,6- dioxo-piperidin bases are (for example, 2,6- dioxo -3- ethyl -3- phenyl Piperidyl)；1,6- dihydro -6- oxo-pyrimidine base；1,6- dihydro -4- oxo-pyrimidine base is (for example, 2- (methyl mercapto) -1,6- dihydro - 4- oxo -5- methylpyrimidine -1- base)；1,2,3,4- tetrahydro -2,4- dioxo pyrimidine radicals is (for example, 1,2,3,4- tetrahydro -2,4- Dioxo -3- ethyl-pyrimidine base)；1,6- dihydro -6- oxo-pyridazinyl is (for example, 1,6- dihydro -6- oxo -3- ethyl pyridazine Base)；1,6- dihydro -6- oxo -1,2,4- triazine radical (for example, 1,6- dihydro -5- isopropyl -6- oxo -1,2,4- triazine radical)； 2,3- dihydro-2-oxo -1H- indyls are (for example, 3,3- dimethyl -2,3- dihydro-2-oxo -1H- indyls and 2,3- bis- Oxo -3 hydrogen -2-, 3 '-spirocyclopropane -1H- indoles -1- bases)；1,3- dihydro -1- oxo -2H- isoindolyl；1,3- dihydro -1, 3- dioxo -2H- isoindolyl；1H- benzopyrene oxazolyl (for example, 1- (ethoxy carbonyl) -1H- benzopyrene oxazolyl)；2,3- bis- Hydrogen -2- oxo -1H- benzimidazolyl (for example, 3- ethyl -2,3- dihydro-2-oxo -1H- benzimidazolyl)；2,3- dihydros- 2- oxo-benzoxazolyl (for example, chloro- 2, the 3- dihydro-2-oxo-benzoxazolyl of 5-)；2,3- dihydro-2-oxos-benzo Oxazolyl；2- oxo -2H- benzopyranyl；Isosorbide-5-Nitrae-benzo dioxane base；1,3- benzo dioxane base；2, 3- dihydro -3- oxo, 4H-1,3- benzothiazine base；3,4- dihydro -4- oxo -3H- quinazolyls are (for example, 2- methyl -3,4- Dihydro -4- oxo -3H- quinazolyl)；1,2,3,4- tetrahydro -2,4- dioxo -3H- quinazolyl (for example, 1- ethyl -1,2, 3,4- tetrahydro -2,4- dioxo -3H- quinazolyls)；1,2,3,6- tetrahydro -2,6- dioxo -7H- purine radicals (for example, 1,2, 3,6- tetrahydro -1,3- dimethyl -2,6- dioxo -7H- purine radicals)；1,2,3,6- tetrahydro -2,6- dioxo -1H- purine radicals (for example, 1,2,3,6- tetrahydro -3,7- dimethyl -2,6- dioxo -1H- purine radicals)；2- oxo benzo [c, d] indyl；1, 1- dioxo -2H- naphtho- [1,8-c, d] isothiazolyl；And 1,8- naphthylene dimethylamido.Other heterocycle includes 3, 3a, 4,5,6,6a- hexahydros-pyrrolo- [3,4-b] pyrroles-(2H)-base and 2,5- diazabicylo [2.2.1] hept- 2- base, high piperazine Piperazine base (or Diazesuberane base), THP trtrahydropyranyl, dithiazole base, benzofuranyl, benzothienyl, oxepane Base, thia cycloheptyl alkyl, Azacyclooctane base, oxocane base (oxecanyl) and thia cyclooctane base.Heterocyclic group It further include the group of following formula
Wherein
E ' is selected from the group being made of-N- and-CH-；F ' is selected from by-N=CH- ,-NH-CH2-、-NH-C(O)-、-NH-、-CH =N- ,-CH2- NH- ,-C (O)-NH- ,-CH=CH- ,-CH2-、-CH2CH2-、-CH2O-、-OCH2,-O- and-S- composition group； And G ' is selected from the group being made of-CH- and-N-.Any heterocycle being mentioned herein can be optionally with one, two, three, four Or five substituent groups replace, the substituent group is independently selected from the group being made up of: (1) C1-7Acyl group (for example, carboxyl aldehyde)； (2)C1-20Alkyl is (for example, C1-6Alkyl, C1-6Alkoxy -C1-6Alkyl, C1-6Alkyl sulphinyl-C1-6Alkyl, amino-C1-6Alkane Base, azido-C1-6Alkyl, (carboxyl aldehyde)-C1-6Alkyl, halogenated-C1-6Alkyl (for example, perfluoroalkyl), hydroxyl-C1-6Alkyl, Nitro-C1-6Alkyl or C1-6Thio alkoxy-C1-6Alkyl)；(3)C1-20Alkoxy is (for example, C1-6Alkoxy, such as perfluoroalkoxy Base)；(4)C1-6Alkyl sulphinyl；(5)C6-10Aryl；(6) amino；(7)C1-6Alkyl-C6-10Aryl；(8) azido；(9) C3-8Naphthenic base；(10)C1-6Alkyl-C3-8Naphthenic base；(11) halogeno-group；(12)C1-12Heterocycle is (for example, C2-12Heteroaryl)； (13)(C1-12Heterocycle) oxygroup；(14) hydroxyl；(15) nitro；(16)C1-20Thio alkoxy is (for example, C1-6Thio alkoxy)； (17)-(CH2)qCO2RA’, the integer that wherein q is 0 to 4, and RA’Selected from the group being made up of: (a) C1-6Alkyl, (b) C6-10 Aryl, (c) hydrogen and (d) C1-6Alkyl-C6-10Aryl；(18)-(CH2)qCONRB’RC’, integer and wherein that wherein q is 0 to 4 RB’And RC’Independently selected from the group being made up of: (a) hydrogen, (b) C1-6Alkyl, (c) C6-10Aryl and (d) C1-6Alkyl-C6-10 Aryl；(19)-(CH2)qSO2RD’, integer and wherein R that wherein q is 0 to 4D’Selected from the group being made up of: (a) C1-6Alkane Base, (b) C6-10Aryl and (c) C1-6Alkyl-C6-10Aryl；(20)-(CH2)qSO2NRE’RF’, wherein q be 0 to 4 integer and Wherein RE’And RF’Respectively independently selected from the group being made up of: (a) hydrogen, (b) C1-6Alkyl, (c) C6-10Aryl and (d) C1-6 Alkyl-C6-10Aryl；(21) mercapto；(22)C6-10Aryloxy group；(23)C3-8Cycloalkyloxy；(24) alkoxy aryl；(25)C1-6 Alkyl-C1-12Heterocycle (such as C1-6Alkyl-C1-12Heteroaryl)；(26) oxo base；(27)(C1-12Heterocycle) imino group；(28) C2-20Alkenyl；And (29) C2-20Alkynyl.In some embodiments, these groups respectively can as described herein further by Replace.For example, C1Alkaryl or C1The alkylidene of alkyl heterocyclic can further be replaced to obtain corresponding aroyl with oxo base (heterocycle) acyl substituent.
As used herein term " (heterocycle) imino group ", which is represented, is connected to parent molecular group by imino group Heterocycle as herein defined.In some embodiments, 1,2,3 or 4 substitution as herein defined can be used in heterocycle Base replaces.
As used herein term " (heterocycle) oxygroup " represent by oxygen atom be connected to parent molecular group as Heterocycle as defined herein.In some embodiments, 1,2,3 or 4 substituent group as herein defined can be used in heterocycle Replace.
As used herein term " (heterocycle) acyl group " represent by carbonyl be connected to parent molecular group as Heterocycle defined in text.In some embodiments, heterocycle can be used 1,2,3 or 4 substituent group as herein defined to take Generation.
As used herein term " hydrocarbon " represents the group being only made of carbon atom and hydrogen atom.
As used herein term " hydroxyl " representative-OH group.
As used herein term " hydroxyalkenyl group " representative is replaced as herein defined by one to three hydroxyl Alkenyl, condition are the single carbon atom that a not more than hydroxyl can be connected to alkyl, and different by dihydroxy acrylic, hydroxyl Pentenyl etc. illustrates.
As used herein term " hydroxy alkyl " representative is replaced as herein defined by one to three hydroxyl Alkyl, condition are the single carbon atom that a not more than hydroxyl can be connected to alkyl, and by hydroxymethyl, dihydroxypropyl Deng illustration.
As used herein term " isomers " means any tautomer of any compound of the invention, solid Isomers, enantiomer or diastereomer.It can be appreciated that the compound of the present invention can have in one or more chiralitys The heart and/or double bond, and therefore spatially isomers such as double bond isomer (that is, geometry E/Z isomers) or diastereoisomer (for example, enantiomter (that is, (+) or (-)) or cis/trans isomers) exists.According to the present invention, the chemistry described herein Structure and therefore the compound of the present invention cover all corresponding stereoisomers, i.e. the pure form of alloisomerism is (for example, geometry Pure, enantiomer-pure or diastereo-isomerism are pure) and enantiomerism and three-dimensional heterogeneous mixture, such as raceme.Change of the invention The enantiomerism and three-dimensional heterogeneous mixture for closing object usually can be by well known methods, such as chiral-phase gas chromatography method, chiral phase High performance liquid chromatography makes that compound crystallization is chiral salt complex or crystalline compounds split into its group in chiral solvent Divide enantiomter or stereoisomer.It can also be by well known method of asymmetric synthesis from alloisomerism or enantiomer-pure Enantiomter and stereoisomer are obtained in mesosome, reagent and catalyst.
As used herein term " amino of N-protected " refer to connection thereon as herein defined one or two The amino as herein defined of N-protected base.
As used herein term " N-protected base " represents undesirable for protecting amino to resist in the synthesis process Those of reaction group.The N-protected base being commonly used is disclosed in Greene, " Protective Groups in Organic Synthesis, " in the 3rd edition (John Wiley & Sons, New York, 1999), it is hereby incorporated herein by.N- Protecting group include acyl group, aroyl or carbamyl for example formoxyl, acetyl group, propiono, valeryl, tertbutylacetyl, 2- chloracetyl, 2- acetyl bromide, trifluoroacetyl group, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4- chlorobenzene formacyl, 4- benzoyl bromide, 4- nitro benzoyl and chiral auxiliary are as protected Or unprotected D, L or D, l-amino acid such as alanine, leucine, phenylalanine etc.；Group containing sulfonyl such as benzene sulfonyl Base, p-toluenesulfonyl etc.；Carbamate formed group such as benzyloxycarbonyl group, to benzyloxycarbonylchloride base, to methbxybenzyl-oxycarbonyl, To nitrobenzyloxycarbonyl, 2- nitrobenzyloxycarbonyl, to bromo-benzyloxycarbonyl, 3,4- dimethoxy-benzyloxycarbonyl, 3,5- dimethoxy Benzyloxycarbonyl group, 2,4- dimethoxy-benzyloxycarbonyl, 4- methbxybenzyl-oxycarbonyl, 2- nitro -4,5- dimethoxy-benzyloxycarbonyl, 3, 4,5- trimethoxy benzyloxycarbonyl groups, 1- (to xenyl) -1- methylethoxycarbonyl, α, alpha-alpha-dimethyl -3,5- dimethoxy benzyl Oxygen carbonyl, benzhydryl Epoxide carbonyl, tertbutyloxycarbonyl, diisopropylmethoxycarbonyl, butyloxycarbonyl, ethoxy carbonyl, Methoxycarbonyl, allyloxy carbonyl, 2,2,2 ,-tri-chloroethoxy base carbonyl, phenyloxycarbonyl, 4-nitrophenoxy carbonyl, fluorenes Base -9- methoxycarbonyl, penta oxygen carbonyl of ring, adamantyloxycarbonyl, hexamethylene oxygen carbonyl, thiophenyl carbonyl etc.；Alkaryl is such as Benzyl, trityl group, benzyloxymethyl etc. and silicyl such as trimethyl silyl etc..Preferred N-protected Base is formoxyl, acetyl group, benzoyl, valeryl, tertbutylacetyl, alanyl, benzenesulfonyl, benzyl, uncle Butoxy carbonyl (Boc) and benzyloxycarbonyl group (Cbz).
As used herein term " nitro " representative-NO2Group.
As used herein term " oxo base " representative=O.
As used herein term " perfluoroalkyl " represent wherein in conjunction with each hydrogen-based of alkyl by fluorine-based substitution as Alkyl as defined herein.Perfluoroalkyl is illustrated by trifluoromethyl, pentafluoroethyl group etc..
As used herein term " perfluoro alkoxy " represents wherein in conjunction with each hydrogen-based of alkoxy by fluorine-based substitution Alkoxy as herein defined.Perfluoro alkoxy is illustrated by trifluoromethoxy, five fluorine ethyoxyls etc..
As used herein term " loop coil base " represents its both ends and combines the identical carbon atoms of precursor group to be formed The C of spiro-cyclic groups2-7Alkylidene diyl, and also represent the C that its both ends combines same atoms1-6Miscellaneous alkylidene diyl.It is formed The miscellaneous alkylidene of loop coil base contains one, two, the three or four miscellaneous original independently selected from the group being made of nitrogen, oxygen and sulphur Son.In some embodiments, loop coil base includes one to seven carbon, does not include the carbon atom for connecting diyl thereon.Of the invention Loop coil base can optionally be taken with the optional substituent group that 1,2,3 or 4 substituent group provided herein is such as used for naphthenic base and/or heterocycle Generation.
As used herein term " stereoisomer " refers to all possible different isomer that compound can possess Form and conformational forms (for example, compound of any formula described herein), specially all possibility of basic molecular structure Spatial chemistry and conformer form, all diastereoisomers, enantiomter and/or conformer.The present invention Some compounds can different tautomeric forms exist, all tautomeric forms are included within the scope of the invention.
As used herein term " sulfonyl alkyl " is represented by-SO3The alkane as herein defined that the sulfo group of H replaces Base.In some embodiments, alkyl can further be replaced with 1,2,3 or 4 substituent group as described herein.
As used herein term " sulfonyl " representative-S (O)2Group.
The chemical substituents of as used herein term " thio alkaryl " representative formula-SR, wherein R is alkaryl.? In some embodiments, alkaryl can further be replaced with 1,2,3 or 4 substituent group as described herein.
The chemical substituents of as used herein term " alkylthio heterocycle " representative formula-SR, wherein R is that alkyl is miscellaneous Ring group.In some embodiments, alkyl heterocyclic can further be replaced with 1,2,3 or 4 substituent group as described herein.
The chemical substituents of as used herein term " thio alkoxy " representative formula-SR, wherein R is such as is determined herein The alkyl of justice.In some embodiments, alkyl can further be replaced with 1,2,3 or 4 substituent group as described herein.
Term " mercapto " representative-SH group.
Compound: it is as used herein, term " compound " mean include the structure of description all stereoisomers, several What isomers, tautomer and isotope.
Compound described herein can be asymmetric (for example, having one or more Stereocenters).Unless in addition referring to Out, all stereoisomers are otherwise intended that, such as enantiomter and diastereoisomer.It can be with optical active forms or racemization The disclosure compound of carbon atom of the form separation containing Asymmetrical substitute.On how to prepare light from optical activity starting material The method for learning active form be it is as known in the art, such as by the fractionation of racemic mixture or pass through stereoselective syntheses. Many geometric isomers of alkene, C=N double bond etc. also are present in compound described herein, and all such stabilizations Isomers be covered by the disclosure.Describe disclosure compound cis geometric isomer and trans- geometric isomer, And it can be separated with isomer mixture or separated isomeric form.
The compound of the disclosure further includes tautomeric form.Tautomeric form exchanged by singly-bound with adjacent double bonds and Associated proton is migrated and is generated simultaneously.Tautomeric form includes prototropy tautomer, for identical empirical formula With the anomeric proton state of total electrical charge.Example prototropy tautomer include keto-enol to, amide-imide acid to, Lactams-lactim is to, amide-imide acid to, enamine-imines pair and annular form, and wherein proton can occupy heterocycle Two or more positions in system, such as 1H- imidazoles and 3H- imidazoles；1H-1,2,4- triazoles, 2H-1,2,4- triazoles and 4H- 1,2,4- triazole；1H- iso-indoles and 2H- iso-indoles；And 1H- pyrazoles and 2H- pyrazoles.Tautomeric form can be at equilibrium-like State is spatially locked as a kind of form by substitution appropriate.
The compound of the disclosure further includes all isotopes for coming across the atom in intermediate or final compound.It is " same Position element " refers to same atoms number but has the atom of different by the neutron population in atomic nucleus and generation different quality number. For example, the isotope of hydrogen includes tritium and deuterium.
The compound and salt of the disclosure can be by conventional method with solvent or moisture sub-portfolio to form solvate and water Object is closed to prepare.
Conservative: as used herein, term " conservative " refers to respectively in the phase of two or more sequences compared With the nucleotide or amino acid residue of the polynucleotide sequence or polypeptide sequence not changed on position.Conservative nucleosides relatively Acid or amino acid are the nucleotide or conservative between more relevant sequence than coming across other places of sequence Those nucleotide or amino acid.
In some embodiments, it if two or more sequences are 100% identical each other, referred to as " protects completely It keeps ".In some embodiments, if two or more sequences are at least 70% identical each other, at least 80% identical, at least 90% is identical or at least 95% is identical, they are referred to as " highly conserved ".In some embodiments, if two or more Multiple sequences are about 70% identical each other, it is about 80% identical, about 90% identical, about 95%, about 98% or about 99% identical, by them Referred to as " highly conserved ".In some embodiments, if two or more sequences are at least 30% identical each other, at least 40% is identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical or extremely Few 95% is identical, they are referred to as " conservative ".In some embodiments, if two or more sequences each other about 30% is identical, about 40% identical, about 50% identical, about 60% identical, about 70% identical, about 80% identical, about 90% identical, about 95% is identical, about 98% identical or about 99% is identical, referred to as " conservative ".The conservative of sequence is applicable to oligonucleotides Polypeptide whole length or be applicable to its part, area or feature.
Control release: as used herein, term " control release " refers to follow and release for realizing the specific for the treatment of results The pharmaceutical composition or compound of mode playback discharge overview.
Cyclic annular or cyclisation: as used herein, term " ring-type " refers to that there are continuous loops.Ring molecule does not need as ring Shape, only connection forms the subunit of continuous open chain.The RNA or mRNA of ring molecule engineering for example of the invention can be single list Member or polymer or one or more components comprising compound or higher structure.
Cell inhibiting: as used herein, " cell inhibiting " refers to inhibition, reduction, compacting cell (for example, lactation Zooblast (for example, people's cell)), bacterium, virus, fungi, protozoan, helminth, prion or combinations thereof growth, point It splits or breeds.
Cytotoxicity: it is as used herein, " cytotoxicity " refer to kill cell (for example, mammalian cell (for example, People's cell)), bacterium, virus, fungi, protozoan, helminth, prion or combinations thereof or it is caused harmful, toxicity Or lethal effect.
Delivering: as used herein, " delivering " refers to delivering compound, substance, entity, part, cargo or payload Movement or mode.
Delivery agents: it is as used herein, " delivery agents " refer at least partly promote polynucleotides, primary construct or Any substance of target cell is delivered in mmRNA body.
Go to stablize: as used herein, term " unstable ", " stabilization removal " or " going to stable region " means to compare same zone Or area or the molecule of the stability difference of the starting wild type or native form of molecule.
Detectable label: as used herein, " detectable label " refer to attachment, be incorporated to or associates that another passes through ability Known method is easy one or more markers, signal or the part of the entity of detection in domain, and the method includes ray photographs Physiognomy, fluorescence, chemiluminescence, enzymatic activity, absorbance etc..Detectable label include radioactive isotope, fluorogen, chromophore, Enzyme, dyestuff, metal ion, ligand such as biotin, avidin, streptavidin and haptens, quantum dot Deng.Detectable label can be located on any position in peptide or protein matter disclosed herein.They can be in amino acid, peptide or protein In matter, or it is located on the end N- or the end C-.
Digestion: as used herein, term " digestion " means to split into smaller or component.Referring to polypeptide or protein When, digestion causes the generation of peptide.
Distally: as used herein, term " distal end " means position far from center or far from target point or area.
Dosage regimen: as used herein, term " dosage regimen " is administration time table or treatment, prevention or mitigates nursing Doctor determine scheme.
The PUD that the dose fractionation factor (DSF)-dosage is separately treated is divided by total daily dosage or the PUD of single unit dose Ratio.The value derives from the comparison of dosage regimen group.
Encapsulating: as used herein, term " encapsulating " means to seal, surround or encase.
The protein cleavage signal of coding: as used herein, " the protein cleavage signal of coding " refers to coding albumen The nucleotide sequence of matter cracking signal.
Engineering: as used herein, embodiment of the present invention is designed to have at them is different from starting point Wild type or natural molecule no matter structure or when feature or characteristic chemically are " engineering ".
Allochthon: as used herein, " allochthon " is the vesica of mammalian cell secretion or is related to answering for RNA degradation Close object.
Expression: as used herein, " expression " of nucleic acid sequence refers to one or more of following event: (1) from DNA Sequence generates RNA template (for example, passing through transcription)；(2) processing RNA transcript by montage, editor, 5 ' caps (for example, formed And/or 3 ' end processing)；(3) RNA translates into polypeptide or protein；And the posttranslational modification of (4) polypeptide or protein.
Feature: as used herein, " feature " refers to feature, characteristic or unique element.
Preparation: as used herein, " preparation " includes at least polynucleotides, primary construct or mmRNA and delivery agents.
Segment: as used herein " segment " refers to part.For example, the segment of protein may include by digesting from training The polypeptide that the full length protein separated in feeding cell obtains.
Functional: as used herein, " functionality " biomolecule is in show can be so as to the characteristic that is characterized to it And/or the biomolecule of active form.
Homology: it is as used herein, term " homology " refer between polymer molecule for example nucleic acid molecules (for example, DNA molecular and/or RNA molecule) between and/or peptide molecule between overall relevance.In some embodiments, if it is poly- The sequence of adduct molecule is at least 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% are same or similar, then it is assumed that they are each other " homologous ".Term " homologous " must Refer to the comparison between at least two sequences (polynucleotide sequence or polypeptide sequence).According to the present invention, if two multicore glycosides The polypeptide of sequences code be at least about 20 amino acid at least at least about the 50% of one section, 60%, 70%, 80%, 90%, 95% or even 99%, then it is assumed that two polynucleotide sequences are homologous.In some embodiments, homologous multicore Nucleotide sequence is characterized in that the ability of coding one section at least 4-5 unique specified amino acid.For less than 60 cores of length The polynucleotide sequence of thuja acid determines homology by the ability of coding one section at least 4-5 unique specified amino acid.Root According to the present invention, if two kinds of protein are at least about 50% at least one section at least about 20 amino acid, 60%, 70%, 80% or 90% is identical, then it is assumed that two protein sequences are homologous.
Identity: as used herein, term " identity " refers to such as oligonucleotide molecules (example between polymer molecule Such as, DNA molecular and/or RNA molecule) between and/or peptide molecule between overall relevance.Two polynucleotide sequences it is same The calculating of one property percentage for example can be by carrying out for best two sequences of omparison purpose comparison (for example, in order to most preferably compare To vacancy being introduced into one or both of the first nucleic acid sequence and second nucleotide sequence and can be ignored for comparative purposes not Identical sequence).In certain embodiments, the length of the sequence compared for comparative purposes be reference sequence length at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or 100%.So Nucleotide on relatively more corresponding nucleotide position afterwards.When the position in First ray is by identical as the corresponding position in the second sequence Nucleotide when occupying, then molecule is identical at the location.Homogeneity percentage between two sequences be by vacancy number and The function of the shared same position number of the sequence that the length in each vacancy is taken into account, the vacancy, which needs to introduce, is used for two sequences The optimal comparison of column.Mathematical algorithm can be used to complete sequence and compare and determine homogeneity percentage between two sequences.Example Such as, it can be used and those of be such as described in following method and determine homogeneity percentage between two nucleotide sequences: Computational Molecular Biology, Lesk, A.M. write, Oxford University Press, New York, 1988；Biocomputing:Informatics and Genome Projects, Smith, D.W. write, Academic Press, New York, 1993；Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987；Computer Analysis of Sequence Data, Part I, Griffin, A.M., and Griffin, H.G. write, Humana Press, New Jersey, and 1994；And Sequence Analysis Primer, Gribskov, M.and Devereux, J. write, M Stockton Press, New York, 1991；The bibliography is respectively hereby incorporated herein by.For example, can be used Meyers and Miller (CABIOS, 1989,4:11-17) algorithm determines the homogeneity percentage between two nucleotide sequences, and the algorithm has been incorporated into ALIGN In program (2.0 version), PAM120 weight residue table, GAP LENGTH PENALTY 12 and gap penalty 4 are used.Or usable GCG GAP program in software package determines the homogeneity percentage between two nucleotide sequences using NWSgapdna.CMP matrix. Determine the warp of the homogeneity percentage between sequence frequently with method include but is not limited in Carillo, H. and Lipman, D., disclosed in SIAM J Applied Math., 48:1073 (1988) those；The bibliography is by reference simultaneously Enter herein.For determining that the technology of identity is programmed in publicly available computer program.It determines between two sequences The exemplary computer software of homology includes but is not limited to GCG program bag, Devereux, J. etc., Nucleic Acids Research, 12 (1), 387 (1984)), BLASTP, BLASTN and FASTA Altschul, S.F. etc., J.Molec.Biol., 215,403 (1990)).
The expression of suppressor: as used herein, phrase " expression of suppressor " means to cause the expression of gene to produce The amount of object is reduced.Expression product can be more to translate from the RNA (for example, mRNA) of genetic transcription or from the mRNA of genetic transcription Peptide.In general, horizontal reduce of mRNA causes to reduce from the horizontal of its polypeptide translated.It can be used for measuring mRNA or protein Standard technique determine expression level.
External: as used herein, term " external ", which refers to, to be occurred in artificial environment, such as test tube or reaction vessel In, in cell culture, petri dish it is medium, rather than the thing in organism (for example, animal, plant or microorganism) Part.
It is internal: it is as used herein, term " internal " refer to occur organism (for example, animal, plant or microorganism or Its cell or tissue) in event.
Separation: as used herein, term " separation " refers to (no matter set from its association in nature or in experiment Set) at least some components in the substance or entity that separate.The substance that isolated substance can associate relative to them has not The purity of same level.Isolated substance and/or entity can from least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, it is separated in about 70%, about 80%, about 90% or more their other components for initially associating with it.In some implementations In scheme, isolated medicament be greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or pure more than about 99%.As used herein, if substance generally not Containing other components, then it is " pure ".Generally separate: " generally separating " means what compound was formed or detected from it It is generally separated in environment.Being partially separated may include the composition for being for example enriched with disclosure compound.It generally separates and may include Contain at least about 50 weight %, at least about 60 weight %, at least about 70 weight %, at least about 80 weight %, at least about 90 weights Measure the combination of the disclosure compound or its salt of %, at least about 95 weight %, at least about 97 weight % or at least about 99 weight % Object.Method for separating compound and their salt is conventional in the art.
Connector: as used herein, connector refers to one group of atom, such as 10-1,000 atom, and may include atom Or group such as, but not limited to, carbon, amino, alkyl amino, oxygen, sulphur, sulfoxide, sulfonyl, carbonyl and imines.Connector can be in first end Place is attached to modified nucleoside or nucleotide in nucleobase or saccharide part, and is attached to payload at second end and for example may be used Detection agent or therapeutic agent.Connector can have enough length to be incorporated into nucleic acid sequence so as not to interfere.It can be for any useful Purpose uses connector, such as (connects two or more polynucleotides, primary building for example, passing through to form mmRNA polymer Body or mmRNA molecule) or mmRNA conjugate and in order to apply payload as described herein.It can be incorporated into connector The example of chemical group includes but is not limited to alkyl, alkenyl, alkynyl, acylamino-, amino, ether, thioether, ester, alkylidene, miscellaneous alkylene Base, aryl or heterocycle respectively can optionally replace as described herein.The example of connector includes but is not limited to unsaturated alkane Hydrocarbon, polyethylene glycol are (for example, ethylene glycol or propanediol monomer unit, such as diethylene glycol, dipropylene glycol, triethylene glycol, 3 the third two Alcohol, tetraethylene glycol or tetraethylene glycol) and dextran polymer and its derivative.Other examples include but is not limited in connector Reducing agent or photodegradation can be used to be split for cleavable part, such as disulfide bond (- S-S-) or azo bond (- N=N-) Solution.The non-limiting example of the key of selective cleavable includes can be for example by using three (2- carboxyethyl) phosphines (TCEP) or other Reducing agent and/or the amido bond of photodegradation cracking, and can for example pass through the ester bond of acid or basic hydrolysis cracking.
Microrna (miRNA) binding site: as used herein, Microrna (miRNA) binding site represents miRNA's At least nucleotide position of the transcribed nucleic acid object of " seed " area combination or area.
Modification: " modification " as used herein refers to the state or structure of the variation of molecule of the invention.It can be permitted Multimode includes mode decorating molecule chemically, in structure and functionally.In one embodiment, mRNA of the invention points Son is modified by introducing non-natural nucleosides and/or nucleotide, such as when it is related to natural ribonucleotide A, U, G and C. Although the non-standard nucleotide of such as cap structure they be different from A, C, G, U ribonucleotide chemical structure, be not considered as " to repair Decorations ".
Mucus: as used herein, " mucus " refers to sticky natural materials and includes mucin glycoprotein.
Naturally occurring: as used herein, " naturally occurring " means that do not need human assistance exists in nature.
Non-human vertebrate: it is as used herein, " non-human vertebrate " include in addition to homo sapiens (Homo sapiens) with Outer all vertebrates, including wild species and raise and train type.The example of non-human vertebrate includes but is not limited to that lactation is dynamic Object, as alpaca, banteng, wild ox, camel, cat, ox, deer, dog, donkey, gayal, goat, cavy, horse, yamma, mule, pig, Rabbit, reinder, sheep, buffalo and yak.
Miss the target: as used herein, " missing the target ", which refers to, appoints any one or more targets, gene or cell transcription object What unexpected effect.
Open reading frame: as used herein, " open reading frame " or " ORF " refers in given reading frame without containing eventually The only sequence of codon.
Be operably connected: as used herein, phrase " being operably connected " refers to two or more molecules, building Function connects between body, transcript, entity, part etc..
Optionally replace: the phrase of form " X optionally replaced " (for example, the alkyl optionally replaced) herein is intended to In " X, wherein X optionally replaces " (for example, " alkyl, wherein the alkyl optionally replaces ").Not to mean spy " X " (for example, alkyl) is levied originally as optional.
Peptide: as used herein, " peptide " is less than or equal to 50 amino acid longs, for example, about 5,10,15,20,25,30, 35,40,45 or 50 amino acid longs.
Paratope: as used herein, " paratope " refers to the antigen binding site of antibody.
Patient: it is as used herein, " patient ", which refers to, can seek or have treatment to need, require treatment, it is positive receive treatment, will Receive the subject for the treatment of, or the subject for specified disease or symptom under the nursing of trained professional.
It is pharmaceutically acceptable: to be referred in scope of sound medical judgment using phrase " pharmaceutically acceptable " herein, fitted For contacting without overdosage toxicity, stimulation, allergic reaction or other problems or complication and reasonable benefit with human and animal Benefit/Hazard ratio those of matches compound, material, composition and/or dosage form.
Pharmaceutically acceptable excipient: as used herein phrase " pharmaceutically acceptable excipient " refer in addition to Any ingredient (for example, can suspend or dissolve the medium of reactive compound) other than compound described herein and suffering from There is generally nontoxic and non-inflammation characteristic in person.Excipient may include for example: antiplastering aid, antioxidant, adhesive, packet Clothing, compression aid, disintegrating agent, dyestuff (pigment), softening agent, emulsifier, filler (diluent), film forming agent or coating, seasoning Agent, fragrance, glidant (flow enhancing agent), lubricant, preservative, printing ink, adsorbent, suspending agent or dispersing agent, sweet taste The water of agent and aquation.Exemplary excipients include but is not limited to: Butylated Hydroxytoluene (BHT), calcium carbonate, calcium phosphate (two alkali ), calcium stearate, Croscarmellose, crosslinked polyvinylpyrrolidone, citric acid, Crospovidone, cysteine, ethyl Cellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, lactose, magnesium stearate, maltitol, mannitol, first sulphur It is propylhomoserin, methylcellulose, methyl p-hydroxybenzoate, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pre- Gelatinized starch, propylparaben, retinyl palmitate, shellac, silica, sodium carboxymethylcellulose, sodium citrate, Sodium starch glycolate, sorbierite, starch (corn), stearic acid, sucrose, talcum, titanium dioxide, VitAVitE, dimension Raw element C and xylitol.
Pharmaceutically acceptable salt: the disclosure further includes the pharmaceutically acceptable salt of compound described herein.Such as this Text is used, and " pharmaceutically acceptable salt " refers to the derivative of disclosed compound, and wherein pass through will be existing for parent compound Acid or alkali are partially converted into its salt form (for example, by making free base groups and suitable organic acid reaction) to modify.Pharmacy The example of upper acceptable salt includes but is not limited to the inorganic acid salt or acylate of alkaline residue such as amine；Acidic residues such as carboxylic acid Alkali salt or organic salt etc..Representative acid-addition salts include acetate, adipate, alginate, ascorbate, asparagus fern ammonia Hydrochlorate, benzene sulfonate, benzoate, disulfate, borate, butyrate, camphor hydrochlorate, camsilate, citrate, ring Pentane propionate, gluconate, lauryl sulfate, esilate, fumarate, gluceptate, glycerophosphate, Hemisulphate, enanthate, caproate, hydrobromate, hydrochloride, hydriodate, 2- hydroxy-ethanesulfonate salt, Lactobionate, lactic acid Salt, laruate, lauryl sulfate, malate, maleate, malonate, mesylate, 2- naphthalene sulfonate, niacin Salt, nitrate, oleate, oxalates, palmitate, embonate, pectate, persulfate, 3- phenylpropionic acid salt, phosphorus Hydrochlorate, picrate, Pivalate, propionate, stearate, succinate, sulfate, tartrate, rhodanate, toluene Sulfonate, undecylate, valerate etc..Representative alkali metal salt or alkali salt include sodium, lithium, potassium, calcium, magnesium etc. and nothing Poison ammonium, quaternary ammonium and amine cation, including but not limited to ammonium, tetramethylammonium, etamon, methylamine, dimethylamine, trimethylamine, triethylamine, Ethamine etc..The pharmaceutically acceptable salt of the disclosure includes the parent compound for example formed from non-toxic inorganic or organic acid Conventional non-toxic salts.It can be by conventional chemical processes from the parent compound synthesis disclosure containing alkaline part or acidic moiety Pharmaceutically acceptable salt.In general, can be by making the amount of these of free acid or free alkali form compound and stoichiometry Appropriate alkali or acid are reacted in water or in organic solvent or in mixture of the water with organic solvent to prepare such salt；It is logical Often, as the non-aqueous medium of ether, ethyl acetate, ethyl alcohol, isopropanol or acetonitrile is preferred.The list of suitable salt is seen Remington ' s Pharmaceutical Sciences, the 17th edition, Mack Publishing Company, Easton, Pa., page 1985,1418, Pharmaceutical Salts:Properties, Selection, and Use, P.H.Stahl It (is write) with C.G.Wermuth, Wiley-VCH, 2008 and Berge etc., Journal of Pharmaceutical Science, 66,1-19 (1977), the bibliography are respectively incorporated herein in its entirety by reference.
Pharmaceutically acceptable solvate: as used herein term " pharmaceutically acceptable solvate " means Wherein the molecule of suitable solvent is incorporated to the compound of the present invention in lattice.Suitable solvent is physiology under institute's applied dose It is upper tolerable.For example, can by from include or mixtures thereof organic solvent, water solution in crystallization, recrystallization or precipitate come Prepare solvate.The example of suitable solvent is ethyl alcohol, water (for example, monohydrate, dihydrate and trihydrate), N- methyl Pyrrolidones (NMP), dimethyl sulfoxide (DMSO), N, N '-dimethyl formamide (DMF), N, N '-dimethyl acetamide (DMAC), 1,3-Dimethyl-2-imidazolidinone (DMEU), 1,3- dimethyl -3,4,5,6- tetrahydro -2- (1H)-pyrimidone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2-Pyrrolidone, Ergol etc..When water is solvent, solvate claims For " hydrate ".
Pharmacokinetics: as used herein, " pharmacokinetics " refers to that ought be related to determination is administered to life living When the destiny of the substance of object, any one or more of characteristic of molecule or compound.Pharmacokinetics is divided into several areas Domain, degree and rate including absorption, distribution, metabolism and excretion.This is commonly referred to as ADME, in which: (A) is absorbed as substance entrance Sanguimotor process；(D) dispersion or distribution of the substance in the body fluid and tissue of entire body are distributed as；(M) be metabolized (or it is raw Object conversion) it is the irreversible sub- metabolin of chemical conversion of parent compound；And (E) excretion (or elimination) refers to that substance disappears from body It removes.In rare cases, some drugs irreversibly accumulate in bodily tissue.
Physical chemistry: as used herein, " physical chemistry " means or about physically and/or chemically characteristic.
Polypeptide/unit medicament (PUD): as used herein, PUD or product/unit medicament are defined as total daily dosage Subdivided portions, the usually product (such as polypeptide) of 1mg, pg, kg measured such as in body fluid or tissue, usually divided by body fluid In magnitude concentration define such as pmol/mL, mmol/mL.
Prevention: as used herein, term " prevention ", which refers to, partially or completely postpones infection, disease, illness and/or symptom Breaking-out；Partially or completely postpone the one or more symptoms, feature or clinical table of specific infection, disease, illness and/or symptom Existing breaking-out；Partially or completely postpone one or more symptoms, feature or the performance of specific infection, disease, illness and/or symptom Breaking-out；Partially or completely postpone the progress of specified disease, illness and/or symptom from infection；And/or reduce development and infect, The risk of disease, illness and/or the relevant pathology of symptom.
Prodrug: the disclosure further includes the prodrug of compound described herein.As used herein, " prodrug " refers to be in change Learn or serve as when physically changed any substance, molecule or the entity of the substance of therapeutic agent, the form of molecule or entity.Prodrug can be with Certain way covalent bonding or chelating and before being applied to mammalian subject, when or discharge or be converted into work later Property drug moiety.It can be being present in chemical combination to modify in a manner of routinely manipulating or so that modification is cracked into parent compound in vivo Functional group in object prepares prodrug.Prodrug include wherein hydroxyl, amino, sulfydryl or carboxyl be bound to mammal by Examination person is cracked respectively when applying to form the compound of any group of free hydroxyl group, amino, sulfydryl or carboxyl.The system of prodrug Standby and purposes is discussed at T.Higuchi and V.Stella, " Pro-drugs as Novel Delivery Systems, " A.C.S. the 14th phase of symposium series and Bioreversible Carriers in Drug Design, write Edward B.Roche, American Pharmaceutical Association and Pergamon Press, in 1987, the ginseng Document is examined to be incorporated hereby hereby.
Proliferation: it is as used herein, term " proliferation " mean growth, amplification or increase or cause fast-growth, amplification or Increase." proliferative " means the ability with proliferation." anti proliferative " means there is counteracting or opposite with multiplication characteristic Characteristic.
Protein cleavage site: as used herein, " protein cleavage site " refers to wherein can be by chemical mode, enzyme Rush mode or photochemical method complete the site of the control cracking of amino acid chain.
Protein cleavage signal: as used herein, " protein cleavage signal " refers to mark or label for cracking At least one amino acid of polypeptide.
Target protein: it is as used herein, term " target protein " or " desired protein " include provided herein is Those of and its segment, mutant, variant and change.
Proximal end: as used herein, term " proximal end " means to be located closer to center or target point or area.
Pseudouridine: as used herein, pseudouridine refers to the C- glucosides isomers of nucleosides uridine." pseudouridine analog " is Any modification, variant, isoform or the derivative of pseudouridine.For example, pseudouridine analog includes but is not limited to 1- carboxymethyl-vacation Uridine, 1- propinyl-pseudouridine, 1- taurine methyl-pseudouridine, 1- taurine methyl -4- be thio-and pseudouridine, 1- methyl be false Uridine (m1ψ), 1- methyl -4- it is thio-pseudouridine (m1s4ψ), the thio -1- methyl-pseudouridine of 4-, 3- methyl-pseudouridine (m3ψ)、 Thio-1- methyl-1-denitrogenation-the pseudouridine of the thio-1- methyl-pseudouridine of 2-, 1- methyl-1-denitrogenation-pseudouridine, 2-, dihydro are false Uridine, 2- be thio-and dihydro pseudouridine, 2- methoxyuridine, 2- methoxyl group -4- be thio-uridine, 4- methoxyl group-pseudouridine, 4- first Oxygroup -2- is thio-pseudouridine, N1- methyl-pseudouridine, 1- methyl -3- (3- amino -3- carboxylic propyl) pseudouridine (acp3ψ) and 2 '-O- methyl-pseudouridine (ψ m).
Purifying: it is as used herein, " purifying ", " purifying ", " purification " mean to be allowed to be generally pure or There is no undesired component, material dirt, mixture or flaw.
Sample: as used herein, term " sample " or " biological sample " refer to the son of its tissue, cell or component part Collection (such as body fluid, including but not limited to blood, mucus, lymph, synovia, celiolymph, saliva, amniotic fluid, amniotic navel cord blood, Urine, vaginal secretion and sperm).Sample further may include from entire organism or its tissue, cell or component part subset or Its fraction or homogenate, lysate or the extract of part preparation, including but not limited to such as blood plasma, serum, spinal fluid, lymph Liquid, skin, respiratory tract, enteron aisle and urogenital tract outer slice, tears, saliva, cream, haemocyte, tumour, organ.Sample into One step refers to culture medium such as nutrient broth or gel containing cellular component such as protein or nucleic acid molecules.
Signal sequence: as used herein, phrase " signal sequence " refers to the sequence that can instruct protein transport or positioning.
Single unit dose: as used herein, " single unit dose " is with dose/primary/single approach/mono- The dosage of any therapeutic agent of a contact point application, that is, single administration event.
Similitude: as used herein, term " similitude " refers to such as polynucleotide molecule (example between polymer molecule Such as, DNA molecular and/or RNA molecule) between and/or peptide molecule between overall relevance.It can be with homogeneity percentage meter The calculating that identical mode carries out the mutual Similarity Percent of polymer molecule is calculated, in addition to Similarity Percent calculating is considered Conservative substitution as understood in the art.
Divided dose: as used herein, " divided dose " is single unit dose or total daily dosage is divided into twice or more Secondary dosage.
Stable: " stable " as used herein refers to firm useful to be subjected to being separated into from reaction mixture enough Purity and be preferably able to be configured to the compound of effective therapeutic agent.
Stablize: it is as used herein, term " stablizing .. ", " stabilizing ", " stable region " mean to make it stable or Become stable.
Subject: it is as used herein, term " subject " or " patient " refer to for example for experiment, diagnosis, prevention and/ Or therapeutic purposes can apply any organism of composition according to the present invention to it.Exemplary subject person includes animal (for example, feeding Newborn animal such as mouse, rat, rabbit, non-human primate and people) and/or plant.
Generally: as used herein, term " generally " refers to the total size or degree for showing target signature or characteristic Or close to total size or the qualitative condition of degree.Biological field ordinarily skilled artisan will understand that, biological phenomenon and chemical phenomenon are very Few (if any) can be completed and/or carry through to the end or reach or avoid absolute results.Therefore, term is used " substantially herein On " lack to obtain intrinsic potential completeness in many biological phenomenons and chemical phenomenon.
Generally equal: as used herein, when it is related to the time difference between dosage, term means positive/negative 2%.
Substantially concurrently: as used herein and when it is related to multiple dosage, term meant in 2 seconds.
By: the individual of " by " disease, illness and/or symptom, which has diagnosed, has or shows disease, illness and/or symptom One or more symptoms.
Susceptible: the not yet diagnosis of the individual of " susceptible " disease, illness and/or symptom has and/or can not show disease, illness And/or the symptom but suspection of symptom have the tendency that developing disease or its symptom.In some embodiments, susceptibility to disease, illness And/or the individual of symptom (for example, cancer) can have one or more features below: (1) with develop disease, illness and/or The relevant gene mutation of symptom；(2) genetic polymorphism relevant to development disease, illness and/or symptom；(3) with disease, illness And/or the expression and/or activity increase and/or reduction of the relevant protein of symptom and/or nucleic acid；(4) with develop disease, illness And/or the relevant habit of symptom and/or life style；(5) family history of disease, illness and/or symptom；And (6) are exposed to And/or infection microorganism relevant to development disease, illness and/or symptom.In some embodiments, susceptibility to disease, illness And/or the individual of symptom will develop disease, illness and/or symptom.In some embodiments, susceptibility to disease, illness and/or disease The individual of shape will not develop disease, illness and/or symptom.
Sustained release: as used herein, term " sustained release " refers to follows rate of release in special time period Pharmaceutical composition or compound discharge overview.
Synthesis: term " synthesis " means by artificially generating, preparing and/or manufacturing.Polynucleotides of the invention Or the synthesis of polypeptide or other molecules can be chemical or enzymatic.
Target cell: as used herein, " target cell " refers to any one or more of target cell.Cell may be present in body Outside, in vivo, in situ or in the tissue or organ of organism.Organism can be animal, preferably mammal, more preferably people And most preferably patient.
Therapeutic agent: term " therapeutic agent " refer to when being applied to subject have treatment, diagnosis and/or prevention effect and/ Or draw any medicament of desired biology and/or pharmacotoxicological effect.
Therapeutically effective amount: it is as used herein, term " therapeutically effective amount " mean to by or susceptible infections, disease, The subject of illness and/or symptom is enough to realize that the treatment of infection, disease, illness and/or symptom, symptom improve, examine when applying The amount of the medicament to be delivered (for example, nucleic acid, drug, therapeutic agent, diagnosticum, prophylactic etc.) of disconnected, prevention and/or breaking-out delay.
Treat effective result: it is as used herein, term " treating effective result " mean by or susceptible infections, disease It is enough to realize that the treatment of infection, disease, illness and/or symptom, symptom improve, examine in the subject of disease, illness and/or symptom The result of disconnected, prevention and/or breaking-out delay.
Total daily dosage: as used herein, " total daily dosage " is to give in 24 hour period or defined amount.It is described Total daily dosage can be used as single unit dose application.
Transcription factor: as used herein, term " transcription factor ", which refers to, for example to be adjusted by activating or inhibiting transcription Transcription from DNA to RNA DNA binding protein.Some transcription factors are implemented separately the adjusting of transcription, and other transcription factors and its Its protein synergism.Some transcription factors can activate under certain conditions and inhibit to transcribe.In general, transcription factor combines spy Targeting sequence or sequence highly similar with the specific consensus sequence in the regulatory region of target gene.Transcription factor can individually or and its The compound transcription to adjust target gene of its molecule.
Treatment: it is as used herein, term " treatment " refer to realize partially or completely specific infection, disease, illness and/ Or the mitigation of the one or more symptoms or feature of symptom, improvement, improvement, alleviation, breaking-out delay, progression inhibiting, seriousness drop Low and/or incidence reduces.For example, " treatment " cancer can refer to inhibit survival, growth and/or the diffusion of tumour.It can be for reduction The purpose for developing the risk of relevant to disease, illness and/or symptom pathology, to not showing disease, illness and/or symptom The subject of sign and/or only show disease, illness and/or symptom early stage sign subject apply treatment.
Unmodified: as used herein, " unmodified " refers to any substance before changing in any way, change Close object or molecule.The unmodified biomolecule that can but simultaneously not always refer to wild type or native form.Molecule can undergo a series of Modification, so that each decorating molecule may act as " unmodified " starting molecule of subsequent modification.
Equivalent and range
Those skilled in the art will recognize that or just can determine using only routine experiment described herein according to this hair Many equivalents of bright specific embodiment.The scope of the present invention is not intended to be limited to above description, but such as appended right Claim is illustrated.
In the claims, unless indicated to the contrary or in addition from context, it is evident that otherwise article such as " one (a) ", " a kind of (an) " and " (the) " can refer to one or more than one.It is unless indicated to the contrary or in addition obvious from context Find out, else if one, more than one or all group memberships come across, be used for or be associated in other ways given production Product or process, it is considered that including that the claims or description of the "or" between one or more members of group is expired Foot.The present invention includes wherein just having a member in group to come across, be used for or be associated in other ways given product Or the embodiment of process.The present invention includes that more than one or all group membership is present in, is used for or closes in other ways The embodiment for being coupled to given product or process.
It is further noted that term "comprising" is it is intended that open and allow but do not require that including other element or step Suddenly.When term "comprising" used herein, thus also cover and open term " by ... form ".
When providing range, endpoint is included.Moreover, it should be understood that unless otherwise noted or in addition from context Understanding with those of ordinary skill in the art is expressed as range it is clear that otherwise in different embodiments of the invention Value, which can be assumed that, to be removed for the subrange in any particular value or the range to 1/10th of the unit of the lower limit of the range It is non-otherwise expressly specified within a context.
In addition, it is to be appreciated that any specific embodiment of the invention in the prior art can from any one or it is more It is clearly excluded in a claim.As it is assumed that such embodiment is known to those of ordinary skill in the art, even if originally Text is not expressly recited exclusion, can also exclude them.Therefore any specific embodiment of composition of the invention is (for example, compile Any nucleic acid or protein of code；Any production method；Any application method etc.) it can be for regardless of whether existing with the prior art Relevant any reason and excluded from any one or more claims.
The source of all references such as bibliography, publication, database, data base entries and herein cited technology are equal It is herein incorporated by reference the application, even if being not expressly set out in the reference.In the source of reference and the statement punching of the application In the case where prominent, the statement that should be subject in the application.
Sub-section titles and form caption are not intended to be limiting.
Embodiment
The generation of the modification of embodiment 1. mRNA
Standard laboratory methods and material can be used to prepare modification mRNA (mmRNA) according to the present invention.Target gene Open reading frame (ORF) can be by containing the 5 ' non-translational regions (UTR) of strong Kozak translation initiation signal and/or may include being used for 3 ' the UTR of α-globin of oligo (dT) sequence of the template addition of poly-A tail is flanked.Modification mRNA can modify to reduce cell Innate immune response.Modification to reduce cell response may include pseudouridine (ψ) and 5- Methyl-Cytidine (5meC, 5mc or m5C).(referring to, Kariko K etc., Immunity 23:165-75 (2005), Kariko K etc., Mol Ther 16:1833-40 (2008), Anderson BR etc., NAR (2010)；The document is integrally incorporated herein each by reference).
ORF may also include a variety of upstreams or downstream additive (such as, but not limited to beta-globin, label etc.), the addition Object can be ordered from optimization service (such as, but not limited to DNA2.0 (Menlo Park, CA)), and can be containing can have XbaI knowledge Other multiple cloning sites.When receiving construct, the construct can be reconstructed and be transformed into Competent Escherichia coli (E.coli) in.
For the present invention, NEB DH5- α competent E.coli has been used.Using 100ng plasmid, according to NEB explanation come Execute conversion.Scheme is as follows:
1 thaws a pipe NEB 5- α competent E.coli cell 10 minutes on ice.
2 add the 1-5 μ l containing 1pg-100ng Plasmid DNA into cell mixture.Carefully flick pipe 4-5 times, it will be thin Born of the same parents mix with DNA.It is not vortexed.
3 place mixture 30 minutes on ice.It does not mix.
4 at 42 DEG C heat shock just 30 seconds.It does not mix.
5 place 5 minutes on ice.It does not mix.
6 move into the room temperature SOC of 950 μ l with pipette into mixture.
7 place 60 minutes at 37 DEG C.Acutely oscillation (250rpm) or rotation.
Selective plate is warmed to 37 DEG C by 8.
9 are sufficiently mixed cell by flicking pipe and overturning.
Every kind of dilution of 50-100 μ l is coated on selective plate, and is incubated overnight at 37 DEG C.Alternatively, It is incubated for 24-36 hours at 30 DEG C or is incubated for 48 hours at 25 DEG C.
Then it is inoculated with the LB growth medium for having used appropriate antibiotic of 5ml using single colonie, and then allows It grows (250RPM, 37 DEG C) 5 hours.Then this is used to be inoculated with 200ml culture medium and it is allowed to grow under the same conditions Overnight.
For separation quality grain (to 850 μ g), Invitrogen PURELINK is usedTMHiPure Maxiprep kit (Carlsbad, CA) illustrates to execute a large amount of preparations according to manufacturer.
In order to generate the cDNA for (IVT) to be transcribed in vitro, use the restriction enzyme of such as XbaI by plasmid (its first Example is shown in FIG. 3) linearisation.It will using the typical restrictive digestion of XbaI include the following: 1.0 μ g of plasmid；10x buffer 1.0μl；XbaI 1.5μl；dH20, until 10 μ l；It is incubated for 1 hour at 37 DEG C.If with laboratory scale (5 μ g of <) execution, that Use the PURELINK of InvitrogenTMPCR Micro kit (Carlsbad, CA) illustrates to clean according to manufacturer Reactant.More massive purifying can be needed using the product (standard of such as Invitrogen with more heavy load capacity PURELINKTMPCR kit (Carlsbad, CA)) it carries out.After the washing, the carrier of linearisation is used into NanoDrop Purifying, and analyzed using agarose gel electrophoresis to confirm linearisation.
Method described herein to prepare modification mRNA can be used for the molecule that generation includes all sizes of long molecule.? The modification mRNA using the method is prepared for for different size of molecule, including acid glucosidase α (GAA) (3.2kb), Cystic fibrosis transmembrane conductance regulator (CFTR) (4.7kb), the VII factor (7.3kb), lysosomal acid lipase (45.4kDa), glucocerebrosidase (59.7kDa) and iduronic acid 2- sulfatase (76kDa).
As non-limiting examples, G-CSF can represent target polypeptides.Sequence used in the step of embodiment 1-5 is summarized It is listed in table 11 and shows.It should be noted that initiation codon (ATG) is added with underscore in each sequence of table 11.
Table 11.G-CSF sequence
Embodiment 2: the PCR generated for cDNA
Use the 2x KAPA HIFI of Kapa Biosystems (Woburn, MA)TMHotStart ReadyMix is executed It is used to prepare the PCR program of cDNA.This system includes 12.5 μ l of 2x KAPA ReadyMix；0.75 μ l of forward primer (10uM)； 0.75 μ l of reverse primer (10uM)；Template cDNA 100ng；And dH20, it is diluted to 25.0 μ l.Reaction condition are as follows: at 95 DEG C Continue 5min；It is recycled with 25: continuing 20sec at 98 DEG C, then continue 15sec at 58 DEG C, then continued at 72 DEG C 45sec then continues 5min, then at 4 DEG C down toward termination at 72 DEG C.
Reverse primer of the invention is the poly-A in mRNA120It is incorporated with poly-T120.With longer or shorter poly (T) other reverse primers of tract can be used for adjusting the length of poly in mRNA (A) tail.
Use the PURELINK of InvitrogenTMPCR Micro kit (Carlsbad, CA) saying according to manufacturer Ming and Qing washes reactant (to 5 μ g).Bigger reactant will need to clean using the product with larger capacity.After cleaning, make Use NANODROPTMCDNA is quantified, and is analyzed by agarose gel electrophoresis, to confirm cDNA as desired size.Then So that cDNA is carried out sequencing analysis, carries out in-vitro transcription reaction later.
(IVT) is transcribed in vitro in embodiment 3.
Reaction is transcribed in vitro and generates the mRNA containing modified nucleoside acid or modification RNA.Inside natural or non-natural NTP Preparation input nucleotide triphosphoric acid (NTP) mixture.
It is typical that reaction solution is transcribed in vitro include the following:
1 template cDNA, 1.0 μ g
2 10x transcription buffers (400mM Tris-HCl pH 8.0,190mM MgCl2, the sub- essence of 50mM DTT, 10mM Amine) 2.0 μ l
3 customization NTP (each 25mM) 7.2 μ l
4RNA enzyme inhibitor 20U
5T7 RNA polymerase 3000U
6dH20 to 20.0 μ l.And
7 are incubated for 3 hours to 5 hours at 37 DEG C.
Crude IVT mixture can store overnight at 4 DEG C, clean for second day.Then using 1U without RNA enzyme DNA enzymatic digests primary template.After being incubated for 15 minutes at 37 DEG C, the MEGACLEAR of Ambion is usedTMKit (Austin, TX) illustrate purifying mRNA according to manufacturer.This kit can purify the RNA up to 500 μ g.It, will using NanoDrop after cleaning RNA is quantitative, and is analyzed by agarose gel electrophoresis, to confirm that RNA is that appropriately sized and RNA does not degrade.
The enzymatic of embodiment 4.mRNA is capped
Following to execute the capped of mRNA, wherein mixture includes: 60 μ g-180 μ g and dH of IVT RNA20, until 72 μ l.It will Mixture is incubated for 5 minutes at 65 DEG C, so that RNA is denaturalized, is transferred to immediately after on ice.
Then scheme is related to 10x and is capped buffer (0.5M Tris-HCl (pH 8.0), 60mM KCl, 12.5mM MgCl2) (10.0 μ l), 20mM GTP (5.0 μ l), 20mM S-adenosylmethionine (2.5 μ l), RNase inhibitor (100U), 2 '-O- transmethylases (400U), cowpox capping enzyme (guanylate transferase) (40U), dH2The mixing of 0 (to 28 μ l)；And 30 minutes (for 60 μ g RNA) are incubated at 37 DEG C or long to 2 hours (for 180 μ g RNA).
Then the MEGACLEAR of Ambion is usedTMKit (Austin, TX) carries out mRNA according to the explanation of manufacturer Purifying.After cleaning, NANODROP is usedTM(ThermoFisher, Waltham, MA) quantifies RNA, and solidifying by agarose Gel electrophoresis is analyzed, to confirm that RNA is that appropriately sized and RNA does not degrade.It can also be by operation reverse transcription PCR with life RNA product is sequenced at the cDNA for sequencing.
Embodiment 5.PolyA tailings reactions
In the case where there is no poly-T in cDNA, it is necessary to execute poly-A tailings reactions before cleaning final product. This is carried out by following: by capped IVT RNA (100 μ l), RNase inhibitor (20U), 10x tailing buffer (0.5M Tris-HCl(pH 8.0)、2.5M NaCl、100mM MgCl2) (12.0 μ l), 20mM ATP (6.0 μ l), Poly-A polymerase (20U)、dH20 (to 123.5 μ l) mixes and is incubated for 30min at 37 DEG C.If having existed poly-A tail in transcript, that It can skip tailings reactions, and use the MEGACLEAR of AmbionTMKit (Austin, TX) is directly cleaned (at most 500μg).Poly-A polymerase is preferably the recombinase expressed in yeast.
For the research for executing and describing herein, poly-A tail is encoded into long including 160 nucleotide in IVT template Degree.It will be appreciated, however, that the machinability or integrality of polyA tailings reactions may not always lead to just 160 nucleotide. Therefore, have about 160 nucleotide, for example, about 150-165,155,156,157,158,159,160,161,162,163, The polyA tail of 164 or 165 nucleotide is within the scope of the present invention.
Natural 5 ' the cap of embodiment 6. and 5 ' cap analogs
Using the chemical RNA cap analog below to generate 5 '-guanosine cap structures, according to the scheme of manufacturer, in body 5 ' that modification RNA is concomitantly completed during outer responsive transcription are capped: 3 '-O-Me-m7G (5 ') ppp (5 ') G [ARCA cap], G (5 ') Ppp (5 ') A, G (5 ') ppp (5 ') G, m7G (5 ') ppp (5 ') A, m7G (5 ') ppp (5 ') G (New England BioLabs, Ipswich, MA).It can be used to generate " Cap0 " structure: the vaccinia virus capping enzyme (New of m7G (5 ') ppp (5 ') G England BioLabs, Ipswich, MA) the 5 ' capped of modification RNA is completed after transcription.Can be used vaccinia virus capping enzyme and Cap1 structure is generated to generate the 2 '-O transmethylases of m7G (5 ') ppp (5 ') G-2 '-O- methyl.Can by Cap1 structure, 5 ' third last nucleotide, 2 '-O is methylated to generate Cap2 structure using 2 '-O transmethylases later.It can be tied by Cap2 Structure is later methylated 5 ' fourth from the last nucleotide, 2 '-O to generate Cap3 structure using 2 '-O transmethylases.Enzyme is preferred From recombinant sources.
When being transfected into mammalian cell, modification mRNA has between 12 hours to 18 hours or small greater than 18 When, such as 24 hours, 36 hours, 48 hours, 60 hours, 72 hours or the stability greater than 72 hours.
Embodiment 7. is capped
A.Protein expression measurement
It can be by human G-CSF of the coding containing ARCA (3 ' O-Me-m7G (5 ') ppp (5 ') G) cap analog or Cap1 structure (cDNA shown in SEQ ID NO:21435；With 5- methyl born of the same parents at each cytimidine shown in SEQ ID NO:21438 Pyrimidine and the mRNA sequence modified completely at each uridine site with pseudouridine substitution, wherein length is not shown in the sequence The polyA tail of about 160 nucleotide) synthesis mRNA be transfected under equal concentrations in people's primary culture keratinocytes. 6 hours after transfection, 12 hours, 24 hours and 36 hours can measure the amount for the G-CSF being secreted into culture medium by ELISA. The synthesis mRNA that higher level G-CSF is secreted into culture medium will correspond to the synthesis with higher translation ability Cap structure mRNA。
B.Purity analysis synthesis
Denaturing agarose-urea gel electrophoresis or HPLC analysis can be used to contain ARCA cap analog to coding for purity Or human G-CSF (the cDNA shown in SEQ ID NO:21435 of the crude synthetic product of Cap1 structure；In SEQ ID NO:21438 What is shown is modified with 5-methylcytosine and at each uridine site at each cytimidine completely with pseudouridine substitution MRNA sequence, wherein the polyA tail that length is about 160 nucleotide is not shown in the sequence) synthesis mRNA be compared. Compared with the synthesis mRNA with multiple bands or the band that trails, have the synthesis mRNA of single unified band corresponding by electrophoresis In higher degree product.Synthesis mRNA with the single peak HPLC will also correspond to higher degree product.With greater efficiency Capping will provide purer mRNA group.
C.Cytokine analysis
It can be by human G-CSF of the coding containing ARCA cap analog or Cap1 structure (shown in SEQ ID NO:21435 cDNA；With 5-methylcytosine and at each uridine site at each cytimidine shown in SEQ ID NO:21438 The mRNA sequence modified completely is substituted with pseudouridine, wherein the polyA that length is about 160 nucleotide is not shown in the sequence Tail) synthesis mRNA be transfected under multiple concentration in people's primary culture keratinocytes.6 hours after transfection, 12 hours, it is 24 small When and 36 hours, the amount of pro-inflammatory cytokine (such as TNF-α and IFN-β) that ELISA measurement is secreted into culture medium can be passed through. The synthesis mRNA that the pro-inflammatory cytokine of higher level is secreted into culture medium, which will correspond to, contains immune activation cap structure Synthesize mRNA.
D.Capping efficiency
ARCA cap can be contained to coding for capping efficiency by LC-MS after capped mRNA nucleic acid enzymatic treatment Human G-CSF (the cDNA shown in SEQ ID NO:21435 of analog or Cap1 structure；Shown in SEQ ID NO:21438 The mRNA sequence modified completely is substituted with 5-methylcytosine at each cytimidine and with pseudouridine at each uridine site Column, wherein the polyA tail that length is about 160 nucleotide is not shown in the sequence) synthesis mRNA analyzed.It is capped The nucleic acid enzymatic treatment of mRNA will generate can be by the LC-MS free nucleotide detected and capped 5 ' -5- triphosphoric acid cap structure Mixture.The amount that product is capped on LC-MS map, which is represented by the percentage for carrying out self-reacting total mRNA and will correspond to, to be added Cap reaction efficiency.Cap structure with higher capping efficiency passes through LC-MS for the capped product with higher amount.
The agarose gel electrophoresis of the modification RNA or RT PCR product of embodiment 8.
The PCR product (200-400ng) of each modification RNA (200-400ng, 20 μ l volumes) or reverse transcription are loaded onto non- In hole on 1.2% agarose E-Gel (Invitrogen, Carlsbad, CA) of denaturation, and run according to the scheme of manufacturer Glue 12-15 minutes.
Embodiment 9.Nanodrop modification RNA is quantified and UV spectroscopic data
Modification RNA (1 μ l) in TE buffer is used for Nanodrop UV absorbance reading, to anti-from being transcribed in vitro The yield of each modification RNA answered is quantified.
Embodiment 10. uses the preparation of the modification mRNA of lipoids
By the way that before being added to cell, mmRNA and lipoids are prepared modification mRNA to set ratio mixing (mmRNA) it is used for experiment in vitro.Preparing in vivo can need to add added ingredient, to be conducive to recycle in entire body.In order to It tests these lipoids and forms the ability for being suitable for the particle to work in vivo, matched using the standard for siRNA- lipoids preparation Method processed is as starting point.Initial mmRNA- lipoids preparation can be by including 42% lipoids, 48% cholesterol and 10%PEG The particle of (ratio may advanced optimize) forms.After particle formulation, adds mmRNA and it is allowed to integrate with compound.Make Measuring method is excluded with standard dyes to be measured encapsulation efficiency.
The material and method of embodiment 11-15
A.Lipid synthesis
By the method summarized in this field synthesize six kinds of lipid DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 and DLin-MC3-DMA, to be prepared together with modification RNA.DLin-DMA and precursor such as Heyes etc., J.Control Release, is synthesized described in 2005,107,276-287.DLin-K-DMA and DLin-KC2-DMA and Precursor such as Semple etc., Nature Biotechnology are synthesized described in 2010,28,172-176.98N12-5 is with before Body such as Akinc etc., Nature Biotechnology are synthesized described in 2008,26,561-569.
C12-200 and precursor are synthesized according to the method summarized in Love etc., PNAS, 2010,107,1864-1869. To containing addition 2- Epoxydodecane in amine 200 (0.723g, 3.36mmol, 1 equivalent) and the bottle of stirring rod (5.10g, 27.7mmol, 8.2 equivalents).Bottle is sealed and is warmed to 80 DEG C.Reaction solution is stirred 4 days at 80 DEG C.Then pass through silica gel Chromatography is used and is purified from absolute dichloromethane (DCM) to DCM: MeOH 98: 2 gradient to mixture.Pass through RP-HPLC pairs Target compound is further purified, to obtain desired compound.
DLin-MC3-DMA and the precursor journey according to described in the WO 2010054401 being incorporated herein by reference in their entirety Sequence is synthesized.By two sub- oil base methanol (1.5g, 2.8mmol, 1 equivalent), N, N- dimethylaminobutyricacid acid (1.5g, 2.8mmol, 1 equivalent), DIPEA (0.73mL, 4.2mmol, 1.5 equivalent) and TBTU (1.35g, 4.2mmol, 1.5 equivalent) exist 10h is stirred at room temperature in mixture in 10mL DMF.Then reaction mixture is diluted and is washed with water in ether.It will be organic Layer is dried over anhydrous sodium sulfate, filters and is concentrated under reduced pressure.The ladder of DCM to DCM: MeOH 98: 2 is used by silica gel chromatograph Degree purifies crude product.Target compound is then set to be subjected to other RP-HPLC purifying, the RP-HPLC purifying uses YMC-Pack C4 column carries out, to obtain target compound.
B.Modify the preparation of RNA nano particle
Prepare synthesis lipid, 1,2- distearyl acyl group -3- phosphatidyl choline (DSPC) in ethanol under the concentration of 50mM (Avanti Polar Lipids, Alabaster, AL), cholesterol (Sigma-Aldrich, Taufkirchen, Germany) And α-[3 '-(1,2- bis- myristoyl -3- propoxyl group)-formamide-propyl]-ω-methoxyl group-polyoxyethylene (PEG-c- DOMG) the solution of (NOF, Bouwelven, Belgium), and stored at -20 DEG C.Lipid is merged, to obtain 50: 10: The molar ratio of 38.5: 1.5 (lipids: DSPC: cholesterol: PEG-c-DOMG), and it is diluted to ethyl alcohol the final lipid of 25mM Concentration.The aqueous solution of modification mRNA under the concentration of 1-2mg/mL is diluted in the 50mM sodium citrate buffer solution of pH 3, MRNA reservoir is modified to be formed.By will synthesize lipid soln and modify mRNA solution with 10: 1,15: 1,20: 1 and 30: 1 it is total Lipid merges with the weight ratio of modification mRNA, to prepare lipid and modify the preparation of mRNA.By lipid ethanol solution fast injection Into the aqueous solution of modification mRNA, to obtain the suspension containing 33% ethyl alcohol.(MI) is injected or by means of syringe pump manually (SP) (Harvard Pump 33 Dual Syringe Pump Harvard Apparatus Holliston, MA) is injected Solution.
In order to remove ethyl alcohol and realize buffer-exchanged, Slide-A-Lyzer box (Thermo Fisher is used Scientific Inc.Rockford, IL) preparation is saturating for 200 times of head product of phosphate buffered saline (PBS) to 7.4 volume of pH Twice, wherein molecular weight cut (MWCO) is 10kD for analysis.Dialysis executes 3 hours at room temperature for the first time, then by preparation at 4 DEG C Lower dialysed overnight.By resulting nano particle suspension by 0.2 μm of sterilizing filter (Sarstedt, N ü mbrecht, Germany it) is filled into vial and is sealed with curling sealing.
C.The characterization of preparation
Using Zetasizer Nano ZS (Malvern Instruments Ltd, Malvern, Worcestershire, UK) measurement modification mRNA nano particle granularity, polydispersity index (PDI) and zeta potential, in 1X PBS measure granularity and Zeta potential is measured in 15mM PBS.
The concentration of modification mRNA nanoparticle formulations is measured using uv-vis spectra.100 μ L are dilute in 1X PBS The preparation released is added in the methanol of 900 μ L and 4: 1 (v/v) mixtures of chloroform.After mixing, in 800 spectrophotometer of DU On (Beckman Coulter, Beckman Coulter, Inc., Brea, CA) between 230nm and 330nm recording solution Absorption spectrum.In nanoparticle formulations modify RNA concentration based on used in preparation modify RNA extinction coefficient and Absorbance at 260nm wavelength and the difference between the baseline value at 330nm wavelength calculate.
Use QUANT-ITTM RNA measures (Invitrogen Corporation Carlsbad, CA) come evaluate by nano particle to modification RNA encapsulating.By sample TE buffer (10mM Tris-HCl, 1mM EDTA, pH 7.5) in be diluted to the concentration of about 5 μ g/mL.The dilute sample of 50 μ L is transferred to 96 hole of polystyrene to put down In plate, the TE buffer of 50 μ L or the 2%Triton X-100 solution of 50 μ L are then added.By plate 37 DEG C at a temperature of incubate It educates 15 minutes.It willReagent 1: 100 dilution in TE buffer, this solution of 100 μ L is added to each Kong Zhong.Use Fluorescence Plate reader (1420 Multilablel Counter of Wallac Victor；Perkin Elmer, Waltham, MA) under the excitation wavelength of~480nm and the launch wavelength of~520nm measure fluorescence intensity.By each sample Fluorescent value subtracts the fluorescent value of reagent blank, and by with the fluorescence intensity of intact sample (not adding Triton X-100) The percentage of free modification RNA is measured divided by the fluorescent value of disrupted sample (causing by adding Triton X-100).
D.Incubated in vitro
By human embryo kidney epithelium (HEK293) cell and hepatocellular carcinoma epithelium (HepG2) cell (LGC standards GmbH, Wesel, Germany) it is inoculated into 96 hole plates (Greiner Bio-one GmbH, Frickenhausen, Germany) On, and the plate for HEK293 cell is pre-coated with 1 Collagen Type VI.HEK293 is connect with the density of 30,000 cells/wells Kind, HepG2 is seeded in the cell culture medium of 100 μ l with the density of 35,000 cells/wells.For HEK293, cell culture medium For DMEM, 10%FCS, add 2mM L-Glutamine, 1mM Sodium Pyruvate and 1x nonessential amino acid (Biochrom AG, Berlin, Germany) and 1.2mg/ml sodium bicarbonate (Sigma-Aldrich, Munich, Germany), and for HepG2, culture medium are MEM (Gibco Life Technologies, Darmstadt, Germany), and 10%FCS adds 2mM L-Glutamine, 1mM Sodium Pyruvate and 1x nonessential amino acid (Biochrom AG, Berlin, Germany.In inoculating cell And after being incubated for, directly addition contains mCherry mRNA (mRNA sequence shown in SEQ ID NO:21439 in quadruplicate Column；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) preparation.With for turning in vitro The mCherry cDNA for recording the T7 promoter of (IVT), 5 ' non-translational regions (UTR) and 3 ' UTR gives in SEQ ID NO:21440 Out.MCherry mRNA with 5meC, and is substituted with pseudouridine in each uridine site and is modified at each cytimidine.
By by medium supernatant be transferred to 96 hole Pro-Bind U base plates (Beckton Dickinson GmbH, Heidelberg, Germany) harvest cell.By trypsase/EDTA of 1/2 volume of cell (Biochrom AG, Berlin, Germany) trypsin digestion, merge with corresponding supernatant, and pass through the PBS/2% of one volume of addition FCS (being Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck, Darmstadt, Germany) is consolidated It is fixed.Then it is excited in LSRII cell instrument (Beckton Dickinson GmbH, Heidelberg, Germany) with 532nm 610/20 filter of laser and PE-Texas Red make sample be subjected to measured by flow cytometry.Give the complete of analyzed sample The standard deviation of average fluorescent strength (MFI) and four separate wells of portion's event.
The purifying of 11. nanoparticle formulations of embodiment
The nanoparticle formulations of DLin-KC2-DMA and 98N12-5 in HEK293 and HepG2 are tested, it is flat to determine Whether equal fluorescence intensity (MFI) depends on lipid and modifies the ratio and/or purifying of RNA.Reach table 12 using syringe pump generation Three kinds of preparations of the DLin-KC2-DMA of the specification and two kinds of preparations of 98N12-5.Pass through SEPHADEXTMDNA grades of G-25 (GE Healthcare, Sweden) purifies the sample of purifying.Every kind of preparation of (aP) exists before and after purifying It is tested in 24 hole plates under the concentration in the 250ng modification hole RNA/.For every kind of preparation and Background Samples, when passing through streaming When Cytometric Analysis, the marker for the channel FL4 is the percentage (%FL4- is positive) and every kind of preparation of positive cell It is shown in table 13 with the MFI of the marker in the channel FL4 of Background Samples.Purified preparation than testing before purification Those preparations have slightly higher MFI.
12. preparation of table
Table 13.HEK293 and HepG2,24 holes, 250ng modify the hole RNA/
12. concentration-response curve of embodiment
To the nanoparticle formulations of 98N12-5 (NPA-005) and DLin-KC2-DMA (NPA-003) under different concentration It is tested, to measure FL4 or mCherry (mRNA sequence shown in SEQ ID NO:21439；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) in dosage range Interior MFI.The preparation tested is summarized in table 14.In order to measure 98N12-5 nanoparticle formulations optium concentration, To the modification RNA of the preparation of various concentration, (100ng, 10ng, 1.0ng, 0.1ng and 0.01ng are every in 24 hole plates of HEK293 Hole) it is tested, and the result of the FL4 MFI of each dosage is shown in table 15.Similarly, in order to measure DLin-KC2-DMA Nanoparticle formulations optium concentration, in 24 hole plates of HEK293 to the modification RNA of the preparation of various concentration (250ng, The every hole 100ng, 10ng, 1.0ng, 0.1ng and 0.01ng) it is tested, and the result of the FL4 MFI of each dosage is in table 16 It shows.It is right with the modification RNA of the preparation of various concentration (the every hole 250ng, 100ng and 30ng) also in 24 hole plates of HEK293 The nanoparticle formulations of DLin-KC2-DMA are tested, and the result of the FL4 MFI of each dosage is shown in table 17.It was found that The dosage in the hole 10ng/ of the dosage and DLin-KC2-DMA in the hole 1ng/ of 98N12-5 is similar with the FL4 MFI of background.
In order to measure concentration level of intimate similar with background, we are utilized with for detecting mCherry expression Optimize the flow cytometer of filter device, and can be to obtain result relative to the increased sensitivity of background level.For 98N12-5 (NPA-005) and DLin-KC2-DMA (NPA-003), analyze the hole 25ng/, the hole 0.25ng/, the hole 0.025ng/ and The dosage in the hole 0.0025ng/, to measure the MFI of mCherry.As shown in Table 18, the concentration in the hole 0.025ng/ or smaller concentration It is horizontal similar to the background MFI of about 386.125 mCherry.
14. preparation of table
The hole table 15.HEK293, NPA-005,24-, n=4
Preparation FL4 MFI
Untreated control 0.246
NPA-005 100ng 2.2175
NPA-005 10ng 0.651
NPA-005 1.0ng 0.28425
NPA-005 0.1ng 0.27675
NPA-005 0.01ng 0.2865
The hole table 16.HEK293, NPA-003,24-, n=4
Preparation FL4 MFI
Untreated control 0.3225
NPA-003 250ng 2.9575
NPA-003 100ng 1.255
NPA-003 10ng 0.40025
NPA-003 1ng 0.33025
NPA-003 0.1ng 0.34625
NPA-003 0.01ng 0.3475
The hole table 17.HEK293, NPA-003,24-, n=4
Preparation FL4 MFI
Untreated control 0.27425
NPA-003 250ng 5.6075
NPA-003 100ng 3.7825
NPA-003 30ng 1.5525
18. concentration of table and MFI
13. hand injection of embodiment and syringe pump preparation
Two kinds of preparations of DLin-KC2-DMA and 98N12-5 are prepared by hand injection (MI) and ejection of syringe pump (SP), And it is analyzed together with Background Samples with mCherry (the mRNA sequence shown in SEQ ID NO:21439 to different preparations Column；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5-methylcytosine and pseudouridine Completely modification) MFI be compared.Table 19 is shown, compared with identical lipid and lipid/RNA ratio hand injection preparation, Syringe pump preparation MFI with higher.
19. preparation of table and MFI
14. lipid nanoparticle preparation of embodiment
By DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200 and DLin-MC3-DMA Preparation is under the concentration in the hole 60ng/ or the hole 62.5ng/ in HepG2 in HEK293 plate and under the concentration in the hole 62.5ng/ It is incubated for 24 hours in the plate of cell, to measure mCherry (the SEQ ID NO:21439 of every kind of preparation；With about 160 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) MFI. The preparation tested is summarized in the following table 20.Such as table 21 for the hole 60ng/ and the table 22,23,24 and 25 for the hole 62.5ng/ Shown, the preparation of NPA-003 and NPA-018 has a highest mCherry MFI, and NPA-008, NPA-010 and NPA-013 Preparation and Background Samples mCherry MFI value are the most similar.
20. preparation of table
Table 21.HEK293,96 holes, 60ng modify the hole RNA/
The hole table 22.HEK293,62.5ng/
Preparation MFI mCherry
It is untreated 871.81
NPA-001 6407.25
NPA-002 14995
NPA-003 29499.5
NPA-005 3762
NPA-006 2676
NPA-007 9905.5
NPA-008 1648.75
NPA-009 2348.25
NPA-010 4426.75
NPA-012 11466
NPA-013 2098.25
NPA-014 3194.25
NPA-015 14524
The hole table 23.HEK293,62.5ng/
The hole table 24.HepG2,62.5ng/
Preparation MFI mCherry
It is untreated 649.94
NPA-001 6006.25
NPA-002 8705
NPA-002-2 15860.25
NPA-003 15059.25
NPA-003-2 28881
NPA-005 1676
NPA-006 1473
NPA-007 15678
NPA-008 2976.25
NPA-009 961.75
NPA-010 3301.75
NPA-012 18333.25
NPA-013 5853
NPA-014 2257
NPA-015 16225.75
The hole table 25.HepG2,62.5ng/
Preparation MFI mCherry
Untreated control 656
NPA-007 16798
NPA-012 21993
NPA-017 20377
NPA-003-2 35651
NPA-018 40154
NPA-010 2496
NPA-015 19741
NPA-019 16373
Preparation research in 15. body of embodiment
The system containing modification mRNA and lipid that is intravenous to rodent (n=5), subcutaneously or intramuscularly applying single dose Agent.The modification mRNA for being applied to rodent is selected from: G-CSF (mRNA sequence shown in SEQ ID NO:21438；With big The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1), hematopoietin (EPO) (SEQ ID NO: MRNA sequence shown in 1638；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1), IX The factor (mRNA sequence shown in SEQ ID NO:1622；PolyA tail with about 160 nucleotide does not show in sequence Out；5 ' caps, Cap 1) or mCherry (mRNA sequence shown in SEQ ID NO:21439；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1).With T7 promoter, the 5 ' non-translational regions for (IVT) to be transcribed in vitro (UTR) it is provided in SEQ ID NO:21441 and SEQ ID NO:21442 with the hematopoietin cDNA of 3 ' UTR.
Every kind of preparation also contain selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA、reLNP、One lipid in DACC and DBTC.It is injected to rodent The modification mRNA of the preparation of 100ug, 10ug or 1ug, and sample is collected in specific time interval.
Modify the rodent of the preparation of mRNA containing human G-CSF from application by specific G-CSF ELISA measurement Serum, and by specificity IX factor ELISA or chromogenic assay to the blood of the mouse from application people IX factor modification RNA It is analyzed clearly.By immunohistochemistry (IHC) or fluorescence-activated cell sorting (FACS) to repairing from applied mCherry The liver and spleen for adoring the mouse of mRNA are analyzed.As control, one group of mouse does not inject any preparation, and collect its serum and Tissue, is analyzed by ELISA, FACS and/or IHC.
A.Time course
Preparation to rodent application containing at least one modification mRNA, with the protein expression to applied preparation Time course is studied.Specified time interval before and after mRNA preparation is modified in application by rodent bloodletting, with Measure protein expression and whole blood count.Also from the application portion of the rodent of subcutaneous and intramuscular administration modification mRNA preparation Sample is collected in position, to measure the protein expression in tissue.
B.Dose response
Preparation to rodent application containing at least one modification mRNA, to measure the dose response of every kind of preparation.? Specified time interval before and after application modification mRNA preparation is by rodent bloodletting, to measure protein expression and whole blood Cell count.Also rodent is put to death, to analyze effect of the modification mRNA preparation to interior tissue.Also applied from subcutaneous and intramuscular Sample is collected with the site of administration of the rodent of modification mRNA preparation, to measure the protein expression in tissue.
C.Toxicity
Preparation to rodent application containing at least one modification mRNA, to study the toxicity of every kind of preparation.It is applying Specified time interval before and after modification mRNA preparation is by rodent bloodletting, to measure protein expression and whole blood cells It counts.Also rodent is put to death, to analyze effect of the modification mRNA preparation to interior tissue.Also repaired from subcutaneous and intramuscular administration The site of administration for adoring the rodent of mRNA preparation collects sample, to measure the protein expression in tissue.
Embodiment 16.PLGA microball preparation
Optimization for preparing the parameter of PLGA microballoon can maintain to be encapsulated in the same of the integrality of the modification RNA in microballoon When, allow adjustable rate of release and high encapsulation efficiencies.Such as, but not limited to the parameter of granularity, the rate of recovery and encapsulation efficiency can be excellent Change, to realize best prepare.
A.The synthesis of PLGA microballoon
Using water as known in the art/oil/water second emulsifying method, using PLGA (Lactel, catalog number (Cat.No.) B6010-2, Intrinsic viscosity 0.55-0.75,50: 50LA: GA), polyvinyl alcohol (PVA) (Sigma, catalog number (Cat.No.) 348406-25G, MW 13-23k) Methylene chloride and hydration are at poly (lactic acid-glycolic acid) (PLGA) microballoon.Briefly, by the water of 0.1ml (W1) be added to 2ml with Range is dissolved in the PLGA of methylene chloride (DCM) (O1) under the concentration of the PLGA of 50-200mg/ml.W1/O1 lotion is existed (IKA Ultra-Turrax Homogenizer, T18) is homogenized under speed 4 (~15,000rpm) 30 seconds.Then by W1/O1 cream Liquid is added in 0.3% to 1% PVA (W2) of 100ml to 200ml, and speed change is homogenized 1 minute.Keep preparation stirring 3 small When, then washed by being centrifuged (20-25min, 4,000rpm, 4 DEG C).Liquid is discarded supernatant, and PLGA is precipitated and is resuspended In the water of 5-10ml, it is repeated 2 times.After wash, it (is indicated by the average particle size that microexamination measures every kind of preparation 20-30 particle).The increase that table 26 shows PLGA concentration leads to the microballoon of larger size.The PLGA concentration of 200mg/mL obtains 14.8 μm of average particle size, 100mg/mL obtains 8.7 μm, and the PLGA of 50mg/mL obtains 4.0 μm of average particle size.
The PLGA concentration that table 26. changes
Table 27 show by homogenization speed from 5 (~20,000rpm) be down to speed 4 (~15,000rpm) cause granularity from 14.8 μm increasing to 29.7 μm.
The homogenization speed that table 27. changes
Table 28 is shown, and increasing W2 volume (that is, by W2: the ratio of O1 increases to 100: 1 from 50: 1) slightly reduces average particle size. PVA concentration, which is changed into 1wt% from 0.3wt%, has minimal effects to PLGA Microsphere Size.
The W2 volume and concentration that table 28. changes
B.Modify the encapsulating of mRNA
By the G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438 of modification；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) with 2mg/ml's Concentration is dissolved in the water (W3).Three batches of PLGA microball preparations are prepared as described above with following parameter: the W3 of the 2mg/ml of 0.1ml, 1% W2 of O1,160ml of the 200mg/ml of 1.6ml, and for first emulsion (W3/O1), it is homogenized under 4 speed, it is right In second emulsion (W3/O1/W2), it is homogenized under 5 speed.After centrifuge washing, by preparation frost in liquid nitrogen, then Freeze-drying 3 days.For the encapsulation efficiency of test formulation, by the material of freeze-drying in DCM depolymerization 6 hours, extracted in water later Night.Then the concentration that RNA is modified in sample is measured by OD260.By obtaining the actual amount of modification RNA, and divided by modification RNA Initial amount carry out computational envelope efficiency.In three batches tested, encapsulation efficiency 59.2,49.8 and 61.3.
C.It is encapsulated in the integrality of the modification mRNA in PLGA microballoon
By the IX factor mRNA (mRNA sequence shown in SEQ ID NO:1622 of modification；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) with different concentration Be dissolved in the water (W4), to change the weight percent (mg modifies RNA/mg PLGA*100) loaded in the formulation and measure Encapsulation efficiency.The PLGA microball preparation of four different batches is prepared using the parameter in table 29, wherein first emulsion (W4/O1) Homogenization speed be 4 and the homogenization speed of second emulsion (W4/O1/W2) is 5.
Table 29.IX factor PLGA microball preparation parameter
After freeze-drying, weighed up in 2ml eppendorf pipe correspond to~10ug modification RNA PLGA microballoon.It was found that freezing The dry overall structure for not destroying PLGA microballoon.In order to increase the weight percent load (wt%) of PLGA microballoon, add into sample Add the modification RNA of the amount of increase.6 hours are vibrated by the DCM of addition 1.0ml into each pipe and then by sample to make PLGA microballoon depolymerization.Modification RNA is extracted, the water of 0.5ml is added in each sample, by sample shaken overnight, later The concentration that RNA is modified in sample is measured by OD260.In order to measure the recycling of extraction process, the IX factor that do not prepare is modified RNA (mRNA sequence shown in SEQ ID NO:1622；PolyA tail with about 160 nucleotide is not shown in sequence； 5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) (depolymerization control) is added in DCM, and is subjected to depolymehzation process. Table 30 shows load and the encapsulation efficiency of sample.All encapsulation efficiency samples are compareed according to depolymerization and are normalized.
The load of 30. weight percent of table and encapsulation efficiency
D.It is encapsulated in the releasing research of the modification mRNA in PLGA microballoon
RNA (mRNA sequence shown in SEQ ID NO:1622 will be modified with the IX factor；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) prepare PLGA it is micro- Ball depolymerization as described above, and the extracted integrality for modifying RNA is measured by autophoresis (Bio-Rad Experion).It will Extracted modification mRNA is compareed with the modification mRNA not prepared and depolymerization to be compared, to test the modification mRNA of encapsulating Integrality.As shown in Figure 4, it for batch ID A, B, C and D, does not prepare for depolymerization control (de- molding control) and pair For (unformed control), most of modRNA are complete.
E.It is encapsulated in the protein expression of the modification mRNA in PLGA microballoon
RNA (mRNA sequence shown in SEQ ID NO:1622 will be modified with the IX factor；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) prepare PLGA it is micro- Ball depolymerization as described above, and be measured by protein expression of the in-vitro transfection measurement to extracted modification RNA.With The IX factor compound with RNAiMAX (Invitrogen) of 250ng modifies RNA reverse transfected HEK 293 in triplicate.
IX factor modification RNA is diluted to the concentration of 25ng/ μ l in the water without nuclease, and RNAiMAX is existed 13.3x is diluted in EMEM without serum.Isometric diluted modification RNA and diluted RNAiMAX are mixed, and And it is allowed to stand 20 to 30 minutes at room temperature.Then, the transfection containing 250ng IX factor modification RNA of 20 μ l is mixed Object is added in the cell suspension containing 30,000 cells of 80 μ l.Then cell is thin in 37 DEG C/5%CO2 of humidification It is incubated for 16h in born of the same parents' incubator, harvests cell culture supernatant later.Pass through the ELISA kit to IX factor-specific (Molecular Innovations, catalog number (Cat.No.) HFIXKT-TOT) expresses the IX factor protein matter in cell supernatant and carries out Analysis, protein expression are shown in table 31 and Fig. 5.In all PLGA microballoon batches tested, IX factor modification RNA exists It is formulated in PLGA microballoon and subsequent depolymerization keeps activity later and expresses the IX factor.
31. protein expression of table
F.It is encapsulated in the releasing research of the modification mRNA in PLGA microballoon
RNA (mRNA sequence shown in SEQ ID NO:1622 will be modified with the IX factor；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) prepare PLGA it is micro- Ball is resuspended in water, until the PLGA microballoon concentration of 24mg/ml.After resuspension, the PLGA microsphere suspension of 150ul is distributed to In eppendorf pipe.During research process, sample is maintained at 37 DEG C and is incubated for and vibrates.In the 0.2nd, 1,2,8,14 and 21 days, sample was extracted in triplicate.In order to measure the amount of the modification RNA discharged from PLGA microballoon, sample is centrifuged, is removed Supernatant, and the concentration that RNA is modified in supernatant is measured by OD 260.Release percentage shown in table 32 is based on each sample The total amount that RNA is modified in product is calculated.After 31 days, 96% IX factor modification RNA is released from PLGA microball preparation.
Table 32. discharges percentage
Time (day) % release
0 0.0
0.2 27.0
1 37.7
2 45.3
4 50.9
8 57.0
14 61.8
21 75.5
31 96.4
G.The granularity reproducibility of PLGA microballoon
Using shown in table 29 be directed to batch D described in the same terms (W4,2.0ml's of the 4mg/ml of 0.4ml 1% W2 of O1,200ml of 200mg/ml, and for W4/O1/W2 lotion, it is homogenized under 5 speed) it is prepared for three batches The secondary IX factor modifies RNA (mRNA sequence shown in SEQ ID NO:1622；PolyA with about 160 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) PLGA microballoon.In order to improve The uniformity of PLGA microsphere suspension is incorporated to filtering before centrifugation.After stirring 3 hours and before centrifugation, make institute There is the material of preparation to pass through 100 μm of nylon net filter devices (Fisherbrand Cell Strainer, catalog number (Cat.No.) 22-363- 549), to remove biggish aggregation.After washing and being resuspended with water, using the PLGA microsphere sample of 100-200 μ l, pass through The granularity of laser diffraction (Malvern Mastersizer2000) measurement preparation.The granularity of sample is shown in table 33.
33. granularity of table summarizes
The result of 3 PLGA microballoon batches of filtering and PLGA prepared without filtering under the same conditions will be used Microballoon batch compares.It include that filtration step reduces average particle size and shows 3 PLGA microballoon batches before washing Between consistent size distribution.
H.The serum stability of IX factor PLGA microballoon
It will be in the IX factor mRNA RNA in buffer (TE) or 90% serum (Se) (shown in SEQ ID NO:1622 MRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5-methylcytosine and Pseudouridine is modified completely), or IX factor mRNA in the PLGA in buffer, 90% serum or 1% serum is in total volume It is incubated under the mRNA concentration of 50ng/ul in the buffer of 70ul, 90% serum or 1% serum.At 0 minute, 30 minutes, 60 points Clock or 120 minutes whens, remove sample.By 4x proteinase K buffer (the 0.4ml 1M TRIS-HCl (pH for adding 25ul 7.5), 0.1ml 0.5M EDTA, 0.12ml 5M NaCl and 0.4ml 10%SDS) and 8ul 20mg/ml Proteinase K, use Proteinase K digests 20 minutes at 55 DEG C inactivates RNA enzyme.Making IX factor mRNA precipitating, (addition 95% ethyl alcohol of 250ul, continues 1 hour, it is centrifuged 10min at 13k rpm and removes supernatant, 70% ethyl alcohol of 200ul is added to precipitating, again in 13k rpm Lower centrifugation 5min simultaneously removes supernatant and precipitating is resuspended in 70ul water) or from PLGA microballoon extract (at 13k rpm from Heart 5min simultaneously removes supernatant, is precipitated with 1ml water washing, and 5min is centrifuged at 13k rpm and removes supernatant, is added to precipitating 280ul methylene chloride simultaneously vibrates 15 minutes, adds 70ul water, then vibrates 2 hours, and remove water phase), pass through biology later Analyzer is analyzed.PLGA microballoon protects IX factor modification mRNA non-degradable after 2 hours in 90% and 1% serum.IX The factor modify mRNA in 90% serum at the beginning between put it is degradable.
17. lipidic nanoparticles In vivo study of embodiment
G-CSF (had into T7 promoter, 5 ' non-translational regions (UTR) and the 3 ' UTR for in-vitro transcription using injection syringe pump method CDNA provided in SEQ ID NO:21437.MRNA sequence shown in SEQ ID NO:21438；With about 160 cores The polyA tail of thuja acid is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) and the IX factor (there is the cDNA of the T7 promoter for in-vitro transcription, 5 ' UTR and 3 ' UTR to provide in SEQ ID NO:21443.In SEQ MRNA sequence shown in ID NO:1622；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) modification mRNA be formulated as lipidic nanoparticles (LNP).With 20: 1 Total lipid and the weight ratio of modification mRNA prepare LNP, and final lipid molar ratios are 50: 10: 38.5: 1.5 (DLin-KC2- DMA: DSPC: cholesterol: PEG-c-DOMG).The preparation listed in table 34 is characterized by granularity, zeta potential and encapsulating.
34. preparation of table
LNP preparation is intravenously applied to mouse (n=5) with the modification mRNA dosage of 100ug, 10ug or 1ug.In administration 8 Mouse is put to death after hour.It is collected from the mouse that applied G-CSF or IX factor modification mRNA preparation by cardiac puncture Serum.Protein expression is measured by ELISA.
Without significant body weight loss (< 5%) in G-CSF or IX factor dosage group.By ELISA, surveyed by standard curve Determine the protein expression of G-CSF or IX factor dosage group.By serum samples diluted (for G-CSF, about 20-2500x, and right In the IX factor, about 10-250x), to ensure sample in the range of linearity of standard curve.As shown in table 35, it is measured by ELISA G-CSF protein expression 17ng/ml, 1200ng/ml and 4700ng/ are respectively about for 1ug, 10ug and 100ug dosage group ml.As shown in table 36,1ug, 10ug and 100ug dosage group are distinguished by the IX factor protein matter expression of ELISA measurement It is about 36ng/ml, 380ng/ml and 3000-11000ng/ml.
Table 35.G-CSF protein expression
Dosage (ug) Concentration (ng/ml) Dilution gfactor Sample volume
1 17.73 20x 5ul
10 1204.82 2500x 0.04ul
100 4722.20 2500x 0.04ul
The expression of table 36.IX factor protein matter
Dosage (ug) Concentration (ng/ml) Dilution gfactor Sample volume
1 36.05 10x 5ul
10 383.04 10x 5ul
100* 3247.75 50x 1ul
100* 11177.20 250x 0.2ul
As shown in table 37, with application same dose modification mRNA intravenous (IV)-lipid composite preparation and flesh The modification mRNA in salt water of interior (IM) or subcutaneous (SC) application same dose is compared, the protein output of above-mentioned LNP preparation Increase about 10,000-100,000 times.As used in table 37, symbol "~" refers to about.
37. protein output of table
G-CSF Dosage (ug) 8-12 hours serum-concentrations (pg/ml) after application
IM 100 ~20-80
SC 100 ~10-40
IV (fat complexes) 100 ~30
IV(LNP) 100 ~5,000,000
IV(LNP) 10 ~1,000,000
IV(LNP) 1 ~20,000
The IX factor Dosage (ug) 8-12 hours serum-concentrations (ng/ml) after application
IM 2x100 ~1.6ng/ml
IV(LNP) 100 ~3,000-10,000ng/ml
IV(LNP) 10 ~400ng/ml
IV(LNP) 1 ~40ng/ml
The material and method of embodiment 18-23
Using injection syringe pump method by G-CSF (mRNA sequence shown in SEQ ID NO:21438；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) and EPO (SEQ MRNA sequence shown in ID NO:1638；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) modification mRNA be formulated as lipidic nanoparticles (LNP).With 20: 1 Total lipid and the weight ratio of modification mRNA prepare LNP, and final lipid molar ratios are 50: 10: 38.5: 1.5 (DLin-KC2- DMA: DSPC: cholesterol: PEG-c-DOMG).The preparation listed in table 38 is characterized by granularity, zeta potential and encapsulating.
38. preparation of table
Embodiment 18. carries out the In vivo study of lipidic nanoparticles using modification mRNA
MRNA dosage is modified to rat (n=5) intravenous (IV), intramuscular (IM) or subcutaneous with the single of 0.05mg/kg (SC) LNP preparation shown in table 38 (above) is applied.Rat control group (n=4) is untreated.2 hours, 8 hours, 24 hours, 48 hours and 96 hours and rat applied G-CSF or EPO modification mRNA preparation after by rat bloodletting, to use ELISA measures protein expression.The rat of intravenous application EPO modification mRNA is also in bloodletting in the 7th day.
As shown in table 39, it is that can examine that the epo protein in the rat of the EPO mRNA of intravenous application modification, which was expressed to the 5th day, It measures.G-CSF to the 7th days in the rat of the G-CSF mRNA of intravenous application modification are detectable.EPO modifies mRNA Subcutaneous and intramuscular administration at least 24 hours be it is detectable, and G-CSF modify mRNA at least 8 hours be detectable 's.In table 39, " OSC " refers to the value outside standard curve, and " NT ", which refers to, not to be tested.
Table 39.G-CSF and epo protein expression
Approach Time EPO serum-concentration (pg/ml) G-CSF serum-concentration (pg/ml)
IV 2 hours 36,981.0 31,331.9
IV 8 hours 62,053.3 70,532.4
IV 24 hours 42,077.0 5,738.6
IV 48 hours 5,561.5 233.8
IV 5 days 0.0 60.4
IV 7 days 0.0 NT
IM 2 hours 1395.4 1620.4
IM 8 hours 8974.6 7910.4
IM 24 hours 4678.3 893.3
IM 48 hours NT OSC
IM 5 days NT OSC
SC 2 hours 386.2 80.3
SC 8 hours 985.6 164.2
SC 24 hours 544.2 OSC
SC 48 hours NT OSC
SC 5 days NT OSC
It is untreated Whole blood 0 0
19. time course In vivo study of embodiment
It is intravenous to mouse (n=5) that mRNA dosage is modified with the single of 0.5mg/kg, 0.05mg/kg or 0.005mg/kg (IV) LNP preparation shown in table 38 (above) is applied.8 is small after it applied G-CSF or EPO modification mRNA preparation to mouse When, 24 hours, 72 hours and 6 days by mouse bloodletting, to use ELISA to measure protein expression.
As shown in table 40, EPO the and G-CSF protein expression in the mouse of intravenous application modification mRNA for It to 72 hours was detectable for the mouse of the modification mRNA administration of 0.005mg/kg and 0.05mg/kg, and for applying It to the 6th day was detectable for mouse with EPO modification mRNA.In table 40, " > ", which refers to, to be greater than, and " ND " refers to It does not detect.
40. protein expression of table
Dosage (mg/kg) Time EPO serum-concentration (pg/ml) G-CSF serum-concentration (pg/ml)
0.005 8 hours 12,508.3 11,550.6
0.005 24 hours 6,803.0 5,068.9
0.005 72 hours ND ND
0.005 6 days ND ND
0.05 8 hours 92,139.9 462,312.5
0.05 24 hours 54,389.4 80,903.8
0.05 72 hours ND ND
0.05 6 days ND ND
0.5 8 hours 498,515.3 > 1,250,000
0.5 24 hours 160,566.3 495,812.5
0.5 72 hours 3,492.5 1,325.6
0.5 6 days 21.2 ND
LNP preparation In vivo study in 20. rodent of embodiment
A.LNP preparation in mouse
MRNA dosage, which is modified, with the single of 0.05mg/kg or 0.005mg/kg applies table to mouse (n=4) intravenous (IV) LNP preparation shown in 38 (above).There is also 3 untreated control mice groups (n=4).It applied G-CSF or EPO is repaired Adorn mRNA preparation after 2 hours, 8 hours, 24 hours, 48 hours and 72 hours by mouse bloodletting, to measure protein expression.G- The protein expression of CSF and EPO is measured using ELISA.
As shown in table 41, EPO the and G-CSF protein expression in mouse is for receiving 0.005mg/kg modification RNA dosage It to as little as 48 hours was detectable for mouse, and for the mouse for receiving 0.05mg/kg modification RNA dosage extremely As little as 72 hours are detectable.In table 41, " OSC " refers to the value outside standard curve, and " NT, which refers to, not to be tested."
Protein expression in 41. mouse of table
B.LNP preparation in rodent
MRNA dosage is modified to shown in rat (n=4) intravenous (IV) application table 38 (above) with the single of 0.05mg/kg LNP preparation.There is also 1 untreated rat control groups (n=4).2 is small after application G-CSF or EPO modification mRNA preparation When, 8 hours, 24 hours, 48 hours, 72 hours, 7 days and 14 days by rat bloodletting, to measure protein expression.G-CSF and EPO Protein expression measured using ELISA.
The SMS message process study of embodiment 21.LNP
It is intravenous to mammal with the single modification mRNA dosage of 0.5mg/kg, 0.05mg/kg or 0.005mg/kg (IV), LNP preparation shown in intramuscular (IM) or subcutaneous (SC) application table 38 (above).Mammal control group is untreated.It is applying With 5 minutes, 10 minutes, 20 minutes, 30 minutes, 45 minutes, 1 hour, 1.5 hours and/or 2 hours after modification mRNA LNP preparation By mammal bloodletting, to use ELISA to measure protein expression.Also by mammal bloodletting, to measure whole blood count As granulocyte is horizontal and erythrocyte counts.
22. non-human primate In vivo study of embodiment
Use hypodermic needle using LNP preparation shown in table 38 (above) as bullet formula intravenous injection (IV) after about It is applied within 30 seconds non-human primate (NHP) (machin) (n=2), the hypodermic needle can be attached to injection as needed Device/trochar (abbocath) or butterfly valve.Single modification with from the dose volume of 0.5mL/kg to NHP application 0.05mg/kg The EPO or G-CSF of mRNAIV dosage or the EPO of 0.005mg/kg.NHP was put in 5-6 days before giving modification mRNALNP preparation Blood, to measure protein expression and the baseline whole blood cell count in serum.It is small the 8th after mRNA preparation is modified in application When, the 24th hour, the 48th hour and the 72nd hour by NHP bloodletting, to measure protein expression.24 hours and 72 small after application When, also measure the whole blood count of NHP.The protein expression of G-CSF and EPO is measured by ELISA.It was tested entirely The urine from NHP is collected in journey and is analyzed to evaluate clinical safety.MRNA system is modified in application G-CSF or EPO After agent, sample is collected from NHP, to use ELISA to measure protein expression.Also to the clinical chemistry of non-human primate, Hematology, urinalysis and cell factor are analyzed.
As shown in table 42, it is detectable for applying the epo protein in the NHP of 0.05mg/kg and expressing to 72 hours, and It is detectable that the 0.005mg/kg of EPO preparation, which gave to 48 hours,.In table 42, " < " refers to less than given value.It is applying With modification mRNA preparation after observe G-CSF protein expression within 24 hours.Preliminarily, the NHP after mRNA preparation is modified in application In observe the increase of granulocyte and reticulocyte level.
Protein expression in 42. non-human primate of table
The non-human primate In vivo study of embodiment 23.G-CSF and EPO
Non-human primate (NHP) is applied to using LNP preparation shown in table 38 (above) as intravenous injection (IV) (machin) (n=2).0.5mg/kg, 0.05mg/kg or 0.005mg/kg are applied to NHP with the dose volume of 0.5mL/kg The G-CSF or EPO of single modification mRNAIV dosage.By NHP bloodletting before giving modification mRNA LNP preparation, to measure serum In protein expression and baseline whole blood cell count.After application G-CSF modification mRNA preparation, the 8th hour, it is the 24th small When, the 48th hour and the 72nd hour by NHP bloodletting, to measure protein expression.After application EPO modification mRNA preparation, 8th hour, the 24th hour, the 48th hour, the 72nd hour and the 7th day by NHP bloodletting, to measure protein expression.
The sample collected in the NHP after modifying mRNA preparation from application G-CSF or EPO is analyzed by ELISA, with Measure protein expression.Before administration, 24 hours, 3 days, 7 days, 14 days and 18 days after G-CSF the or EPO preparation of application modification, Also neutrophil leucocyte and reticulocyte count are measured.
As shown in table 43, more than 72 hours after G-CSF protein expression is not detected.In table 43, " < 39 ", which refers to, to be lower than The value of the Monitoring lower-cut of 39pg/ml.
Table 43.G-CSF protein expression
As shown in table 44, more than 7 days after be not detected epo protein expression.In table 44, " < 7.8 ", which refers to, to be lower than The value of the Monitoring lower-cut of 7.8pg/ml.
Table 44.EPO protein expression
As shown in table 45, the neutrophil leucocyte of all G-CSF groups increased relative to level before being administered.
Pharmacotoxicological effect of the table 45.G-CSF mRNA in NHP
As shown in table 46, upon administration 3 days to 14/18 day, the granulophilocyte of all EPO groups was relatively 24 small after administration When reticulocyte level increased.
Pharmacotoxicological effect of the table 46.EPO mRNA to neutrophil count
As shown in table 47-49, EPO modifies the application of RNA to including hemoglobin (HGB), hematocrit (HCT) and red Other hematopoietin parameters that haemocyte (RBC) counts have an impact.
Pharmacotoxicological effect of the table 47.EPO mRNA to hemoglobin
Pharmacotoxicological effect of the table 48.EPO mRNA to hematocrit
Pharmacotoxicological effect of the table 49.EPO mRNA to red blood cell
As shown in table 50 and 51, the application of RNA is modified to including alanine aminotransferase (ALT) and aspartate transaminase (AST) Serum Chemical Parameter has an impact.
Pharmacotoxicological effect of the table 50.EPO mRNA to alanine aminotransferase
Pharmacotoxicological effect of the table 51.EPO mRNA to aspartate transaminase
As shown in table 52, application of lipidic nanoparticles-preparation modification RNA at high dose (0.5mg/kg) causes to repair Adorn the increase of cell factor, interferon-' alpha ' (IFN-α) after mRNA is applied.
Pharmacotoxicological effect of the table 52.EPO mRNA to alanine aminotransferase
Intramuscular and/or subcutaneous administration the research in non-human primate of embodiment 24.
By the EPO mRNA (mRNA sequence shown in SEQ ID NO:1638 containing modification in salt water；With big The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is modified completely with 5-methylcytosine and pseudouridine) Or G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide, sequence It is not shown in column；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) preparation intramuscular (IM) or subcutaneous (SC) It is applied to non-human primate (machin) (NHP).The single modification mRNA dosage of 0.05mg/kg or 0.005mg/kg is in In the dose volume of 0.5mL/kg.5-6 days by non-human primate bloodletting before administration, with measure serum protein concentrations and Baseline whole blood cell count.Application modify mRNA preparation after, the 8th hour, the 24th hour, the 48th hour, the 72nd hour, 7th day and the 14th day by NHP bloodletting, to measure protein expression.The protein expression of G-CSF and EPO is surveyed by ELISA It is fixed.24 hours after application, 72 hours, 7 days and 14 days are also measured the whole blood count of NHP.It was tested entirely The urine from NHP is collected in journey and is analyzed to evaluate clinical safety.Tissue also near collection injection site is simultaneously It is analyzed, to measure protein expression.
The transport of the modification of embodiment 25. mRNA
In order to measure the positioning and/or transport of modification mRNA, research can be executed as follows.
SiRNA is prepared according to known in the art and/or method described herein and modifies the LNP preparation of mRNA.LNP preparation It may include at least one modification mRNA, codified protein, such as G-CSF, EPO, VII factor and/or as described herein any Protein.Intramuscular or subcutaneous injection can be used locally to be applied to preparation in the muscle of mammal.Modify the dosage of mRNA With the variable dimension of LNP, with measure to the effect transported in the mammalian body and/or assessment to biological respinse (such as but Be not limited to inflammation) influence.Can put mammal bloodletting in different times, with measure be present in serum by being applied Modification mRNA coding protein expression and/or measurement mammal in whole blood count.
For example, can the modification of the VII factor expressing and be secreted into serum in liver of intramuscular and/or subcutaneous administration coding mRNA.Modify mRNA application while or before, apply siRNA to knock out the endogenous VII factor.By intramuscular and/or subcutaneous note The VII factor that applied modification mRNA is generated is penetrated to measure in blood.Also, the level of the VII factor is in injection site It is measured in neighbouring tissue.If the VII factor is expressed in blood, there is the transport of modification mRNA.If VII because Son is expressed in the tissue without expressing in blood, then there is only the local expressions of the VII factor.
Embodiment 26. has the preparation of a variety of modification mRNA
The LNP preparation of modification mRNA is prepared according to known in the art and/or as described herein or methods known in the art. LNP preparation may include at least one modification mRNA, codified protein, such as G-CSF, EPO, thrombopoietin and/or sheet Any protein described in text.At least one modification mRNA may include 1,2,3,4 or 5 kind of modification mRNA molecule.Contain at least one Kind modification mRNA preparation can with single or multiple dosage regimens intravenous, intramuscular or subcutaneous administration.It can be at least one in application It modifies before or after mRNA preparation, point collects the biological sample for being such as, but not limited to blood and/or serum in different times Product are simultaneously analyzed.It is described after the preparation that mammal applied at least one modification mRNA containing coding protein The expression of protein 50-200pg/ml in the biological sample is considered as biologically effective.
27. polyethylene glycol ratio research of embodiment
A.The preparation and characterization of PEG LNP
Lipidic nanoparticles (LNP) is prepared using injection syringe pump method.With the G-CSF mRNA of 20: 1 total lipid and modification (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide is not shown in sequence；5' Cap, Cap1；Modified completely with 5-methylcytosine and pseudouridine) weight ratio prepare LNP.The molar ratio range of preparation is in table It is shown in 53.
53. molar ratio of table
To two kinds of PEG lipid 1, bis- myristoyl-sn- glycerol methoxy of 2- at 1.5mol% or 3.0mol% Base polyethylene glycol (PEG-DMG, NOF catalog number (Cat.No.)GM-020) and 1,2- distearyl acyl group-sn- glycerol first Oxygroup polyethylene glycol (PEG-DSG, NOF catalog number (Cat.No.)GS-020 it) is tested.LNP preparation and repair After the encapsulating of the G-CSF mRNA of decorations, LNP preparation is characterized by granularity, zeta potential and encapsulating percentage, is as a result existed It is shown in table 54.
The characterization of table 54.LNP preparation
B.The internal screening of PEG LNP
The preparation of PEG LNP described in table 55 is intravenously applied to mouse (n=5) with the dosage of 0.5mg/kg.It is applying Serum is collected from mouse within 2 hours, 8 hours, 24 hours, 48 hours, 72 hours and 8 days after preparation.By ELISA to serum into Row analysis, to measure the protein expression of G-CSF, expression is shown in table 55.It is used using the LNP preparation ratio of PEG-DMG The LNP preparation of PEG-DSA provides significantly higher protein expression level.
55. protein expression of table
The research of 28. Cationic Lipid Formulations of embodiment
A.The preparation and characterization of cation lipid nano particle
Lipidic nanoparticles (LNP) is prepared using injection syringe pump method.Matched with 20: 1 total lipid and the weight ratio of modification mRNA LNP processed.Cation lipid, DSPC, cholesterol and PEG-c-DOMG final lipid molar ratios range summarized in table 56.
56. molar ratio of table
The ethanol solution of 25mM lipid and the modification RNA in the citrate of 50mM pH 3 are mixed, it is spontaneous to generate Vesica is formed.Keep vesica stable in ethanol, removes ethyl alcohol later, and buffer-exchanged is carried out by dialysis.Then pass through Granularity, zeta potential and encapsulating percentage characterize LNP.Table 57 is described using DLin-MC3-DMA, DLin-DMA or C12- 200 as cation lipid encapsulating EPO modification mRNA (mRNA sequence shown in SEQ ID NO:1638；With about 160 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) or G-CSF Modify mRNA (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide, in sequence It is not shown；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) LNP characterization.
The characterization of 57. Cationic Lipid Formulations of table
B.The internal screening of cationic LNP preparation
The preparation of Cationic Lipid Formulations described in table 57 is intravenously applied to mouse (n=5) with the dosage of 0.5mg/kg. Serum is collected from mouse within 2 hours, 24 hours, 72 hours and/or 7 days after applying preparation.Serum is divided by ELISA Analysis, to measure the protein expression of EPO or G-CSF, expression is shown in table 58.
58. protein expression of table
It is seen in mouse of the application with the LNP preparation of cation lipid C12-200 (NPA-075-1 and NPA-076-1) It observes toxicity and put to death the mouse at the 24th hour, because mouse shows as fur is short and small, cowers behavior and body Loss is greater than 10% symptom again.It is expected that C12-200 is more toxic, but also there is high expression level in the short period. Cation lipid DLin-DMA (NPA-073-1 and NPA-074-1) has minimum table in the three kinds of cation lipids tested It reaches.For EPO preparation, DLin-MC3-DMA (NPA-071-1 and NPA-072-1) to third day shows good representation and extremely It is higher than Background Samples within 7th day.
The screening technique of 29. protein expression of embodiment
A.Electrospray ionisation
It is prepared for may include by the biological sample for the protein for being applied to the modification RNA coding of subject and according to EFI Manufacturer's scheme of mist ionization (ESI) is analyzed it using 1,2,3 or 4 mass-synchrometer.Also using series connection ESI mass spectrum System analyzes biological sample.
The mode of protein fragments or entire protein is compared with the known control of given protein, and is passed through Compare determining identity.
B.Substance assistant laser desorpted/ionization
Preparation may include the biological sample by being applied to the protein for modifying RNA coding of subject, and auxiliary according to matrix Manufacturer's scheme of laser desorption/ionization (MALDI) is helped to analyze it.
The mode of protein fragments or entire protein is compared with the known control of given protein, and is passed through Compare determining identity.
C.Liquid chromatography-tandem mass spectrometry
It can will may include wherein being wrapped by the biological sample trypsin treatment of the protein of modification RNA coding with digestion The protein contained.Resulting peptide is analyzed by Liquid chromatography-tandem mass spectrometry (LC/MS/MS).In a mass spectrometer by peptide Fragmentation can be matched via computerized algorithm with protein sequence database with generating diagnostic mode.It can will be digested Sample dilution, to realize the starting material of the given protein of 1ng or less.Containing simple buffering liquid background (for example, water or Volatile salts) biological sample be suitable for directly digesting in the solution；More complicated background is (for example, detergent, non-volatile Salt, glycerol) need other cleaning step in order to sample analysis.
The mode of protein fragments or entire protein is compared with the known control of given protein, and is passed through Compare determining identity.
30. lipidic nanoparticles In vivo study of embodiment
Using injection syringe pump method by mCherry mRNA (mRNA sequence shown in SEQ ID NO:21444；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) match It is made as lipidic nanoparticles (LNP).LNP is prepared with the weight ratio of 20: 1 total lipid and modification mRNA, and final lipid rubs You are than being 50: 10: 38.5: 1.5 (DLin-KC2-DMA: DSPC: cholesterol: PEG-c-DOMG).By granularity, zeta potential and Encapsulating characterizes the mCherry preparation listed in table 59.
Table 59.mCherry preparation
LNP preparation is intravenously applied to mouse (n=5) with the modification mRNA dosage of 100ug.It upon administration 24 hours will be small Mouse is put to death.By immunohistochemistry (IHC), Western blotting or fluorescence-activated cell sorting (FACS) to from applied The liver of the mouse of mCherry modification mRNA preparation and spleen are analyzed.
The histology of liver shows consistent mCherry expression in entire slice, and untreated animal is not expressed mCherry.Expression of the mCherry in handled animal is also confirmed using Western blotting, and in untreated animal not Detect mCherry.Use tubulin as control marker, and is detected in processing and untreated mouse Tubulin shows that the normal protein expression in liver cell is uninfluenced.
FACS and IHC is executed also on mCherry and the spleen of untreated mice.By facs analysis, all leucocytes are thin Born of the same parents group is negative for mCherry expression.Through IHC, between the mouse of mCherry processing and the spleen of untreated mice There is no Observable difference yet.
31. syringe pump In vivo study of embodiment
MCherry is modified into mRNA (mRNA sequence shown in SEQ ID NO:21439 using injection syringe pump method；With big The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is formulated as lipidic nanoparticles (LNP).With 20: 1 Total lipid and the weight ratio of modification mRNA prepare LNP, and final lipid molar ratios are 50: 10: 38.5: 1.5 (DLin- KC2-DMA: DSPC: cholesterol: PEG-c-DOMG).MCherry preparation is characterized by granularity, zeta potential and encapsulating.
LNP preparation is intravenously applied to mouse (n=5) with the modification mRNA dosage of 10ug or 100ug.24 is small upon administration When mouse is put to death.By immunohistochemistry (IHC), Western blotting and/or fluorescence-activated cell sorting (FACS) to next It is analyzed from the liver for the mouse that applied mCherry modification mRNA preparation and spleen.
The expression of 32. in vitro and in vivo of embodiment
A.Use vivoexpression of the lipoids preparation in people's cell
In different lipoids: under mmRNA ratio, to the ratio of the mmRNA and lipidoid of the test for in-vitro transfection Rate is empirically tested.2.5: 1,5: 1,10: 1 and 15: 1 class is utilized using the Previous work of siRNA and lipoids Lipid: wt: wt ratio of siRNA.Relative to siRNA, in the longer situation of the length of mmRNA, lower lipoids and mmRNA Wt: wt ratio can be effective.In addition, also using RNAIMAX to compareTM(Invitrogen, Carlsbad, CA) or TRANSIT-mRNA (Mirus Bio, Madison, WI) cation lipid delivery vehicle prepares mmRNA.
Lipoids-preparation luciferase (IVT cDNA sequence shown in SEQ ID NO:21445；SEQ ID NO: MRNA sequence shown in 21446, the polyA tail (being not shown in sequence) with about 160 nucleotide, 5 ' cap Cap1, Substitute modification completely with pseudouridine with 5-methylcytosine and at each uridine site at each cytimidine), green fluorescence Albumen (GFP) (IVT cDNA sequence shown in SEQ ID NO:21447；MRNA sequence shown in SEQ ID NO:21448 Column, the polyA tail (being not shown in sequence) with about 160 nucleotide, 5 ' cap Cap1, with 5- methyl at each cytimidine Cytimidine and substitute modification completely with pseudouridine at each uridine site), G-CSF mRNA is (in SEQ ID NO:21439 The mRNA sequence shown；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) and EPO MRNA (mRNA sequence shown in SEQ ID NO:1638；PolyA tail with about 160 nucleotide does not show in sequence Out；5 ' caps, Cap1) it expresses and it is expected that the ability of protein product can be confirmed by following: for luciferase expression, pass through hair Light；GFP is expressed, flow cytometry is passed through；And G-CSF and hematopoietin (EPO) are secreted, passed through ELISA。
B.Internal expression after intravenous injection
Use a variety of different lipoids, the including but not limited to systematicness of 98N12-5, C12-200 and MD1 realization preparation Intravenous application.
Lipoids preparation containing mmRNA is injected intravenously into animal body.Modify the albumen of mRNA (mmRNA) coding The expression of matter is assessed from the blood and/or other organ samples (such as, but not limited to liver and spleen) collected in animal. Carry out the persistence that single dose intravenously studies the magnitude, dose response degree and expression that will also allow assessment desired product.
In one embodiment, using the system based on lipoids of 98N12-5, C12-200, MD1 and other lipoids Agent by luciferase, green fluorescent protein (GFP), mCherry fluorescin, secretion alkaline phosphatase (sAP), people G- CSF, the people IX factor or human forcing erythrogenin (EPO) mmRNA are delivered in animal.It is being prepared as discussed previously with lipid After mmRNA, by animal packet, to receive containing selected from luciferase, GFP, mCherry, sAP, human G-CSF, people's IX factor And one saline formulation or lipoids preparation in the different mmRNA of people EPO.Before being injected into animal, it will contain The lipoids preparation of mmRNA dilutes in PBS.Then to the mmRNA of the preparation of animal application single dose, the dosage exists The dosage of 10mg/kg is to down to changing in the dosage range of 1ng/kg, and wherein preferred scope is 10mg/kg to 100ng/kg, The dosage of middle mmRNA depends on the weight of animals, and such as 20 grams of mouse maximums receive the preparation of 0.2ml, and (administration is based on mmRNA/kg Weight).After applying mmRNA- lipoids preparation, acquisition serum, tissue and/or Tissue Lysates, and mmRNA coding The horizontal time interval in single time interval or a range of product is measured.The luciferase of lipoids preparation, GFP, mCherry, sAP, G-CSF, IX factor and EPO mmRNA express the ability for it is expected protein product by following come really Recognize: the expression for luciferase, by shining；Expression for GFP and mCherry, passes through flow cytometry；For sAP, Pass through enzymatic activity；Or the slice for G-CSF, IX factor and/or EPO, pass through ELISA.
The further research of multidose scheme is performed, also to measure the maximum expression of mmRNA, evaluation mmRNA driving Expression saturability (by parallel or sequentially giving control and activity mmRNA preparation), and measurement repeats medicament administration Whether feasibility is (by give mmRNA by week or the dosage separated the moon, then measuring expression by such as immunogenicity The influence of factor).As the protein of G-CSF and EPO physiologic function assessment also by analyzing come the sample of self-test animal Product simultaneously detect the increase of granulocyte and erythrocyte counts respectively to determine.The expressed protein product such as IX factor is in animal In activity can also pass through IX factor enzymatic activity (as activation partial thromboplastin time measurement) analysis and blood coagulation The influence of time is assessed.
C.Vivoexpression after intramuscular and/or subcutaneous injection
It needs to deliver the oligonucleotides including mRNA via intramuscular injection approach or subcutaneous routes to lipoids preparation Purposes evaluated because not had been reported to it previously.The intramuscular of mmRNA and/or subcutaneous injection are evaluated, with true Whether the fixed lipoids preparation containing mmRNA can generate part and the systematicness expression of desired protein.
It is the lipoids preparation of 98N12-5, C12-200 and MD1 containing mmRNA is intramuscular and/or be subcutaneously injected into animal In, the mmRNA be selected from luciferase, green fluorescent protein (GFP), mCherry fluorescin, secretion alkaline phosphatase (sAP), human G-CSF, the people IX factor or human forcing erythrogenin (EPO) mmRNA.In muscle or subcutaneous tissue, and The expression of the protein of mmRNA coding is systematically assessed in blood and other organs (such as liver and spleen).Single dose research is permitted Perhaps the magnitude of desired product, the persistence of dose response degree and expression are assessed.
By animal packet to receive saline formulation or the preparation containing modification mRNA.Before injection, it will contain mmRNA's Lipoids preparation dilutes in PBS.The mmRNA of the preparation of dosage into animal application single dose intravenous, the dosage is in 50mg/kg To down to changing in the dosage range of 1ng/kg, wherein preferred scope is 10mg/kg to 100ng/kg.It is intramuscular to apply for mouse Maximum dose is about 1mg mmRNA, or for being intramuscularly injectied into lower limb after mouse, and maximum dose is down to 0.02ng mmRNA.For subcutaneous administration, to the mmRNA of the preparation of animal application single SC dosage, the dosage is in 400mg/kg to low Change in the dosage range of 1ng/kg, wherein preferred scope is 80mg/kg to 100ng/kg.For mouse, subcutaneous administration Maximum dose is about 8mg mmRNA or down to 0.02ng mmRNA.
For 20 grams of mouse, the volume of single intramuscular injection is up to 0.025ml, and single subcutaneous injection is up to 0.2ml.The optimal dose of applied mmRNA is calculated by the weight of animal.Time point after applying mmRNA- lipoids Difference obtains serum, tissue and Tissue Lysates, and measures the level of mmRNA coded product.The fluorescence that lipoids is prepared Plain enzyme, green fluorescent protein (GFP), mCherry fluorescin, the alkaline phosphatase (sAP) of secretion, human G-CSF, people's IX factor Or the ability of human forcing erythrogenin (EPO) mmRNA expression expectation protein product is confirmed by following: for fluorescence Plain expression of enzymes, by shining；GFP and mCherry is expressed, by flow cytometry, for sAP, by enzymatic activity, and G-CSF, IX factor and hematopoietin (EPO) are secreted, ELISA is passed through.
The other research of multidose scheme is also performed, to measure the maximum expression using mmRNA, evaluation mmRNA is driven The saturability (being realized by parallel or sequentially giving control and activity mmRNA preparation) of dynamic expression, and measurement repeat drug Application feasibility (by give mmRNA by week or the dosage that separates the moon, and then measurement expression whether by such as The influence of the factor of immunogenicity).It also uses and utilizes the research of multiple subcutaneously or intramuscularly injection sites a time point, with It further increases the exposure of mmRNA drug and improves protein generation.As GFP, mCherry, sAP, human G-CSF, the people IX factor with And the assessment of the physiologic function of the protein of people EPO by analyze come the sample of self-test animal and detect granulocyte and/or The change of erythrocyte counts is measured.Activity of the expressed protein product such as the IX factor in animal can also pass through IX The analysis and the influence in clotting time of factor enzymatic activity (such as the partial thromboplastin time measurement of activation) are assessed.
The difunctional mmRNA of embodiment 33.
Using teachings described herein and synthetic method, modification RNA is designed and synthesized to be difunctional, thus coding one Kind or various kinds of cell toxic protein molecule and synthesized using cytotoxic nucleoside.
The application of difunctional modification mRNA is realized using salt water or lipid carrier.Once application, just translates difunctional modification MRNA is to generate the cytotoxic peptide of coding.In the modification mRNA degradation of delivering, cytotoxic nucleoside is released, this is also influenced To the treatment benefit of subject.
The transfection of the modification of embodiment 34. mRNA
A.Reverse transfection
For the experiment executed in the tissue culture plate that 24 hole collagens are coated with, with 1x105Cell density be inoculated with angle Matter forms cell.For the experiment executed in the tissue culture plate that 96 hole collagens are coated with, with 0.5x105Cell density connect Kind keratinocyte.For every kind of modification mRNA (mmRNA) to be transfected, mRNA:RNAIMAX is modified in such as described preparationTM And before cell adherence to tissue culture plate, by itself and cell in the cell inoculation period (such as 6 in porous flat plate Hour) in mixing.
B.Forward direction transfection
In the tissue culture plate of 24 hole collagens coating, with 0.7x105Cell density be inoculated with keratinocyte.It is right In the experiment executed in the tissue culture plate that 96 hole collagens are coated with, with 0.3x105Cell density inoculation cutin formed carefully Born of the same parents.Keratinocyte is grown to > 70% for 24 hours and is converged.For every kind of modification mRNA (mmRNA) to be transfected, such as institute Description preparation modification mRNA:RNAIMAXTMAnd in cell inoculation and after being adhered to tissue culture plate, through 24 hours by its It is transfected on the cell in porous flat plate.
C.Modify mRNA translation screening: G-CSF ELISA
Keratinocyte is in the EPILIFE culture with replenishers S7 from Invitrogen (Carlsbad, CA) Converge lower growth in > 70% in base.By one group of keratinocyte 300ng and RNAIMAX from InvitrogenTM MRNA (mmRNA) reverse transfection of compound chemical modification.By another group of keratinocyte with 300ng with come from The RNAIMAX of InvitrogenTMCompound modification mRNA forward direction transfection.By first by RNA with without replenishersCulture medium is incubated at room temperature 10 minutes in 5X volume dilution object to form modification mRNA:RNAIMAXTMIt is multiple Close object.
In second bottle, by RNAIMAXTMReagent with without replenishersCulture medium is in 10X volume It is incubated at room temperature in dilution 10 minutes.Then by RNA bottle and RNAIMAXTMBottle mixing, and be incubated at room temperature 20-30 minutes, it is added in cell in a manner of dropwise later.18 hours after triplicate every kind of chemical modification mRNA transfection Human granular leukocyte-colony stimulating factor (G-CSF) concentration secreted in culture medium is measured.
Human G-CSF from the secretions of the Human keratinocytes of transfection using from Invitrogen ELISA kit or R&D system (Minneapolis, MN) illustrates to be quantified according to what manufacturer was recommended.
D.Modify mRNA dosage and duration: G-CSF ELISA
Keratinocyte has replenishers S7's from InvitrogenIn > in culture medium 70% converges lower growth.With 0ng, 46.875ng, 93.75ng, 187.5ng, 375ng, 750ng or 1500ng with come from The RNAIMAX of Invitrogen (Carlsbad, CA)TMCompound modification mRNA reverse transfection keratinocyte.It forms and repairs Adorn mRNA:RNAIMAXTMCompound, as described.0 after the transfection of every kind of modification mRNA of triplicate each concentration, 6, the human G-CSF concentration secreted in culture medium was measured in 12,24 and 48 hours.Human G-CSF is formed from people's cutin of transfection The secretion of cell uses ELISA kit or R&D system from Invitrogen to illustrate to be determined according to what manufacturer was recommended Amount.
The research of 35. divided dose of embodiment
It designs and executes and utilize the research of multiple subcutaneously or intramuscularly injection sites a time point, increased with studying The exposure of mmRNA drug and the method for improving protein generation.Other than the detection of expressed protein product, also by pair The sample for carrying out self-test animal is analyzed to be measured come the assessment of the physiologic function to protein.
, it is surprising that the protein output and phenotypic response that the divided doses of mmRNA generate are greater than logical by measurement Cross those of single unit administration or multiple dosage regimen generation.
The design of single unit dose, multidose and divided dose experiment is related to promoting using the people only applied in buffer Erythropoietin(EPO) (EPO) mmRNA (mRNA shown in SEQ ID NO:1638；PolyA with about 160 nucleotide Tail is not shown in sequence；5 ' caps, Cap1).Administration medium (F. buffer) is made up of: 150mM NaCl, 2mM CaCl2、2mM Na+Phosphate (1.4mM sodium dihydrogen phosphate；0.6mM disodium hydrogen phosphate) and 0.5mM EDTA, pH 6.5.Make PH is adjusted with sodium hydroxide and final solution is sterile filtered.With 5meC at each cytimidine, and in each uridine position MmRNA is modified with pseudouridine substitution at point.
For the single unit dose of 100ug, by (intramuscular) injection of animal (n=5) IM.For multiple dosing, two are used A timetable, the dosage of 3 100ug and the dosage of 6 100ug.For divided doses scheme, using two timetables, 3 times The dosage of the dosage of 33.3ug mmRNA and 6 16.5ug mmRNA.Control administration is related to that buffering is used only under 6 dosage Liquid.Control mmRNA is related to the luciferase mmRNA (IVT shown in SEQ ID NO:21445 of 6 administrations at 100ug CDNA sequence；MRNA sequence shown in SEQ ID NO:21446, the polyA tail with about 160 nucleotide is (in sequence It is not shown), 5 ' cap Cap1 are substituted with 5-methylcytosine at each cytimidine and at each uridine site with pseudouridine Completely modification) use.Blood and musculature were evaluated in 13 hours after injection.
13h after the I.M. single of EPO mmRNA in buffer, multiple or divided doses, measures in mice serum People's epo protein.Seven groups of mouse (n=5 mouse/group) are handled and evaluated.As a result it is shown in table 60.
The research of 60. divided dose of table
Splitting factor is defined as the product of per unit drug divided by the single dose product of per unit drug (PUD).For example, For processing group 2, by the product (EPO) of value 28 or per unit drug (mmRNA) divided by the single dose product of per unit drug 0.14.It as a result is 2.Similarly, for processing group 4, by the product (EPO) of value 1.1 or per unit drug (mmRNA) divided by every list The single dose product 0.14 of position drug.It as a result is 7.9.Therefore, the dose fractionation factor (DSF) can be used as divided doses scheme The indicant of efficiency.For any single administration of total daily dosage, DSF should be equal to 1.Therefore, big in divided doses scheme In the instruction that any DSF of this value is efficiency increase.
In order to determine the influence of dose response trend, injection site and inject the influence of timing, research is performed.At these In research, dose response knot is measured using the various dose of 1ug, 5ug, 10ug, 25ug, 50ug and intervenient value Fruit.The divided doses of 100ug accumulated dose include three times or six 1.6ug, 4.2ug, 8.3ug, 16.6ug or or be equal to application institute Select the value of accumulated dose and the dosage of accumulated dose.
Injection site is selected from limbs or shows any body surface of the enough area suitable for injection.This may also include The selection of injection depth, to target corium (intradermal), epidermis (epidermis), subcutaneous tissue (SC) or muscle (IM).Injection angles will Changed based on target site of delivery, wherein target the injection at intradermal position and the plane of skin surface into 10-15 degree angle, for Subcutaneous injection, the plane with skin surface is at the angle between 20 degree to 45 degree, and for being largely injected into muscle, at 60 degree Angle between 90 degree.
The quantifying in allochthon of embodiment 36.
The amount of mmRNA of the invention and positioning can be by measuring (initial, the time course of the amount in separated allochthon Or remaining benchmark) be determined.In our current research, due to mmRNA be usually codon optimization and in sequence with it is endogenous MRNA is different, so by using Gibbings, PCT/IB2009/005878's (its content is incorporated herein by reference in their entirety) Method quantifies the level of mmRNA compared with natural or wild type mRNA endogenous levels.
In these researchs, pass through the method performed below: first preferably from previously with polynucleotides of the invention, Allochthon or vesica are separated in primary construct or the patient body fluid of mmRNA processing, then passes through mRNA microarray, qRT-PCR, Or one in other means (including suitable antibody or immunohistochemical method) in this field for measuring RNA is in institute It states and measures polynucleotides, primary construct or mmRNA level in allochthon.
Embodiment 37. modifies effect of the mRNA to cell viability, cytotoxicity and Apoptosis
This experiment shows the cell viability of the Human keratinocytes of different modification mRNA in-vitro transfections, cytotoxicity And Apoptosis.Keratinocyte has Human keratinocytes growth from Invitrogen (Carlsbad, CA) Replenishers, there is no hydrocortisonesConverge lower growth in > 70% in culture medium.With 0ng, 46.875ng, 93.75ng, 187.5ng, 375ng, 750ng, 1500ng, 3000ng or 6000ng with the RNAIMAX from InvitrogenTM Compound modification mRNA reverse transfection keratinocyte.Form modification mRNA:RNAIMAXTMCompound.Triplicate every The human G-CSF concentration secreted in culture medium was carried out in 0,6,12,24 and 48 hour after the transfection of every kind of modification mRNA of a concentration Measurement.Human G-CSF uses ELISA kit or R&D from Invitrogen from the secretion of the Human keratinocytes of transfection System illustrates to be quantified according to what manufacturer was recommended.
Use the APOTOX-GLO for coming from Promega (Madison, WI)TMKit is turning according to the explanation of manufacturer 0 after dye, cell viability, cytotoxicity and Apoptosis were measured in 12,48,96 and 192 hours.
Embodiment 38. is using ELISA measurement detection to the cell innate immune response of modification mRNA
It is dry to the human tumor necrosis factor-alpha (TNF-α) for being secreted from the Human keratinocytes of in-vitro transfection, people The enzyme linked immunosorbent assay (ELISA) (ELISA) for disturbing element-β (IFN-β) and human granular leukocyte-colony stimulating factor (G-CSF) is tested, For detecting cell innate immune response.Keratinocyte has people from Invitrogen (Carlsbad, CA) Keratinocyte growth replenishers, there is no hydrocortisonesConverge lower life in > 70% in culture medium It is long.As described in triplicate with 0ng, 93.75ng, 187.5ng, 375ng, 750ng, 1500ng or 3000ng with come from The RNAIMAX of InvitrogenTMTNF-α cutin secreted by compound chemical modification mRNA (mmRNA) reverse transfection is formed carefully Born of the same parents.Using the ELISA kit from Invitrogen, according to the scheme of manufacturer, after every kind of chemical modification mRNA is transfected 24 hours, the TNF-α of the secretion in culture medium is measured.
Turned according to the scheme of manufacturer in every kind of chemical modification mRNA using the ELISA kit from Invitrogen 24 hours after dye, the TNF-beta secreted in same medium is measured.24 hours after every kind of chemical modification mRNA is transfected, The human G-CSF concentration secreted in same medium is measured.Use ELISA kit or R&D from Invitrogen System (Minneapolis, MN), according to the explanation that manufacturer is recommended, to human G-CSF from point of the Human keratinocytes of transfection It secretes and is quantified.By measuring exemplary 1 cytokines TNF-α and IFN-β, these tables of data are illustrated and natural or otherization It learns modification polynucleotides or reference compound is compared, which kind of modification mRNA (mmRNA) can cause reduced cell is congenital to exempt from Epidemic disease response.
The cell proliferating determining of the mRNA- induction of 39. human granular leukocytes of embodiment-colony stimulating factor (G-CSF) modification
Human keratinocytes are had replenishers S7's from InvitrogenIn culture medium, Under the converging of > 70%, in the coating of 24 hole collagens(Coming, Lowell, MA) co-cultures tissue training It supports and is grown in plate.It is triplicate with compound indicated of 750ng and the RNAIMAX from Invitrogen as described Chemical modification mRNA (mmRNA) reverse transfection keratinocyte.Modification mRNA:RNAIMAX compound is formed, it is such as described 's.6-8 hours replacement keratinocyte culture mediums after transfection.42 hours after transfection, it is permeable that 0.4 μm-hole half will be inserted with 24 holes of polyester filmPlate is placed on the keratinocyte containing human G-CSF modification mRNA transfection In culture plate.
By people myeloblast, Kasumi-1 cell or KG-1 (0.2x105Cell) it is inoculated into and inserts in the hole, and in total training Support start after 42 hours, using CyQuant Direct Cell Proliferation Assay (Invitrogen, Carlsbad, CA) cell proliferation is quantified in 96 hole plates in 100-120 μ l volume.By repairing for encoding human G-CSF The myeloblast proliferation of decorations mRNA- induction is expressed as the keratinocyte according to untransfected/myeloblast and co-cultures control wells Normalized cell Proliferation percentage.42 hours after the co-cultivation of every kind of triplicate modification mRNA starts, to cutin shape It is measured at the human G-CSF concentration that cell and myeloblast insertion co-culture the secretion in hole.Using from Invitrogen ELISA kit, according to manufacturer recommend explanation the secretion of human G-CSF is quantified.
Do not turn by RT-PCR detection Human keratinocytes feeder cells (keratinocyte feeder cell) and The human G-CSF of transfection in the people myeloblast of dye modifies mRNA.It usesKit (Qiagen, Valencia, CA) according to manufacturer illustrate extract and crack the total serum IgE from sample cell.The total serum IgE of extraction is carried out RT-PCR, for usingM-MuLV Taq RT-PCR kit (New England BioLabs, Ipswich, MA) according to the explanation of manufacturer, the specificity that user G-CSF specific primer carries out modification mRNA-G-CSF expands Increase.Show RT-PCR product by 1.2% agarose gel electrophoresis.
Embodiment 40: measurement is co-cultured
The modification of chemically distinct modified nucleoside acid including encoding human granulocyte-colony stimulating factor (G-CSF) MRNA can transfect the cell Proliferation of non-competent cell co-culturing environment moderate stimulation.Co-culturing includes the cell that can highly transfect Type (such as Human keratinocytes) and the non-competent cell type (such as whole blood cells (WBC)) of transfection.Repairing for G-CSF will be encoded Decorations mRNA is transfected into height and can transfect in cell, thus allow to generate G-CSF albumen and be secreted into extracellular environment, wherein G-CSF is worked in a manner of similar paracrine, to stimulate the whole blood cells proliferation of expression G-CSF receptor.The WBC group expanded Body can be used for treating the WBC group of immune function depression patient or partial reconfiguration immunosuppressed patients, and to reduce machine The risk of opportunistic infections.
In another example, cell such as fibroblast can be transfected with certain growth factors transfection height, to support simultaneously Stimulation can transfecting difference embryonic stem cell or induction multipotential stem cell growth, maintenance or differentiation.
Embodiment 41: the detection assay of human IgG antibody
A.The ELISA of human IgG antibody is detected
This example describes the ELISA for human IgG, and the human IgG comes from human IgG modification mRNA (mmRNA) transfection Chinese hamster ovary (CHO) and human embryo kidney (HEK, HER-2 negative) 293 cells.Coming from human embryo kidney (HEK) 293 It is grown in 293 culture medium of CD with L-Glutamine replenishers of Invitrogen, until it reaches the remittance of 80%-90% It closes.Grow Chinese hamster ovary celI in the CD CHO culture medium with L-Glutamine, hypoxanthine and thymidine replenishers.In a side Face, in 7ml culture medium in the 75cm2 culture flask from Corning with 24 μ g with from Invitrogen's RNAIMAXTM compound modification mRNA transfects 2x106 cell.On the other hand, in 24 hole plates with 1 μ g with come from The RNAIMAXTM of Invitrogen compound modification mRNA transfects 80,000 cells.By by mmRNA in the vial with 5X body Product dilution with CD 293 or CD CHO culture medium be incubated at room temperature 10 minutes come formed modification mRNA:RNAIMAXTM it is compound Object.In second bottle, by RNAIMAXTM reagent with 10X volume dilution degree 293 culture medium of CD or CD CHO culture medium It is incubated at room temperature 10 minutes.Then mmRNA bottle is mixed with RNAIMAXTM bottle, and is incubated at room temperature 20-30 points Clock is added in CHO or HEK cell later in a manner of dropwise.Culture supernatants are stored in 4 degrees Celsius.24μg mmRNA The concentration of the human IgG of secretion in transfection object in culture medium measures for 12,24,36 hours after transfection, and 1 μ g mmRNA Transfection object was measured at 36 hours.Recommended using the ELISA kit from Abcam (Cambridge, MA) according to manufacturer Explanation Herceptin is quantified from the secretion of 293 cell of HEK of transfection.Data show the IgG antibody of humanization (such as Herceptin) mmRNA can be translated in HEK cell, and Herceptin is secreted cell and is discharged into born of the same parents In external environment.In addition, statistics indicate that, generating the protein of secretion with the mmRNA transfection cell of coding Herceptin can be by Ratio is extended to bioreactor or big cell culture condition.
B.Modify the Western detection for the human IgG antibody that mRNA is generated
The heavy chain and light chain of the Western blotting of CHO-K1 cell Herceptin modification mRNA (mmRNA) of 1 μ g are each From cotransfection.Chinese hamster ovary celI is grown in 24 hole plates using standard scheme.Cell supernatant or cell pyrolysis liquid are being transfected It collects within 24 hours, separated on 12%SDS-Page gel and using Invitrogen (Carlsbad, CA) afterwards It is transferred on nitrocellulose filter.By cell and it is conjugated to the anti-of DYLIGHT594 (ab96904, abcam, Cambridge, MA) First conjugate of the rabbit polyclonal antibody of human IgG and the goat polyclonal antibodies for the anti-Rb IgG for being conjugated to alkaline phosphatase Second conjugate is incubated with.After incubation, using Invitrogen's (Carlsbad, CA)Alkaline phosphatase Chromogenic substrate detects antibody.
C.Modify the cellular immunity dyeing of Herceptin and Rituximab that mRNA is generated
Respectively by the CHO-K1 cell Herceptin of 500ng or the heavy chain of any of Rituximab and light chain Cotransfection.Coming from cellIt is grown in the F-12K culture medium and 10%FBS of (Grand Island, NY).It will Cell is fixed in PBS with 4% paraformaldehyde, the 0.1%Triton X-100 in PBS is permeabilized 5-10 at room temperature Minute, and cell is washed 3 times with room temperature PBS.Using being conjugated to594 (ab96904, abcam, Cambridge, MA) anti-human igg rabbit polyclonal antibody, Herceptin and benefit are executed according to the dilution that manufacturer is recommended Appropriate former times monoclonal antibody dyeing.Core DNA dyeing is executed with the DAPI dyestuff from Invitrogen (Carlsbad, CA).In modification mRNA When transfection, the protein of Herceptin and Rituximab is translated and positions to cytoplasm.It obtains within 13 hours after transfection Figure.
D.Modify the combination immunoblotting measurement of Herceptin and Rituximab that mRNA is generated
Herceptin and Rituximab are detected using in conjunction with immune-blotting method measurement.By various concentration (the antigen and CD20 of the ErB2 peptide (ab40048, abeam, Cambridge, MA) of 100ng/ul to 0ng/ul), Herceptin Peptide (ab97360, abeam, Cambridge, MA), Rituximab antigen glue is run on 12%SDS-Page gel, and It is transferred on film using the iBlot from Invitrogen.By its respective cell supernatant of film from CHO-K1 cell It is incubated for 1 hour, the respective corotation of the CHO-K1 cell Herceptin of 500ng or the heavy chain of Rituximab and light chain Dye.Film is closed with 1%BSA, and adds the second level anti-human igg for being conjugated to alkaline phosphatase (abcam, Cambridge, MA) Antibody.Antibody test is carried out using the NOVEX alkaline phosphatase chromogenic substrate of Invitrogen (Carlsbad, CA).Data are aobvious Show that the humanization IgG antibody generated by modification mRNA can identify and be bound to its corresponding antigen.
E.Cell proliferating determining
The adherent cell system SK-BR-3 cell line for being overexpressed the derived from human adenocarcinoma of breast of HER2/neu receptor can be used for comparing Compared with the antiproliferative properties for the modification mRNA (mmRNA) for generating Herceptin.Being generated by modification mRNA for various concentration is pure Change Herceptin and Herceptin is added in cell culture medium, and it is triplicate to acting through for cell growth Cytotoxicity and viability measure to assess.
Embodiment 42: modification mRNA is largely transfected into cell culture
A.Cation lipid delivery vehicle
Use RNAIMAXTM(Invitrogen, Carlsbad, CA) or TRANSIT-mRNA (Mirus Bio, Madison, WI) cation lipid delivery vehicle carries out RNA transfection.First by RNA and reagent in Opti-MEM basal medium Dilution in (Invitrogen, Carlsbad, CA).100ng/uL RNA is diluted into 5x, and by the RNAIMax/ μ g RNA of 5 μ L Dilute 10x.Diluted component is merged, and is incubated at room temperature 15 minutes, is scattered in culture medium later.For TRANSIT-mRNA transfection, dilutes 10x for 100ng/uL RNA, and add BOOST reagent (in 2 μ L/ μ g in Opti-MEM Under the concentration of RNA), add TRANSIT-mRNA (under the concentration of 2 μ L/ μ g RNA), be then incubated at room temperature 2 minutes it Afterwards, the compound of RNA- lipid is delivered to culture medium.For RiPS derivative, cultivated in Nutristem xenofree hES RNA transfection is executed in base (Stemgent, Cambridge, MA), keratinocyte is tested, in Dermal Cell RNA transfection is executed in the additional Keratinocyte Growth Kit (ATCC) of Basal Medium, and for all other reality It tests, executes RNA transfection outside Opti-MEM plus in 2%FBS.Modification mRNA (mmRNA) is successfully introduced into host cell to be made It is monitored with a variety of known methods, such as fluorescent marker (such as green fluorescent protein (GFP)).Modify the Successful transfection of mRNA It can be determined by the protein expression level via such as Western blotting or immunocytochemistry measurement target drone polypeptide.It can It is used for according to similar RNA- lipid complex ratio according to similar approach by large volume popularization to more liters (5-10,000L) Culture medium form.
B.The electroporation delivering of exogenous synthesis mRNA transcript
By with modification mRNA (mmRNA) transcript that synthesizes in vitro transfect MRC-5 fibroblast and by using It designs specifically to detect the quantitative RT-PCR of the primer of foreign transcripts measurement transfection efficiency and to carry out Electroporation parameters Optimization.The 150uF capacitor discharge of F be will charge to the Opti- for 50 μ l being suspended in standard electroporation cup (gap 2mm) The 2.5x10 of MEM (Invitrogen, Carlsbad, CA)6Modification in a cell for repeating to be delivered over 10,000 copies MRNA transcript/hole is sufficient (as measured using standard curve method), while maintaining high viability (> 70%). In addition experiment can disclose, and voltage needed for effectively transfecting cell with mmRNA transcript may depend on the cell during electroporation Density.Cell density can be from 1x106Cell/50 μ l are changed to 2.5x106Cell/50 μ l density, and need 110V to 145V To transfect cell with similar efficiency measured in the transcription copy in every hole.It is similar electroporation strategy can be flowed with large volume Ground executes big more liters (5-10,000L) electroporation, the strategy it is similar with the method described in above-mentioned bacterial strains (Li et al., 2002；Geng etc., 2010).
Embodiment 43: delivering in lipid complex body is used
A.People EPO modifies RNA lipid complex
Human forcing erythrogenin mRNA (the mRNA shown in SEQ ID NO:1638 that will be modified containing 100 μ g；Have The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) (EPO；The 5-methylcytosine modified completely； N1- methyl-pseudouridine) preparation and 30 volume % RNAIMAXTM(Lipoplex-h-Epo-46；2nd generation or Gen2) lipid It is compound, four C57/BL6 mouse are delivered to so that 50-70uL is intramuscular.The other groups of luciferases by receiving the compound modification of lipid MRNA (compound-luc of lipid) (IVT cDNA sequence shown in SEQ ID NO:21445；It is shown in SEQ ID NO:21446 MRNA sequence, with about 160 nucleotide polyA tail (being not shown in sequence), 5 ' cap Cap1, in each cytimidine position Substitute modification completely with pseudouridine with 5-methylcytosine and at each uridine site at point) injection mouse or Receive the mouse composition of the injection of the preparation buffer as negative control, the compound modification of the lipid under 65ul dose volume Luciferase mRNA served as control, contain the RNAiMAX with 30 volume % of 100 μ gTMThe fluorescence of the compound modification of lipid Plain enzyme mRNA.13 hours after intramuscular injection, serum is collected from every mouse, to measure mice serum by people EPO ELISA The amount of middle people's epo protein, as a result shows in table 61.
61. people EPO yield (IM injecting pathway) of table
B.Human G-CSF modifies RNA lipid complex
By the RNAIMAX with 30 volume % containing 100 μ gTMThe human G-CSF of the modification of two kinds of compound patterns of lipid MRNA (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide does not show in sequence Out；5 ' caps, Cap1) (G-CSF (G-CSF) that is modified completely with 5-methylcytosine and pseudouridine or with 5-methylcytosine and The G-CSF (G-CSF-N1) that N1- methyl-pseudouridine is modified completely) in one preparation with 150uL intramuscular (I.M) delivering, with 150uL subcutaneous (S.C) is delivered and is delivered to C57/BL6 mouse with 225uL intravenous (I.V).
The luciferase mRNA of the modification of three 100 μ g of control group intramuscular administration is (shown in SEQ ID NO:21445 IVT cDNA sequence；MRNA sequence shown in SEQ ID NO:21446, the polyA tail (sequence with about 160 nucleotide It is not shown in column), 5 ' cap Cap1 use pseudouridine with 5-methylcytosine at each cytimidine and at each uridine site Substitution modification completely) (Luc-unsp I.M.), or the luciferase mRNA (Luc-unsp of the intravenous modification for applying 150 μ g ) or the preparation buffer (buffer I.M.) of intramuscular administration 150uL I.V..6 hours after application preparation, received from every mouse Collect serum, to measure the amount of human G-CSF albumen in mice serum by human G-CSF ELISA, is as a result shown in table 62.
These results indicate that when passing through the delivering of I.V. or I.M. administration method in lipid composite preparation, 5- methyl Cytimidine/pseudouridine and 5-methylcytosine/N1- methyl-pseudouridine modification human G-CSF mRNA can lead to human G-CSF egg It is white specific expressed in serum.
Human G-CSF (I.M., I.V., S.C. injecting pathway) in 62. serum of table
Preparation Approach G-CSF(pg/ml)
G-CSF I.M. 85.6
G-CSFN1 I.M. 40.1
G-CSF S.C. 3.9
G-CSF N1 S.C. 0.0
G-CSF I.V. 31.0
G-CSF N1 I.V. 6.1
Luc-unsp I.M. 0.0
Luc-unsp I.V. 0.0
Buffer I.M. 0.0
C.The comparison of human G-CSF modification RNA lipid complex
By the RNAIMAX with 30 volume % containing 100 μ gTMLipid it is compound have it is 5-methylcytosine (5mc) and false The human G-CSF mRNA (G-CSF-Gen1- lipid complex) of the modification of uridine (ψ) modification, in salt water there is 5mc and ψ to repair The human G-CSF mRNA (G-CSF-Gen1- salt water) of the modification of decorations, there is N1-5- methylcystein (N1-5mc) and ψ to modify With the RNAIMAX of 30 volume %TMThe human G-CSF mRNA (G-CSF-Gen2- lipid complex) of the compound modification of lipid, in salt The human G-CSF mRNA (G-CSF-Gen2- salt water) of the modification modified with N1-5mc and ψ in water, it is modified with 5mc and ψ With the RNAIMAX of 30 volume %TMThe luciferase (Luc- lipid complex) of the compound modification of lipid, or having in salt water The preparation intramuscular (I.M.) or subcutaneous (S.C.) of the luciferase mRNA (Luc- salt water) of the modification of 5mc and ψ modification is delivered to C57/BL6 mouse, and the control group of every kind of method of administration gives the preparation buffer (F. of 80uL dosage to C57/BL6 mouse Buffer).13 hours after injection, serum and tissue from injection site are collected from every mouse, and pass through G-CSF ELISA is analyzed, to be compared to human G-CSF protein level.The knot of human G-CSF albumen in the mice serum of intramuscular administration Fruit and subcutaneous administration result are shown in table 63.
These results indicate that no matter ought be applied on the way with saline formulation or with lipid composite preparation by I.M. or S.C. When diameter delivers, 5-methylcytosine/pseudouridine and 5-methylcytosine/N1- methyl-pseudouridine modification human G-CSF mRNA are equal It is specific expressed in serum to can lead to human G-CSF albumen.As shown in table 63,5-methylcytosine/N1- methyl-pseudouridine The human G-CSF mRNA of modification substantially shows relative to 5-methylcytosine/pseudouridine modification human G-CSF mRNA, human G-CSF Albumen generates increase.
Human G-CSF albumen in 63. mice serum of table
D.The comparison of the RNA lipid complex of mCherry modification
Intramuscular and subcutaneous administration
By the RNAIMAX with 30 volume % containing 100 μ gTMCompound modification mCherry mRNA (the SEQ ID of lipid MRNA sequence shown in NO:21439；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) Or the modification mCherry mRNA in salt water preparation is intramuscular and subcutaneous delivery is to mouse.It will also prepare that buffer is intramuscular or skin Under be applied to control group mice.The injection site that mouse can be collected for 17 hours after injection, responsible generation is determined for being sliced One or more cell types of protein.
Intravitreal administration
Can by containing 10 μ g's and RNAIMAXTMCompound modification mCherry mRNA of lipid, repairing in buffer is being prepared Adorn mCherry mRNA and RNAIMAXTMThe luciferase mRNA of the compound modification of lipid, the modification in buffer is being prepared The preparation of luciferase mRNA is applied with 5 μ l/ dose volumes by intravitreal injection (IVT) in rats.Also pass through IVT is applied to control rats with 5 μ l/ dose volumes and is prepared buffer.Can after injection 18 hours, it collects handled big The eyes of mouse, for being sliced and cracking, to measure, whether mmRNA can effectively be delivered to eyes in vivo and protein generates As a result, and also determine be responsible for produce protedogenous one or more cell types in vivo.
Intranasal administration
Intranasal delivery contains the RNAIMAX with 30 volume % of 100 μ gTMCompound modification mCherry mRNA of lipid, Modification mCherry mRNA, the RNAIMAX with 30 volume % in salt waterTMThe luciferase mRNA of the compound modification of lipid or The preparation of the luciferase mRNA of modification in salt water.Also buffer is prepared to control group intranasal administration.It can be about 13 after instillation Hour collects lung, for being sliced (for receiving those of mCherry mRNA) or being homogenized (for receiving luciferase mRNA Those of).These samples will be used to measure the knot that whether mmRNA can effectively be delivered to lung in vivo and protein generates Fruit, and also determine and be responsible for producing protedogenous one or more cell types in vivo.
Embodiment 44: the internal delivering of different lipid ratios is used
Modification mRNA is delivered to C57/BL6 mouse, to evaluate different lipid ratio and resulting protein expression.It will tool There are 100 μ g and 10%, 30% or 50%RNAIMAXTMThe people EPO mRNA of the compound modification of lipid (shows in SEQ ID NO:1638 MRNA out；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5-methylcytosine and Pseudouridine is modified completely), 100 μ g and 10%, 30% or 50%RNAIMAXTMThe luciferase mRNA of the compound modification of lipid (IVT cDNA sequence shown in SEQ ID NO:21445；MRNA sequence shown in SEQ ID NO:21446 has about The polyA tail (being not shown in sequence) of 160 nucleotide, 5 ' cap Cap1, at each cytimidine with 5-methylcytosine and At each uridine site with pseudouridine substitute completely modification) preparation or prepare buffer with 70 μ l dosage intramuscular administration of single To mouse.13 hours collection serum after injection, to carry out people EPO ELISA, to measure people's epo protein water in every mouse It is flat.The ELISA's the results show that RNAIMAX for different weight percentage of people EPO shown in table 64TMRespectively, it is expressed in muscle The people EPO of modification be secreted into serum.
People's epo protein (IM injecting pathway) in 64. mice serum of table
Preparation EPO(pg/ml)
Epo+10%RNAiMAX 11.4
Luc+10%RNAiMAX 0
Epo+30%RNAiMAX 27.1
Luc+30%RNAiMAX 0
Epo+50%RNAiMAX 19.7
Luc+50%RNAiMAX 0
F. buffer 0
Embodiment 45: the intramuscular and subcutaneous delivering in vivo in mammal
By people EPO mRNA (the mRNA sequence shown in SEQ ID NO:1638 for the modification prepared in preparing buffer Column；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5-methylcytosine and pseudouridine Modification completely) it is delivered to C57/BL6 mouse or Sprague-Dawley rat, to evaluate the dose dependent generated to people EPO. The people EPO mRNA (h-EPO) of the modification of 50 μ l, luciferase mRNA (Luc) (SEQ ID of modification are injected to rat intramuscular IVT cDNA sequence shown in NO:21445；MRNA sequence shown in SEQ ID NO:21446 has about 160 cores The polyA tail (being not shown in sequence) of thuja acid, 5 ' cap Cap1, with 5-methylcytosine and in each urine at each cytimidine Modification completely is substituted with pseudouridine at glycosides site) or buffer (F. buffer) is prepared, as described in administration graphics table 65.
To in Mouse Muscle or subcutaneous injection 50 μ l modification people EPO mRNA (h-EPO), modification luciferase mRNA (Luc) or buffer (F. buffer) is prepared, as described in administration graphics table 66.13 hours after injection, blood is collected, and to blood It is analyzed clearly, to measure the amount of people EPO in every mouse or rat.The average value and geometrical mean (pg/ of rat studies Ml it) is also shown in table 65.
65. rat studies of table
66. mice study of table
Embodiment 46: active duration after intramuscular internal delivering
By people EPO mRNA (the mRNA sequence shown in SEQ ID NO:1638 for the modification prepared in preparing buffer Column；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5-methylcytosine and pseudouridine Modification completely) it is delivered to Sprague-Dawley rat, to measure the duration of dose response.50 μ l are injected to rat intramuscular Modification people EPO mRNA (h-EPO), modification luciferase mRNA (IVT cDNA shown in SEQ ID NO:21445 Sequence；MRNA sequence shown in SEQ ID NO:21446, the polyA tail with about 160 nucleotide (do not show in sequence Out), 5 ' cap Cap1 is substituted complete with 5-methylcytosine at each cytimidine and at each uridine site with pseudouridine Modification) (Luc) or preparation buffer (F. buffer), as described in administration graphics table 67.2 after intramuscular injection, 6,12,24, 48 and 72 hours, by rat bloodletting, to measure the concentration of people EPO in serum at given time.The average value and geometry of this research Average value (pg/ml) is also shown in table 67.
Chart is administered in table 67.
Embodiment 47: administration method
Further research is executed to investigate the administration for using different administration approach.According to the scheme summarized in embodiment 35, By the administration chart summarized in table 68, (I.M.), intravenous (IV) or subcutaneous (S.C.) administration into every group of 4 Mouse Muscles. Serum is collected from all mouse within 13 hours after injection, collect tissue from injection site that is intramuscular and subcutaneously organizing, and from vein Interior group of collection spleen, liver and kidney.Intramuscular group and the result subcutaneously organized are shown in table 69.
Chart is administered in table 68.
People's epo protein (IM injecting pathway) in 69. mice serum of table
The research of the quick elimination type lipidic nanoparticles (reLNP) of embodiment 48.
A.Modify the preparation of RNA reLNP
Be prepared for synthesis lipid, 1,2- distearyl acyl group -3- phosphatidyl choline (DSPC) (Avanti Polar Lipids, Alabaster, AL), cholesterol (Sigma-Aldrich, Taufkirchen, Germany) and α-[3 '-(1,2- bis- Pork and beans Cool acyl group -3- propoxyl group)-formamide-propyl]-ω-methoxyl group-polyoxyethylene (PEG-c-DOMG) (NOF, Bouwelven, Belgium solution), and it is stored in -20 DEG C.It synthesizes lipid and is selected from DLin-DMA with lactone, with terminal ester DLin-DMA, DLin-MC3-DMA- lactone and DLin-MC3-DMA with terminal ester.ReLNP is merged, to obtain 50: The molar ratio of 10: 38.5: 1.5 (reLNP: DSPC: cholesterol: PEG-c-DOMG).By the way that lipid soln and modification mRNA is molten Liquid is merged with 10: 1,15: 1,20: 1 and 30: 1 total lipid with the weight ratio of modification mRNA, to prepare reLNP and modification mRNA Preparation.
B.The characterization of preparation
Using Zetasizer Nano ZS (Malvern Instruments Co., Ltd, Malvern, Worcestershire, UK) granularity, polydispersity index (PDI) and the zeta potential for modifying mRNA nano particle are measured, in 1X PBS Middle measurement granularity and zeta potential is measured in 15mM PBS.
The concentration of modification mRNA nanoparticle formulations is measured using uv-vis spectra.After mixing, it is divided in DU 800 Remember between 230nm and 330nm on photometer (Beckman Coulter, Beckman Coulter Co., Ltd, Brea, CA) Record the absorption spectrum of solution.Modification RNA concentration in nanoparticle formulations is based on the extinction coefficient for modifying RNA used in preparation And the absorbance at 260nm wavelength and the difference between the baseline value at 330nm wavelength calculate.
Use QUANT-ITTM RNA measurement (Invitrogen Corporation Carlsbad, CA) come evaluate nano particle to modification RNA encapsulating.By sample dilution, it is transferred to 96 hole plate of polystyrene, then adds TE Buffer or 2%Triton X-100 solution.It is incubated for plate and incites somebody to actionReagent is dilute in TE buffer It releases, and this solution is added in each hole.Use Fluorescence Plate reader (1420 Multilablel of Wallac Victor Counter；Perkin Elmer, Waltham, MA) measurement fluorescence intensity.The fluorescence of reagent blank is subtracted from each sample Value, and the percentage by measuring free modification RNA divided by the fluorescent value of disrupted sample with the fluorescence intensity of intact sample Than.
C.Incubated in vitro
By human embryo kidney epithelium (HEK293) cell and hepatocellular carcinoma epithelium (HepG2) cell (LGC standards GmbH, Wesel, Germany) it is inoculated into 96 hole plates (Greiner Bio-one GmbH, Frickenhausen, Germany) On, and the plate for HEK293 cell is pre-coated with 1 Collagen Type VI.By HEK293 with the density of about 30,000 cells/wells Inoculation, HepG2 are seeded in 100 μ l cell culture mediums with the density of about 35,000 cells/wells.In inoculating cell and it is incubated for it Afterwards, directly addition contains mCherry mRNA (mRNA sequence shown in SEQ ID NO:21439；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1) preparation.With for be transcribed in vitro (IVT) T7 promoter, 5 ' The mCherry cDNA of non-translational region (UTR) and 3 ' UTR are provided in SEQ ID NO:21440.
By by medium supernatant be transferred to 96 hole Pro-Bind U base plates (Beckton Dickinson GmbH, Heidelberg, Germany) harvest cell.By trypsase/EDTA of 1/2 volume of cell (Biochrom AG, Berlin, Germany) trypsin digestion, merge with corresponding supernatant, and pass through the PBS/2% of one volume of addition FCS (being Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck, Darmstadt, Germany) is consolidated It is fixed.Then in the LSRII cell instrument (Beckton Dickinson GmbH, Heidelberg, Germany) with excitation laser and 610/20 filter of PE-Texas Red makes sample be subjected to measured by flow cytometry.Give whole events of analyzed sample Average fluorescent strength (MFI) and four separate wells standard deviation.
D.Internal preparation research
The preparation containing modification mRNA and reLNP of single dose is applied into mouse vein.It is applied to the modification of mouse MRNA is selected from: G-CSF (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide, It is not shown in sequence；5 ' caps, Cap1), the IX factor (mRNA shown in SEQ ID NO:1622；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1) or mCherry (mRNA sequence shown in SEQ ID NO:21439；Tool There is the polyA tail of about 160 nucleotide, is not shown in sequence；5 ' caps, Cap1).
To the modification mRNA of the preparation of mouse injection 100ug, 10ug or 1ug, and after applying preparation 8 hours by mouse It puts to death.It is carried out by serum of the specific G-CSF ELISA to the mouse for the preparation for modifying mRNA containing human G-CSF from application Measurement, and the serum by specificity IX factor ELISA or chromogenic assay to the mouse for modifying RNA from the application people IX factor It is analyzed.By immunohistochemistry (IHC) or fluorescence-activated cell sorting (FACS) to from applied mCherry modification The liver of the mouse of mRNA and spleen are analyzed.As control, one group of mouse does not inject any preparation, and collects its serum and group It knits, is analyzed by ELISA, FACS and/or IHC.
The in-vitro transfection of embodiment 49.VEGF-A
Human vascular endothelial growth factor isoform A (VEGF-A) is modified by reverse transfection mRNA (SEQ ID NO: MRNA sequence shown in 1672；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) 24 It is transfected into Human keratinocytes in porous flat plate.Make Human keratinocytes from Invitrogen (Carlsbad, CA) with replenishers S7It is grown in culture medium, until it reaches converging for 50%-70%.With 0ng, 46.875ng, 93.75ng, 187.5ng, 375ng, 750ng and 1500ng with from Invitrogen (Carlsbad, CA RNAIMAX)TMThe modification mRNA (mmRNA) of compound coding VEGF-A transfects cell.By first by RNA with 5X volume Dilution is used without replenishersCulture medium is incubated at room temperature 10 minutes to form RNA:RNAIMAXTMIt is compound Object.In second bottle, with 10X volume dilution degree by RNAIMAXTMReagent with without replenishersCulture Base is incubated at room temperature 10 minutes together.Then by RNA bottle and RNAIMAXTMBottle mixing, and it is incubated at room temperature 20- 30 minutes, it is added in cell in a manner of dropwise later.
Coding VEGF-A (the mRNA sequence shown in SEQ ID NO:1672 being transfected into Human keratinocytes；Tool There is the polyA tail of about 160 nucleotide, is not shown in sequence；5 ' caps, Cap1) the mRNA optimized completely include translation phase Between modification, such as natural nucleus glycoside triphosphoric acid (NTP), the pseudouridine at each uridine site and at each cytimidine site 5-methylcytosine (vacation-U/5mC), and N1- methyl-pseudouridine at each uridine site and in each cytimidine site The 5-methylcytosine (N1- methyl-vacation-U/5mC) at place.Cell is transfected with the mmRNA of coding VEGF-A, for each concentration, 6,12,24 and 48 hours after transfection, using the ELISA kit from Invitrogen (Carlsbad, CA) according to manufacture The explanation that quotient recommends measures the VEGF-A concentration (ρ g/ml) secreted in culture medium.It is shown in table 70 and Fig. 6 A, 6B and 6C These data show that the modification mRNA for encoding VEGF-A can be translated in Human keratinocytes, and VEGF-A quilt It is transported out cell and is discharged into extracellular environment.
Table 70.VEGF-A administration and Protein secretion
The In vivo study of the embodiment 50.IX factor
It will prepare and preparing the people IX factor mmRNA (Gen1 in buffer；The 5-methylcytosine and false urine modified completely Glycosides) mouse is delivered to by intramuscular injection.The result shows that the IX factor protein in serum increases, as after application 13 hours survey Amount.
In this research, with 2x 100ug/ mouse to mouse (for IX factor N=5, for luciferase or buffer Compare N=3) intramuscular injection 50 μ l IX factor mmRNA (mRNA sequence shown in SEQ ID NO:1622；With about 160 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1), luciferase (IVT shown in SEQ ID NO:21445 CDNA sequence；MRNA sequence shown in SEQ ID NO:21446, the polyA tail with about 160 nucleotide is (in sequence It is not shown), 5 ' cap Cap1 are substituted with 5-methylcytosine at each cytimidine and at each uridine site with pseudouridine Modification completely) or prepare buffer (F. buffer).After intramuscular injection 13 hours by mouse bloodletting, it is more to measure people in serum The concentration (pg/mL) of peptide.As a result it disclosing, the application of IX factor mmRNA leads to the level of the 1600pg/mL at 13 hours, with Luciferase or the IX factor less than 100pg/mL of buffer control application are contrasted.
51. multiple location of embodiment application: intramuscular and subcutaneous
Gen1 or Gen2 (5-methylcytosine (5mc) and pseudouridine (ψ) modification, G-CSF-Gen1 will be modified to；Or N1- 5-methylcytosine (N1-5mc) and ψ modification, G-CSF-Gen2) and prepare buffer in prepare human G-CSF modification MRNA (the mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide is not shown in sequence； 5 ' caps, Cap1) pass through intramuscular (IM) or subcutaneous (SC) injected delivery to mouse.Four dosage or 2x 50ug (two are executed daily Position) injection, continue three days (24 hours be spaced).6 hours the 4th dosage of application before blood collection and CBS analysis.It is right According to including luciferase (IVT cDNA sequence shown in SEQ ID NO:21445；Shown in SEQ ID NO:21446 MRNA sequence, the polyA tail (being not shown in sequence) with about 160 nucleotide, 5 ' cap Cap1 are used at each cytimidine 5-methylcytosine and at each uridine site with pseudouridine substitute completely modification) or prepare buffer (F. buffer). The first mRNA injection (last time modification mRNA dosage after 6 hours) afterwards 72 hours by mouse bloodletting, to measure mRNA coding Effect of the human G-CSF to neutrophil count.Dosage regimen is shown in table 71, resulting neutrophil count (thousand/ UL) same.In table 71, asterisk (*) indicates the statistical significance of p < 0.05.
For intramuscular administration, data are disclosed, and for Gen1 G-CSF mRNA, neutrophil count is than control on day 3 It is 4 times high, it is 2 times high for Gen2 G-CSF mmRNA.For subcutaneous administration, data are disclosed, for Gen2 G-CSF mRNA, 3rd day, neutrophil count was 2 times higher than compareing.
These statistics indicate that, 5-methylcytosine/pseudouridine and 5-methylcytosine/N1- methyl-pseudouridine-modification MRNA can be active for biology, and the specificity increase counted such as blood neutrophil proves.
71. dosage regimen of table
Embodiment 52. is intravenously applied
It will be carried out modifying or not having modification with 5-methylcytosine (5mc) and pseudouridine (ψ) modification (Gen 1), and The human G-CSF modification mRNA (mRNA shown in SEQ ID NO:21438 being formulated in 10% lipid complex (RNAiMax) Sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) with the dosage of 50ug RNA and Pass through intravenously (IV) to be injected at the 0th day, the 2nd day and the 4th day with the volume of 100ul and is delivered to mouse.On day 1, the 5th day With the 8th day measurement neutrophil leucocyte.Control includes non-specific mammalian rna or only includes preparing buffer (F. buffering Liquid).On day 1, by mouse bloodletting, the human G-CSF encoded with measurement modification mRNA is thin to neutral grain is increased within the 5th day and the 8th day The effect that born of the same parents count.Dosage regimen is shown in table 72, resulting neutrophil count (thousand/uL；K/uL) same.
For intravenously applying, data are disclosed, in the case where G-CSF modifies mRNA, in the 5th day neutrophil count It is four to five times higher than compareing, and do not have in the case where unmodified G-CSF mRNA or non specific control.Finally injecting Four days later, blood count returned to baseline.Other changes of leucocyte group are not observed.
In table 72, asterisk (*) indicates the statistical significance of p < 0.001 compared with buffer.
These statistics indicate that, when being delivered by I.V. administration method, lipid is compound -5-methylcytosine/vacation for preparing Uridine-modification mRNA can be active for biology, as the specificity increase counted by blood neutrophil proves.It is other thin Born of the same parents' subgroup does not have significant changes.The unmodified G-CSF mRNA similarly applied shows do not have pharmacology to neutrophil count Effect.
72. dosage regimen of table
53. saline formulation of embodiment: intramuscular administration
A.Protein expression
Human G-CSF is modified into mRNA (mRNA sequence shown in SEQ ID NO:21438；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1) and people EPO mmRNA (mRNA sequence shown in SEQ ID NO:1638 Column；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1), G-CSF modification mRNA (use 5- first Base cytimidine (5mc) and pseudouridine (ψ) modification) and EPO modification mRNA (with N1-5- methylcystein (N1-5mc) and ψ modification Modified) it is formulated in and prepares buffer (150mM sodium chloride, 2mM calcium chloride, 2mM phosphate, 0.5mM EDTA (pH 6.5)) In, and injected by intramuscular (IM) with the dose delivery of 100ug to mouse.
Control includes luciferase (IVT cDNA sequence shown in SEQ ID NO:21445；SEQ ID NO:21446 Shown in mRNA sequence, with about 160 nucleotide polyA tail (being not shown in sequence), 5 ' caps, Cap1, each Modification completely is substituted with 5-methylcytosine at cytimidine and with pseudouridine at each uridine site) or preparation buffer (F. buffer).13 hours by mouse bloodletting after injection, to measure the concentration (pg/mL) of human polypeptides in serum.(G-CSF group Human G-CSF is measured in mice serum and EPO group measures people EPO in mice serum).Data are shown in table 73.
73. dosage regimen of table
B.Dose response
People EPO is modified into mRNA (mRNA sequence shown in SEQ ID NO:1638；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) it is formulated in preparation buffering In liquid and pass through intramuscular (IM) injected delivery to mouse.
Control includes luciferase (mRNA sequence shown in SEQ ID NO:21446；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) or preparation buffer (F. buffer).13 hours by mouse bloodletting after injection, to measure the concentration (pg/mL) of human polypeptides in serum.Dosage and table It is shown up in table 74.
74. dosage regimen of table and expression
Embodiment 54.EPO multiple dosing/multiple applications
It designs and executes and utilize the research of multiple intramuscular injection sites a time point.
The design of single multiple dosing experiment is related to using the human forcing erythrogenin applied in preparing buffer (EPO) mmRNA (mRNA sequence shown in SEQ ID NO:1638；PolyA tail with about 160 nucleotide, in sequence It is not shown；5 ' caps, Cap1) or G-CSF mmRNA (mRNA sequence shown in SEQ ID NO:21438；With about 160 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1).Medium (F. buffer) is administered and is used as control.In each born of the same parents EPO and G-CSF modification mRNA is carried out with 5-methylcytosine at pyrimidine and with pseudouridine substitution at each uridine site Modification.
For the single unit dose of 100ug, (passed to (intramuscular) injection of animal (n=5) Sprague-Dawley rat IM It send to a thigh).For multiple dosing, EPO and G-CSF mmRNA uses the dosage of 6 100ug (to be delivered to two big Leg).Control administration is related to using buffer with single dose.People's EPO blood level was evaluated in 13 hours after injection.
13 hours after intramuscular injection, people's epo protein matter is measured in rat blood serum.Five groups of rats are handled and commented Valence.As a result it is shown in table 75.
The research of 75. multiple dosing of table
The exchange research of 55. signal sequence of embodiment
It is stimulated using modified nucleoside acid pseudouridine and 5-methylcytosine (vacation-U/5mC) composite coding human granulocyte colony The factor (G-CSF) (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide, sequence In be not shown；5 ' caps, Cap1) mmRNA several variants.These variants include encoding wild type N-terminal secreting signal peptide sequence Arrange (MAGPATQSPMKLMALQLLLWHSALWTVQEA；SEQ ID NO:95), non-secreting signal peptide sequence or be derived from other The G-CSF construct of the secretion signal peptide sequence of mRNA.These include wherein wild type G-CSF signal peptide sequence by following The sequence of either signal peptide sequence substitution: people α -1- antitrypsin (AAT) (MMPSSVSWGILLLAGLCCLVPVSLA；SEQ ID NO:94), the people IX factor (FIX) (MQRVNMIMAESPSLITICLLGYLLSAECTVFLDHENANKILNRPKR；SEQ ID NO:96), people's prolactin (Prolac) (MKGSLLLLLVSNLLLCQSVAP；SEQ ID NO:97) or human albumin (Alb) (MKWVTFISLLFLFSSAYSRGVFRR；SEQ ID NO:98).
Using the Lipofectamine 2000 (Life Technologies) of 1ul, in 24 hole plates, by 250ng's The modification mRNA for encoding each G-CSF variant is transfected into HEK293A (293A in table), mouse muscle-forming cell (MM in table) In (C2C12, CRL-1772, ATCC) and rat myoblasts (RM in table) (L6 system, CRL-1458, ATCC) cell line, often Contain 300,000 cells in a hole.Supernatant, and user's G-CSF ELISA kit (Life are harvested at 24 hours later Technologies it) is analyzed by G-CSF albumen of the ELISA to secretion.Data shown in table 76 disclose, with the white egg of coding The G-CSF albumen of the cell secretion of the G-CSF mmRNA transfection of white signal peptide is 12 times up to fewer than its wild type counterparts.
The exchange of 76. signal peptide of table
56. cell factor research of embodiment: PBMC
A.PBMC separation and culture
From in heparin sodium pipe research blood constitutent (Research Blood Components) (lot number KP30928 and KP30931 human blood of the 50mL from two donors is received in).For each donor, blood is merged, with DPBS (SAFC Bioscience 59331C, lot number 071M8408) it is diluted to 70mL and is uniformly distributed between two 50mL conical pipes. The Ficoll Paque (GE Healthcare 17-5442-03, lot number 10074400) of 10mL is gently dispersed in blood layer Lower section.In the case where low acceleration and braking, pipe is centrifuged 30 minutes at 2000rpm.Remove pipe and by erythrocyte sedimentation rate palm fibre Yellow PBMC layers is gently transferred to fresh 50mL conical pipe and is washed with DPBS.Pipe is centrifuged 10 minutes at 1450rpm.
Supernatant is sucked out and precipitates PBMC and is resuspended and is washed in the DPBS of 50mL.By pipe at 1250rpm from The heart 10 minutes.This washing step is repeated, and PBMC precipitating is resuspended in Optimem I (Gibco 11058, the lot number of 19mL 1072088) it in and counts.Cell suspension is adjusted to 3.0x10^6 cell/mL living cells concentration.
Then the round bottom for these cells being coated on the processing of five (each donor) 96 hole tissue culture medium (TCM)s with the hole 50uL/ is flat On plate (Costar 3799).In 30 minutes, transfection mixture is added in each hole with the volume in the hole 50uL/.It is transfecting 4 hours later, with fetal calf serum (Gibco 10082, lot number 1012368) supplementing culture medium of 10uL.
B.Transfect object preparation
By the mmRNA (mRNA sequence shown in SEQ ID NO:21438 of encoding human G-CSF；With about 160 cores The polyA tail of thuja acid is not shown in sequence；5 ' caps, Cap1) (contain (1) natural NTP, (2) have 5-methylcytosine and vacation The 100% of uridine replaces, or (3) there is 5-methylcytosine and N1- methyl-pseudouridine 100% to replace)；Coding fluorescence element MmRNA (the IVT cDNA sequence shown in SEQ ID NO:21445 of enzyme；MRNA sequence shown in SEQ ID NO:21446 Column, the polyA tail (being not shown in sequence) with about 160 nucleotide, 5 ' caps, Cap1, with 5- first at each cytimidine Base cytimidine and substitute modification completely with pseudouridine at each uridine site) (there is 5- first containing (1) natural NTP or (2) The 100% of base cytimidine and pseudouridine replaces) and TLR agonist R848 (Invivogen tlrl-r848) in final volume 38.4ng/uL is diluted in the Optimem I of 2500uL.
Individually, with the 2000 (Invitrogen of Lipofectamine of 13.1mL Optimem I dilution 432uL 11668-027, lot number 1070962).In 96 hole plates, by each mmRNA of the 135uL of nine equal portions, positive control (R-848) Or negative control (Optimem I) is added to the diluted Lipofectamine 2000 of 135uL.Material to be transfected will be contained Plate is incubated for 20 minutes.Then transfection mixture is transferred to everyone PBMC plate with the hole 50uL/.Then it is incubated at 37 DEG C Plate.At the 2nd hour, the 4th hour, the 8th hour, the 20th hour and the 44th hour, each plate is removed from incubator, and And freezing supernatant.
After removing the last one plate, user G-CSF ELISA kit (Invitrogen KHC2032) and HumanIFN-α's ELISA kit (Thermo Scientific 41105-2) is measured supernatant.Each condition repeat into Row is twice.
C.As a result
The ability for generating coded protein to unmodified and modification mRNA (mmRNA) over time is commented Estimate, equally the ability of mRNA triggering congenital immunity identification is assessed, as measured by being generated by interferon-' alpha '.In vitro The use of PBMC culture be to measure the accepting method of the immunostimulatory potential of oligonucleotides (Robbins etc., Oligonucleotides 200919:89-102；It is incorporated herein by reference in their entirety).
The standard curve for each ELISA plate that result control is fitted using four parameter logistic curves is subjected to interpolation. Be shown in table 77 and 78 as measured by specific ELISA as time goes by G-CSF and IFN-α yield from 2 The average value of a independent PBMC donor.
In G-CSF ELISA, at every point of time, the back from the untreated condition of Lipofectamine 2000 is subtracted Scape signal.Statistics indicate that replacing or having 5- methyl containing natural NTP, 100% with 5-methylcytosine and pseudouridine In the case where the G-CSF mRNA that cytimidine and N1- methyl-pseudouridine 100% replace, observe through human peripheral monokaryon Cell-specific real estate stranger's G-CSF albumen.G- is significantly increased by using modification mRNA relative to unmodified mRNA The generation of CSF, wherein showing the G- of highest level containing 5-methylcytosine and N1- methyl-pseudouridine G-CSF mmRNA CSF is generated.For congenital immunity identification, unmodified mRNA causes significant IFN-α to generate, and modifies mRNA and hinder significantly Interferon-' alpha ' generation is stopped.It is not dramatically increased with the G-CSF mRNA that 5-methylcytosine and N1- methyl-pseudouridine are modified completely Cell factor, and G-CSF mRNA induction IFN-α, TNF-α and the IP10 modified completely with 5-methylcytosine and pseudouridine.Perhaps Mostly other cell factors are not influenced by any modification.
Table 77.G-CSF signal
Table 78.IFN- alpha signal
The chemical modification range of the modification of embodiment 57. mRNA
The modified nucleoside acid of such as, but not limited to chemical modification 5-methylcytosine and pseudouridine has shown that dynamic in lactation Innate immune response is reduced in object cell and increases the expression of RNA.It is astonishing and not previously known, work as chemical modification Amount be less than 100% when, the effect that chemical modification is showed is titratable.Previously it is believed that modification is for causing chemical modification completely Beneficial effect is required and enough, and mRNA's has minimum effect less than 100% modification.However, having shown now Show, can be used less than completely modification obtain chemical modification benefit and effect be target, concentration and modification dependence.
A. the modification RNA transfected in PBMC
By the G-CSF mRNA or unmodified G- of 960ng modified with 5-methylcytosine (5mC) and pseudouridine (false U) The Lipofectamine 2000 of CSF mRNA and 0.8uL is transfected into together from three normal blood donors (D1, D2, D3) In peripheral blood mononuclear cells (PBMC).G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438；With about 160 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1) with 5mC and vacation U (100% modifies) is modified completely, do not have to 5mC and vacation U modification (0% modifies) is modified with the part 5mC and vacation U, so that mRNA will be modified containing 50%, 25% modifies, 10% modification, 5% modification, 1% modification or 0.1% modification.For G-CSF express, also to luciferase (SEQ ID NO: MRNA sequence shown in 21446；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is complete The control sample of the 5meC modified entirely and vacation U) are analyzed.For TNF-α and IFN-α, also to Lipofectamine2000, LPS, R-848, luciferase (mRNA sequence shown in SEQ ID NO:21446；PolyA with about 160 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；The 5mC modified completely and vacation) and the control sample of P (I) P (C) analyzed.? 22 hours after transfection, harvests supernatant and pass through elisa assay, to measure protein expression.The expression of G-CSF is in table 79 It shows, and the expression of IFN-α and TNF-α is shown in table 80.The expression of IFN-α and TNF-α can turn for G-CSF mRNA The second order effect of dye.Table 79 and 80 is shown, when mRNA is not to modify completely, the chemical modification of G-CSF, IFN-α and TNF-α Amount be it is titratable, and the titratable trend of each target is not identical.
Table 79.G-CSF expression
Table 80.IFN- α and TNF-α expression
B.The modification RNA transfected in HEK293
Human embryo kidney epithelium (HEK293) cell is connect in 100ul cell culture medium with the density of 30,000 cells/wells Kind is in 96 hole plates.Xiang Kongzhong addition and RNAiMAXTMThe 250ng's that (Invitrogen, Carlsbad, CA) is prepared together G-CSF mRNA (the mRNA sequence shown in SEQ ID NO:21438 of modification；PolyA with about 160 nucleotide Tail is not shown in sequence；5 ' caps, Cap1).G-CSF modifies (100% modification) with 5mC and vacation U completely, repairs without 5mC and vacation U Decorations (0% modification), or modified with the part 5mC and vacation U, so that mRNA will contain 75% modification, 50% modification or 25% modification.Also To control sample (AK 5/2, mCherry (SEQ ID NO:21439；PolyA tail with about 160 nucleotide, in sequence It is not shown；5 ' caps, Cap1；The 5mC and vacation U modified completely) and untreated) analyzed.With 5-methylcytosine and vacation The half-life period for the G-CSF mRNA that uridine is modified completely is about 8-10 hours.Supernatant is harvested after 16 hours and is passed through ELISA analyzes the G-CSF protein of secretion.Table 81 shows that when mRNA is not to modify completely, the chemistry of G-CSF is repaired The amount of decorations is titratable.
Table 81.G-CSF expression
G-CSF expresses (ng/ml)
100% modification 118.4
75% modification 101.9
50% modification 105.7
25% modification 231.1
0% modification 270.9
AK 5/2 166.8
mCherry 0
It is untreated 0
Embodiment 58: the internal delivering of modification mRNA (mmRNA)
To modify RNA it is intramuscular, it is subcutaneous or intravenous be delivered to C57/BL6 mouse, with use luciferase evaluation modification RNA Bio distribution.Using sodium hydroxide pair be used together with all delivering methods containing 150mM sodium chloride, 2mM calcium chloride, 2mM Na+- phosphate (including 1.4mM sodium dihydrogen phosphate and 0.6mM disodium hydrogen phosphate) and 0.5mM ethylenediamine tetra-acetic acid (EDTA) preparation buffer is adjusted, and to reach 6.5 final pH, is filtered and sterilizes later.Made using 1X concentration To deliver buffer.In order to form the compound solution of lipid for being delivered to mouse, in a bottle, by the RNA of 50 μ g in room It is balanced 10 minutes in delivering buffer under temperature, and in the second bottle, by 10 μ l RNAiMAXTMIt is slow in delivering at room temperature It is balanced 10 minutes in fliud flushing.After balance, bottle is merged, and adds the final volume for delivering buffer to reach 100 μ l, Then it is incubated at room temperature 20 minutes.In phase plateau of the fluorescein exposure curve between 15 minutes and 30 minutes Between, before imaging, fluorescein is applied to every mouse with 150mg/kg by intraperitoneal injection (IP).In order to generate fluorescence The D- fluorescein potassium or sodium salt of 1g are dissolved in the phosphate buffer solution (DPBS) of 66.6ml distillation (without Mg2+ or Ca2 by element +) in, to prepare 15mg/ml solution.Solution is gently mixed to and is made it through 0.2 μm of injection filter, is blown later with nitrogen It sweeps, equal part is simultaneously frozen in -80 DEG C, while being protected from light as far as possible.It, will using water-bath (if fluorescein is insoluble) on the day of administration Solution thaws, and is gently mixed and keeps on ice.
The whole body images of every mouse are obtained within 2,8 and 24 hours upon administration.It is received upon administration from every mouse within 24 hours Collect organization chart picture and serum.By the liver,spleen,kidney of the mouse of intravenous administration dosage, lung, the heart, perinephric fat tissue and thymus gland Imaging.By the liver,spleen,kidney of the mouse of intravenously or subcutaneously administration dosage, lung, perinephric fat tissue and injection site muscle Imaging.For each administration method and dosage regimen, bioluminescence is measured by whole body images, in terms of photons/second.
A.Intramuscular administration
For every kind of preparation, with the single dose of the 50 μ g modification RNA in 50 μ l volume injecteds in right hind, and With the single dose of the 5 μ g modification RNA in 50 μ l volume injecteds, into Mouse Muscle (I.M.) is applied and is used 5- first in left hind It is modification luciferase mRNA (naked-Luc) that base cytimidine and pseudouridine are modified completely, complete with 5-methylcytosine and pseudouridine The lipid of modification compound modification luciferase mRNA (compound-luc of lipid) (IVT shown in SEQ ID NO:21445 CDNA sequence；MRNA sequence shown in SEQ ID NO:21446, the polyA tail with about 160 nucleotide is (in sequence It is not shown), 5 ' caps, Cap1 is replaced with 5-methylcytosine at each cytimidine and at each uridine site with pseudouridine Generation modification completely), granulocyte colony stimulating factor (G-CSF) mRNA of the compound modification of lipid (shows in SEQ ID NO:21438 MRNA sequence out；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is phonetic with 5- methyl born of the same parents Pyridine and pseudouridine are modified completely) (lipid compound-cell factor) or prepare buffer.2,8 and 24 hours upon administration, every group The bioluminescence average value of luciferase expression signal is shown in table 82.Contain and be free of lipid complex in 5 μ g and 50 μ g Modification RNA preparation injection site, bioluminescence positive signals.
82. vivo biodistribution photon imaging of table (I.M. injecting pathway)
B.Subcutaneous administration
It is subcutaneous to mouse with the single dose of the 50 μ g modification mRNA in 100 μ l volume injecteds for every kind of preparation (S.C.) the luciferase mRNA (naked-Luc) of application modification, the compound modification of lipid luciferase mRNA (lipid is compound- Luc), the G-CSF mRNA (compound-G-CSF of lipid) of the compound modification of lipid or preparation buffer.2,8 and 24 are small upon administration When, the bioluminescence average value of every group of luciferase expression signal is shown in table 83.Contain in 50 μ g and multiple without lipid Close the injection site of the modification mRNA preparation of object, bioluminescence positive signals.
83. vivo biodistribution photon imaging of table (S.C. injecting pathway)
C.Intravenous application
For every kind of preparation, with the single dose of the 50 μ g modification mRNA in 100 μ l volume injecteds, into mouse vein (I.V.) the luciferase mRNA (naked-Luc) of application modification, the compound modification of lipid luciferase mRNA (lipid is compound- Luc), the G-CSF mRNA (compound-G-CSF of lipid) of the compound modification of lipid or preparation buffer.2 hours upon administration, come It is shown in table 84 from the bioluminescence average value of the luciferase expression signal in every group of spleen.It is compound to contain lipid in 50 μ g In the spleen of the modification mRNA preparation of object, bioluminescence positive signals.
84. vivo biodistribution photon imaging of table (I.V. injecting pathway)
59. buffer formulation research of embodiment
With the volume injected of 50 μ l with the modification mRNA dosage of the 200ug/ rat as described in table 85 to rat (n=5) G-CSF of the intramuscular administration in buffer solution modifies mRNA (mRNA sequence shown in SEQ ID NO:21438；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is repaired completely with N1- pseudouridine and 5-methylcytosine Decorations) or IX factor modification mRNA (mRNA sequence shown in SEQ ID NO:1622；PolyA with about 160 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；It is modified completely with N1- pseudouridine and 5-methylcytosine).MRNA will be modified in water Freeze-drying continues 1-2 days.Then it is reconstructed in the buffer being listed below to the aimed concn of 6mg/ml.Pass through OD 260 Measure concentration.Sample is diluted to 4mg/ml in buffer appropriate before administration.
In order to make to modify mRNA precipitating, respectively to modify 4 times of addition pH 5.5 of 1/10 and total volume of mRNA total volume 3M sodium acetate and straight alcohol.Material is placed in -80C, continues minimum 1 hour.Then by material at 4000rpm, 4C from The heart 30 minutes.Remove supernatant and will precipitating centrifugation and with 75% ethanol washing 3x.Finally, by precipitating with buffer reconstruct to The aimed concn of 6mg/ml.Concentration is measured by OD 260.Sample is diluted in buffer appropriate before administration 4mg/ml.Unless being illustrated below, otherwise all samples are prepared by desivac.
85. buffer administration group of table
Blood serum sample is collected from rat in different time intervals, and is directed to G- using G-CSF or IX factor ELISA The blood serum sample is analyzed in the expression of CSF or IX factor protein matter.
The research of 60. multiple dosing of embodiment
8 times (biweekly) was injected intravenously to Sprague-Dawley rat (n=8) through 28 days.It is injected to rat The human G-CSF modification of 0.5mg/kg, 0.05mg/kg, 0.005mg/kg or 0.0005mg/kg being formulated in lipidic nanoparticles Human G-CSF modification mRNA, 0.2mg/kg's in salt water of mRNA or luciferase modification mRNA, 0.5mg/kg is formulated in rouge Human G-CSF protein N eupogen or not interpretable human G-CSF in matter nano particle modify mRNA.Between the scheduled time Serum is collected every period, to evaluate G-CSF protein expression (8,24 and 72 hours after this week is administered for the first time), whole blood cells meter Several and white blood cell count (24 and 72 hours after this week is administered for the first time) and clinical chemistry are (24 after this week is administered for the first time With 72 hours).Rat is put to death at the 29th day (after the last administration 4 days), with measure whole blood count, white blood cell count, Clinical chemistry, protein expression and the effect by histopathology and ptomatopsia evaluation to major organs.In addition, Antibody determination is carried out on rat within 29 days.
Embodiment 61.LNP In vivo study
Luciferase is modified into mRNA (mRNA sequence shown in SEQ ID NO:21446 using injection syringe pump method；With big The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is modified completely with 5-methylcytosine and pseudouridine) It is formulated as lipidic nanoparticles (LNP).LNP, and final lipid are prepared with the weight ratio of 20: 1 total lipid and modification mRNA Molar ratio is 50: 10: 38.5: 1.5 (DLin-KC2-DMA: DSPC: cholesterol: PEG-c-DMG).As shown in table 86, pass through Granularity, zeta potential and encapsulating characterize luciferase LNP preparation.
86. luciferase preparation of table
If table 87 is summarized, intravenous and subcutaneous administration luciferase LNP preparation intramuscular to Balb-C mouse (n=3) is simultaneously And the luciferase modification RNA being formulated in PBS is applied into mouse vein.
87. luciferase preparation of table
Intravenous and intramuscular administration luciferase LNP preparation mouse was imaged at 2,8,24,48,120 and 192 hours, And the mouse of subcutaneous administration luciferase LNP preparation was imaged at 2,8,24,48 and 120 hours, to measure luciferase table It reaches, as shown in table 88.In table 88, " NT ", which is meant, not to be tested.20 minutes before imaging, mouse abdomen was given with 150mg/kg D- luciferin solution is injected in film.Then by Animal Anesthesia and use IVIS Lumina II imaging system (Perkin Elmer) Acquire image.Bioluminescence is measured as the total flow (photons/second) of whole mouse.
88. luciferase expression of table
One mouse of intravenous application LNP preparation was put to death at the 8th hour, to measure the luciferase table in liver and spleen It reaches.Equally, a mouse of intramuscular administration LNP preparation was put to death at the 8th hour, with measure muscle around injection site and Luciferase expression in liver and spleen.As shown in table 89, after intravenous and intramuscular administration liver and spleen in and intramuscular note It penetrates and observes expression in the muscle around position.
Luciferase expression in the tissue of table 89.
62. cell factor research of embodiment: PBMC
A.PBMC separation and culture
50mL is received from the research blood constitutent (lot number KP30928 and KP30931) in heparin sodium pipe from two confessions The human blood of body.For each donor, blood is merged, with DPBS (SAFC Bioscience 59331C, lot number It 071M8408) is diluted to 70mL and is uniformly distributed between two 50mL conical pipes.By the Ficoll Paque (GE of 10mL Healthcare 17-5442-03, lot number 10074400) it is gently dispersed in below blood layer.In the feelings of low acceleration and braking Under condition, pipe is centrifuged 30 minutes at 2000rpm.It removes pipe and is gently transferred to PBMC layers of buffy fresh 50mL conical pipe and washed with DPBS.Pipe is centrifuged 10 minutes at 1450rpm.
Supernatant is sucked out and precipitates PBMC and is resuspended and is washed in the DPBS of 50mL.By pipe at 1250rpm from The heart 10 minutes.This washing step is repeated, and PBMC precipitating is resuspended in Optimem I (Gibco 11058, the lot number of 19mL 1072088) it in and counts.Cell suspension is adjusted to 3.0x10^6 cell/mL living cells concentration.
Then the round bottom for these cells being seeded in the processing of five (each donor) 96 hole tissue culture medium (TCM)s with the hole 50uL/ is flat On plate (Costar 3799).In 30 minutes, transfection mixture is added in each hole with the volume in the hole 50uL/.It is transfecting 4 hours afterwards, with fetal calf serum (Gibco 10082, lot number 1012368) supplementing culture medium of 10uL.
B.Transfect object preparation
By the modification mRNA (mRNA sequence shown in SEQ ID NO:21438 of encoding human G-CSF；With about 160 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1) (contain (1) natural NTP, (2) have 5-methylcytosine and The 100% of pseudouridine replaces, or (3) there is 5-methylcytosine and N1- methyl-pseudouridine 100% to replace)；Coding fluorescence MRNA (the IVT cDNA sequence shown in SEQ ID NO:21445 of plain enzyme；MRNA sequence shown in SEQ ID NO:21446 Column, the polyA tail (being not shown in sequence) with about 160 nucleotide, 5 ' caps, Cap1, with 5- first at each cytimidine Base cytimidine and substitute modification completely with pseudouridine at each uridine site) (there is 5- first containing (1) natural NTP or (2) The 100% of base cytimidine and pseudouridine replaces) and TLR agonist R848 (Invivogen tlrl-r848) in final volume 38.4ng/uL is diluted in the Optimem I of 2500uL.
Individually, with the 2000 (Invitrogen of Lipofectamine of 6.76mL Optimem I dilution 110uL 11668-027, lot number 1070962).In 96 hole plates, by each mRNA of the 135uL of nine equal portions, positive control (R-848) or Negative control (Optimem I) is added to the diluted Lipofectamine 2000 of 135uL.It will be flat containing material to be transfected Plate is incubated for 20 minutes.Then transfection mixture is transferred to everyone PBMC plate with the hole 50uL/.Then it is incubated at 37 DEG C flat Plate.At the 2nd hour, the 4th hour, the 8th hour, the 20th hour and the 44th hour, each plate is removed from incubator, and Freeze supernatant.
After removing the last one plate, user G-CSF ELISA kit (Invitrogen KHC2032) and HumanIFN-α's ELISA kit (Thermo Scientific 41105-2) is measured supernatant.Each condition repeat into Row is twice.
C.Protein and innate immune response analysis
The unmodified and modification mRNA ability for generating coded protein is assessed over time, it is same right The ability of mRNA triggering congenital immunity identification is assessed, as measured by being generated by interferon-' alpha '.External PBMC culture The use of object be to measure the accepting method of the immunostimulatory potential of oligonucleotides (Robbins etc., 2009 19:89-102 of Oligonucleotides).
The standard curve for each ELISA plate that result control is fitted using four parameter logistic curves is subjected to interpolation. Be shown in table 90 and 91 as measured by specific ELISA as time goes by G-CSF, interferon-' alpha ' (IFN-α) and The average value from 3 individual PBMC donors of tumor necrosis factor α (TNF-α) yield.
In G-CSF ELISA, at every point of time, subtract untreated from Lipofectamine 2000 (LF2000) The background signal of condition.Statistics indicate that replacing or having containing natural NTP, 100% with 5-methylcytosine and pseudouridine In the case where the G-CSF mRNA for thering is 5-methylcytosine and N1- methyl-pseudouridine 100% to replace, observe outside by people All blood monocytes specifically generate human G-CSF albumen.Relative to the mRNA that 5-methylcytosine and pseudouridine are modified, pass through Using 5-methylcytosine and N1- methyl-pseudouridine modification mRNA, the generation of G-CSF is significantly increased.
For congenital immunity identification, although two kinds of modification mRNA chemical substances are both with respect to positive control (R848, p (I) p (C)) prevent IFN-α and TNF-α to generate significantly, but there is significant differences really between chemical substance.5- first Base cytimidine and the mRNA of pseudouridine modification cause the low still IFN-α of detectable level and TNF-α to generate, and 5- methyl born of the same parents Pyrimidine and N1- methyl-pseudouridine modification mRNA cause undetectable IFN-α and TNF-α to generate.
Therefore, it has been determined that, the more than one cell factor marker of the activation in addition to needing to examine closely innate immune response Except, have also surprisingly found that the combination of modification provides the cell response of different level (protein generates and immune activation). Modification N1- methyl-pseudouridine in this research has shown that the 5-methylcytosine/vacation urine for imparting and being studied beyond other people Additional Protection except the standard combination of glycosides, so as to cause nearly 150 times of twice of protein and immune activation (TNF-α) Reduction.
Assuming that PBMC contains big congenital immunity RNA identification sensor array, and protein translation can also be carried out, So it provides the system for being suitable for testing the interdependency of the two approach.Known mRNA translation can be by described congenital Negative effect (Kariko etc. Immunity (2005) 23:165-175 of the activation of property immunization route；The Cell such as Warren Stem Cell (2010) 7:618-630).Use PBMC as external test system, may establish translation (is in the case G-CSF Albumen generates) correlation between (be illustrated as in the case IFN-α and TNF-α protein generation) is generated with cell factor. Better protein generation is related to the induction of lower congenital immunity activated pathway, and can advantageously be sentenced based on this ratio New chemical substance of breaking (table 92).
In this research, compared with the 9944/1=9944 of cell factor IFN-α, two kinds of 5-methylcytosine are all had Chemical modification pseudouridine and N1- methyl-pseudouridine PC ratio are 4742/141=34.For cytokine TNF-α, two kinds of chemistry Substance has respectively 153 and 1243 PC ratio, shows that, for any cell factor, N1- methyl-pseudouridine is excellent modification. In table 90 and 91, " NT ", which is meant, not to be tested.
Table 90.G-CSF
Table 91.IFN- α and TNF-α
The ratio of table 92.G-CSF and cell factor
The external PBMC of embodiment 63. research: modification percentage
By the G-CSF mRNA or unmodified G- of 480ng modified with 5-methylcytosine (5mC) and pseudouridine (false U) The Lipofectamine 2000 of CSF mRNA and 0.4uL is transfected into together from three normal blood donors (D1, D2 and D3) Peripheral blood mononuclear cells (PBMC) in.G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1) with 5mC and vacation U (100% modifies), no is modified completely Modified with 5mC and vacation U modification (0% modification) or with the part 5mC and vacation U so that mRNA will contain 75% modify, 50% modify or 25% modification.It is expressed also directed to G-CSF, to luciferase (mRNA sequence shown in SEQ ID NO:21446；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1；The 5meC that modifies completely and vacation U) control sample into Row analysis.For TNF-α and IFN-α, also to Lipofectamine 2000, LPS, R-848, luciferase (SEQ ID NO: MRNA sequence shown in 21446；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is complete The 5mC modified entirely is analyzed with vacation U) and the control sample of P (I) P (C).22 hours after transfection, harvest supernatant is simultaneously By elisa assay, to measure protein expression.The expression of G-CSF is shown in table 93, and the expression of IFN-α and TNF-α It is shown in table 94.The expression of IFN-α and TNF-α can be the second order effect of the transfection of G-CSF mRNA.The display of table 93 and 94, when MRNA be not modify completely when, the chemical modification of G-CSF, interferon-' alpha ' (IFN-α) and tumor necrosis factor-alpha (TNF-α) Amount is titratable, and the titratable trend of each target is not identical.
By using PBMC as external test system, translation (being in the case the generation of G-CSF albumen) may be established With the correlation between cell factor generation (being illustrated as IFN-α protein in the case to generate).Better protein generates It is related to the induction of lower congenital immunity activated pathway, and the modification of chemical substance can be advantageously judged based on this ratio Percentage (table 95).As calculated in table 93 and 94 and table 95 as shown in, it is complete with 5-methylcytosine and pseudouridine Modification show protein more much better than no any modification (natural G-CSF mRNA)/cell factor generation than (for IFN-α is 100 times, is 27 times for TNF-α).The linear relationship for partially modifying the modification for showing and gradually decreasing, to lead Cause lower protein/cell factor ratio.
Table 93.G-CSF expression
Table 94.IFN- α and TNF-α expression
Table 95.PC is than the influence with modification percentage
The modification RNA that embodiment 64. transfects in PBMC
By the G-CSF mRNA or unmodified G- of 500ng modified with 5-methylcytosine (5mC) and pseudouridine (false U) The Lipofectamine 2000 of CSF mRNA and 0.4uL is transfected into together from three normal blood donors (D1, D2 and D3) Peripheral blood mononuclear cells (PBMC) in.G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1) with 5mC and vacation U (100% modifies), no is modified completely It is modified with 5mC and vacation U modification (0% modifies) or with the part 5mC and vacation U, so that mRNA will be modified containing 50%, 25% modifies, 10% modification, 5% modification, 1% modification or 0.1% modification.Also directed to G-CSF, tumor necrosis factor-alpha (TNF-α) and interference The expression of element-α (IFN-α), to mCherry (mRNA sequence shown in SEQ ID NO:21439；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1；The 5meC and pseudouridine modified completely) control sample, with 5- methyl The G-CSF (control G-CSF) and untreated control that cytimidine and pseudouridine are modified completely are analyzed.6 after transfection Hour and 18 hours harvest supernatant simultaneously by elisa assay, to measure protein expression.The G-CSF of donor 1, IFN-α and The expression of TNF-α is shown in table 96, and donor 2 is shown in table 97, and donor 3 is shown in table 98.
Complete 100% modification with 5-methylcytosine and pseudouridine causes to generate in all three human PBMC's donors Most of protein translation (G-CSF) and minimal amount of cell factor.The amount for reducing modification causes more cell factors to produce Raw (IFN-α and TNF-α), therefore further highlight modification completely to reduction cell factor and improve the important of protein translation Property (as G-CSF herein generate prove).
96. donor 1 of table
97. donor 2 of table
98. donor 3 of table
Innate immune response research in embodiment 65.BJ fibroblast
A.Single transfection
From American type culture collection (American Type Culture Collection, ATCC) (catalogue Number CRL-2522) it obtains people's foreskin primary fibroblast (BJ fibroblast) and it is made to be supplemented with 10% fetal calf serum In 37 DEG C, 5%CO in Eagle ' s minimum essential medium (ATCC, catalog number (Cat.No.) 30-2003)2Lower growth.By BJ fibroblast It is seeded in 24 hole plates in the culture medium of 0.5ml with the density of 300,000 cells/wells.Use Lipofectamine 2000 (Invitrogen, catalog number (Cat.No.) 11668-019) according to manufacturer scheme, transfect 250ng with 5-methylcytosine and It is that pseudouridine modifies (Gen1) completely or modify having for (Gen2) completely with 5-methylcytosine and N1- methyl-pseudouridine Cap0, Cap1 or modification G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438 without cap；With about The polyA tail of 140 nucleotide is not shown in sequence；5 ' caps, Cap1).Also transfect poly I:C (PIC), Lipofectamine 2000 (Lipo), natural fluoresence element enzyme mRNA (mRNA sequence shown in SEQ ID NO:21446；Tool There is the polyA tail of about 160 nucleotide, is not shown in sequence；5 ' caps, Cap1) and natural G-CSF mRNA control sample. Cell is harvested after 18 hours, separates total serum IgE and using RNeasy trace quantity reagent kit (catalog number (Cat.No.) 74004) according to manufacturer Scheme carries out the total serum IgEProcessing.Use High Capacity cDNA Reverse Transcription kit (catalog number (Cat.No.) 4368814) carries out cDNA points using the total serum IgE of 100ng according to the scheme of manufacturer Analysis.Then SybrGreen is used, according to the scheme of manufacturer, to pass through quantitatively real-time PCR, needle in 384 instrument of Biorad CFX CDNA is analyzed in the expression of innate immune response gene.Table 99 shows innate immune response transcript relative to pipe The expression of family gene HP RT (hypoxanthine phosphoribosyltransferase), and it is expressed as the induction times relative to HPRT Number.In the table, the panel of gauge include: RIG-I be retinoic acid inducible gene 1, IL-6 be interleukin-6, OAS-1 be oligoadenylate synthetase 1, IFNb is interferon-beta, and AIM2 is not present in melanoma -2, IFIT1-1 be with The interferon-induced protein of thirty-four peptide repetitive sequence 1, PKR be protein kinase R, TNF α be tumor necrosis factor α and IFN α is interferon-' alpha '.
99. innate immune response transcriptional level of table
B.It repeats to transfect
From American type culture collection (American Type Culture Collection, ATCC) (catalogue Number CRL-2522) it obtains people's foreskin primary fibroblast (BJ fibroblast) and it is made to be supplemented with 10% fetal calf serum In 37 DEG C, 5%CO in Eagle ' s minimum essential medium (ATCC, catalog number (Cat.No.) 30-2003)2Lower growth.By BJ fibroblast It is seeded in 24 hole plates in the culture medium of 0.5ml with the density of 300,000 cells/wells.According to the scheme of manufacturer, daily Transfection 250ng it is unmodified, modify completely with 5-methylcytosine and pseudouridine (Gen1) or with 5-methylcytosine with N1- methyl-pseudouridine modifies the modification G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438 of (Gen2) completely； PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), continue 5 days.To also Lipofectamine 2000 (L2000) and mCherry mRNA (mRNA sequence shown in SEQ ID NO:21439；Have The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is modified completely with 5- methylcytidine and pseudouridine) Control sample transfect daily, continue 5 days.As a result it is shown in table 100.
After one day, unmodified mRNA show the cell of interferon-beta (IFN-β) and interleukin-6 (IL-6) because Sub- response.At least show cytokine response after 2-3 days with the mRNA that pseudouridine is modified, and with 5-methylcytosine with N1- methyl-pseudouridine modification mRNA shows reduced response after 3-5 days.
100. cytokine response of table
The vivo detection of 66. innate immune response of embodiment
In order to distinguish importance of the different chemical modifications to vivo protein generation and internal cytokine response of mRNA, There is the G-CSF mRNA (unmodified G-CSF mRNA) of 5 ' cap Cap1 to (n=5) intramuscular injection of female BAl BIc/C mice (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide is not shown in sequence；), The G-CSF mRNA (G-CSF mRNA 5mc/pU) that is modified completely with 5-methylcytosine and pseudouridine, there is (G-CSF mRNA Being urinated with 5-methylcytosine and N1- methyl-vacation 5mc/N1pU) or without 5 ' caps (G-CSF mRNA 5mc/N1 pU is non-cap) The control of G-CSF mRNA or R848 or 5% sucrose that glycosides is modified completely, as described in table 101.
Chart is administered in table 101.
8 hours collection blood upon administration.Using ELISA, the albumen of G-CSF, TNF-α and IFN-α are measured by ELISA Matter is horizontal.8 hours upon administration, muscle was collected from injection site and is surveyed using quantitative real-time polymerase chain reaction (QPCR) Determine the mRNA of RIG-1, PKR in muscle, AIM-2, IFIT-1, OAS-2, MDA-5, IFN-β, TNF-α, IL-6, G-CSF, CD45 It is horizontal.
The vivo detection research of 67. innate immune response of embodiment
There is G-CSF mRNA (the unmodified G-CSF of 5 ' cap Cap1 to (n=5) intramuscular injection of female BAl BIc/C mice MRNA) (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide does not show in sequence Out；), the G-CSF mRNA (G-CSF mRNA 5mc/pU) that is modified completely with 5-methylcytosine and pseudouridine, there is (G-CSF MRNA 5mc/N1pU) or do not have 5 ' caps (G-CSF mRNA 5mc/N1 pU is non-cap) use 5-methylcytosine and N1- first The control of G-CSF mRNA or R848 or 5% sucrose that base-pseudouridine is modified completely, as described in table 102.Upon administration 8 Hour collects blood, and uses ELISA, by the protein level of ELISA measurement G-CSF and interferon-' alpha ' (IFN-α), and It is shown in table 102.
As shown in table 102, the G-CSF mRNA of unmodified, 5mc/pU and 5mc/N1pU modification causes in mice serum Human G-CSF expression.The G-CSF mRNA of not capped 5mC/N1pU modification does not show that the human G-CSF in serum is expressed, and dashes forward The importance with 5 ' cap structures for protein translation is gone out.
As expected, in R848, only in 5% sucrose and untreated group without expression human G-CSF albumen.Importantly, The significant difference generated as measured by the mouse IFN-α in serum, observed cell factor.As expected, it does not repair The G-CSF mRNA of decorations shows steady internal cytokine response (being greater than R848 positive control).The G- of 5mc/pU modification CSF mRNA shows low but detectable internal cytokine response really, and the mRNA of 5mc/N1pU modification is in serum Detectable IFN-α is not shown (medium or untreated animal are same).
In addition, the response of the mRNA of 5mc/N1pU modification is identical, no matter whether it is capped.These vivo results are reinforced Such conclusion: 1) unmodified mRNA generates steady innate immune response, 2) it is incorporated to and subtracts by the 100% of 5mc/pU modification Few response, but do not disappear and 3) being incorporated to for 5mc/N1pU modification does not lead to detectable cytokine response.
It is carried out finally, due to which these are injected in 5% sucrose (itself is without influence), these results should accurately reflect these and repair The immunostimulatory potential of decorations.
By the data, it will therefore be apparent that the molecule of N1pU modification generates greater protein matter, and concomitantly expresses IFN-α and have Have minimum or does not influence.It will also be clear that capped is needed for protein generates for this chemical modification.With it is unmodified PC (PC=9) ratio of mRNA is compared, 748 protein: cell factor ratio means this chemical modification just work relevant to IFN-α With or biological significance for be very excellent.
Human G-CSF and mouse IFN-α in 102. serum of table
Embodiment 68: the internal delivering of RNA is modified
Pass through the IX factor of G-CSF mRNA or modification to the delivering modification of female Sprague Dawley rat (n=6) MRNA generates to evaluate the protein of modification mRNA.To rat injection 400ug in 100ul by lyophilized form in 5% sucrose The G-CSF mRNA of middle reconstruct modified completely with 5-methylcytosine and pseudouridine is (shown in SEQ ID NO:21438 MRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) (G-CSF Gen1), use The G-CSF mRNA (G-CSF Gen2) or use 5-methylcytosine that 5-methylcytosine and N1- methyl-pseudouridine are modified completely The IX factor mRNA (mRNA sequence shown in SEQ ID NO:1622 modified completely with pseudouridine；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1) (IX factor Gen1).8 hours collection blood after injection, and lead to Cross the G-CSF protein level in ELISA measurement serum.G-CSF protein level after table 103 is shown 8 hours in serum.
These results indicate that after single intramuscular injection, the mRNA of G-CSF Gen1 and G-CSF Gen2 modification Human G-CSF albumen is generated in rats, and compared with Gen1 chemical substance, when using Gen2 chemical substance, human G-CSF egg White generation is improved.
G-CSF albumen (IM injecting pathway) in 103. rat blood serum of table
Preparation G-CSF albumen (pg/ml)
G-CSF Gen1 19.37
G-CSF Gen2 64.72
IX factor Gen 1 2.25
69. chemical modification of embodiment: in vitro study
A.The in-vitro screening of PBMC
By G-CSF (the SEQ ID NO:21438 of 500ng modified completely with the chemical modification summarized in table 104 and 105 Shown in mRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) mRNA with 0.4uL Lipofectamine 2000 is transfected into the peripheral blood mononuclear cells (PBMC) from three normal blood donors together In.Also to LPS, R848, P (I) P (C) and mCherry (mRNA sequence shown in SEQ ID NO:21439；With about 160 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) pair Product are analyzed in the same old way.Supernatant and stored frozen are harvested, until passing through elisa assay, to measure G-CSF protein expression, And the induction of cytokines interferon-α (IFN-α) and tumor necrosis factor α (TNF-α).The protein expression of G-CSF is in table It is shown in 104, and the expression of IFN-α and TNF-α is shown in table 105.
It is in table 104 statistics indicate that, it is many but and not all chemical modification can be used for the effective real estate stranger in PBMC G-CSF.It is worth noting that, 100%N1- methyl-pseudouridine replaces the human G-CSF for showing highest level to generate (than false urine Glycosides itself is almost higher by 10 times).When N1- methyl-pseudouridine is used in combination with 5- methylcytidine, high-caliber people G- is also generated Csf protein (this is high when being also used in combination than pseudouridine with 5 methylcytidines).
In view of protein in PBMC generate and cell factor generate between inverse relationship, also show in table 105 it is similar become Gesture, wherein have the substitution of N1- methyl-pseudouridine 100% do not cause cytokine induction (be similar to only transfection control) and Pseudouridine shows the detectable cytokine induction higher than background.
Other modifications (such as N6- methyladenosine and α-thiacydidine) seem to increase cell factor stimulation.
104. chemical modification of table and G-CSF protein expression
105. chemical modification of table and cytokine-expressing
B.In-vitro screening in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 83ng had into table 106 Described in chemical modification luciferase modify RNA (mRNA sequence shown in SEQ ID NO:21446；With about 160 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).
It is used as transfection reagent using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY), And 0.2ul is diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, two kinds of solution is merged, And it is incubated for again at room temperature 15 minutes.Then solution 20ul merged is added to the cell training that 100ul contains HeLa cell It supports in base and is incubated at room temperature.
After being incubated for 18 to 22 hours, with the Passive Lysis Buffer of 100ul (Promega, Madison, WI) according to the explanation of manufacturer by the cell cracking of expressing luciferase.It is impermeable that the aliquot of lysate is transferred to white Bright 96 hole plate of polystyrene (Corning, Manassas, VA), and the complete luciferase assay solution with 100ul (Promega, Madison, WI) merges.Lysate volume is adjusted or dilutes, until the sample detection for generating peak signal It is shown in table 106 to the RLU for being no more than 2mio relative light unit (RLU)/hole, the every kind of chemical substance tested.Plate is read Taking device is BioTek Synergy H1 (BioTek, Winooski, VT).The background signal for not having the plate of reagent is about 200 phases To light unit/hole.
These results indicate that it is many but and not all chemical modification can be used for the effective real estate stranger G- in HeLa cell CSF.It is worth noting that, 100%N1- methyl-pseudouridine replaces the human G-CSF for showing highest level to generate.
The relative light unit of 106. luciferase of table
Chemical modification RLU
N6- methyladenosine (m6a) 534
5- methylcytidine (m5c) 138,428
N4- acetyl group cytidine (ac4c) 235,412
5- formoxyl cytidine (f5c) 436
5-methylcytosine/pseudouridine tests A1 48,659
5-methylcytosine/N1- methyl-pseudouridine tests A1 190,924
Pseudouridine 655,632
1- methyl pseudouridine (m1u) 1,517,998
2- thio uridine (s2u) 3387
5- methoxyuridine (mo5u) 253,719
5-methylcytosine/pseudouridine, test b 1 317,744
5-methylcytosine/N1- methyl-pseudouridine, test b 1 265,871
The bromo- uridine of 5- 43,276
5 (2 carbonyl vinyl) uridines 531
5 (3-1E allylamino) uridines 446
5-methylcytosine/pseudouridine tests A2 295,824
5-methylcytosine/N1- methyl-pseudouridine tests A2 233,921
5-methyl-uridin 50,932
α-is thio-cytidine 26,358
5-methylcytosine/pseudouridine, test b 2 481,477
5-methylcytosine/N1- methyl-pseudouridine, test b 2 271,989
5-methylcytosine/pseudouridine tests A3 438,831
5-methylcytosine/N1- methyl-pseudouridine tests A3 277,499
Unmodified luciferase 234,802
C.In-vitro screening in rabbit reticulocyte lysate
With the chemical modification listed in table 107 to luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446 Column；PolyA tail with 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is modified, and it is being free of into nucleic acid The final quantity of the 250ng in 10ul is diluted in the sterile water of enzyme.Diluted luciferase is added to the fresh preparation of 40ul Rabbit reticulocyte lysate in, and the 1.5mL polypropylene reaction tube of standard (Thermo Fisher Scientific, Waltham, MA) at 30 DEG C in dry heat block carry out In Vitro Translation reaction.With Rabbit Reticulocyte Lysate (nuclease is processed) kit (Promega, Madison, WI) according to manufacturer illustrate carry out translation measurement. Reaction buffer is supplemented with one to one blending of the provided amino acid primary liquid for lacking any of leucine or methionine Object, so that the reaction mixture for containing two kinds of enough amino acid is generated, to allow effective In Vitro Translation.
After being incubated for 60 minutes, stop reaction by the way that reaction tube to be put on ice for.RNA will be modified containing luciferase In Vitro Translation reaction solution aliquot be transferred to White-opalescent 96 hole plate of polystyrene (Corning, Manassas, VA), and merge with the complete luciferase assay solution (Promega, Madison, WI) of 100ul.Adjust or The volume for diluting In Vitro Translation reaction solution, until the sample detection for generating peak signal is to no more than 2mio relative light unit (RLU) RLU in/hole, the every kind of chemical substance tested is shown in table 107.Plate reader is BioTek Synergy H1 (BioTek, Winooski, VT).The background signal for not having the plate of reagent is about 200 relative light units/hole.
It is highly dependent that these not celliferous translation results and protein in HeLa generate result, and usually in two germlines It works in system or inoperative identical modification is highly dependent.One noticeable exception is that 5- formoxyl cytidine is modified Luciferase mRNA works in not celliferous translation system, and does not rise in the transfection system based on HeLa cell Effect.The similar difference between two kinds of measurements is also observed in the case where the G-CSF mRNA of 5- formoxyl cytidine modification.
The relative light unit of 107. luciferase of table
Chemical modification RLU
N6- methyladenosine (m6a) 398
5- methylcytidine (m5c) 152,989
N4- acetyl group cytidine (ac4c) 60,879
5- formoxyl cytidine (f5c) 55,208
5-methylcytosine/pseudouridine tests A1 349,398
5-methylcytosine/N1- methyl-pseudouridine tests A1 205,465
Pseudouridine 587,795
1- methyl pseudouridine (m1u) 589,758
2- thio uridine (s2u) 708
5- methoxyuridine (mo5u) 288,647
5-methylcytosine/pseudouridine, test b 1 454,662
5-methylcytosine/N1- methyl-pseudouridine, test b 1 223,732
The bromo- uridine of 5- 221,879
5 (2 carbonyl vinyl) uridines 225
5 (3-1E allylamino) uridines 211
5-methylcytosine/pseudouridine tests A2 558,779
5-methylcytosine/N1- methyl-pseudouridine tests A2 333,082
5-methyl-uridin 214,680
α-is thio-cytidine 123,878
5-methylcytosine/pseudouridine, test b 2 487,805
5-methylcytosine/N1- methyl-pseudouridine, test b 2 154,096
5-methylcytosine/pseudouridine tests A3 413,535
5-methylcytosine/N1- methyl-pseudouridine tests A3 292,954
Unmodified luciferase 225,986
70. chemical modification of embodiment: In vivo study
A.The internal screening of G-CSF modification mRNA
In every leg to Balb-C mouse (n=4) intramuscular injection be formulated in 1x PBS with the change summarized in table 108 Learn the modification G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438 that modification is modified completely；With about 160 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1).MRNA (SEQ ID NO:21446 also is modified to luciferase Shown in mRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；Use pseudouridine Modified completely with 5-methylcytosine) control and the control of PBS tested.Serum is collected, after 8 hours to pass through ELISA measures G-CSF protein level and cytokine levels.
Table 108.G-CSF
mRNA Chemical modification
G-CSF Pseudouridine
G-CSF 5-methyl-uridin
G-CSF 2- thio uridine
G-CSF 4-thiourdine
G-CSF 5- methoxyuridine
G-CSF 2 '-floxuridines
G-CSF 5- Broxuridine
G-CSF 5- [3 (1-E- allylamino) uridines)
G-CSF α-is thio-cytidine
G-CSF 5- methylcytidine
G-CSF N4- acetyl group cytidine
G-CSF Pseudouridine and 5-methylcytosine
G-CSF N1- methyl-pseudouridine and 5-methylcytosine
Luciferase Pseudouridine and 5-methylcytosine
PBS Nothing
B.The internal screening of luciferase modification mRNA
To the use containing the 42ug to 103ug being formulated in 1x PBS of Balb-C mouse (n=4) subcutaneous injection 200ul Modification luciferase mRNA (the mRNA sequence shown in SEQ ID NO:21446 that the chemical modification summarized in table 109 is modified completely Column；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1).Also the control of PBS is surveyed Examination.The dosage of the luciferase mRNA of modification is also summarized in table 109.After administration 8 hours, mouse is imaged to measure luciferase Expression.20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then simultaneously by Animal Anesthesia Image is acquired using IVIS Lumina II imaging system (Perkin Elmer).Bioluminescence is measured as total stream of whole mouse It measures (photons/second).
As indicated in table 109, the chemical substance of all luciferase mRNA modifications shows activity in vivo, 2 ' fluorine urine Except glycosides.In addition, the mRNA of 1- methyl pseudouridine modification shows very high luciferase expression, (ratio is containing pseudouridine The expression that 5 times of mRNA high).
The screening of 109. luciferase of table
Embodiment 71. combines the internal screening of luciferase modification mRNA
Being summarized in table 110 for the 100ug of 200ul being formulated in 1xPBS is subcutaneously injected to Balb-C mouse (n=4) The modification luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446 modified completely of chemical modification；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1).Also the control of PBS is tested.Administration 8 hours Afterwards, mouse is imaged to measure luciferase expression.20 minutes before imaging, D- is injected into mouse peritoneum with 150mg/kg Luciferin solution.Then image is acquired by Animal Anesthesia and using IVIS Lumina II imaging system (Perkin Elmer). Bioluminescence is measured as the total flow (photons/second) of whole mouse.
As indicated in table 110, the chemical substance (combination) of all luciferase mRNA modifications shows activity in vivo.This Outside, modify in mRNA (have N4- acetyl group cytidine or 5- methylcytidine) N1- methyl-pseudouridine there are ratio with pseudouridine It is used together like combinations when being tested and shows higher expression.Generally speaking, these statistics indicate that, contain N1- first No matter base-pseudouridine luciferase mRNA is used alone (table 109) or (table is being applied in combination with other modified nucleoside acid 110) when, improved vivo protein is caused to be expressed.
The screening combination of 110. luciferase of table
The fibroblastic innate immune response of embodiment 72.BJ
It is primary at fiber finer that people's foreskin is obtained from American type culture collection (ATCC) (catalog number (Cat.No.) CRL-2522) Born of the same parents (BJ fibroblast) simultaneously make it in Eagle ' s minimum essential medium (ATCC, the catalog number (Cat.No.) for being supplemented with 10% fetal calf serum It is grown at 37 DEG C, 5%CO2 in 30-2003).By BJ fibroblast with 130,000 cells/wells in the culture medium of 0.5ml Density be seeded in 24 hole plates.Using Lipofectamine 2000 (Invitrogen, catalog number (Cat.No.) 11668-019) according to It is (Gen1) or phonetic with 5- methyl born of the same parents to transfect being modified completely with 5-methylcytosine and pseudouridine for 250ng for the scheme of manufacturer Pyridine and N1- methyl-pseudouridine modify the modification G-CSF mRNA (mRNA shown in SEQ ID NO:21438 of (Gen2) completely Sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1).Also transfect Lipofectamine The control sample of 2000 and unmodified G-CSF mRNA (natural G-CSF).Cell is transfected, five Consecutive Days are continued.In every wheel 4 hours removal transfection composites after transfection.
After transfection, according to the scheme of manufacturer, daily by ELISA for G-CSF (R&D system, the catalogue of secretion Number DCS50), tumor necrosis factor-alpha (TNF-α) and interferon-' alpha ' (IFN-α) be measured culture supernatants.In the first round After transfection 6 hours and 18 hours and then every other day, use CELL TITER(Promega, catalog number (Cat.No.) G7570) cell is analyzed for viability.Total serum IgE is separated from the cell harvested simultaneously and uses RNAEASY micro Kit (catalog number (Cat.No.) 74004) is used according to the scheme of manufacturerThe total serum IgE is handled.Use High Capacity cDNA Reverse Transcription kit (Applied Biosystems, catalog number (Cat.No.) 4368814) is pressed According to the scheme of manufacturer, cDNA analysis is carried out using the total serum IgE of 100ng.Then using SybrGreen in Biorad CFX 384 According to the scheme of manufacturer in instrument, by quantitatively real-time PCR, for the expression of innate immune response gene, to cDNA into Row analysis.
Embodiment 73. uses the in-vitro transcription of wild type T7 polymerase
As it was earlier mentioned, using wild type T7 polymerase, with the different chemical substances and chemistry listed in table 111-114 Substance is combined to luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) and G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438； PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is modified completely.
The yield of translation reaction is measured by spectrophotometry (OD260), the yield of luciferase is in table 111 In show, and the yield of G-CSF is shown in table 113.
Luciferase and the mRNA of G-CSF modification are also subject to enzymatic and cover reaction, and pass through spectrophotometry (OD260) each modification mRNA yield for covering reaction evaluate and assess correct size using biological analyser.It is glimmering The yield of the capping reaction of light element enzyme is shown in table 112, and the yield of the capping reaction of G-CSF is shown in table 114.
The in-vitro transcription chemistry of 111. luciferase of table
112. luciferase of table modifies the capped chemistry and yield of mRNA
Table 113.G-CSF modifies the in-vitro transcription chemistry and yield of mRNA
Table 114.G-CSF modifies the capped chemistry and yield of mRNA
Embodiment 74. uses the in-vitro transcription of 7 polymerase of mutation T
Using 7 polymerase of mutation T (T7 Transcription kit (catalog number (Cat.No.) DS010925) (Madison, WI), with the different chemical substances and combination of chemicals listed in table 115-118 to fluorescence Plain enzyme mRNA (mRNA sequence shown in SEQ ID NO:21446；PolyA tail with about 160 nucleotide, in sequence It is not shown；5 ' caps, Cap1) and G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438；With about 160 cores The polyA tail of thuja acid is not shown in sequence；5 ' caps, Cap1) it is modified completely.
The yield of translation reaction is measured by spectrophotometry (OD260), the yield of luciferase is in table 115 In show, and the yield of G-CSF is shown in table 117.
Luciferase and G-CSF modification mRNA are also subject to enzymatic and cover reaction, and pass through spectrophotometry (OD260) Each modification mRNA yield for covering reaction evaluate and assesses correct size using biological analyser.Luciferase The yield for covering reaction is shown in table 116, and the yield of the capping reaction of G-CSF is shown in table 118.
115. luciferase of table modifies the in-vitro transcription chemistry and yield of mRNA
116. luciferase of table modifies the capped chemistry and yield of mRNA
Chemical modification Yield (ug)
2 ' Flucytosines 19.2
2 ' floxuridines 16.7
5-methylcytosine/pseudouridine tests A 7.0
5-methylcytosine/N1- methyl-pseudouridine tests A 21.5
N1- acetyl group cytidine/2- floxuridine 47.5
5- methylcytidine/2- floxuridine 53.2
2- Flucytosine/pseudouridine 58.4
2- Flucytosine/N1- methyl-pseudouridine 26.2
2- Flucytosine/2- thio uridine 12.9
2- Flucytosine/5- Broxuridine 26.5
2- Flucytosine/2- floxuridine 35.7
2- fluorine guanine/5-methylcytosine 24.7
2- fluorine guanine/5-methylcytosine/pseudouridine 32.3
2- fluorine guanine/5- methylcytidine/N1- methyl-pseudouridine 31.3
2- fluorine guanine/pseudouridine 20.9
2- fluorine guanine/N1- methyl-pseudouridine 29.8
5- methylcytidine/pseudouridine, test b 58.2
5- methylcytidine/N1- methyl-pseudouridine, test b 44.4
Table 117.G-CSF modifies the in-vitro transcription chemistry and yield of mRNA
Table 118.G-CSF modifies the capped chemistry and yield of mRNA
Chemical modification Yield (ug)
2 ' Flucytosines 16.9
2 ' floxuridines 17.0
5-methylcytosine/pseudouridine tests A 10.6
5-methylcytosine/N1- methyl-pseudouridine tests A 22.7
N1- acetyl group cytidine/2- floxuridine 19.9
5- methylcytidine/2- floxuridine 21.3
2- Flucytosine/pseudouridine 65.2
2- Flucytosine/N1- methyl-pseudouridine 58.9
2- Flucytosine/2- thio uridine 41.2
2- Flucytosine/5- Broxuridine 35.8
2- Flucytosine/2- floxuridine 36.7
2- fluorine guanine/5-methylcytosine 36.6
2- fluorine guanine/5-methylcytosine/pseudouridine 37.3
2- fluorine guanine/5- methylcytidine/N1- methyl-pseudouridine 30.7
2- fluorine guanine/pseudouridine 29.0
2- fluorine guanine/N1- methyl-pseudouridine 22.7
5- methylcytidine/pseudouridine, test b 60.4
5- methylcytidine/N1- methyl-pseudouridine, test b 33.0
Embodiment 75. 2 ' O- methyl and 2 ' fluoro based compounds
By luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is prepared as the pattern of the chemical substance for having in table 119 modified completely, and Using 7 polymerase of mutation T (T7 Transcription kit (catalog number (Cat.No.) DS010925) (Madison, WI) it is transcribed.Contain the mRNA of 2 ' fluoro bases using Durascribe T7 preparation, however, The unusable Durascribe T7 of the mRNA for containing 2 ' O methyl is transcribed.
Other 7 polymerases of mutation T (Nat Biotechnol. (2004) 22:1155-1160 may can be used；Nucleic Acids Res. (2002) 30:e138 or United States Patent (USP) 7,309,570, wherein the content of each is integrally incorporated this by reference Text) realize that the mRNA of 2 ' O methyl modification is incorporated to.Alternatively, enzymatic means can be used to introduce 2 ' OMe modification after transcription.
Modification is introduced on 2 ' groups of sugar has many potential advantages.Known 2 ' OMe replaces (substitution of such as 2 ' fluoro bases) Nuclease-resistant, and have also shown out, when being incorporated into other nucleic acid (such as siRNA and antisense), eliminate congenital exempt from Epidemic disease identification (is integrally incorporated, Crooke is edited, Antisense Drug Technology, second edition；Boca Raton:CRC is published Society).
Then 2 ' fluoro bases-modification mRNA can be transfected into HeLa cell, to assess the albumen in cellular context Matter generates, and also assesses in not celliferous rabbit reticulocyte system identical mRNA.Using unmodified The control (natural fluoresence element enzyme) of luciferase carries out two kinds of transcription experiments, transfects to untreated and simulation not also directed to HeLa (only Lipofectamine 2000) control of transfection is analyzed, and is directed to control of the rabbit granulophilocyte to not RNA It is analyzed.
For HeLa transfection experiment, in the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd； Manassas, VA) and be inoculated in the EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax) of total volume 100ul/ In the 96 hole cel culture plates (Corning, Manassas, VA) in hole.Make cell at 37 DEG C in 5%CO2It is grown in atmosphere Overnight.Second day, the luciferase for containing 2 ' fluoro bases with chemical modification described in table 119 of 83ng is modified into RNA (mRNA sequence shown in SEQ ID NO:21446；PolyA tail with about 160 nucleotide is not shown in sequence；5' Cap, Cap1) it is diluted in the OPTI-MEM (LifeTechnologies, Grand Island, NY) of 10ul final volume.It uses Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by 0.2ul It is diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, two kinds of solution is merged, and at room temperature It is incubated for again 15 minutes.Then solution 20ul merged be added to 100ul contain in the cell culture medium of HeLa cell and It is incubated at room temperature.After being incubated for 18 to 22 hours, with the Passive Lysis Buffer of 100ul (Promega, Madison, WI) according to the explanation of manufacturer by the cell cracking of expressing luciferase.The aliquot of lysate is transferred to 96 hole plate of polystyrene (Corning, Manassas, VA) of White-opalescent, and surveyed with the complete luciferase of 100ul Determine solution (Promega, Madison, WI) merging.Lysate volume is adjusted or dilutes, until the sample for generating peak signal Product examine is measured no more than 2mio relative light unit (RLU)/hole, and the RLU for the every kind of chemical substance tested is shown in table 119. Plate reader is BioTek Synergy H1 (BioTek, Winooski, VT).The background signal for not having the plate of reagent is About 200 relative light units/hole.
Rabbit reticulocyte lysate is measured, the luciferase mRNA of 2 '-fluoro bases will be contained without nuclease Sterile water in be diluted to the final quantity of 250ng in 10ul, and the freshly prepared rabbit net for being added to 40ul knit it is red thin In cellular lysate object, in the 1.5mL polypropylene reaction tube (Thermo Fisher Scientific, Waltham, MA) of standard In Vitro Translation reaction is carried out at 30 DEG C in dry heat block.With Rabbit Reticulocyte Lysate (at nuclease Managed) kit (Promega, Madison, WI) according to manufacturer illustrate carry out translation measurement.Reaction buffer supplement One to one blend of the provided amino acid primary liquid for lacking any of leucine or methionine is provided, is contained to generate The reaction mixture of two kinds of enough amino acid, to allow effective In Vitro Translation.After being incubated for 60 minutes, by that will react Pipe is put on ice for stopping reaction.
The aliquot of In Vitro Translation reaction solution containing luciferase modification RNA is transferred to the polyphenyl of White-opalescent 96 hole plate of ethylene (Corning, Manassas, VA), and with the complete luciferase assay solution of 100ul (Promega, Madison, WI) merge.The volume for adjusting or diluting In Vitro Translation reaction solution, until the sample detection for generating peak signal It is shown in table 120 to the RLU for being no more than 2mio relative light unit (RLU)/hole, the every kind of chemical substance tested.Plate is read Taking device is BioTek Synergy H1 (BioTek, Winooski, VT).The background signal for not having the plate of reagent is about 160 phases To light unit/hole.
Such as table 119 and 120 as it can be seen that a variety of compounds containing 2 ' fluoro bases are active and generate fluorescein in vitro Zymoprotein.
Table 119.HeLa cell
Chemical modification Concentration (ug/ml) Volume (ul) Yield (ug) RLU
2 ' fluorine adenosines 381.96 500 190.98 388.5
2 ' Flucytosines 654.56 500 327.28 2420
2 ' fluorine guanines 541,795 500 270.90 11,705.5
2 '-floxuridines 944.005 500 472.00 6767.5
Natural fluoresence element enzyme It is not applicable It is not applicable It is not applicable 133,853.5
Simulation It is not applicable It is not applicable It is not applicable 340
It is untreated It is not applicable It is not applicable It is not applicable 238
120. rabbit granulophilocyte of table
Chemical modification RLU
2 ' fluorine adenosines 162
2 ' Flucytosines 208
2 ' fluorine guanines 371,509
2 '-floxuridines 258
Natural fluoresence element enzyme 2,159,968
Without RNA 156
Embodiment 76. uses the luciferase in the combined HeLa cell of modification
In order to evaluate 2 ' fluoro bases-mRNA of modification and being used in combination for other modifications, used as described in embodiment 75 Wild type T7 polymerase (compound without containing fluoro base) uses 7 polymerase of mutation T (compound containing fluoro base) will A series of mRNA transcriptions.All modification mRNA are tested in HeLa cell by in-vitro transfection.
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 83ng had into table 121 Described in chemical modification luciferase modify RNA (mRNA sequence shown in SEQ ID NO:21446；With about 160 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 0.2ul is diluted in the OPTI-MEM of 10ul final volume.In room temperature After five minutes, two kinds of solution are merged, and is incubated for again at room temperature 15 minutes for lower incubation.Then solution 20ul merged adds It is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.
After being incubated for 18 to 22 hours, with the Passive Lysis Buffer of 100ul (Promega, Madison, WI) according to the explanation of manufacturer by the cell cracking of expressing luciferase.It is impermeable that the aliquot of lysate is transferred to white Bright 96 hole plate of polystyrene (Corning, Manassas, VA), and the complete luciferase assay solution with 100ul (Promega, Madison, WI) merges.Lysate volume is adjusted or dilutes, until the sample detection for generating peak signal It is shown in table 121 to the RLU for being no more than 2mio relative light unit (RLU)/hole, the every kind of chemical substance tested.Plate is read Taking device is BioTek Synergy H1 (BioTek, Winooski, VT).The background signal for not having the plate of reagent is about 200 phases To light unit/hole.
As proved in table 121, most of combinations of modification obtain generating the mRNA of functional luciferase protein, including All compounds without fluoro base and many combinations for containing the modification of 2 ' fluoro bases.
121. luciferase of table
Embodiment 77.G-CSF is transcribed in vitro
In order to assess activity of our all different chemical modifications under the second open reading frame background, our employments The experiment that previously used luciferase mRNA is carried out is repeated in G-CSF mRNA.Using wild type T7 polymerase (for it is all not The compound of the base containing fluoro) or 7 polymerase of mutation T (for all compounds containing fluoro base), in table 122 and 123 Chemical substance is to G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is modified completely.Commercially-available 7 polymerase of mutation T ( T7 Transcription kit (catalog number (Cat.No.) DS010925) (Madison, WI).
Modification RNA in-vitro transfection in table 122 and 123 into HeLa cell or is added to rabbit granulophilocyte (250ng Modification mRNA) in, as indicated.Also to untreated, simulation transfection (only transfection reagent) with 5-methylcytosine and The control for the G-CSF that N1- methyl-pseudouridine is modified completely is modified completely with 5-methylcytosine and N1- methyl-pseudouridine Luciferase control (mRNA sequence shown in SEQ ID NO:21446；PolyA tail with about 160 nucleotide, sequence It is not shown in column；5 ' caps, Cap1) it is analyzed.The expression of G-CSF albumen is measured by ELISA, value is shown in table 122 and 123 Out.In table 122, " NT ", which is meant, not to be tested.
As shown in table 123, it is many but and not all chemical modification cause human G-CSF albumen to generate.From based on cell and These results of not celliferous translation system with usually work in two kinds of systems or it is inoperative it is identical modification very It is related.One noticeable exception is the G-CSF mRNA of 5- formoxyl cytidine modification, in not celliferous translation system In work, and do not work in the transfection system based on HeLa cell.Also in the luciferase of 5- formoxyl cytidine modification The similar difference between two kinds of measurements is observed in the case where mRNA.
As indicated in table 123, it is many but and not all G-CSF mRNA modification chemical substance (when used in combination) Show activity in vivo.In addition, N1- methyl-pseudouridine in modification mRNA (there is N4- acetyl group cytidine or 5- methylcytidine) In the presence of than showing higher expression being used together like combinations when being tested with pseudouridine.Generally speaking, these are counted According to the external protein expression for showing to cause to improve containing N1- methyl-pseudouridine G-CSF mRNA.
Table 122.G-CSF expression
Combinatorial chemistry substance in table 123.HeLa cell
The screening of 78. chemical substance of embodiment
The table (table 124-126) being listed below summarizes the major part using the different compounds presented in preceding embodiment External and in-vitro screening data.Based on cell, there are good correlations between not celliferous translation measurement.Either exist The background of luciferase mRNA is still tested under the background of G-CSF mRNA, and identical chemical substitute usually shows good Consistency.Finally, very high protein expression level is shown in vitro and in vivo containing N1- methyl-pseudouridine mRNA, And it is seldom stimulated to no detectable cell factor, and be superior to the mRNA containing pseudouridine in vitro and in vivo.
Combinatorial chemistry described in the natural or non-naturally occurring chemical substance described in table 124 and 125 or table 126 Substance is to luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446；PolyA with about 160 nucleotide Tail is not shown in sequence；5 ' caps, Cap1) and G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438；With big The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is modified, and use method described herein pair It is tested.
In table 125 and 126, " * " refer to using 7 polymerase of mutation T ( T7 Transcription Kit (catalog number (Cat.No.) DS010925) (Madison, WI) in-vitro transcription reaction；" * * " refers to using mutation T7 polymerase (T7 Transcription kit (catalog number (Cat.No.) DS010925) ( Madison, WI) the outer responsive transcription of the second effective aspect；" * * * " refers to that (rabbit granulophilocyte is split in not celliferous translation object Solution object) in the generation observed；The protein generation of HeLa is judged by "+", " +/- " and "-", when mentioning G-CSF When PBMC, " ++++", refer to greater than 6,000pg/ml G-CSF, " +++ " refers to that, greater than 3,000pg/ml G-CSF, " ++ " refers to Greater than 1,500pg/ml G-CSF, "+" refers to greater than 300pg/ml G-CSF, and " +/- " refers to 150-300pg/ml G-CSF, and And background is about 110pg/ml；When mentioning cell factor PBMC, " ++++" refer to greater than 1,000pg/ml interferon-' alpha ' (IFN- α), " +++ " refers to greater than 600pg/ml IFN-α, and " ++ " refers to that, greater than 300pg/ml IFN-α, "+" refers to greater than 100pg/ml IFN-α, "-" refers to less than 100pg/ml and background is about 70pg/ml；And " NT ", which refers to, not to be tested.In table 125, Using 7 polymerase of mutation T (T7 Transcription kit (catalog number (Cat.No.) DS010925) (Madison, WI) evaluation protein generation.
Table 124. is naturally occurring
Table 125. is non-naturally occurring
In table 126, the generation of the protein of HeLa is judged by "+", " +/- " and "-", when mentioning G-CSF PBMC When, " ++++" refer to greater than 6,000pg/ml G-CSF, " +++ " refers to greater than 3,000pg/ml G-CSF, and " ++ ", which refers to, to be greater than 1,500pg/ml G-CSF, "+" refer to that, greater than 300pg/ml G-CSF, " +/- " refers to 150-300pg/ml G-CSF, and carries on the back Scape is about 110pg/ml；When mentioning cell factor PBMC, " ++++" refer to greater than 1,000pg/ml interferon-' alpha ' (IFN-α), " +++ " refers to greater than 600pg/ml IFN-α, and " ++ " refers to that, greater than 300pg/ml IFN-α, "+" refers to greater than 100pg/ml IFN-α, "-" refers to less than 100pg/ml and background is about 70pg/ml；" WT " refers to that wild type T7 polymerase, " MT " refer to 7 polymerase of mutation T (T7 Transcription kit (catalog number (Cat.No.) DS010925) ( Madison, WI), and " NT " refers to and does not test.
126. combinatorial chemistry substance of table
2 ' fluoro base chemical substances in embodiment 79.PBMC
G-CSF is measured by measurement interferon-' alpha ' (IFN-α) and tumor necrosis factor-alpha (TNF-α) yield modifies mRNA (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 160 nucleotide is not shown in sequence；5' Cap, Cap1) triggering innate immune response ability.The use of external PBMC culture is to measure the immune of oligonucleotides Stimulate the accepting method (2009 19:89-102 of Robbins etc., Oligonucleotides) of potentiality, and transfection method It is described herein.It is interferon-' alpha ' (IFN-α) and tumor necrosis factor α (TNF-α) as time goes by shown in table 127 The average value from 2 or 3 independent PBMC donors of yield, as measured by specific ELISA.Also to R848, P (I) control of P (C), LPS and Lipofectamine 2000 (L2000) are analyzed.
For congenital immunity identification, although relative to positive control (R848, P (I) P (C)), two kinds of modification mRNAization Learn substance prevents IFN-α and TNF-α from generating significantly, but IFN-α and TNF-α generation are reduced very by 2 ' fluoro based compounds It is extremely lower than other combinations, and the combination of N4- acetyl group cytidine rises Cytokines characteristic spectrum.
Table 127.IFN- α and TNF-α
Embodiment 80. has the modification mRNA of 5 ' UTR of marmor erodens
It can be used as flanking region and 5 ' non-translational regions (UTR) be provided.It may include multiple 5 ' UTR and the multiple 5 ' in flanking region UTR can be identical or with different sequences.Any part (including not having) of flanking region can be codon optimization, and appoint What one can be before or after codon optimization independently containing one or more different structures or chemical modification.
5 ' UTR may include the 5 ' UTR from marmor erodens (TEV).Using 5 ' UTR variant, one of them or Multiple nucleotide are added or removed to end, including A, T, C or G.
The expression for the mRNA that embodiment 81.PLGA is prepared
A. the synthesis and characterization of luciferase PLGA microballoon
It will be modified completely with 5-methylcytosine and N1- methyl-pseudouridine, replace 25% uridine simultaneously to 2- thio uridine It is replacing that 25% cytimidine modified with 5-methylcytosine, modified completely with N1- methyl-pseudouridine, or used pseudouridine Luciferase mRNA (the mRNA sequence shown in SEQ ID NO:21446 modified completely；With about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it reconstructs in 1x TE buffer and is then formulated in PLGA microballoon.Make With water as known in the art/oil/water second emulsifying method, using PLGA- ester cap (Lactel, catalog number (Cat.No.) B6010-2, inherently Viscosity 0.55-0.75,50: 50LA: GA), polyvinyl alcohol (PVA) (Sigma, catalog number (Cat.No.) 348406-25G, MW 13-23k) dichloro Methane and hydration are at PLGA microballoon.Briefly, the mRNA (W1) by the 0.4ml of 4mg/ml in TE buffer is added to 2ml It is dissolved under the PLGA concentration of 200mg/ml in the PLGA (O1) of methylene chloride (DCM).By W1/O1 lotion speed 5 (~ 19,000rpm) (IKA Ultra-Turrax Homogenizer, T18) is homogenized under 30 seconds.Then W1/O1 lotion is added to In 250ml 1%PVA (W2), and it is homogenized 1 minute at speed 5 (~19,000rpm).It stirs preparation 3 hours, then makes It is larger to remove by 100 μm of nylon net filter devices (Fisherbrand Cell Strainer, catalog number (Cat.No.) 22-363-549) Aggregation, and it is washed eventually by centrifugation (10min, 9,250rpm, 4 DEG C).Liquid is discarded supernatant, and will PLGA precipitating is resuspended in the water of 5-10ml, is repeated 2 times.After washing and being resuspended with water, the PLGA using 100-200 μ l is micro- Ball sample to measure the granularity of preparation by laser diffraction (Malvern Mastersizer2000).By the preparation by washing It freezes in liquid nitrogen, is then lyophilized 2-3 days.
After freeze-drying ,~PLGA the MS of 10mg is weighed up in 2ml eppendorf pipe, and pass through addition 1ml's Sample is simultaneously vibrated 2-6 hours to make its depolymerization by DCM.By addition 0.5ml water and by sample shaken overnight come from de- preparation MRNA is extracted in PLGA microballoon.DCM is added in the luciferase mRNA (control that do not prepare) not prepared in TE buffer In, and it is subjected to depolymehzation process (depolymerization control), to be used as control in transfection measurement.Encapsulation efficiency, weight percent load and Granularity is shown in table 128.Encapsulation efficiency is calculated as the mg of the mRNA of the de- preparation from PLGA microballoon divided by being added to preparation In mRNA primary quantity.The mg for the mRNA that weight percent load in preparation is calculated as the de- preparation from PLGA microballoon is removed Primary quantity with the PLGA being added in preparation.
Table 128.PLGA feature
B.It is encapsulated in the protein expression of the modification mRNA in PLGA microballoon
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA in).Grow cell overnight in 5%CO2 atmosphere at 37 DEG C.Second day, by the luciferase of the de- preparation of 83ng The luciferase mRNA that mRNA PLGA microsphere sample, the de- luciferase mRNA prepared compare (de- molding control) or do not prepare Control (unformed control) be diluted in 10ul final volume OPTI-MEM (LifeTechnologies, Grand Island, NY in).It is used as transfection reagent using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY), and And 0.2ul is diluted in the OPTI-MEM of 10ul final volume.After being incubated at room temperature 5min, two kinds of solution are merged, and It is incubated for 15min again at room temperature.Then solution 20ul merged is added to the cell culture medium that 100ul contains HeLa cell In.Then it is incubated for plate as described above.
After being incubated for 18 to 22 hours, with the Passive Lysis Buffer of 100ul (Promega, Madison, WI) according to the explanation of manufacturer by the cell cracking of expressing luciferase.It is impermeable that the aliquot of lysate is transferred to white Bright 96 hole plate of polystyrene (Corning, Manassas, VA), and the complete luciferase assay solution with 100ul (Promega, Madison, WI) merges.The background signal for not having the plate of reagent is about 200 relative light units/hole.Plate is read Taking device is BioTek Synergy H1 (BioTek, Winooski, VT).
The bioluminescence (in terms of relative light unit RLU) of harvest cell and each sample is shown in table 129.These samples The transfection of product confirmed that the different chemical substances of luciferase mRNA still are able to expression fluorescein after the preparation of PLGA microballoon Zymoprotein.
129. chemical modification of table
The in vitro study of the embodiment 82.IX factor
A.Culture medium without serum
The transfected with human IX factor mRNA (mRNA sequence shown in SEQ ID NO:1622 in the culture medium without serum； PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is complete with 5-methylcytosine and pseudouridine Full modification).It collects cell culture supernatant and it is made to carry out trypsin digestion, carry out 2 dimension HPLC separation of peptide later.Make Peptide is detected with substance assistant laser desorpted/ionization.Detect 7 kinds in 8 kinds of peptides and peptide detected by the IX factor spy Have.These are the result shows that the mRNA transfected in the culture medium without serum can express overall length IX factor protein.
B.Human embryo kidney (HEK) 293A cell
Using Lipofectamine 2000 in DMEM, in the presence of 10%FBS, by the codon optimization of 250ng People's IX factor mRNA (mRNA sequence shown in SEQ ID NO:1622；It is modified completely with 5-methylcytosine and pseudouridine； PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) be transfected into HEK 293A cell (150, 000 cells/well) in.3 hours removal transfection composites after transfection.3,6,9,12,24,48 and 72 hours harvests are thin after transfection Born of the same parents.It separates total serum IgE and is analyzed for cDNA.Using the IX cytokine specific primers group of codon optimization, pass through quantitatively real-time PCR CDNA is analyzed.The level of human hypoxanthine's phosphoribosyl transferase 1 (HPRT) is for normalizing.Using data as can The percentage of detection mRNA is drawn, and the mRNA level in-site at 3 hour time point is considered as 100%.With 5-methylcytosine and Half-life period of the IX factor modification mRNA that pseudouridine is modified completely in human embryo kidney 293 (HEK293) cell is that about 8-10 is small When.
83. saline formulation of embodiment: subcutaneous administration
Human G-CSF is modified into mRNA (mRNA sequence shown in SEQ ID NO:21438；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) and people EPO modification MRNA (mRNA sequence shown in SEQ ID NO:1638；PolyA tail with about 160 nucleotide does not show in sequence Out；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) it is formulated in salt water and flesh is passed through with the dosage of 100ug Interior (IM) injected delivery is to mouse.
Control includes luciferase (mRNA sequence shown in SEQ ID NO:21446；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) or preparation buffer (F. buffer).13 hours by mouse bloodletting after injection, to measure the concentration (pg/mL) of human polypeptides in serum.(G-CSF group Human G-CSF is measured in mice serum and EPO group measures people EPO in mice serum).Data are shown in table 130.
In the case where preparation is not present, mRNA fast degradation in serum shows to deliver mRNA to continue in systems The best method of long period is by preparing mRNA.As shown in table 130, it can be used only and prepare buffer subcutaneous delivery mRNA.
130. dosage regimen of table
84. Intravitreal delivery of embodiment
The mCherry being formulated in salt water is modified into mRNA (mRNA sequence shown in SEQ ID NO:21439；Have The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is repaired completely with 5-methylcytosine and pseudouridine Decorations) and luciferase modification mRNA (mRNA sequence shown in SEQ ID NO:21446；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) Intravitreal delivery in Rat, as described in table 131.Sample is compared with the only control with salt water of Intravitreal delivery.
Chart is administered in table 131.
On day 1, while making Animal Anesthesia, preparation is applied to the left eye or right eye of every animal.In application The previous day gives gentamicin ophthalmic ointment or solution twice to two eyes.Also gives and infusing immediately after injection Give gentamicin ophthalmic ointment or solution in second day penetrated.Before administration, it is given to each eye and expands pupil drops (1% support Pyrrole card amine and/or 2.5% phenylephrine).
18 hours upon administration, mCherry will be received and deliver the eyes extraction of the dosage of buffer, and by every eye Eyeball is separately positioned in the pipe containing 4% paraformaldehyde of 10mL in room temperature to be fixed for tissue overnight.Second day, by eye Eyeball is individually transferred in the pipe of 30% sucrose containing 10mL and is stored in 21 DEG C until they are processed and are sliced.In F- It is evaluated under microscope by the glass slide of different slice preparations.In the glass slide of the eyes preparation with application mCherry modification mRNA In observe positive expression, and compare and do not show expression.
Cytokine-expressing is studied in 85. body of embodiment
To mouse intramuscular injection 200ug without modified with 5 ' cap Cap1 (unmodified), with 5-methylcytosine and Pseudouridine and 5 ' cap Cap1 modify completely (Gen1) or with 5-methylcytosine and N1- methyl-pseudouridine and 5 ' caps Cap1 (Gen2 cap) or the G-CSF modification mRNA modified completely without cap (not capped Gen2) (show in SEQ ID NO:21438 MRNA sequence out；PolyA tail with about 160 nucleotide is not shown in sequence).Also to R-848,5% sucrose and The control of untreated mouse is analyzed.After 8 hours, serum is collected from mouse and is expressed for interferon-' alpha ' (IFN-α) It is analyzed.As a result it is shown in table 132.
Table 132.IFN- alpha expression
Preparation IFN-α(pg/ml)
Unmodified G-CSF 67.012
G-CSF Gen1 8.867
G-CSF Gen2 cap 0
Not capped G-CSF Gen2 0
R-848 40.971
5% sucrose 1.493
It is untreated 0
The vivoexpression of embodiment 86.VEGF modification mRNA
With concentration shown in table 133, with and from Invitrogen (Carlsbad, CA) Lipofectamine2000 compound modification mRNA (mmRNA) VEGF-A (mRNA sequence shown in SEQ ID NO:1672； PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is complete with 5-methylcytosine and pseudouridine Full modification) transfected HEK 293.Protein expression is detected by ELISA, and protein (pg/ml) is in table 133 and Fig. 7 It shows.
133. protein expression of table
In-vitro screening of the embodiment 87.GFP in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA in).Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, by having in table 134 for 37.5ng or 75ng The green fluorescent protein (GFP) of the chemical modification modifies RNA (mRNA sequence shown in SEQ ID NO:21448；Have The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 0.2ul is diluted in the OPTI-MEM of 10ul final volume.In room temperature After five minutes, two kinds of solution are merged, and is incubated for again at room temperature 15 minutes for lower incubation.Then solution 20ul merged adds It is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.
After being incubated for 18 to 22 hours, with the Passive Lysis Buffer of 100ul (Promega, Madison, WI) according to the explanation of manufacturer by the cell cracking of expressing luciferase.It is impermeable that the aliquot of lysate is transferred to white Bright 96 hole plate of polystyrene (Corning, Manassas, VA), and the complete luciferase assay solution with 100ul (Promega, Madison, WI) merges.It measures the median fluorescence intensity (MFI) of every kind of chemical substance and is shown in table 134.
These results indicate that being repaired completely compared with other chemical substances with N1- methyl-pseudouridine and 5-methylcytosine The GFP of decorations generates greater protein matter in HeLa cell.In addition, the GFP for being applied to the higher doses of cell leads to highest MFI Value.
134. average fluorescent strength of table
Embodiment 88. is homogenized
To different luciferase mRNA solution (as described in table 135, wherein " X " refers to the solution comprising the component) (mRNA sequence shown in SEQ ID NO:21446；PolyA tail with about 160 nucleotide is not shown in sequence；5' Cap, Cap1；Modified completely with 5-methylcytosine and pseudouridine) it is evaluated, to test the yield percentage of different solutions, lead to It crosses the integrality of biological analyser test mRNA and tests the protein expression of mRNA by in-vitro transfection.Such as meaning in table 135 Out, mRNA solution is prepared with 4mg/ml in water, 1x TE buffer, and is added to methylene chloride (DCM) or contains 200mg/ml poly- (lactic-co-glycolic acid) (PLGA) (Lactel, catalog number (Cat.No.) B6010-2, intrinsic viscosity 0.55-0.75,50: 50 LA: GA) in DCM, to realize the final mRNA concentration of 0.8mg/ml.To need the solution that is homogenized speed 5 (about 19, 000rpm) it is homogenized 30 seconds under (IKA Ultra-Turrax Homogenizer, T18).In water, methylene chloride and poly- (lactic acid- Co- glycolic) the mRNA sample in (PLGA) is expendable (NR).All samples other than NR sample keep mRNA Integrality, as measured by biological analyser (Bio-rad Experion).
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Grow cell overnight in 5%CO2 atmosphere at 37 DEG C.It second day, will be from recyclable sample The luciferase mRNA of 250ng is diluted in OPTI-MEM (LifeTechnologies, the Grand of 10ul final volume Island, NY) in.Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) as transfection Reagent, and 0.2ul is diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, by two kinds of solution Merge, and is incubated for again at room temperature 15 minutes.Then solution 20ul merged is added to 100ul and contains the thin of HeLa cell In born of the same parents' culture medium.Then it is incubated for plate as described above.Also to the control luciferase mRNA (luciferase prepared in salt water MRNA) (Control) and untreated cell (Untreat.) are evaluated.The bioluminescence for harvesting cell and each signal is flat Mean value (in terms of photons/second) (biolum. (p/s)) is also shown in table 135.In analysis, recyclable sample all shows glimmering The activity of light element enzyme mRNA.
After being incubated for 18 to 22 hours, with the Passive Lysis Buffer of 100ul (Promega, Madison, WI) according to the explanation of manufacturer by the cell cracking of expressing luciferase.It is impermeable that the aliquot of lysate is transferred to white Bright 96 hole plate of polystyrene (Corning, Manassas, VA), and the complete luciferase assay solution with 100ul (Promega, Madison, WI) merges.The background signal for not having the plate of reagent is about 200 relative light units/hole.Plate is read Taking device is BioTek Synergy H1 (BioTek, Winooski, VT).
Harvest the bioluminescence average value (in terms of relative light unit RLU) (biolum. (RLU)) of cell and each signal Also it is shown in table 135.In analysis, recyclable sample all shows the activity of luciferase mRNA.
135. solution of table
Embodiment 89.TE buffer and water quality evaluation
As in table 136 general introduction by luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446；With big The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is modified completely with 5-methylcytosine and pseudouridine) In water or TE buffer then reconstruct is simultaneously formulated in PLGA microballoon.Use water as known in the art/oil/water second emulsifying Method uses PLGA (Lactel, catalog number (Cat.No.) B6010-2, intrinsic viscosity 0.55-0.75,50: 50 LA: GA), polyvinyl alcohol (PVA) (Sigma, catalog number (Cat.No.) 348406-25G, MW 13-23k) methylene chloride and hydration are at PLGA microballoon.Briefly, will MRNA (W1) of the 0.2ml to 0.6ml of 2mg/ml to 6mg/ml in water or TE buffer is added to 2ml in 100mg/ml It is dissolved under PLGA concentration in the PLGA (O1) of methylene chloride (DCM).At speed 5 (~19,000rpm) by W1/O1 lotion Change (IKA Ultra-Turrax Homogenizer, T18) 30 seconds.Then W1/O1 lotion is added to 250ml 1%PVA (W2) it in, and is homogenized 1 minute at speed 5 (~19,000rpm).
It stirs preparation 3 hours, then makes it through 100 μm of nylon net filter device (Fisherbrand Cell Strainer, catalog number (Cat.No.) 22-363-549) to remove biggish aggregation, and eventually by centrifugation (10min, 9,250rpm, 4 DEG C) it is washed.Liquid is discarded supernatant, and PLGA precipitating is resuspended in the water of 5-10ml, is repeated 2 times.It will be by washing The preparation washed freezes in liquid nitrogen, is then lyophilized 2-3 days.After freeze-drying, weighed up in 2ml eppendorf pipe~10mg PLGA MS, and pass through the DCM for adding 1ml and so that sample is vibrated 2-6 hours to make its depolymerization.By addition 0.5ml water and make Sample shaken overnight from the PLGA microballoon of de- preparation extracts mRNA.The luciferase that will not be prepared in water or TE buffer MRNA (the de- control prepared) is added in DCM, and is subjected to depolymehzation process, to be used as control in transfection measurement.
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Grow cell overnight in 5%CO2 atmosphere at 37 DEG C.Second day, by the de- preparation of 100ng Luciferase mRNA sample be diluted in 10ul final volume OPTI-MEM (LifeTechnologies, Grand Island, NY in).It is used as transfection reagent using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY), and And 0.2ul is diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, two kinds of solution is merged, and It is incubated for again at room temperature 15 minutes.Then solution 20ul merged is added to the cell culture that 100ul contains HeLa cell In base.Then it is incubated for plate as described above.
After being incubated for 18 to 22 hours, with the Passive Lysis Buffer of 100ul (Promega, Madison, WI) according to the explanation of manufacturer by the cell cracking of expressing luciferase.It is impermeable that the aliquot of lysate is transferred to white Bright 96 hole plate of polystyrene (Corning, Manassas, VA), and the complete luciferase assay solution with 100ul (Promega, Madison, WI) merges.The background signal for not having the plate of reagent is about 200 relative light units/hole.Plate is read Taking device is BioTek Synergy H1 (BioTek, Winooski, VT).In order to measure the luciferase from every kind of preparation The relative light unit (RLU) of every kind of preparation is taken off the RLU of preparations. Control (in water or TE divided by mRNA appropriate by the activity of mRNA MRNA in buffer).Table 136 shows the activity of luciferase mRNA.By being formulated in TE buffer (relative to water), show Work improves the activity of luciferase mRNA in PLGA microball preparation (Form.).
136. preparation of table
Chemical modification on embodiment 90.mRNA
In the day before transfection, by with trypsin-EDTA solutions (Life Technologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 83ng had into table 137 Described in chemical modification luciferase modify RNA (mRNA sequence shown in SEQ ID NO:21446；With about 140 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 0.2ul is diluted in the OPTI-MEM of 10ul final volume.In room temperature After five minutes, two kinds of solution are merged, and is incubated for again at room temperature 15 minutes for lower incubation.Then solution 20ul merged adds It is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.
After being incubated for 18 to 22 hours, with the Passive Lysis Buffer of 100ul (Promega, Madison, WI) according to the explanation of manufacturer by the cell cracking of expressing luciferase.It is impermeable that the aliquot of lysate is transferred to white Bright 96 hole plate of polystyrene (Corning, Manassas, VA), and the complete luciferase assay solution with 100ul (Promega, Madison, WI) merges.Lysate volume is adjusted or dilutes, until the sample detection for generating peak signal It is shown in table 137 to the RLU for being no more than 2mio relative light unit (RLU)/hole, the every kind of chemical substance tested.Plate is read Taking device is BioTek Synergy H1 (BioTek, Winooski, VT).The background signal for not having the plate of reagent is about 200 phases To light unit/hole.
137. chemical modification of table
The intramuscular and subcutaneous administration of the modification of embodiment 91. mRNA
To be formulated in PBS (pH 7.4) modified completely with 5-methylcytosine and pseudouridine (5mC/pU), use 5- It is that methylcystein and N1- methyl-pseudouridine (5mC/N1mpU) are modified completely, being modified completely with pseudouridine (pU), use N1- It is that methyl-pseudouridine (N1mpU) is modified completely or in which replace 25% cytimidine with 5-methylcytosine and with the thio urine of 2- The luciferase modification mRNA that glycosides is modified instead of 25% uridine (5mC/s2U) is (shown in SEQ ID NO:21446 MRNA sequence；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) with the dosage of 2.5mg/kg Intramuscular or subcutaneous administration is to Balb-C mouse.For intramuscular delivering, the 2nd hour, the 8th hour, the 24th hour, the 48th hour, 72nd hour, the 96th hour, the 120th hour and the 144th hour, and for subcutaneous delivery, the 2nd hour, the 8th hour, Mouse was imaged in 24 hours, the 48th hour, the 72nd hour, the 96th hour and the 120th hour.20 minutes before imaging, with 150mg/kg injects D- luciferin solution into mouse peritoneum.Then it is by Animal Anesthesia and using IVIS Lumina II imaging System (Perkin Elmer) acquires image.Bioluminescence is measured as the total flow (photons/second) of whole mouse.Intramuscular administration is put down Equal total flow (photons/second) is shown in table 138, and the average overall flow rate (photons/second) of subcutaneous administration shows in table 139 Out.Background signal is 3.79E+05 (p/s).For all chemical substances, flesh is observed between the 24th hour and the 48th hour The peak value of interior application is expressed, and still detects expression at the 144th hour.For subcutaneous delivery, at the 2nd hour to the 8th hour It observes that peak value is expressed, and detected expression at the 72nd hour.
138. intramuscular administration of table
139. subcutaneous administration of table
92. osmotic pumps research of embodiment
Before implantation, to osmotic pumps (Osmotic Pump 2001D, DURECT Corp.Cupertino, CA) load 0.2ml 1X PBS (pH 7.4) (PBS load pump) or 0.2ml in 1x PBS (pH 7.4) luciferase of the 1mg/ml in modifies mRNA (mRNA sequence shown in SEQ ID NO:21446；With about 140 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1；It is repaired completely with 5-methylcytosine and N1- methyl-pseudouridine Decorations) it (luciferase load pump) and is incubated overnight at 37 DEG C in 1x PBS (pH 7.4).
It is subcutaneously implanted PBS load pump or luciferase load pump to Balb-C mouse (n=3), and the 2nd hour, the 8th Hour and the 24th hour are imaged.As control, PBS load pump is subcutaneously implanted, and to mouse subcutaneous injection in 1x PBS Luciferase modify mRNA (PBS load pump；SC luciferase) or not implantable osmotic pump and to mouse subcutaneous injection in 1x Luciferase in PBS modifies mRNA (SC luciferase).Luciferase preparation is summarized in table 140.
140. luciferase preparation of table
The external osmotic pumps research of embodiment 93.
To external osmotic pumps (Osmotic Pump 2001D, DURECT Corp.Cupertino, CA) it is negative Carry the fluorescein of the 1mg/ml in 1x PBS (pH 7.4) of the 1X PBS (pH 7.4) (PBS load pump) or 0.2ml of 0.2ml Enzyme modification mRNA (mRNA sequence shown in SEQ ID NO:21446；PolyA tail with about 140 nucleotide, sequence In be not shown；5 ' caps, Cap1；Modified completely with 5-methylcytosine and N1- methyl-pseudouridine) (luciferase load pump) simultaneously And it is incubated overnight at 37 DEG C in 1x PBS (pH 7.4).
Using the conduit for being connected to external PBS load pump or luciferase load pump, applied to Balb-C mouse (n=3) Preparation.Mouse is imaged at the 2nd hour, the 8th hour and the 24th hour.As control, using external PBS load pump, and give Luciferase of the mouse subcutaneous injection in 1x PBS modifies mRNA (PBS load pump；SC luciferase) or without using external pump And only the luciferase to mouse subcutaneous injection in 1x PBS modifies mRNA (SC luciferase).20 points before imaging Clock injects D- luciferin solution into mouse peritoneum with 150mg/kg.Then by Animal Anesthesia and use IVIS Lumina II Imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as the total flow (photons/second) of whole mouse.Fluorescein Enzyme preparation is summarized in table 141, and average overall flow rate (photons/second).
141. luciferase preparation of table
The research of 94. fibrin sealant of embodiment
Fibrin sealant is formed in dual barrel syringe by fibrinogen and fibrin ferment, such as Tisseel (Baxter Healthcare Corp., Deerfield, IL).In mixing, fibrinogen is converted to fiber in about 10 seconds to 30 seconds Albumen is to form fibrin clot.The natural clotting mechanism of this grumeleuse analog body.In addition, fibrin hydrogel is can It is potentially served as the three-dimensional structure of Sustained release delivery.Currently, fibrin sealant is approved for stopping blooding and what is sealed answers To replace conventional surgical technique (such as suture, ligature and burn).
Fibrin ferment and fibrinogen component are individually loaded into dual barrel syringe.It is subcutaneous to Balb-C mouse (n=3) Inject the fibrinogen of 50ul, the fibrin ferment of 50ul, and the fluorescein also modified in same area to mouse injection Enzyme mRNA (mRNA sequence shown in SEQ ID NO:21446；PolyA tail with about 140 nucleotide, in sequence not It shows；5 ' caps, Cap1；Modified completely with 5-methylcytosine and N1- methyl-pseudouridine) (Tisseel+ luciferase), 50ul Fibrinogen and 50ul fibrin ferment (Tisseel) or modification luciferase mRNA (luciferase).Use double barrel injection Device carries out the injection of fibrinogen and fibrin ferment simultaneously.15 minutes progress fluoresceins after fibrinogen/Thrombin The SC of enzyme is injected, to allow fibrin hydrogel polymeric (Tisseel+ luciferase group).Also to the control of untreated mice Group is evaluated.Mouse is imaged at the 5th hour and the 24th hour.20 minutes before imaging, with 150mg/kg to mouse abdomen D- luciferin solution is injected in film.Then by Animal Anesthesia and use IVIS Lumina II imaging system (Perkin Elmer) Acquire image.Bioluminescence is measured as the total flow (photons/second) of whole mouse.Luciferase preparation is summarized in table 142, and And average overall flow rate (photons/second) is shown in table 143.It was found that fibrin sealant do not interfere imaging and luciferase and The injection of Tisseel shows the expression of luciferase.
142. luciferase preparation of table
143. total flow of table
Fibrin sealant research of the embodiment 95. containing mRNA
A.Modify mRNA and calcium chloride
Before reconstruct, it will be modified completely with 5-methylcytosine and N1- methyl-pseudouridine or with N1- methyl-pseudouridine Luciferase mRNA (the mRNA sequence shown in SEQ ID NO:21446 modified completely；With about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is added in calcium chloride.Then fibrin ferment is reconstructed using calcium chloride.Use fiber Protein dissolution inhibitor solution illustrates reconstruct fibrinogen according to manufacturer.The blood coagulation containing modification mRNA that will be reconstructed Enzyme and fibrinogen are loaded into dual barrel syringe.It is modified to mouse subcutaneous injection 50ul fibrinogen and containing for 50ul The PBS of the fibrin ferment of mRNA or the modification luciferase mRNA containing equivalent dose to mouse injection 50ul.It is also right The control group of untreated mice is evaluated.Mouse is imaged to measure average overall flow rate (photons/second) in predetermined space.
B.The modification mRNA and calcium chloride that lipidic nanoparticles are prepared
It is complete with 5-methylcytosine and N1- methyl-pseudouridine in lipidic nanoparticles by being formulated in front of reconstruct Modification or the luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446 modified completely with N1- methyl-pseudouridine； PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) it is added in calcium chloride.Then chlorine is used Change calcium and reconstructs fibrin ferment.Illustrate reconstruct fibrinogen according to manufacturer with fibrinolysis inhibitor solution.It will weigh The fibrin ferment containing modification mRNA and fibrinogen of structure are loaded into dual barrel syringe.To mouse subcutaneous injection 50ul fiber The fibrin ferment containing modification mRNA or the modification containing equivalent dose to mouse injection 50ul of proteinogen and 50ul The PBS of luciferase mRNA.Also the control group of untreated mice is evaluated.Mouse is imaged in predetermined space flat to measure Equal total flow (photons/second).
C.Modify mRNA and fibrinogen
Before reconstruct, it will be modified completely with 5-methylcytosine and N1- methyl-pseudouridine or with N1- methyl-pseudouridine Luciferase mRNA (the mRNA sequence shown in SEQ ID NO:21446 modified completely；With about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is added in fibrinolysis inhibitor solution.Then fiber egg is used White dissolution inhibition agent solution reconstructs fibrinogen.Illustrate reconstruct fibrin ferment according to manufacturer with calcium chloride solution.It will weigh The fibrinogen containing modification mRNA and fibrin ferment of structure are loaded into dual barrel syringe.To mouse subcutaneous injection 50ul blood coagulation The fibrinogen containing modification mRNA or the modification containing equivalent dose to mouse injection 50ul of enzyme and 50ul The PBS of luciferase mRNA.Also the control group of untreated mice is evaluated.Mouse is imaged in predetermined space flat to measure Equal total flow (photons/second).
D.The modification mRNA and fibrinogen that lipidic nanoparticles are prepared
It is complete with 5-methylcytosine and N1- methyl-pseudouridine in lipidic nanoparticles by being formulated in front of reconstruct Modification or the luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446 modified completely with N1- methyl-pseudouridine； PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) it is added to fibrinolysis inhibitor In solution.Then fibrinogen is reconstructed using fibrinolysis inhibitor solution.With calcium chloride solution according to manufacturer Illustrate to reconstruct fibrin ferment.The fibrinogen containing modification mRNA and fibrin ferment that are reconstructed are loaded into dual barrel syringe. It is injected to the fibrinogen containing modification mRNA of mouse subcutaneous injection 50ul fibrin ferment and 50ul or to the mouse The PBS of the modification luciferase mRNA containing equivalent dose of 50ul.Also the control group of untreated mice is evaluated.Pre- Mouse is imaged to measure average overall flow rate (photons/second) in fixed interval.
E.Modify mRNA and fibrin ferment
Before reconstruct, it will be modified completely with 5-methylcytosine and N1- methyl-pseudouridine or with N1- methyl-pseudouridine Luciferase mRNA (the mRNA sequence shown in SEQ ID NO:21446 modified completely；With about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is added to reconstructed fibrin ferment (in the saying according to manufacturer with calcium chloride After the bright reconstruct fibrin ferment) in.Then illustrate that reconstruct is fine according to manufacturer using fibrinolysis inhibitor solution Fibrillarin is former.The fibrinogen reconstructed and the fibrin ferment containing modification mRNA are loaded into dual barrel syringe.To mouse The fibrin ferment and 50ul fibrinogen or inject containing for 50ul to the mouse that subcutaneous injection 50ul contains modification mRNA The PBS of the modification luciferase mRNA of equivalent dose.Also the control group of untreated mice is evaluated.In predetermined space to small Mouse is imaged to measure average overall flow rate (photons/second).
F.The modification mRNA and fibrin ferment that lipidic nanoparticles are prepared
It is complete with 5-methylcytosine and N1- methyl-pseudouridine in lipidic nanoparticles by being formulated in front of reconstruct Modification or the luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446 modified completely with N1- methyl-pseudouridine； PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) be added to reconstructed fibrin ferment (with Calcium chloride according to manufacturer illustrate to reconstruct the fibrin ferment after) in.Then it is pressed using fibrinolysis inhibitor solution Illustrate reconstruct fibrinogen according to manufacturer.The fibrinogen reconstructed and the fibrin ferment containing modification mRNA are loaded into In dual barrel syringe.Contain the fibrin ferment and 50ul fibrinogen or to described of modification mRNA to mouse subcutaneous injection 50ul Mouse injects the PBS of the modification luciferase mRNA containing equivalent dose of 50ul.Also the control group of untreated mice is carried out Evaluation.Mouse is imaged to measure average overall flow rate (photons/second) in predetermined space.
The Cationic Lipid Formulations of 96. 5-methylcytosine of embodiment and N1- methyl-pseudouridine modification mRNA
By the luciferase mRNA modified completely with 5-methylcytosine and N1- methyl-pseudouridine (SEQ ID NO: MRNA sequence shown in 21446；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) match It is formed in cation lipid described in table 144.With the dosage of 0.05mg/kg into Balb-C mouse vein (I.V.), intramuscular (I.M.) or subcutaneous (S.C.) applies preparation.
144. Cationic Lipid Formulations of table
20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by animal fiber crops Liquor-saturated and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as whole mouse Total flow (photons/second).Mouse is imaged in 2 hours upon administration, 8 hours and 24 hours, and to every kind of administration method and sun The average overall flow rate (photons/second) of cationic lipid formulations measures.Background traffic is about 4.17E+05p/s.The result of imaging It is shown in table 145.In table 145, " T ", which refers to, not to be tested.
145. flow of table
97. lipidic nanoparticles of embodiment are intravenously studied
By luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) it is formulated in containing 50% The DLin-MC3-DMA as described in table 146 or DLin-KC2-DMA, 38.5% cholesterol, 10%DSPC and 1.5%PEG In lipidic nanoparticles.With the dosage of 0.5mg/kg, 0.05mg/kg, 0.005mg/kg or 0.0005mg/kg to Balb-C mouse Intravenously (I.V.) applies preparation.20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg. Then image is acquired by Animal Anesthesia and using IVIS Lumina II imaging system (Perkin Elmer).Bioluminescence measurement For the total flow (photons/second) of whole mouse.
146. preparation of table
For DLin-KC2-DMA, 2 hours upon administration, 8 hours, 24 hours, 72 hours, 96 hours and 168 hours are right Mouse imaging, and the average overall flow rate of every kind of administration method and Cationic Lipid Formulations (photons/second) is measured.Background Flow is about 3.66E+05p/s.The result of imaging is shown in table 147.At the 8th hour to imaging organs, and measure liver, The average overall flow rate (photons/second) of spleen, lung and kidney.Also the control of every kind of organ is analyzed.As a result it is shown in table 147. The peak signal of all dosage levels is in after application 8 hours.In addition, by increased or decrease LNP dosage can control to The distribution of Different Organs (liver, spleen, lung and kidney).
147. flow of table
148. organ flow of table
For DLin-MC3-DMA, 2 hours upon administration, 8 hours, 24 hours, 48 hours, 72 hours and 144 hours are right Mouse imaging, and the average overall flow rate of every kind of administration method and Cationic Lipid Formulations (photons/second) is measured.Background Flow is about 4.51E+05p/s.The result of imaging is shown in table 149.At the 8th hour to imaging organs, and measure liver, The average overall flow rate (photons/second) of spleen, lung and kidney.Also the control of every kind of organ is analyzed.As a result it is shown in table 150. The peak signal of all dosage levels is in after application 8 hours.In addition, by increased or decrease LNP dosage can control to The distribution of Different Organs (liver, spleen, lung and kidney).
149. flow of table
150. organ flow of table
98. lipidic nanoparticles of embodiment are subcutaneously studied
By luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) it is formulated in containing 50% The DLin-KC2-DMA as described in table 151,385% cholesterol, 10%DSPC and 1.5%PEG lipidic nanoparticles in. Preparation is applied to Balb-C mouse subcutaneous (S.C.) with the dosage of 0.5mg/kg, 0.05mg/kg or 0.005mg/kg.
Table 151.DLin-KC2-DMA preparation
20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by animal fiber crops Liquor-saturated and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as whole mouse Total flow (photons/second).Mouse is imaged in 2 hours upon administration, 8 hours, 24 hours, 48 hours, 72 hours and 144 hours, And the average overall flow rate of every kind of administration method and Cationic Lipid Formulations (photons/second) is measured.The lower limit of detection is About 3E+05p/s.The result of imaging is shown in table 152.At the 8th hour to imaging organs, and measure liver, spleen, lung and kidney Average overall flow rate (photons/second).Also the control of every kind of organ is analyzed.As a result it is shown in table 153.All dosage levels Peak signal be in application after 8 hours.In addition, by increased or decrease LNP dosage can control to Different Organs (liver, Spleen, lung and kidney) distribution.At high doses, LNP preparation moves to the outside of subcutaneous infusion sites because liver, spleen, lung and High-caliber luciferase expression is detected in kidney.
152. flow of table
153. organ flow of table
99. cation lipid nano particle of embodiment is subcutaneously studied
By luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) it is formulated in containing 50% DLin-MC3-DMA, 38.5% cholesterol, 10%DSPC and 1.5%PEG lipidic nanoparticles in.With 0.5mg/kg, The dosage of 0.05mg/kg or 0.005mg/kg applies preparation to Balb-C mouse subcutaneous (S.C.).
Mouse is imaged in 2 hours upon administration, 8 hours, 24 hours, 48 hours, 72 hours and 144 hours, and to every The average overall flow rate (photons/second) of kind administration method and Cationic Lipid Formulations measures.At the 8th hour to imaging organs, And measure the average overall flow rate (photons/second) of liver, spleen, lung and kidney.Also the control of every kind of organ is analyzed.
The research of 100. luciferase lipid complex of embodiment
With 5-methylcytosine and pseudouridine (5mC/pU) completely modification, with 5-methylcytosine and N1- methyl-pseudouridine (5mC/N1mpU) completely modification or in which with 5-methylcytosine replace 25% cytimidine and replace 25% with 2- thio uridine Compound luciferase mRNA (the mRNA sequence shown in SEQ ID NO:21446 of the lipid modified of uridine (5mC/s2U) Column；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1).With the dosage of 0.10mg/kg to (I.V.), intramuscular (I.M.) or subcutaneous (S.C.) applies preparation in Balb-C mouse vein.
20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by animal fiber crops Liquor-saturated and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as whole mouse Total flow (photons/second).Mouse is imaged in 8 hours upon administration, 24 hours and 48 hours, and to every kind of administration method and change The average overall flow rate (photons/second) for learning modification measures.Background signal is about 3.91E+05p/s.The result of imaging is in table 154 In show.At the 6th hour to imaging organs, and measure the average overall flow rate (photons/second) of liver, spleen, lung and kidney.Also to every kind The control of organ is analyzed.As a result it is shown in table 155.
154. flow of table
155. organ flow of table
The Cationic Lipid Formulations of the modification of embodiment 101. mRNA
25% cytimidine wherein will be replaced with 5-methylcytosine and replace with 2- thio uridine 25% uridine (5mC/ S2U the luciferase mRNA (mRNA sequence shown in SEQ ID NO:21446) modified；With about 140 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1) it is formulated in cation lipid described in table 156.With 0.05mg/ The dosage of kg (I.V.), intramuscular (I.M.) or subcutaneous (S.C.) into Balb-C mouse vein applies preparation.
156. Cationic Lipid Formulations of table
20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by animal fiber crops Liquor-saturated and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as whole mouse Total flow (photons/second).Mouse is imaged in 2 hours upon administration, 8 hours and 24 hours, and to every kind of administration method and sun The average overall flow rate (photons/second) of cationic lipid formulations measures.Background traffic is about 3.31E+05p/s.The result of imaging It is shown in table 157.In table 157, " NT ", which refers to, not to be tested.The average flow rate that untreated mouse shows is small the 2nd When be 3.14E+05, be 3.33E+05 at the 8th hour and be 3.46E+05 at the 24th hour.For all three tested Approach observed that peak value is expressed at the 8th hour.DLin-KC2-DMA has expression more better than DLin-MC3-DMA, and right In all approach evaluated, DODMA all shows expression.
157. flow of table
The preparation of embodiment 102.5- methylcystein and N1- methyl-pseudouridine modification mRNA
By the luciferase mRNA modified completely with 5-methylcytosine and N1- methyl-pseudouridine (SEQ ID NO: MRNA sequence shown in 21446；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) match It is formed in PBS (pH 7.4).With the dosage of 2.5mg/kg, into Balb-C Mouse Muscle, (I.M.) or subcutaneous (S.C.) applies preparation.
20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by animal fiber crops Liquor-saturated and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as whole mouse Total flow (photons/second).Mouse is imaged in 5 minutes upon administration, 30 minutes, 60 minutes and 120 minutes, and applies to every kind The average overall flow rate (photons/second) of approach and Cationic Lipid Formulations measures.Background traffic is about 3.78E+05p/s.At The result of picture is shown in table 158.Under two kinds of route of delivery, the expression of luciferase was had observed that at the 30th minute. The peak value expression of subcutaneous administration appears between the 30th minute to the 60th minute.Intramuscular expression was still increasing at the 120th minute Greatly.
158. flow of table
The intramuscular and subcutaneous administration of 103. chemical modification mRNA of embodiment
With the dosage of 2.5mg/kg into Balb-C Mouse Muscle or subcutaneous administration is formulated in PBS (pH 7.4) and uses N4- Acetyl group cytidine is modified completely, is modified completely with 5- methoxyuridine, is complete with N4- acetyl group cytidine and N1- methyl-pseudouridine Modification modifies mRNA (SEQ ID NO:21446 with the luciferase that 5-methylcytosine and 5- methoxyuridine are modified completely Shown in mRNA sequence；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1).It is being imaged 20 minutes before, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by Animal Anesthesia and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence be measured as whole mouse total flow (photon/ Second).Mouse is imaged at the 2nd hour, the 8th hour and the 24th hour.The average overall flow rate (photons/second) of intramuscular administration is in table It is shown in 159, and the average overall flow rate (photons/second) of subcutaneous administration is shown in table 160.Background signal is 3.84E+05 (p/s).For all chemical substances, the peak value expression of intramuscular administration is observed between the 24th hour and the 48th hour, and Expression was still detected at the 120th hour.For subcutaneous delivery, observed that peak value is expressed at the 2nd hour to the 8th hour, and Expression is arrived in detection in 72nd hour.
159. intramuscular administration of table
160. subcutaneous administration of table
104. In vivo study of embodiment
The luciferase modification mRNA containing at least one chemical modification is formulated as lipid nanometer using injection syringe pump method Grain (LNP), and it is characterized by granularity, zeta potential and encapsulating.
As summarized in table 161, into Balb-C Mouse Muscle, (I.M.), intravenous (I.V.) and subcutaneous (S.C.) application is glimmering Light element enzyme LNP preparation.As control, the luciferase modification RNA being formulated in PBS is applied into mouse vein.
161. luciferase preparation of table
Mouse is imaged at the 2nd, 8,24,48,120 and 192 hour, (is measured as entire mouse to measure bioluminescence Total flow (photons/second)).Injection the 8th hour and the 192nd hour, to liver,spleen,kidney and subcutaneous administration and intramuscular administration Image areas, to measure bioluminescence.
The Cationic Lipid Formulations of 105. chemical modification mRNA of embodiment are studied
It will be complete with 5-methylcytosine and pseudouridine (5mC/pU), pseudouridine (pU) or N1- methyl-pseudouridine (N1mpU) Luciferase mRNA (the mRNA sequence shown in SEQ ID NO:21446 modified entirely；With about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is formulated in cation lipid described in table 162.With 0.05mg/kg's Dosage (I.V.), intramuscular (I.M.) or subcutaneous (S.C.) into Balb-C mouse vein applies preparation.
162. Cationic Lipid Formulations of table
20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by animal fiber crops Liquor-saturated and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as whole mouse Total flow (photons/second).Mouse is imaged in 2 hours upon administration, 8 hours and 24 hours, and to every kind of administration method and sun The average overall flow rate (photons/second) of cationic lipid formulations measures.Background traffic is about 4.11E+05p/s.The result of imaging It is shown in table 163.For all three approach tested, observed that peak value is expressed at the 8th hour.
163. flow of table
The research of the mRNA of 106. chemical modification of embodiment
It will be modified completely with N4- acetyl group cytidine (N4- acetyl group), and be modified, used completely with 5- methoxyuridine (5meth) N4- acetyl group cytidine and N1- methyl-pseudouridine (N4- acetyl group/N1mpU) modification completely or with 5-methylcytosine and 5- first Luciferase mRNA (the mRNA sequence shown in SEQ ID NO:21446 that oxygroup uridine (5mC/5-meth) is modified completely；Tool There is the polyA tail of about 140 nucleotide, is not shown in sequence；5 ' caps, Cap1) it is formulated in DLin-MC3-DMA, such as table Described in 164.
(I.V.), intramuscular (I.M.) or subcutaneous (S.C.) application into Balb-C mouse vein with the dosage of 0.05mg/kg Preparation.
164. Cationic Lipid Formulations of table
20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by animal fiber crops Liquor-saturated and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as whole mouse Total flow (photons/second).Mouse is imaged in 2 hours upon administration, 6 hours and 24 hours, and to every kind of administration method and sun The average overall flow rate (photons/second) of cationic lipid formulations measures.Background traffic is about 2.70E+05p/s.The result of imaging It is shown in table 165.
165. flow of table
Embodiment 107. is containing there are many lipidic nanoparticles of modification mRNA
By EPO mRNA (mRNA sequence shown in SEQ ID NO:1638；PolyA with about 140 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and N1- methyl-pseudouridine), G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 140 nucleotide is not shown in sequence；5' Cap, Cap1；Modified completely with 5-methylcytosine and N1- methyl-pseudouridine) and IX factor mRNA (SEQ ID NO:1622 Shown in mRNA sequence；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5- methyl Cytimidine and N1- methyl-pseudouridine are modified completely) it is formulated in DLin-MC3-DMA, as described in table 166.With 0.05mg/kg Dosage into Balb-C mouse vein (I.V.), intramuscular (I.M.) or subcutaneous (S.C.) apply preparation.Also applied with equivalent dose With the control LNP preparation for only containing a kind of mRNA.
Table 166.DLin-MC3-DMA preparation
Serum is collected from mouse within 8 hours, 24 hours, 72 hours and/or 7 days after applying preparation.Pass through elisa assay Serum is to measure the protein expression of EPO, G-CSF and IX factor.
The Cationic Lipid Formulations research of 108. 5-methylcytosine of embodiment and N1- methyl-pseudouridine modification mRNA
By EPO mRNA (mRNA sequence shown in SEQ ID NO:1638；PolyA with about 140 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and N1- methyl-pseudouridine) or G-CSF mRNA (mRNA sequence shown in SEQ ID NO:21438；PolyA tail with about 140 nucleotide is not shown in sequence；5' Cap, Cap1；Modified completely with 5-methylcytosine and N1- methyl-pseudouridine) it is formulated in DLin-MC3-DMA and DLin-KC2- In DMA, as described in table 167.With the dosage of 0.05mg/kg into Balb-C mouse vein (I.V), intramuscular (I.M.) or subcutaneous (S.C.) preparation is applied.
Table 167.DLin-MC3-DMA and DLin-KC2-DMA preparation
Serum is collected from mouse within 8 hours, 24 hours, 72 hours and/or 7 days after applying preparation.Pass through elisa assay Serum, to measure the protein expression of EPO and G-CSF.
The external VEGF PBMC of embodiment 109. research
500ng 5-methylcytosine and pseudouridine are modified into (VEGF 5mC/pU) completely, with 5-methylcytosine and N1- methyl-pseudouridine modifies VEGF mRNA (the SEQ ID of (VEGF 5mC/N1mpU) or unmodified (VEGF unmod) completely MRNA sequence shown in NO:1672；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) It is transfected into the peripheral blood from three normal blood donors (D1, D2 and D3) together with the Lipofectamine 2000 of 0.4uL In monocyte (PBMC).For each donor, control also is used as without processing to cell.22 hours after transfection, receive It obtains supernatant and passes through elisa assay, to measure protein expression and cytokine induction.The expression of VEGF and IFN-α induction It is shown in table 168 and Fig. 8 A and 8B.
168. protein of table and cytokine levels
The vivoexpression of the modification of embodiment 110. mRNA
With concentration shown in table 169,170 and 171, using program as described herein, mRNA (SEQ ID is modified with EPO MRNA sequence shown in NO:1638；PolyA tail with 160 nucleotide is not shown in sequence；5 ' caps, Cap1；Use 5- Methylcystein and pseudouridine are modified completely) transfected HEK 293；The mRNA modified with transforming growth factor β (TGF-β) (mRNA sequence shown in SEQ ID NO:1668；Poly A tail with about 160 nucleotide is not shown in sequence；5' Cap, Cap1；Modified completely with 5-methylcytosine and pseudouridine) forward direction transfection HeLa cell；And with come from The Lipofectamine2000 of Invitrogen (Carlsbad, CA) compound Bactericidal/Permeability Increasing Protein (rBPI-21) MRNA (the SEQ ID NO:21449 of modification；Poly A tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) transfection HepG 2 cell.Protein expression is detected by ELISA, and And protein (pg/ml) is also shown in table 169,170 and 171.In table 169, " > ", which is meant, to be greater than.For TGF-β, also The simulation transfection of control and Lipofectamine2000 to untreated cell is tested.
Table 169.EPO protein expression
Table 170.TGF- β protein expression
Table 171.rBPI-21 protein expression
111. bicistronic mRNA of embodiment modifies mRNA
By human embryo kidney epithelial cell (HEK293) be seeded in 96 hole plates (Greiner Bio-one GmbH, Frickenhausen, Germany) on, with 30,000 density by HEK293 be inoculated in 100 μ l cell culture mediums (DMEM, 10%FCS, addition 2mM L-Glutamine, 1mM Sodium Pyruvate and 1x nonessential amino acid (Biochrom AG, Berlin, Germany) and 1.2mg/ml sodium bicarbonate (Sigma-Aldrich, Munich, Germany)) in, after inoculating cell, The bicistronic mRNA for adding 75ng modifies mRNA (mCherry-2A-GFP) (SEQ ID NO:21450；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine), mCherry repairs Adorn mRNA (mRNA SEQ ID NO:21439；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) or green fluorescent protein (GFP) modification mRNA (SEQ ID NO: MRNA sequence shown in 21448；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5-methylcytosine and pseudouridine are modified completely) and be incubated for.Also the control of untreated cell is evaluated. MCherry-2A-GFP refer to the code area comprising mCherry, 2A peptide and GFP code area modification mRNA sequence.
By by medium supernatant be transferred to 96 hole Pro-Bind U base plates (Beckton Dickinson GmbH, Heidelberg, Germany) harvest cell.By trypsase/EDTA of 1/2 volume of cell (Biochrom AG, Berlin, Germany) trypsin digestion, merge with corresponding supernatant, and pass through the PBS/2% of one volume of addition FCS (being Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck, Darmstadt, Germany) is consolidated It is fixed.Then it is excited in LSRII cell instrument (Beckton Dickinson GmbH, Heidelberg, Germany) with 532nm 610/20 filter of laser and PE-Texas Red make sample be subjected to the measurement of flow cell instrument.The mean fluorecence of all events is strong Degree (MFI) is shown in table 172.The cell for the mRNA transfection modified with bicistronic mRNA can express mCherry and GFP.
The MFI of the modification of table 172. mRNA
Modify mRNA mCherry MFI GFP MFI
mCherry 17746 427
GFP 427 20019
mCherry-2A-GFP 5742 6783
It is untreated 427 219
Embodiment 112. generates the 5-methylcytosine of antibody and the cationic lipid of N1- methyl-pseudouridine modification mRNA Matter preparation research
By Trastuzumab heavy chain (HC) mRNA (mRNA sequence shown in SEQ ID NO:21451；With about 140 cores The polyA tail of thuja acid is not shown in sequence；5 ' caps, Cap1；It is modified completely with 5-methylcytosine and N1- methyl-pseudouridine) With Trastuzumab light chain (LC) (mRNA sequence shown in SEQ ID NO:21452；PolyA with about 140 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and N1- methyl-pseudouridine) it is formulated in DLin- In MC3-DMA and DLin-KC2-DMA, as described in table 173.With the agent of 0.500mg/kg, 0.050mg/kg and 0.005mg/kg (I.V.) application preparation is measured into Balb-C mouse vein.
Table 173.DLin-MC3-DMA and DLin-KC2-DMA preparation
Serum is collected from mouse within 8 hours, 24 hours, 72 hours and/or 7 days after applying preparation.Pass through elisa assay Serum, to measure the protein expression of Trastuzumab.
113. pseudouridine of embodiment and N1- methyl-pseudouridine orientation SAR
With the concern recently to pyrimidine nucleoside pseudouridine, a series of structure-activities researchs are devised to containing pseudouridine Or the mRNA of N1- methyl-pseudouridine modification is studied.
The research is designed to inquire into work as and be modified at N1, C6,2-, 4- and in phosphate backbone When, chain length, the lipophilicity of increase, the presence of ring structure and the influence of hydrophobic or hydrophilic interaction change.Also to stabilization Property is studied.
For this purpose, to being related to alkylation, cycloalkylation, alkyl-cycloalkyl, arylation, alkyl-aryl-group, there is amino Allcylating moiety, the allcylating moiety with carboxylic acid group and the allcylating moiety containing amino acids charged moiety repair Decorations are studied.Alkylated degree is usually C1-C6.The example of chemical modification includes that listed in table 174 and table 175 A bit.
174. pseudouridine of table and N1- methyl pseudouridine SAR
175. pseudouridine of table and N1- methyl-pseudouridine SAR
Embodiment 114. is naturally incorporated to non-naturally occurring nucleosides
Natural and non-naturally occurring nucleosides is incorporated into the mRNA of encoding target polypeptide.These example is in table 176 With 177 in provide.Certain commercially available ribonucleoside triphosphotes (NTP) are studied in polynucleotides of the invention.These Selection provided in table 176.Then protein, inducing cytokine are generated for gained mRNA and/or generates treatment achievement Ability it is detected.
Table 176. naturally and non-naturally occurring nucleosides
Chemical modification Compound number It is naturally occurring
N4- methyl-cytosine 1 Y
N4, the N4--OMe- cytimidine of dimethyl -2 ' 2 Y
5- ethoxyacetic acid-methyl esters-uridine 3 Y
N3- methyl-vacation-uridine 4 Y
5- methylol-cytimidine 5 Y
5- trifluoromethyl-cytimidine 6 N
5- trifluoromethyl-uridine 7 N
5- Methyl-amino-methyl-uridine 8 Y
5- carboxy-methyl-amino-methyl-uridine 9 Y
5- carboxymethyl group amino methyl -2 '-OMe- uridine 10 Y
5- carboxymethyl group amino methyl -2- is thio-uridine 11 Y
5- Methylaminomethyl -2- is thio-uridine 12 Y
5- methoxy-carbonyl-methyl-uridine 13 Y
5--OMe- the uridine of methoxy-carbonyl-methyl -2 ' 14 Y
5- ethoxyacetic acid-uridine 15 Y
3- (3- amino -3- carboxylic propyl)-uridine 16 Y
5- (carboxyl hydroxymethyl) uridine methyl esters 17 Y
5- (carboxyl hydroxymethyl) uridine 18 Y
The non-naturally occurring ribonucleoside triphosphote of table 177.
Embodiment 115. is incorporated to modification to nucleobase and carbohydrate (sugar)
Natural and non-naturally occurring nucleosides is incorporated into the mRNA of encoding target polypeptide.It is incorporated into mRNA for it And protein, inducing cytokine and/or the ability for generating treatment achievement are generated, to nucleobase and carbohydrate (sugar) The commercially available nucleosides and NTP of modification detected.The example of these nucleosides provides in table 178 and 179.
The combination modification of table 178.
The naturally occurring combination of table 179.
Title Compound number It is naturally occurring
5- Methoxycarbonylmethyl -2- thio uridine TP 1 Y
5- Methylaminomethyl -2- thio uridine TP 2 Y
5- carbamo, lmethyl uridine TP 3 Y
5- carbamo, lmethyl -2 '-O- methyluridine TP 4 Y
1- methyl -3- (3- amino -3- carboxylic propyl) pseudouridine TP 5 Y
5- Methylaminomethyl -2- seleno uridine TP 6 Y
5- carboxymethyl group uridine TP 7 Y
5- methyldihydrouridine TP 8 Y
Lysidine TP 9 Y
5- taurine methyluridine TP 10 Y
5- taurine methyl -2- thio uridine TP 11 Y
5- (isopentene group amino methyl) uridine TP 12 Y
5- (isopentene group amino methyl) -2- thio uridine TP 13 Y
5- (isopentene group amino methyl) -2 '-O- methyluridine TP 14 Y
N4- acetyl group -2 '-O- methylcytidine TP 15 Y
N4,2 '-O- dimethyl cytidine TP 16 Y
5- formoxyl -2 '-O- methylcytidine TP 17 Y
2 '-O- methyl pseudouridine TP 18 Y
Thio -2 '-O- methyluridine TP of 2- 19 Y
3,2 '-O- dimethyl uridine TP 20 Y
In the table, " UTP " represents uridine triphosphate, and " GTP " represents guanosine triphosphate, and " ATP " represents adenosine triphosphate, " CTP " represents cytimidine triphosphoric acid, and " TP " represents triphosphoric acid and " Bz " represents benzyl.
Protein output of 116. vascular endothelial growth factor of embodiment in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 180 Described in chemical modification vascular endothelial growth factor (VEGF) modification RNA (mRNA shown in SEQ ID NO:1672 Sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in 10ul final volume In OPTI-MEM (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by the lipofectamine of 0.2ul 2000 are diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, two kinds of solution is merged, and in room It is incubated for again under temperature 15 minutes.Then solution 20ul merged is added to 100ul and contains in the cell culture medium of HeLa cell And it is incubated at room temperature.VEGF mRNA is also used, second group of HeLa cell is evaluated according to above procedure.For first Group HeLa cell is also analyzed to untreated cell and only with the control of the cell handled of lipofecatamine 2000.
Be incubated for 18 to 22 hours after, collect the cell culture supernatant of the cell of VEGF expression and 10.000rcf lower centrifugation 2 minutes.Then with VEGF- specific ELISA kit (R&D Systems, Minneapolis, MN), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until the value measured In the range of linearity of ELISA standard curve.The amount of generated protein is shown in table 180.
These results indicate that the VEGF modified completely with 1- methyl pseudouridine is thin in HeLa compared with other chemical substances Greater protein matter is generated in born of the same parents.
180. protein output of table
The comparison of protein output of the vascular endothelial growth factor of 117. chemical modification of embodiment in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 181 Described in chemical modification vascular endothelial growth factor (VEGF) modification RNA (mRNA shown in SEQ ID NO:1672 Sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in 10ul final volume In OPTI-MEM (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by the lipofectamine of 0.2ul 2000 are diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, two kinds of solution is merged, and in room It is incubated for again under temperature 15 minutes.Then solution 20ul merged is added to 100ul and contains in the cell culture medium of HeLa cell And it is incubated at room temperature.Also the control of untreated cell is analyzed.
Be incubated for 18 to 22 hours after, collect the cell culture supernatant of the cell of VEGF expression and 10.000rcf lower centrifugation 2 minutes.Then with VEGF- specific ELISA kit (R&D Systems, Minneapolis, MN), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until the value measured In the range of linearity of ELISA standard curve.The amount of generated protein is shown in table 181 and Fig. 9.
These results indicate that the VEGF modified completely with 1- methyl pseudouridine is thin in HeLa compared with other chemical substances Greater protein matter is generated in born of the same parents.
181. protein output of table
The yield of 118. VEGF protein of embodiment in mammals
The compound vascular endothelial growth factor of lipid (VEGF) mRNA (mRNA sequence shown in SEQ ID NO:1672； PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is unmodified, with 5-methylcytosine and vacation Uridine (5mC/pU) is modified completely, is modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU), wherein uses 5- Methylcystein replaces 25% cytimidine and replaces 25% uridine (5mC/s2U) to be modified with 2- thio uridine, uses 1- Methyl pseudouridine (1mpU) modification completely is modified completely with pseudouridine (pU).It is quiet to mouse with the dosage of 2ug mRNA/ mouse Preparation is applied in arteries and veins.As control, one group of mouse is untreated.It 3 hours after application, is expressed for vegf protein, by special Property VEGF ELISA measure the serum from mouse.As a result it is shown in table 182 and Figure 10.
182. protein output of table
Protein output of 119. granulocyte colony stimulating factor of embodiment in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 183 Described in chemical modification granulocyte colony stimulating factor (G-CSF) modification RNA (shown in SEQ ID NO:21438 MRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the final body of 10ul In long-pending OPTI-MEM (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by the lipofectamine of 0.2ul 2000 are diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, two kinds of solution is merged, and in room It is incubated for again under temperature 15 minutes.Then solution 20ul merged is added to 100ul and contains in the cell culture medium of HeLa cell And it is incubated at room temperature.Also divided to untreated cell and only with the control of the cell handled of lipofecatamine 2000 Analysis.
Be incubated for 18 to 22 hours after, collect expression G-CSF cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then with G-CSF- specific ELISA kit (R&D Systems, Minneapolis, MN), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until the value measured In the range of linearity of ELISA standard curve.The amount of generated protein is shown in table 183 and Figure 11.
183. protein output of table
The yield of 120. granulocyte colony stimulating factor albumen of embodiment in mammals
Granulocyte colony stimulating factor (G-CSF) mRNA (mRNA sequence shown in SEQ ID NO:21438；With big The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) use 5-methylcytosine and pseudouridine (5mC/pU) It modifies completely, is modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU), wherein uses 5-methylcytosine generation For 25% cytimidine and replace 25% uridine (5mC/s2U) to be modified with 2- thio uridine, with 1- methyl pseudouridine (1mpU) modification completely is modified completely with pseudouridine (pU).With 2ug mRNA/ mouse and 2ul lipofectamine 2000 (L2000)/mouse dosage is to mouse (CD1) (n=3) intravenous formulation.As control, one group of mouse is untreated.? 6 hours after application, for G-CSF protein expression, the serum from mouse is measured by specific G-CSF ELISA.As a result exist It is shown in table 184 and Figure 12.
184. protein output of table
Protein output of the IX factor of 121. chemical modification of embodiment in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and be inoculated with (10% fetal calf serum (FCS) and 1x are supplemented in Eagle ' the s minimum essential medium (EMEM) of total volume 100ul Glutamax in 24 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 It is grown overnight in atmosphere.Second day, by 250ng 5-methylcytosine and pseudouridine (5mC/pU) completely modification, with 5- first Base cytimidine and 1- methyl pseudouridine (5mC/1mpU) completely modification, wherein with 5-methylcytosine replace 25% cytimidine simultaneously It replaces 25% uridine (s2U/5mC) to be modified with 2- thio uridine, modified or used completely with 1- methyl pseudouridine (1mpU) The IX factor that pseudouridine (pU) is modified completely modifies mRNA (mRNA sequence shown in SEQ ID NO:1622；With about 140 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul Long-pendingIn.It is incubated at room temperature after five minutes, two kinds of solution is merged, and be incubated for 15 points again at room temperature Clock.Then solution 20ul merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature. Also untreated control is analyzed.
Be incubated for 18 hours after, by with from Pierce Biotechnology (Thermo Scientific, Rockford, IL) immunoprecipitation (IP) buffer lytic cell come collect expression the IX factor cell cell culture on It is simultaneously centrifuged 2 minutes by clear liquid at 10.000rcf.By clarified supernatant 1: 2 (1ml lysate/2 holes/24 hole plates) or 1: 5 (1ml lysate/5 holes/24 hole plates) dilution, then with IX factor-specific ELISA kit according to the explanation pair of manufacturer It is analyzed.All samples are diluted, until the value measured is in the range of linearity of ELISA standard curve.Two The amount of produced protein is shown in table 185 and Figure 13 in research.
185. protein output of table
Protein output of 122. apolipoprotein A-1 of embodiment in HeLa cell
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and be inoculated with (10% fetal calf serum (FCS) and 1x are supplemented in Eagle ' the s minimum essential medium (EMEM) of total volume 100ul Glutamax in 24 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 It is grown overnight in atmosphere.It is second day, the apolipoprotein A-1 of 250ng modified completely with 5-methylcytosine and pseudouridine is wild Raw type (APOA1wt) modifies RNA (mRNA sequence shown in SEQ ID NO:21453；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1), the Apolipoprotein A1 modified completely with 5-methylcytosine and pseudouridine Paris (APOA1 Paris) modifies RNA (mRNA sequence shown in SEQ ID NO:21454；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1) or the Apolipoprotein A1 modified completely with 5-methylcytosine and pseudouridine Milano (APOA1 Milano) modifies RNA (mRNA sequence shown in SEQ ID NO:21455；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1) it is diluted in 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul Long-pendingIn.It is incubated at room temperature after five minutes, two kinds of solution is merged, and be incubated for 15 points again at room temperature Clock.Then solution 20ul merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature. Also to only being analyzed with the control of cell that lipofecatamine 2000 is handled.
Be incubated for 18 hours after, by with from Pierce Biotechnology (Thermo Scientific, Rockford, IL) immunoprecipitation (IP) buffer lytic cell collect expression APOA1wt, APOA1 Paris or APOA1 It is simultaneously centrifuged 2 minutes by the cell culture supernatant of the cell of Milano at 10.000rcf.By clarified supernatant 1: 2 (1ml lysate/2 holes/24 hole plates) or 1: 5 (1ml lysate/5 holes/24 hole plates) dilution, then with APOA1- specificity ELISA kit is analyzed it according to the explanation of manufacturer.All samples are diluted, until the value measured exists In the range of linearity of ELISA standard curve.The amount of generated protein in the research and two researchs is repeated in table 186 It is shown in Figure 14.
186. protein output of table
The detection of 123. apolipoprotein A-1 wild-type protein of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by 1,250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU), use 5- It is that methylcystein and 1- methyl pseudouridine (5mC/1mpU) are modified completely, wherein replace 25% born of the same parents phonetic with 5-methylcytosine Pyridine and replace that 25% uridine (s2U/5mC) modified with 2- thio uridine, repaired completely with 1- methyl pseudouridine (1mpU) Decorations or apolipoprotein A-1 (APOA1) the wild type mRNA modified completely with pseudouridine (pU) (are shown in SEQ ID NO:21453 MRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul most Final volumeIn (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) are used as transfection reagent, and it is final that 5.5ul is diluted in 250ul VolumeIn.It is incubated at room temperature after five minutes, two kinds of solution is merged, and be incubated for 15 points again at room temperature Clock.Then solution 500ul merged is added to 3ml and contains in the cell culture medium of HeLa cell.Then as described above It is incubated for plate.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
By being centrifuged 2 minutes at 3,000rpm for cell precipitation.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).Each lysate sample preparation is to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to be supplemented to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 Anti- APOA1 under dilution in the 1X TBS solution of 3ml 5%BSA ApoA1 rabbit polyclonal antibody (Abcam, Cambridge, MA), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With stirring gently, 1X is used TBS/0.1%Tween washs film 3 times, every time five minutes.Goat antirabbit HRP conjugate (Abcam, Cambridge, MA) is sewed It is bonded to horseradish peroxidase and is bound to ApoA1 rabbit polyclonal antibody.The antibody of conjugation is in the 1X TBS solution of 5%BSA It dilutes 1: 1000 to 1: 5000 and is incubated at room temperature 3 hours.From containing there are many chemical modifications in HeLa cell lysate APOA1 wild type mRNA APOA1 protein Western blotting detection be shown in FIG. 15.
The detection of 124. apolipoprotein A-1 Paris albumen of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by 1,250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU), use 5- It is that methylcystein and 1- methyl pseudouridine (5mC/1mpU) are modified completely, wherein replace 25% born of the same parents phonetic with 5-methylcytosine Pyridine and replace that 25% uridine (s2U/5mC) modified with 2- thio uridine, repaired completely with 1- methyl pseudouridine (1mpU) Decorations or apolipoprotein A-1 (APOA1) the Paris mRNA modified completely with pseudouridine (pU) (are shown in SEQ ID NO:21454 MRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul most Final volumeIn (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) are used as transfection reagent, and it is final that 5.5ul is diluted in 250ul VolumeIn.It is incubated at room temperature after five minutes, two kinds of solution is merged, and be incubated for 15 points again at room temperature Clock.Then solution 500ul merged is added to 3ml and contains in the cell culture medium of HeLa cell.Then as described above It is incubated for plate.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
By being centrifuged 2 minutes at 3,000rpm for cell precipitation.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).Each lysate sample preparation is to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 Anti- APOA1 under dilution in the 1X TBS solution of 3ml 5%BSA ApoA1 rabbit polyclonal antibody (Abcam, Cambridge, MA), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With stirring gently, 1X is used TBS/0.1%Tween washs film 3 times, every time five minutes.Goat antirabbit HRP conjugate (Abcam, Cambridge, MA) is sewed It is bonded to horseradish peroxidase and is bound to ApoA1 rabbit polyclonal antibody.By the antibody of conjugation 5%BSA 1X TBS solution It is middle to dilute 1: 1000 to 1: 5000 and be incubated at room temperature 3 hours.The chemistry containing there are many is come from HeLa cell lysate to repair The Western blotting detection of the APOA1 protein of the APOA1 Paris mRNA of decorations is shown in FIG. 16.
The detection of 125. apolipoprotein A-1 Milano albumen of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by 1,250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU), use 5- It is that methylcystein and 1- methyl pseudouridine (5mC/1mpU) are modified completely, wherein replace 25% born of the same parents phonetic with 5-methylcytosine Pyridine and replace that 25% uridine (s2U/5mC) modified with 2- thio uridine, repaired completely with 1- methyl pseudouridine (1mpU) Decorations or apolipoprotein A-1 (APOA1) the Milano mRNA modified completely with pseudouridine (pU) (are shown in SEQ ID NO:21455 MRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul most Final volumeIn (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) are used as transfection reagent, and it is final that 5.5ul is diluted in 250ul VolumeIn.It is incubated at room temperature after five minutes, two kinds of solution is merged, and be incubated for 15 points again at room temperature Clock.Then solution 500ul merged is added to 3ml and contains in the cell culture medium of HeLa cell.Then as described above It is incubated for plate.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
By being centrifuged 2 minutes at 3,000rpm for cell precipitation.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 points at 4 DEG C in 10,000rpm Clock clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).Each lysate sample preparation is to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 Anti- APOA1 under dilution in the 1X TBS solution of 3ml 5%BSA ApoA1 rabbit polyclonal antibody (Abcam, Cambridge, MA), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With stirring gently, 1X is used TBS/0.1%Tween washs film 3 times, every time five minutes.Goat antirabbit HRP conjugate (Abcam, Cambridge, MA) is sewed It is bonded to horseradish peroxidase and is bound to ApoA1 rabbit polyclonal antibody.By the antibody of conjugation 5%BSA 1X TBS solution It is middle to dilute 1: 1000 to 1: 5000 and be incubated at room temperature 3 hours.The chemistry containing there are many is come from HeLa cell lysate to repair The Western blotting detection of the APOA1 protein of the APOA1 Milano mRNA of decorations is shown in FIG. 17.
The detection of 126. fibrinogen A albumen of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by being modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) for 1,250ng Fibrinogen A (FGA) mRNA (mRNA sequence shown in SEQ ID NO:21456；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 5.5ul is diluted in 250ul final volumeIn. It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then 500ul is merged Solution is added to 3ml and contains in the cell culture medium of HeLa cell.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
By being centrifuged 2 minutes at 3,000rpm for cell precipitation.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).Each lysate sample preparation is to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 The fibrinogen A goat polyclonal antibodies of antifibrin original A under dilution in the 1X TBS solution of 3ml 5%BSA (Abcam, Cambridge, MA) is kept for 3 hours at room temperature and is gently mixed on orbital shaker.Adjoint stirring gently, Film is washed 3 times, every time five minutes with 1X TBS/0.1%Tween.The anti-goat HRP conjugate of donkey (Abcam, Cambridge, MA it) is conjugated to horseradish peroxidase and is bound to fibrinogen A goat polyclonal antibodies.By the antibody of conjugation in 5%BSA 1X TBS solution in dilute 1: 1000 to 1: 5000 and be incubated at room temperature 3 hours.As used shown in the frame in Figure 18, fiber Desired size of the Western blotting detection of proteinogen A close to 95kd.
Protein output of the plasminogen of 127. chemical modification of embodiment in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, by 250ng with 5- methyl born of the same parents The plasminogen that pyrimidine and 1- methyl pseudouridine are modified completely modifies RNA (mRNA sequence shown in SEQ ID NO:21457； PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI- of 10ul final volume In MEM (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by the lipofectamine of 0.2ul 2000 are diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, two kinds of solution is merged, and in room It is incubated for again under temperature 15 minutes.Then solution 20ul merged is added to 100ul and contains in the cell culture medium of HeLa cell And it is incubated at room temperature.MRNA (mRNA sequence shown in SEQ ID NO:1638 also is modified to hematopoietin (EPO) Column；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5-methylcytosine and pseudouridine Modification completely) control and the control of untreated cell analyzed.
Be incubated for 18 to 22 hours after, collect expression plasminogen cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then with plasminogen-specific ELISA kit (R&D Systems, Minneapolis, MN), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, directly To the value measured in the range of linearity of ELISA standard curve.The amount of generated protein is shown in table 187 and Figure 19 Out.
187. protein output of table
The detection of 128. plasminogen protein of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by being modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) for 1250ng Plasminogen mRNA (mRNA sequence shown in SEQ ID NO:21457；PolyA tail with about 160 nucleotide, It is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume(LifeTechnologies, Grand Island, NY) in.Made using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) For transfection reagent, and 5.5ul is diluted in 250ul final volumeIn.It is incubated at room temperature 5 minutes Afterwards, two kinds of solution are merged, and be incubated for again at room temperature 15 minutes.Then solution 500ul merged is added to 3ml and contains In the cell culture medium of HeLa cell.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 Antiplasmin original under dilution in the 1X TBS solution of 3ml 5%BSA plasminogen goat polyclonal antibodies (Abcam, Cambridge, MA), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With stirring gently, 1X is used TBS/0.1%Tween washs film 3 times, every time five minutes.The anti-goat HRP conjugate (Abcam, Cambridge, MA) of donkey is sewed It is bonded to horseradish peroxidase and is bound to plasminogen goat polyclonal antibodies.By the antibody of conjugation 5%BSA 1X TBS 1: 1000 to 1: 5000 are diluted in solution and are incubated at room temperature 3 hours.Figure 20, which is shown, detects plasminogen close to 95kd Desired size.
The detection of 129. galactose-1-phosphate uridyl transferase protein of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by the galactolipin-of 1250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU) 1- phosphate uridyl-transferase (GALT) mRNA (mRNA sequence shown in SEQ ID NO:21458；With about 160 cores The polyA tail of thuja acid is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 5.5ul is diluted in 250ul final volumeIn. It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then 500ul is merged Solution is added to 3ml and contains in the cell culture medium of HeLa cell.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 GALT mouse monoclonal antibody (the Novus of anti-GALT under dilution in the 1X TBS solution of 3ml 5%BSA Biological, Littleton CO), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With stirring gently It mixes, is washed film 3 times, every time five minutes with 1X TBS/0.1%Tween.Donkey anti-mouse HRP conjugate (Abcam, Cambridge, MA) it is conjugated to horseradish peroxidase and is bound to GALT mouse monoclonal antibody.By the antibody of conjugation 5% 1: 1000 to 1: 5000 are diluted in the 1X TBS solution of BSA and are incubated at room temperature 3 hours.As shown in the frame in Figure 21, The Western blotting of GALT is detected as about 42kd.
The detection of 130. argininosuccinase albumen of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by the smart amino of 1250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU) Succinic acid lyases (ASL) mRNA (mRNA sequence shown in SEQ ID NO:21459；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 5.5ul is diluted in 250ul final volumeIn. It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then 500ul is merged Solution is added to 3ml and contains in the cell culture medium of HeLa cell.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 ASL mouse monoclonal antibody (the Novus of anti-ASL under dilution in the 1X TBS solution of 3ml 5%BSA Biological, Littleton CO), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With stirring gently It mixes, is washed film 3 times, every time five minutes with 1X TBS/0.1%Tween.Donkey anti-mouse HRP conjugate (Abcam, Cambridge, MA) it is conjugated to horseradish peroxidase and is bound to ASL mouse monoclonal antibody.By the antibody of conjugation 5% 1: 1000 to 1: 5000 are diluted in the 1X TBS solution of BSA and are incubated at room temperature 3 hours.As shown in the frame in Figure 22, ASL Western blotting be detected as about 50kd.
The detection of 131. tyrosine transaminase albumen of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by being modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) for 1250ng Tyrosine transaminase (TAT) mRNA (mRNA sequence shown in SEQ ID NO:21460；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 5.5ul is diluted in 250ul final volumeIn. It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then 500ul is merged Solution is added to 3ml and contains in the cell culture medium of HeLa cell.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 TAT rabbit polyclonal antibody (the Novus of anti-TAT under dilution in the 1X TBS solution of 3ml 5%BSA Biologicals), kept for 3 hours and be gently mixed on orbital shaker at room temperature.With stirring gently, with 1X TBS/ 0.1%Tween washs film 3 times, every time five minutes.Goat antirabbit HRP conjugate (Abcam, Cambridge, MA) is conjugated to Horseradish peroxidase is simultaneously bound to TAT rabbit polyclonal antibody.The antibody of conjugation is diluted 1 in the 1X TBS solution of 5%BSA : it 1000 to 1: 5000 and is incubated at room temperature 3 hours.As shown in the frame in Figure 23, the Western blotting of TAT is detected as about 50kd, wherein chemiluminescence band is shown with white.
Embodiment 132.1,4- alpha-glucans-branch's zymoprotein detection: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by being modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) for 1250ng Isosorbide-5-Nitrae-alpha-glucans-branching enzyme (GBE1) mRNA (mRNA sequence shown in SEQ ID NO:21461；With about 160 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 5.5ul is diluted in 250ul final volumeIn. It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then 500ul is merged Solution is added to 3ml and contains in the cell culture medium of HeLa cell.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 GBE1 rabbit polyclonal antibody (the Novus of anti-GBE1 under dilution in the 1X TBS solution of 3ml 5%BSA Biological, Littleton CO), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With stirring gently It mixes, is washed film 3 times, every time five minutes with 1X TBS/0.1%Tween.Goat antirabbit HRP conjugate (Abcam, Cambridge, MA) it is conjugated to horseradish peroxidase and is bound to GBE1 rabbit polyclonal antibody.By the antibody of conjugation 5% 1: 1000 to 1: 5000 are diluted in the 1X TBS solution of BSA and are incubated at room temperature 3 hours.As shown in the frame in Figure 24, The desired size that the Western blotting detection of GBE1 is about 70kd.
Protein output of 133. factor of embodiment in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, by 250ng with 5- methyl born of the same parents The factor that pyrimidine and 1- methyl pseudouridine are modified completely modifies RNA (mRNA sequence shown in SEQ ID NO:21462； PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI- of 10ul final volume In MEM (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by the lipofectamine of 0.2ul 2000 are diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, two kinds of solution is merged, and in room It is incubated for again under temperature 15 minutes.Then solution 20ul merged is added to 100ul and contains in the cell culture medium of HeLa cell And it is incubated at room temperature.MRNA (mRNA sequence shown in SEQ ID NO:1638 also is modified to hematopoietin (EPO) Column；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5-methylcytosine and pseudouridine Completely modification) and the control of untreated cell analyzed.
Be incubated for 18 to 22 hours after, collect expression plasminogen cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then with factor-specific ELISA kit (R&D Systems, Minneapolis, MN), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, directly To the value measured in the range of linearity of ELISA standard curve.The amount of generated protein is shown in table 188 and Figure 25 Out.
188. protein output of table
Protein output of the factor of 134. chemical modification of embodiment in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and be inoculated with (10% fetal calf serum (FCS) and 1x are supplemented in Eagle ' the s minimum essential medium (EMEM) of total volume 100ul Glutamax in 24 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 It is grown overnight in atmosphere.Second day, by 250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU), use 5- It is that methylcystein and 1- methyl pseudouridine (5mC/1mpU) are modified completely, wherein replace 25% born of the same parents phonetic with 5-methylcytosine Pyridine and replace that 25% uridine (s2U/5mC) modified with 2- thio uridine, repaired completely with 1- methyl pseudouridine (1mpU) Decorations modify RNA (mRNA sequence shown in SEQ ID NO:21462 with the factor that pseudouridine (pU) is modified completely；Tool There is the polyA tail of about 140 nucleotide, is not shown in sequence；5 ' caps, Cap1) it is diluted in 10ul final volumeIn (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by the lipofectamine of 0.2ul 2000 are diluted in 10ul final volumeIn.It is incubated at room temperature after five minutes, two kinds of solution is merged, and It is incubated for again at room temperature 15 minutes.Then solution 20ul merged is added to the cell culture that 100ul contains HeLa cell In base and it is incubated at room temperature.Also untreated control is analyzed.
Be incubated for 18 hours after, by with from Pierce Biotechnology (Thermo Scientific, Rockford, IL) immunoprecipitation (IP) buffer lytic cell come collect expression factor cell cell culture It is simultaneously centrifuged 2 minutes by supernatant at 10.000rcf.By clarified supernatant 1: 2 (1ml lysate/2 holes/24 hole plates) or 1 : then factor-specific ELISA kit saying according to manufacturer is used in 5 (1ml lysates/5 holes/24 hole plates) dilution It is bright to analyze it.All samples are diluted, until the value measured is in the range of linearity of ELISA standard curve. The amount of generated protein is shown in table 189 and Figure 26 in two researchs.
189. protein output of table
The detection of 135. ceruloplasmin of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2Gas It is grown overnight in atmosphere.Second day, by being modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) for 1250ng Ceruloplasmin (CP or CLP) mRNA (mRNA sequence shown in SEQ ID NO:1621；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 5.5ul is diluted in 250ul final volumeIn. It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then 500ul is merged Solution is added to 3ml and contains in the cell culture medium of HeLa cell.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 Ceruloplasmin rabbit polyclonal antibody (the Novus of anti-CP albumen under dilution in the 1X TBS solution of 3ml 5%BSA Biological, Littleton CO), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With stirring gently It mixes, is washed film 3 times, every time five minutes with 1X TBS/0.1%Tween.Goat antirabbit HRP conjugate (Abcam, Cambridge, MA) it is conjugated to horseradish peroxidase and is bound to ceruloplasmin rabbit polyclonal antibody.By the anti-of conjugation Body dilutes 1: 1000 to 1: 5000 in the 1X TBS solution of 5%BSA and is incubated at room temperature 3 hours.Such as the frame in Figure 27 Shown, the Western blotting of ceruloplasmin (CLP) is detected as about 148kd.
Protein output of 136. transforminggrowthfactor-β1 of embodiment in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, by 83ng, 250ng or Transforminggrowthfactor-β1 (TGF-β 1) modification RNA (the SEQ ID of 750ng modified completely with 5-methylcytosine and pseudouridine MRNA sequence shown in NO:1668；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) It is diluted in the OPTI-MEM (LifeTechnologies, Grand Island, NY) of 10ul final volume.It uses Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by 0.2ul Lipofectamine 2000 be diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, by two kinds Solution merges, and is incubated for again at room temperature 15 minutes.Then solution 20ul merged is added to 100ul and contains HeLa cell Cell culture medium in and be incubated at room temperature.Also to the cell (L2000) and untreated of the processing of lipofecatmine 2000 The control of cell is analyzed.
Be incubated for 18 to 22 hours after, collect expression TGF-β 1 cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then with TGF-β 1- specific ELISA kit (R&D Systems, Minneapolis, MN), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until the value measured In the range of linearity of ELISA standard curve.The amount of generated protein is shown in table 190 and Figure 28.
190. protein output of table
The detection of 137. ornithine transcarbamylase albumen of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2Gas It is grown overnight in atmosphere.Second day, by the ornithine of 1250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU) Carbamylrtansferase (OTC) mRNA (mRNA sequence shown in SEQ ID NO:1659；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 5.5ul is diluted in 250ul final volumeIn. It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then 500ul is merged Solution is added to 3ml and contains in the cell culture medium of HeLa cell.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 OTC rabbit polyclonal antibody (the Novus of anti-OTC albumen under dilution in the 1X TBS solution of 3ml 5%BSA Biological, Littleton CO), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With stirring gently It mixes, is washed film 3 times, every time five minutes with 1X TBS/0.1%Tween.Goat antirabbit HRP conjugate (Abcam, Cambridge, MA) it is conjugated to horseradish peroxidase and is bound to OTC rabbit polyclonal antibody.By the antibody of conjugation in 5%BSA 1X TBS solution in dilute 1: 1000 to 1: 5000 and be incubated at room temperature 3 hours.As shown in the frame in Figure 29, OTC's The expection size that Western blotting detection is about 40kd.
The In vivo study of embodiment 138.LDLR in mammals
It is final to 0.2mL by mixing 8.0 μ g mRNA and Dulbecco ' s improvement Eagle ' s culture medium (DMEM) Volume makes low-density lipoprotein (LDL) receptor (LDLR) mRNA (mRNA shown in SEQ ID NO:21463；With 5- methyl born of the same parents Pyrimidine and pseudouridine are modified completely；5 ' caps, Cap1；PolyA tail with 160 nucleotide is not shown in sequence) with Lipofectamine 2000 is compound.
By Lipofectamine 2000 with DMEM dilute 12.5 times and with isometric diluted ldl receptor mRNA solution Mixing.Sample is incubated at room temperature 5 minutes, and the compound mRNA mixture of 0.1mL volume is injected into three In the tail vein of each in C57BL/6 mouse.Every animal receives the ldl receptor mRNA of 2.0 μ g of accumulated dose.It, will after 6 hours Animal puts to death and takes out spleen.According to standardization program separating Morr. cell (not cracking red blood cell in advance) and with equal quantities to people The IgG of ldl receptor specificity or non-immunity IgG as control are dyed.
By the expression of hybridoma supematant assesse ldl receptor, wherein being gated to CD11b+ splenocyte group.Such as Figure 30 institute Show, compared with the cell (non-immunity IgG) dyed with non-immunity IgG, with ldl receptor IgG dyeing (LDLR IgG) to The presence at the peak that right avertence is moved shows each middle ldl receptor in three individual mices in vivo in CD11b+ splenocyte group Expression.
For the mouse only handled with Lipofectamine, ldl receptor specific peak is not observed and dyes similar Observed by the case where non-immunity IgG.
Embodiment 139. modifies UGT1A1 mRNA research
According to standardization program, HEK293 cell is seeded in 500,000 cells/6 hole plates density containing 10% tire In the DMEM of cow's serum and make cells grew overnight.Second day, by 1 family of UDP glucuronyl transferase of 800ng, more Peptide A1 (UGT1A1) mRNA (mRNA shown in SEQ ID NO:21464；It is modified completely with 5-methylcytosine and pseudouridine； 5 ' caps, Cap1；PolyA tail with 160 nucleotide is not shown in sequence) it is diluted in the Optimum buffer of 0.3mL, The sample of the Lipofectamine 2000 of 4.0 μ L is also diluted in the Optimum buffer of 0.3mL, and by by two Kind solution mixing keeps mRNA and Lipofectamine 2000 compound.
After being incubated at room temperature 15 minutes, cell is transfected with 2000 mixture of UGT1A1 mRNA/lipofectamine. After being incubated for 18 hours at 37 DEG C, culture medium is sucked out and washs cell with PBS.By harvesting cell and will be thin with cell shovel scraping Born of the same parents are centrifuged 3 minutes at 3,000 rpm.Cell precipitation is washed with PBS, is centrifuged as described, and passes through addition The supplement of 0.25mL has the RIPA buffer of protease inhibitor cocktail to crack cell precipitation.By cell extract 12, It is centrifuged 10 minutes under 210rpm, and supernatant fraction (lysate) is freezed in -80 DEG C.
Using Simple Simon Western capillary electrophoresis, carried out using the antibody to hCCSP T1A1 specificity Western blotting.Three parts of race glue of lysate sample of 0.005mg.As shown in figure 31, UGT1A1 is detected as about 60kDa's Single band.In contrast, band is not detected using come the lysate of the control cell for uncorrelated mRNA transfection of using by oneself.
Internal expression of the embodiment 140.LDLR in mouse
Use the internal expression of LDLR-/- mouse test LDLR mmRNA.LDL is applied to LDLR-/- mouse by injection mmRNA.The tissue from mouse is detected for LDLR expression.The western blot analysis of mouse tissue is carried out, to seek Look for the LDLR protein expression as caused by LDLR mmRNA application.Real-time RT-PCR is carried out on mouse tissue to find LDLR base Because of expression.
The yield of embodiment 141.LDLR in mammals
Ornithine transcarbamylase (OTC) is the mitochondrial protein expressed in Matrix attachment region.The feature of OTC defect It is the cumulative toxicity of ammonia and leads to urea cycle disorder.
The modification mRNA of coding OTC is applied to mouse described in embodiment 140.Point collects serum, group in different times It knits and/or organ, to measure protein expression.
Also carry out the further research wherein to the mouse of embodiment 140 application LDLR modification mRNA.In different times Point collects serum, tissue and/or organ, to measure protein expression.
Yield of the XI factor of 142. chemical modification of embodiment in HEK293 cell
Human embryo kidney epithelial cell (HEK293) (LGC standards GmbH, Wesel, Germany) is inoculated in pre- It is first coated in 24 hole plates (Greiner Bio-one GmbH, Frickenhausen, Germany) of 1 Collagen Type VI.It will HEK293 is seeded in 100 μ l cell culture mediums with the density of about 100,000 cells/wells.By cell inoculation and it is being incubated for it Afterwards, XI factor mRNA (modRNA FXI) (mRNA shown in SEQ ID NO:1625 containing 350ng or 750 is directly added Sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) preparation.Also to untreated thin The control of born of the same parents is evaluated.
By by medium supernatant be transferred to 96 hole Pro-Bind U base plates (Beckton Dickinson GmbH, Heidelberg, Germany) harvest cell.By trypsase/EDTA of 1/2 volume of cell (Biochrom AG, Berlin, Germany) trypsin digestion, merge with corresponding supernatant, and pass through the PBS/2% of one volume of addition FCS (being Biochrom AG, Berlin, Germany)/0.5% formaldehyde (Merck, Darmstadt, Germany) is consolidated It is fixed.After being incubated for 12 hours, collects the cell culture supernatant of the cell of the expression XI factor and be centrifuged at 10.000rcf 2 minutes.Then the XI factor-specific ELISA kit (Innovative Research, Novi, MI) is used, according to manufacturer Explanation clear supernatant is analyzed.The amount of generated protein is shown in FIG. 32.
The detection of 143. Aquaporin-5 albumen of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by 1250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU), with 5- first It is that base cytimidine and 1- methyl pseudouridine (5mC/1mpU) are modified completely, with the uridine of 2- thio uridine modification 25% and with 5- first The cytimidine (s2U and 5mC) of base cytimidine modification 25%, with pseudouridine (pU) modification completely or with 1- methyl pseudouridine (1mpU) Aquaporin-5 mRNA (the mRNA sequence shown in SEQ ID NO:1617 modified completely；With about 160 nucleotide PolyA tail, be not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 5.5ul is diluted in 250ul final volume In.It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then 500ul is closed And solution be added to 3ml and contain in the cell culture medium of HeLa cell.Then it is incubated for plate as described above.Also to untreated The control of cell is evaluated.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 The Aquaporin-5 rabbit polyclonal of anti-Aquaporin-5 albumen under dilution in the 1X TBS solution of the 5%BSA of 3ml Antibody (Abcam, Cambridge, MA) is kept for 3 hours at room temperature and is gently mixed on orbital shaker.With stirring gently It mixes, is washed film 3 times, every time five minutes with 1X TBS/0.1%Tween.Goat antirabbit conjugate (Abcam, Cambridge, MA it) is conjugated to horseradish peroxidase and is bound to Aquaporin-5 rabbit polyclonal antibody.By the antibody of conjugation in 5%BSA 1X TBS solution in dilute 1: 1000 to 1: 5000 and be incubated at room temperature 3 hours.As shown in the frame in Figure 33, protein Trace detects protein in the every kind of chemical substance evaluated.
Protein output of the embodiment 144.VII factor in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 191 Described in chemical modification the VII factor modify RNA (mRNA sequence shown in SEQ ID NO:1623；With about 140 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul In long-pending OPTI-MEM.It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then The solution that 20ul is merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.Also to not The control of processing cell is analyzed.
Be incubated for 18 to 22 hours after, collect expression the VII factor cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then Hyphen Biomed Chromogenic kit (Aniara, West are used Chester OH) clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until being surveyed Fixed value is in the range of linearity of ELISA standard curve.The amount of generated protein compared with untreated sample exists It is shown in table 191 and Figure 34.
191. protein output of table
Protein output of the insulin glargine of 145. chemical modification of embodiment in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and be inoculated with (10% fetal calf serum (FCS) and 1x are supplemented in Eagle ' the s minimum essential medium (EMEM) of total volume 100ul Glutamax in 24 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 It is grown overnight in atmosphere.Second day, by 250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU), use 5- It is that methylcystein and 1- methyl pseudouridine (5mC/1mpU) are modified completely, wherein replace 25% born of the same parents phonetic with 5-methylcytosine Pyridine and replace that 25% uridine (s2U/5mC) modified with 2- thio uridine, repaired completely with 1- methyl pseudouridine (1mpU) Decorations modify RNA (mRNA sequence shown in SEQ ID NO:21465 with the insulin glargine that pseudouridine (pU) is modified completely； PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in 10ul final volumeIn (LifeTechnologies, Grand Island, NY).Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by the lipofectamine of 0.2ul 2000 are diluted in 10ul final volumeIn.It is incubated at room temperature after five minutes, two kinds of solution is merged, and It is incubated for again at room temperature 15 minutes.Then solution 20ul merged is added to the cell culture that 100ul contains HeLa cell In base and it is incubated at room temperature.Also untreated control is analyzed.
Be incubated for 18 hours after, by with from Pierce Biotechnology (Thermo Scientific, Rockford, IL) immunoprecipitation (IP) buffer lytic cell come collect expression insulin glargine cell cell culture It is simultaneously centrifuged 2 minutes by object supernatant at 10.000rcf.By clarified supernatant 1: 2 (1ml lysate/2 holes/24 hole plates) Or 1: 5 (1ml lysate/5 holes/24 hole plates) dilution, then with insulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden) it is analyzed it according to the explanation of manufacturer.All samples are diluted, until the value measured In the range of linearity of ELISA standard curve.The amount of generated protein is shown in table 192 and Figure 35 in two researchs Out.In table 192, " > ", which is meant, to be greater than.
192. protein output of table
Protein output of 146. tissue factor of embodiment (factor 3) in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 193 Described in chemical modification tissue factor (factor 3) modify RNA (mRNA sequence shown in SEQ ID NO:21466；Have The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul In long-pending OPTI-MEM.It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then The solution that 20ul is merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.Also to not The control of processing cell is analyzed.
Be incubated for 18 to 22 hours after, collect the expression tissue factor cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then Hyphen Biomed Chromogenic kit (Aniara, West are used Chester OH) clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until being surveyed Fixed value is in the range of linearity of ELISA standard curve.The amount of generated protein compared with untreated sample exists It is shown in table 193 and Figure 36.In table 193, " > ", which is meant, to be greater than.
193. protein output of table
Protein output of the XI factor of 147. chemical modification of embodiment in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 194 Described in chemical modification the XI factor modify RNA (mRNA sequence shown in SEQ ID NO:1625；With about 140 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul In long-pending OPTI-MEM.It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then The solution that 20ul is merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.Also to not The control of processing cell is analyzed.
Be incubated for 18 to 22 hours after, collect expression the XI factor cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then with the XI factor-specific ELISA kit (Innovative Research, Novi, MI), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until the value measured In the range of linearity of ELISA standard curve.The amount of generated protein compared with untreated sample in table 194 and It is shown in Figure 37.In table 194, " > ", which is meant, to be greater than.
194. protein output of table
Protein output of the embodiment 148.XI factor in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 20,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, by 250ng with 5- methyl born of the same parents The XI factor that pyrimidine and 1- methyl pseudouridine are modified completely modifies RNA (mRNA sequence shown in SEQ ID NO:1625；Have The polyA tail of about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul In long-pending OPTI-MEM.It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then The solution that 20ul is merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.Also to rush Erythropoietin(EPO) (EPO) modifies mRNA (mRNA sequence shown in SEQ ID NO:1638；With about 160 nucleotide PolyA tail, be not shown in sequence；Cap, Cap1；Modified completely with 5-methylcytosine and pseudouridine) and untreated cell Control is analyzed.
Be incubated for 18 to 22 hours after, collect expression plasminogen cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then with the XI factor-specific ELISA kit (Innovative Research, Novi, MI), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until the value measured In the range of linearity of ELISA standard curve.The amount of generated protein is shown in table 195 and Figure 38.
195. protein output of table
Protein output of 149. insulin aspart of embodiment in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 196 Described in chemical modification insulin aspart modify RNA (mRNA sequence shown in SEQ ID NO:21467；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul In long-pending OPTI-MEM.It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then The solution that 20ul is merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.Also to not The control of processing cell is analyzed.
Be incubated for 18 to 22 hours after, collect expression insulin aspart cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then with insulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until being measured Value in the range of linearity of ELISA standard curve.The amount of generated protein compared with untreated sample is in table It is shown in 196 and Figure 39.In table 196, " > ", which is meant, to be greater than.
196. protein output of table
Protein output of 150. insulin lispro of embodiment in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 197 Described in chemical modification insulin lispro modify RNA (mRNA sequence shown in SEQ ID NO:21468；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul In long-pending OPTI-MEM.It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then The solution that 20ul is merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.Also to not The control of processing cell is analyzed.
Be incubated for 18 to 22 hours after, collect expression insulin lispro cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then with insulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until being measured Value in the range of linearity of ELISA standard curve.The amount of generated protein compared with untreated sample is in table It is shown in 197 and Figure 40.In table 197, " > ", which is meant, to be greater than.
197. protein output of table
151. paddy of embodiment relies protein output of the insulin in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 198 Described in chemical modification paddy rely insulin modify RNA (mRNA sequence shown in SEQ ID NO:21469；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul In long-pending OPTI-MEM.It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then The solution that 20ul is merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.Also to not The control of processing cell is analyzed.
Be incubated for 18 to 22 hours after, collect expression insulin lispro cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then with insulin-specific ELISA kit (Mercodia AB, Uppsala, Sweden), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until being measured Value in the range of linearity of ELISA standard curve.The amount of generated protein compared with untreated sample is in table It is shown in 198 and Figure 41.In table 198, " > ", which is meant, to be greater than.
198. protein output of table
Protein output of 152. human growth hormone (HGH) of embodiment in HeLa cell
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng had into table 199 Described in chemical modification human growth hormone (HGH) (hGH) modify RNA (mRNA sequence shown in SEQ ID NO:1648；Have The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in the OPTI-MEM of 10ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and the lipofectamine of 0.2ul 2000 is diluted in the final body of 10ul In long-pending OPTI-MEM.It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then The solution that 20ul is merged is added in the cell culture medium that 100ul contains HeLa cell and is incubated at room temperature.Also to not The control of processing cell is analyzed.
Be incubated for 18 to 22 hours after, collect expression human growth hormone (HGH) cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then human growth hormone (HGH) ELISA kit (catalog number (Cat.No.) DGH00 is used；R&D Minneapolis, MN), clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, directly To the value measured in the range of linearity of ELISA standard curve.Generated protein compared with untreated sample Amount shown in table 199 and Figure 42.In table 199, " > ", which is meant, to be greater than.
199. protein output of table
The detection of 153. oncoprotein of embodiment, 53 albumen: Western blotting
To CD1 mouse (Harlan Laboratories, South Easton, MA), intravenously application is phonetic with 5- methyl born of the same parents It is that pyridine and pseudouridine (5mC/pU) are modified completely, modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) , with the uridine of 2- thio uridine modification 25% and with the cytimidine of 5-methylcytosine modification 25% (s2U and 5mC), with vacation Uridine (pU) the compound oncoprotein 53 of modification or the lipid modified completely with 1- methyl pseudouridine (1mpU) completely (TP53 or P53) mRNA (mRNA sequence shown in SFQ ID NO:1670；PolyA tail with about 140 nucleotide, in sequence not It shows；5 ' caps, Cap1).To mouse be applied in 100ul sterilized base DMEM culture medium (without additive, LifeTechnologies, Grand Island, NY) in 2ul Lipofectamine 2000 The mRNA of (LifeTechnologies, Grand Island, NY) compound 2ug dosage.
After 6 hours, animal is put to death and obtains serum and spleen.Spleen is transferred to 6 hole plates, and depositing in 1ml PBS It is kept on ice lower.It repeatedly uses scalpel blend rubber cell scraper to cut a spleen, squeezes out splenocyte until PBS is because thin Born of the same parents discharge and become muddy.
Cell is transferred to be placed on 12 hole cel culture plates 100um cell filter (BD Biosciences, San Jose, CA), and leave fibre fractionation.By gravity, cell passes through cell filter and collects to be cultivated in 12 holes of lower section In disk.The PBS of 1ml is transferred to Eppendorf to manage and rotate 5min at 2000rpm together with the splenocyte freely floated. PBS is discarded, and cell precipitation is merged with the fresh PBS of 500ul.By vortex 5min of short duration at 2000rpm by splenocyte It is resuspended.It discards PBS and 1ml BD Pharmlyse is added in cell precipitation.Splenocyte is resuspended by of short duration vortex.It will Cell is incubated at room temperature 3 minutes, is then rotated 5 minutes at 200 rpm.Cell simultaneously institute as above twice is washed with 500ul PBS It states and is rotated.Cell is resuspended with the PBS of 500ul and is rotated as described.
The splenocyte of 250ul is merged with 1x Pharmlyse buffer, it is of short duration to be vortexed or be resuspended with pipette, then exist It is rotated 2 minutes under 2000rpm.
In a pipe, cell precipitation is resuspended in the protease inhibitor cocktail with mammalian cell In 500ul RIPA buffer (BostonBioproducts, Ashland, MA) and by lysate freeze or immediately continue with into Row BCA measurement.In the second pipe, 250ul FACS staining kit fixed solution (4% formaldehyde is added；R and D Systems, Minneapolis, MN), it is then incubated at room temperature 10 minutes.Cell is washed twice with 500ul PBS and as above It is described to be rotated.Cell precipitation is resuspended in 500PBS and is stored in 4 DEG C.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60min, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 minutes in the 1X TBS solution of 5%BSA, then in the 1X TBS+ of 5%BSA It is incubated for again in 0.1%Tween solution 15 minutes.It is applied under 1: 500 to 1: 2000 dilution in the 1X of 3ml 5%BSA The primary antibody of anti-target proteins in TBS solution is kept for 3 hours at room temperature and is gently mixed on orbital shaker.With gently Stirring, film is washed 3 times, every time five minutes with 1X TBS/0.1%Tween.Secondary antibody [[which kind of antibody? ]] it is conjugated to horseradish Be an antiantibody [[which kind of antibody simultaneously bound to for peroxidase? ]].Secondary antibody is diluted 1 in the 1X TBS solution of 5%BSA: It 1000 to 1: 5000 and is incubated at room temperature 3 hours.Primary antibody and secondary antibody are purchased from Abcam (Cambridge, MA), Novus Biologicals (Littleton, CO), Thermo Fisher (Rockford, IL), Millipore (Billerica, MA) Or R and D systems (Minneapolis, MD).
At the end of incubation time, with being gently mixed, film is washed 3 times, every time 5 points with 1X TBS/0.1%Tween Clock.According to guidance by film 5ml Pierce WestPico Chemiluminescent Subtrate (Thermo Fisher, Rockford, IL) in development.
As shown in the frame in Figure 43 A and 43B, respectively for 2 samples being evaluated for every kind of chemical substance, protein Trace detects the protein near the expection size of 53kd.
The detection of 154. tuftelin of embodiment, 1 albumen: Western blotting
By human embryo kidney epithelial cell (HEK293) be seeded in 96 hole plates (Greiner Bio-one GmbH, Frickenhausen, Germany) on and for the plate of HEK293 cell be pre-coated with 1 Collagen Type VI.By HEK293 with 35,000 density is seeded in 100 μ l cell culture mediums, and (DMEM, 10%FCS add 2mM L-Glutamine, 1mM Sodium Pyruvate With 1x nonessential amino acid (Biochrom AG, Berlin, Germany) and 1.2mg/ml sodium bicarbonate (Sigma- Aldrich, Munich, Germany)) in.
With 20 μ l sample buffers (20% glycerol, 4%SDS, 100mM Tris-HCl pH 6.8,0.2% bromophenol blue, 5% beta -mercaptoethanol)/hole is by the cell cracking of transfection.Make supernatant liquid precipitate with the frost acetone of 4 volumes and is dissolved in sample In buffer.95 DEG C are heated the sample to, continues 5 minutes, and in the SDS- polyacrylamide gel containing 10% acrylamide Upper race glue.
By semi-drying trace by Protein transfer to nitrocellulose filter.The TBS solution of 5% skimmed milk power of film is sealed It closes, TUFT1 rabbit polyclonal antibody (the Novus Biologicals, catalog number (Cat.No.) NBP1- being subsequently used under 1: 500 dilution 87446) it is incubated for.The secondary antibody (St.Cruz Biotech, Heidelberg, Germany) that is conjugated using donkey anti-rabbit HRP- and Super Signal West Pico detection reagent (Pierce) detects signal.As shown in figure 44, Western blotting is in 2ug (swimming Road 2) and 200ng (swimming lane 3) sample in detect protein.In Figure 44, swimming lane 1 is marker.
The detection of 155. galactokinase of embodiment, 1 albumen: Western blotting
To CD1 mouse (Harlan Laboratories, South Easton, MA), intravenously application is phonetic with 5- methyl born of the same parents It is that pyridine and pseudouridine (5mC/pU) are modified completely, modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) , with the uridine of 2- thio uridine modification 25% and with the cytimidine of 5-methylcytosine modification 25% (s2U and 5mC), with vacation Uridine (pU) the compound galactokinase 1 (GALK1) of modification or the lipid modified completely with 1- methyl pseudouridine (1mpU) completely MRNA (mRNA sequence shown in SEQ ID NO:21470；PolyA tail with about 140 nucleotide does not show in sequence Out；5 ' caps, Cap1).To mouse be applied in 100ul sterilized base DMEM culture medium (without additive, LifeTechnologies, Grand Island, NY) in 2ul Lipofectamine 2000 The mRNA of (LifeTechnologies, Grand Island, NY) compound 2ug dosage.
After 6 hours, animal is put to death and obtains serum and spleen.Spleen is transferred to 6 hole plates, and depositing in 1ml PBS It is kept on ice lower.It repeatedly uses scalpel blend rubber cell scraper to cut a spleen, squeezes out splenocyte until PBS is because thin Born of the same parents discharge and become muddy.
Cell is transferred to be placed on 12 hole cel culture plates 100um cell filter (BD Biosciences, San Jose, CA), and leave fibre fractionation.By gravity, cell passes through cell filter and collects to be cultivated in 12 holes of lower section In disk.The PBS of 1ml is transferred to Eppendorf to manage and rotate 5min at 2000rpm together with the splenocyte freely floated. PBS is discarded, and cell precipitation is merged with the fresh PBS of 500ul.5 minutes are vortexed by splenocyte by of short duration at 2000rpm It is resuspended.It discards PBS and 1ml BD Pharmlyse is added in cell precipitation.Splenocyte is resuspended by of short duration vortex.It will Cell is incubated at room temperature 3 minutes, is then rotated 5 minutes at 200 rpm.Cell simultaneously institute as above twice is washed with 500ul PBS It states and is rotated.Cell is resuspended with the PBS of 500ul and is rotated as described.
The splenocyte of 250ul is merged with 1x Pharmlyse buffer, it is of short duration to be vortexed or be resuspended with pipette, then exist It is rotated 2 minutes under 2000rpm.
In a pipe, cell precipitation is resuspended in the protease inhibitor cocktail with mammalian cell In 500ul RIPA buffer (BostonBioproducts, Ashland, MA) and by lysate freeze or immediately continue with into Row BCA measurement.In the second pipe, 250ul FACS staining kit fixed solution (4% formaldehyde is added；R and D Systems, Minneapolis, MN), it is then incubated at room temperature 10 minutes.Cell is washed twice with 500ul PBS and as above It is described to be rotated.Cell precipitation is resuspended in 500PBS and is stored in 4 DEG C.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60min, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 minutes in the 1X TBS solution of 5%BSA, then in the 1X TBS+ of 5%BSA It is incubated for again in 0.1%Tween solution 15 minutes.It is applied under 1: 500 to 1: 2000 dilution in the 1X of 3ml 5%BSA The GALK1 rabbit polyclonal antibody (Abcam, Cambridge, MA) of anti-GALK1 albumen in TBS solution, holding 3 is small at room temperature When and be gently mixed on orbital shaker.With stirring gently, film is washed 3 times with 1X TBS/0.1%Tween, every time five Minute.Goat antirabbit HRP conjugate (Abcam, Cambridge, MA) is conjugated to horseradish peroxidase and is bound to GALK1 rabbit Polyclonal antibody.The antibody of conjugation is diluted 1: 1000 to 1: 5000 in the 1X TBS solution of 5%BSA and is incubated at room temperature It educates 3 hours.Primary antibody and secondary antibody purchased from Abcam (Cambridge, MA), Novus Biologicals (Littleton, CO), Thermo Fisher (Rockford, IL), Millipore (Billerica, MA) or R and D systems (Minneapolis, MD).
At the end of incubation time, with stirring gently, film is washed 3 times with 1X TBS/0.1%Tween, every time 5 Minute.According to guidance by film in 5ml Pierce WestPico Chemiluminescent Subtrate (Thermo Fisher, Rockford, IL) in development.
As shown in the frame in Figure 45 A and 45B, respectively for 2 samples being evaluated for every kind of chemical substance, protein Trace detects the protein near the expection size of 30kd and 42kd.
The detection of 156. alexin β 103A albumen of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by the alexin β of 1250ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU) 103A (DEFB103A) mRNA (mRNA sequence shown in SEQ ID NO:1631；PolyA with about 140 nucleotide Tail is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volume In (LifeTechnologies, Grand Island, NY).Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) it is used as transfection reagent, and 5.5ul is diluted in 250ul final volumeIn. It is incubated at room temperature after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then 500ul is merged Solution is added to 3ml and contains in the cell culture medium of HeLa cell.Then it is incubated for plate as described above.Also to untreated cell Control evaluated.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 The DEFB103A rabbit polyclonal antibody of anti-DEFB103A albumen under dilution in the 1X TBS solution of 3ml 5%BSA (Abcam, Cambridge, MA) is kept for 3 hours at room temperature and is gently mixed on orbital shaker.Adjoint stirring gently, Film is washed 3 times, every time five minutes with 1X TBS/0.1%Tween.Donkey anti-rabbit NL557 conjugate (R&D Systems, Minneapolis, MN) it is conjugated to horseradish peroxidase and is bound to DEFB103A rabbit polyclonal antibody.By the antibody of conjugation 1: 1000 to 1: 5000 are diluted in the 1X TBS solution of 5%BSA and are incubated at room temperature 3 hours.Such as the frame institute in Figure 46 Show, Western blotting detects protein in the every kind of chemical substance evaluated.
The confirmation of 157. peptide identity of embodiment
Can be used with quantitative LC- multiple-reaction monitoring (MRM) with the concatenated liquid chromatography-mass spectrography of mass spectrum (LC-MS/MS) Come evaluate protein to confirm that peptide identity.
Can be used, there is quantitative LC- multiple-reaction monitoring (MRM) to measure (Biognosys AG, Schlieren Switzerland) evaluate any protein target as described herein with the concatenated liquid chromatography-mass spectrography of mass spectrum (LC-MS/MS) Target identity.It is commented using the LC-MS/MS that there is quantitative LC-MRM to measure (Biognosys, Schlieren Switzerland) Valence contain by modification mRNA expression protein HeLa cell lysate to confirm that in cell lysate peptide identity.Make The method described in known and/or this field compares the peptide fragment identified with the known protein for including isoform Compared with.
A.Sample preparation
By being incubated for 1 hour at 37 DEG C with (2- carboxyethyl) phosphine of 5mM tri- (TCEP) come each in reductive cleavage buffer Protein in sample.In the dark at room temperature, it is alkylated effect using 10mM iodoacetamide, continues 30 minutes.Make Protease with trypsase (sequence-level, PromegaCorporation, Madison, WI) 1: 50: will under protein rate Protein digestibility is at peptide.Digestion carries out (total digestion time is 12 hours) overnight at 37 DEG C.Peptide is cleaned, for using C18 Column spinner (The Nest Group, Southborough, MA) is analyzed by mass spectrometry according to the explanation of manufacturer.Peptide is dry extremely It is completely dried and is resuspended in LC solvent A (1% acetonitrile, 0.1% formic acid (FA)).All solvents be fromThe HPLC- grade of (St.Louis, MO), and unless otherwise stated, otherwise all chemicals are equal It is obtained from(St.Louis, MO).
B.LC-MS/MS and LC-MRM
For all mass spectral analyses, peptide is injected into the filling on nLC nanometers of liquid chromatographic systems of Proxeon Easy C18 column (Magic AQ, 3um granularity,Aperture, Michrom Bioresources, Inc (Auburn, CA)；11cm column It is long, 75um internal diameter, New Objective (Woburn, MA)) in.LC solvent is A: 1% second in the water with 0.1%FA Nitrile；B: 3% water in the acetonitrile with 0.1%FA.LC gradient for air gun analysis is the 5%-35% in 120 minutes Solvent B is that (total gradient length is by the 35%-100% solvent B in 2 minutes and the 100% solvent B for continuing 8 minutes later 130 minutes).LC-MS/MS shotgun experiment for peptide discovery is in the Thermo equipped with standard nano-electrospray source It is carried out on Scientific (Thermo Fisher Scientific) (Billerica, MA) Q Exactive mass spectrograph.For The LC gradient of LC-MRM is 5%-35% solvent B in 30 minutes, be the 35%-100% solvent B in 2 minutes later with And the 100% solvent B for continuing 8 minutes (total gradient length is 40 minutes).Thermo Scientific(Thermo Fisher Scientific) (Billerica, MA) TSQ Vantage triple quadrupole mass spectrograph is equipped with standard nano-electrospray source.With In the non-predetermined MRM mode of recalibration, operated under 20ms residence time/transformation.For peptide in entire sample Relative quantification operates TSQ Vantage with scheduled MRM mode, and wherein acquisition window length is 4 minutes.LC eluent is existed The Q1 peak width of electron spray and use 0.7Da execute MRM and analyze under 1.9kV.By linear regression, counted according to the specification of supplier Calculate the collision energy of TSQ Vantage.
C.Measurement design, data processing and analysis
For the generation of LC-MRM measurement, 12 analyze from LC-MS/MS with the measurement of scheduled LC-MRM mode are most by force Fragment ions, and using mProphet scoring part(Cluetec, Karlsruhe, Germany) comes Handle data (Reiter etc., mProphet:Automated data processing and statistical Validation for large-scale SRM experiments, Nature Methods, 2011 (8), 430-435；Institute The content for stating document is incorporated herein by reference).Manual verification is carried out to measurement, measures accurate segment intensity, and opposite IRT (indexation the retention time) (Using such as Escher iRT, a normalized is specified in the iRT- peptide of Biognosys Retention time for more targeted measurement of peptides, Proteomics, 2012 (12), 1111-1121；The content of the document is incorporated herein by reference).
For the relative quantification of the peptide of entire sample series, in 8 most strong transformation of the entire sample series to each measurement It measures.Use SpectroDiveTM(Biognosys, Schlieren Switzerland) carries out data analysis.For choosing Determine peptide, compares total peak area, and the False discovery rate of application 0.05.With lower than 0.05 Q value peptide be not included and It is considered as and is not detected in corresponding sample.
The confirmation of peptide identity of the embodiment 158. from the modification mRNA containing chemical modification
Using the LC-MS/MS evaluation with quantitative LC-MRM containing by low-density lipoprotein receptor as described in embodiment 157 Body (LDLR) modifies mRNA (mRNA sequence shown in SEQ ID NO:21463；PolyA with about 140 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine), ornithine transfer Enzyme (OTC) modifies mRNA (mRNA sequence shown in SEQ ID NO:1659；PolyA tail with about 160 nucleotide, It is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine), wild-type apoliproteins A-I (APOA1wt) mRNA (mRNA sequence shown in SEQ ID NO:21453 is modified；PolyA with about 160 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine), hepcidin (HEPC) modification MRNA (mRNA sequence shown in SEQ ID NO:21471；PolyA tail with about 160 nucleotide does not show in sequence Out；5 ' caps, Cap1；Modified completely with 5-methylcytosine and 1- methyl pseudouridine), 2 type hemochromatosis (HFE2) modification MRNA (mRNA sequence shown in SEQ ID NO:21472；PolyA tail with about 160 nucleotide does not show in sequence Out；5 ' caps, Cap1；Modified completely with 5-methylcytosine and 1- methyl pseudouridine) or fumarylacetoacetate hydrolase (FAH) mRNA (mRNA sequence shown in SEQ ID NO:21473 is modified；PolyA tail with about 160 nucleotide, sequence It is not shown in column；5 ' caps, Cap1；Modified completely with 5-methylcytosine and 1- methyl pseudouridine) generate protein cell Lysate.The peptide fragment of evaluated protein identified is shown in table 200.All peptides are specificity for Parent Protease , LDLR and HFE2 are specific for Parent Protease and its isoform.In table 200, " Uniprot ID " refers to and works as Protein mark from UniProt database when peptide fragment sequences being compared for all evaluation protein in database Know symbol.House keeping protein for evaluating the protein in cell lysate is shown in table 201.
200. protein and peptide fragment sequence of table
201. house keeping protein of table
The confirmation of peptide identity of the embodiment 159. from chemical modification mRNA
As described in embodiment 157 using the LC-MS/MS with quantitative LC-MRM to containing by with 5-methylcytosine and That pseudouridine (5mC and pU) is modified completely, being modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC and 1mpU), 25% uridine wherein is modified with 2- thio uridine and is modified with 25% cytimidine of 5-methylcytosine modification (s2U and 5mC) , with pseudouridine (pU) modification completely or the cytotoxic t-lymphocyte modified completely with 1- methyl pseudouridine (1mpU) correlation Albumen 4 (CTLA4) modifies mRNA (mRNA sequence shown in SEQ ID NO:21521；With about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1), serpin peptidase inhibitors clade A (α -1 antiprotease, anti-tryptose Enzyme) member 1 (SERPINA1) modification mRNA (mRNA sequence shown in SEQ ID NO:21522；With about 140 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1), sphingomyelin phosphodiesterase 1 (SMPD1) modify mRNA (SEQ ID MRNA sequence shown in NO:21523；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), hypoxanthine-guanine phosphoribosyl transferase (HPRT1) modifies mRNA (shown in SEQ ID NO:21524 MRNA sequence；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), 4- hydroxyphenylpyruvic acid it is double Oxygenase (HPD) modifies mRNA (mRNA sequence shown in SEQ ID NO:21525；With about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1), bone morphogenesis protein-7 (BMP7) modification mRNA (SEQ ID NO: MRNA sequence shown in 21526；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), ovum Phosphatide-cholesterol acyltransferase (LCAT) modifies mRNA (mRNA sequence shown in SEQ ID NO:21527；With about The polyA tail of 140 nucleotide is not shown in sequence；5 ' caps, Cap1), the short isoform 1 (AIFsh) of apoptosis inducing factor Modify mRNA (mRNA sequence shown in SEQ ID NO:21528；PolyA tail with about 140 nucleotide, in sequence It is not shown；5 ' caps, Cap1), oncoprotein 53 (TP53 or P53) modify mRNA (mRNA sequence shown in SEQ ID NO:1670 Column；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), argininosuccinate synthetase (ASS1) mRNA (mRNA sequence shown in SEQ ID NO:21529 is modified；PolyA tail with about 160 nucleotide, It is not shown in sequence；5 ' caps, Cap1), KIT ligand/stem cell factor (KITLG) modification mRNA (shows in SEQ ID NO:21530 MRNA sequence out；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), nerve modulation egg White 1 β 2a isoform (NRG1) modifies mRNA (cDNA sequence shown in SEQ ID NO:21531；For (IVT) to be transcribed in vitro T7 promoter, 5 ' non-translational regions (UTR) and 3 ' UTR, show in the sequence), vasoactive intestinal peptide (VIP) modification mRNA (SEQ MRNA sequence shown in ID NO:21532；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), aryl sulfatase B (ARSB) modifies mRNA (mRNA sequence shown in SEQ ID NO:1618；With about 140 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1), 6- pyruvoyl tetrahydro pterin synzyme (PTS) modify mRNA (mRNA sequence shown in SEQ ID NO:1609；PolyA tail with about 140 nucleotide is not shown in sequence；5' Cap, Cap1), protein 5 (FNDC5 or irisin) containing type III fibronectin domain modification mRNA (SEQ ID NO: CDNA sequence shown in 21533；For T7 promoter, 5 ' non-translational regions (UTR) and the 3 ' UTR of (IVT) to be transcribed in vitro, in sequence Shown in column), amelogenin (AMELY) modification mRNA (mRNA sequence shown in SEQ ID NO:1613；With about 140 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1), aldolase A (ALDOA) modification mRNA (SEQ ID NO: MRNA sequence shown in 21534；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), mind MRNA (mRNA sequence shown in SEQ ID NO:21535 is modified through growth factor (NGF)；With about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1), Glycogensynthase 2 (GYS2) modification mRNA (shown in SEQ ID NO:21536 MRNA sequence；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), GTP cyclohydrolase 1 (GCH1) mRNA (mRNA sequence shown in SEQ ID NO:1649 is modified；PolyA tail with about 140 nucleotide, sequence It is not shown in column；5 ' caps, Cap1), hepatocyte growth factor (HGF) modify mRNA (mRNA shown in SEQ ID NO:21537 Sequence；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), outer M-band-A (EDA) modification MRNA (mRNA sequence shown in SEQ ID NO:1636；PolyA tail with about 140 nucleotide does not show in sequence Out；5 ' caps, Cap1), arginase (ARG1) modify mRNA (mRNA sequence shown in SEQ ID NO:21538；With about The polyA tail of 140 nucleotide is not shown in sequence；5 ' caps, Cap1), Serum amyloid P (APCS) modify mRNA (mRNA sequence shown in SEQ ID NO:21539；PolyA tail with about 140 nucleotide is not shown in sequence；5' Cap, Cap1), phosphorylase kinase γ 2 (PHKG2) modify mRNA (mRNA sequence shown in SEQ ID NO:21540；Have The polyA tail of about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), 685 deoxyribonuclease Is (DNASE1) repair Adorn mRNA (mRNA sequence shown in SEQ ID NO:1632；PolyA tail with about 140 nucleotide does not show in sequence Out；5 ' caps, Cap1), Exenatide modify mRNA (mRNA sequence shown in SEQ ID NO:21541；With about 140 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1), glycogen generate albumen -1 (PYGL) modify mRNA (SEQ ID MRNA sequence shown in NO:21542；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), alpha-galactosidase (GLA) modifies mRNA (mRNA sequence shown in SEQ ID NO:1640；With about 140 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1), α-L- iduronase (IDUA) modify mRNA (SEQ ID MRNA sequence shown in NO:1652；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), Galactokinase -1 (GALK1) modifies mRNA (mRNA sequence shown in SEQ ID NO:21470；With about 140 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1) cell lysate of protein that generates evaluated.Evaluated egg The peptide fragment of white matter identified is shown in table 202.
202. protein and peptide fragment sequence of table
The confirmation of 160. peptide identity of embodiment and the comparison of recombinant protein
Liquid chromatogram (LC)-mass spectrum (MS) analysis, LC-MS/MS trypsase peptide mapping/sequencing analysis and 2 is carried out Fluorescent differences gel electrophoresis (2-D DIGE) is tieed up to confirm the ornithine ammonia first modified completely with 5-methylcytosine and pseudouridine Acyltransferase (OTC) modifies mRNA (mRNA sequence shown in SEQ ID NO:1659；With about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) or the Portugal UDP modified completely with 5-methylcytosine and 1- methyl pseudouridine 1 family polypeptides A1 (UGT1A1) of grape uronic acid based transferase modifies mRNA (mRNA sequence shown in SEQ ID NO:21464； PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) what is transfected with any one transcript Expression in HeLa cell.Following material: hplc grade water (EMD Chemicals Inc., Gibbstown, NJ) is used；First Sour (90%) (J.T.Baker, Phillipsburg, NJ)；Iodoacetamide (Sigma Aldrich, St.Louis, MO)；Methanol (EMD Chemicals Inc., Gibbstown, NJ)；Tris (2- carboxyethyl) phosphonium salt acid salt solution (TCEP) of 0.5M (Sigma Aldrich, St.Louis, MO)；Tris (methylol) aminomethane buffer substance (Tris buffer) pH 8.0 (Fluka-Sigma Aldrich, St.Louis, MO)；Calcium chloride is dehydrated ACS reagent (CaCl2) (Sigma Aldrich, St.Louis, MO)；OTC reference protein [the recombinant full-lenght people OTC albumen (Abcam, Cambridge, MA) of purifying]；UGT1A1 Reference protein [the rhCC16 T1A1 albumen (OriGene Technologies, Rockville, MD) of purifying.
Hela cell is transfected with the mRNA of coding OTC and UGT1A1.After transfection, by cell cracking and it is centrifuged to form cell Precipitating.The total protein concentration of each sample is measured by dicinchonine acidity test (BCA measurement).From untransfected Total protein concentration of the cell precipitation of HeLa cell with 1707 μ g/mL, and the HeLa cell transfected from OTC and UGT1A1 Cell precipitation be respectively provided with the total protein concentration of 3947 μ g/mL and 2609 μ g/mL.
For trypsase peptide mapping/sequencing, recombinant protein and cell precipitation is made to be subjected to trypsin digestion.Have The OTC and UGT1A1 of preparation and reorganization in the 100mM Tris buffer of 3mM calcium chloride.Protein (each 5 μ g) is used into 2mM TCEP Reduction, is then denaturalized 5 minutes at 100 DEG C.Sample is cooled to room temperature, 2mM iodoacetamide subsequently is then used.Use 0.5mM The sample of the further reductive alkylation of TCEP.Sample is handled overnight in a water bath at 37 DEG C with trypsase.It will be by digestion Sample take out from water-bath and be quenched with the aqueous solution of 2% formic acid.In the 100mM Tris with 3mM calcium chloride of pH 8.0 Cell precipitation is prepared in buffer.The cell precipitation of reconstruct and supernatant samples (each 50 μ g) 2mM TCEP are restored, then It is denaturalized 5 minutes at 100 DEG C.Sample is cooled to room temperature, 100mM iodoacetamide subsequently is then used.With 0.5mM TCEP into The sample of one step reductive alkylation.Sample is handled overnight in a water bath at 37 DEG C with trypsase.By the sample by digestion It takes out from water-bath and is quenched with the aqueous solution of 2% formic acid.
The tryptic peptide of recombinant protein, cell precipitation and supernatant samples is injected into 150x2.1mm Xbridge On BEH300 C18 column.Column heater is set as 35 DEG C.Mobile phase A is the aqueous solution of 0.1% formic acid.Mobile phase B is 90/ 0.1% formic acid in 10 acetonitrile/waters (v/v).With ABSciex API QSTAR ELITE quadrupole time of-flight mass spectrometer (AB Sciex, Foster City, CA) acquisition digestion product mass spectrogram.Using positive ionization electrospray ionization (ESI) in the flight time (TOF) data are acquired in MS and MS/MS mode.It is 500 DEG C by Turbo IonSpray interface setting and maintains 5.0kV's Under turbospray voltage, wherein removing cluster potential is 35V.In the sprayer and ion for being respectively set as 40 and 35 (arbitrary units) It is ionized under the auxiliary of spray gas (nitrogen).It is adopted using 2.0 software of Analyst QS (AB Sciex, Foster City, CA) Collect and handles data.
Using BioAnalyst software (AB Sciex, Foster City, CA) by LC-MS data and OTC and UGT1A1 egg White prediction/theory tryptic digests compare.Use the digestion product of recombinant protein heavy as the cell in digestion The reference peptide to form sediment with matched peptide in supernatant.Also LC-MS and LC-MS/MS data are mapped and are sequenced, to point out HeLa The tryptic digests of cell-derived protein.Peptide based on LC-MS/MS product ion map confirmation tryptic peptide Sequence.
There are OTC albumen in the cell precipitation of the HeLa cell transfected with OTC mRNA.Peptide mapping is in cell precipitation 16 kinds of OTC peptides are identified in tryptic digest.The product ion map of cell-derived peptide and the map of recombinant protein are good Good matching.The database search of human protein points out that the sequence of 13 kinds of unmodified peptides is only specific to people OTC.Pass through use The HeLa cell of UGT1A1 mRNA transfection expresses UGT1A1 albumen.Based on accurate mass distribution and retention time, in cell precipitation Tryptic digest in identify 7 kinds of UGT1A1 peptides matching Tooth-Lid Factor T1A1 albumen.It is matched by LC-MS/MS confirmation Peptide identity.Search based on human protein, 4 kinds in tryptic peptide observed by 7 kinds are special to hCCSP T1A albumen Property, and a kind in tryptic peptide observed by 7 kinds is only specific to UGT1A1.Table 203 shows from UGT1A1 and modifies The peptide identified in the HeLa cell lysate of mRNA, and table 204 shows the peptide identified from OTC modification mRNA.
Table 203.UGT1A1 peptide fragment
Peptide fragment sequences SEQ ID NO
AMAIADALGKIPQTVLWR 21618
WLPQNDLLGHPMTR 21619
IPQTVLWR 21620
TYPVPFQR 21621
AMAIADALGK 21622
ENIMR 21623
SDFVK 21624
Table 204.OTC peptide fragment
The protein overview of cell precipitation sample is further characterized by two-dimensional fluoroscopic difference gel electrophoresis.With different glimmering Photoinitiator dye handles test sample.Isoelectric focusing (IEF) is used in the first dimension and uses dodecyl sulphate in the second dimension Sodium polyacrylamide gel electrophoresis (SDS-PAGE) separates the content of sample.To the albumen of OTC and UGT1A1 cell precipitation Matter overview is compared.DeCyder variance analysis software (GE Healthcare, Buckinghamshire, UK) is used for data Analysis.Analyzing result confirms that there are OTC and UGT1A1 in HeLa cell.
The confirmation of peptide identity of the embodiment 161. from the modification mRNA containing chemical modification
As described in embodiment 157, contained using having the LC-MS/MS of quantitative LC-MRM to evaluate by phonetic with 5- methyl born of the same parents Pyridine and pseudouridine (5mC and pU) completely modification, with 5-methylcytosine and 1- methyl pseudouridine (5mC and 1mpU) completely modification, Wherein modify 25% uridine with 2- thio uridine and modified with 25% cytimidine of 5-methylcytosine modification (s2U and 5mC), The Catherine rhzomorph antimicrobial peptide modified with pseudouridine (pU) or modified completely with 1- methyl pseudouridine (1mpU) completely (CAMP) mRNA (mRNA sequence shown in SEQ ID NO:1621 is modified；PolyA tail with about 140 nucleotide, sequence It is not shown in column；5 ' caps, Cap1), CAP18 modify mRNA (mRNA sequence shown in SEQ ID NO:21638；With about The polyA tail of 140 nucleotide is not shown in sequence；5 ' caps, Cap1), ciliary neurotrophic factor (CNTF) modify mRNA (mRNA sequence shown in SEQ ID NO:21639；PolyA tail with about 140 nucleotide is not shown in sequence；5' Cap, Cap1), follicle-stimulating hormone (FSH) beta polypeptides (FSHB) modify mRNA (mRNA sequence shown in SEQ ID NO:21640；With big The polyA tail of about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), interferon-' alpha ' 2 (IFNA2) modify mRNA (SEQ ID MRNA sequence shown in NO:1654；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), 1 fibroblast of interferon beta (IFNB1) modifies mRNA (mRNA sequence shown in SEQ ID NO:1655；With about 140 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1), mitochondria methylmalonyl-CoA mutase (MUTA) Modify mRNA (mRNA sequence shown in SEQ ID NO:1658；PolyA tail with about 140 nucleotide, in sequence not It shows；5 ' caps, Cap1), Galectin-3 (LEG3) modify mRNA (mRNA sequence shown in SEQ ID NO:21641；Have The polyA tail of about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), transforming growth factor beta-3 (TGFB3) modification MRNA (mRNA sequence shown in SEQ ID NO:21642；PolyA tail with about 140 nucleotide does not show in sequence Out；5 ' caps, Cap1) or tuftelin (TUFT1) modification mRNA (mRNA sequence shown in SEQ ID NO:1669；With big The polyA tail of about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) generate albuminous cell lysate.Evaluated egg The peptide fragment of white matter identified is shown in table 205.In table 205, " Uniprot ID " refers to when in database Protein identifier from UniProt database when peptide fragment sequences are compared in all evaluation protein.
205. protein and peptide fragment sequence of table
The detection of Expression of LDL Receptor in 162. cell culture of embodiment
A.HeLa cell transfecting
HeLa cell is coated on Eagles minimum essential medium (EMEM, Life Technologies, Grand Island, NY) in 24 porose discs (Corning Life Sciences, Tewksbury, MA) (7.5x104Cells/well) on simultaneously The overnight incubation under standard cell culture conditions, the culture medium are supplemented with 10% fetal calf serum (FCS, Life Technologies, Grand Island, NY) and 1X glutamax reagent (Life Technologies, Grand Island, NY).By by the LDL receptor of 250ng modified completely with 5-methylcytosine and pseudouridine (LDLR) mRNA (mRNA sequence shown in SEQ ID NO:21463 is modified；PolyA tail with about 140 nucleotide, It is not shown in sequence；5 ' caps, Cap1), mCherry modify mRNA (mRNA sequence shown in SEQ ID NO:21439；Have The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is repaired completely with 5-methylcytosine and pseudouridine Decorations) or luciferase modification mRNA (mRNA sequence shown in SEQ ID NO:21446；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) and 50 μ l Opti-MEM Reagent (Life Technologies, Grand Island, NY) merges in the first pipe, and transfects and try with the L2000 of 1 μ l Agent (Life Technologies, Grand Island, NY) merges in the Opti-MEM of 50 μ l in the second pipe to prepare use Transfection solution in each hole to be processed.After preparation, the first pipe and the second pipe are incubated at room temperature 5 minutes, later will Respective content merges.Combined Transfection solution is incubated at room temperature 15 minutes.Then 100 μ l are added into each hole Transfection solution.Cell is further cultured for 16 hours, continues to analyze later.
B. pass through Flow cytometry LDLR
After transfection, the 0.25% trypsase (Life of 60 μ l is added from removal culture medium in cell and into each hole Technologies, Grand Island, NY).By cell trypsin digestion 2 minutes, the pancreas egg in 240 holes μ l/ was added later White enzyme inhibitor (Life Technologies, Grand Island, NY).Resulting cell solution is transferred to 96 hole plates (Corning Life Sciences, Tewksbury, MA) makes cell precipitation (800x gravity continues 5 minutes) simultaneously by centrifugation Discard supernatant liquid.Cell precipitation is washed with PBS and is resuspended in Foxp3 and fixes/permeabilization solution (eBioscience, San Diego, CA) in, it is kept for 45 minutes.Cell is precipitated again by centrifugation (800x gravity continues 5 minutes), and is resuspended in Change in buffer (eBiosciences, San Diego, CA), is kept for 10 minutes.Pass through centrifugation (800x gravity continues 5 minutes) It precipitates cell again, and is washed in permeabilization buffer.Then it uses the primary antibody for LDLR, be that phycoerythrin marks later Two process resistant cells.Then the cell of label and FACS buffer solution (had into 1% bovine serum albumin(BSA) and 0.1% sodium azide PBS) merge, and be transferred to gathering pipe.As shown in Figure 47, BD Accuri (BD Biosciences, San are then used Jose, CA) it is analyzed by cell of the flow cytometry to label.
C. pass through Immunofluorescence test LDLR
20 points are handled at room temperature by the cells rinsed with PBS of transfection, and with fixed solution (PBS with 4% formaldehyde) Clock.Then it is washed with PBS and uses permeabilization/lock solution (Tris with 5% bovine serum albumin(BSA) together with 0.1%Tween-20 Buffered saline) processing cell.With stirring gently, cell is incubated at room temperature 2 hours, later with containing 0.05% The PBS of Tween-20 is washed 3 times.Then by cell with or without primary antibody (goat anti-LDLR, R&D Systems, Minneapolis, MN) or normally IgG compares processing 2 hours at room temperature, washs 3 with the PBS containing 0.05%Tween-20 It is secondary, and with contain 1: 200 dilution donkey anti goat igg and fluorescent marker (R&D Systems, Minneapolis, MN) two Anti- solution processing.Cell is washed with the PBS containing 0.05%Tween-20 again and is detected by fluorescent microscopic imaging.Without glimmering Light immunostaining detects transient expression luciferase or the cell of mCherry by fluorescence microscopy.
Embodiment 163. passes through Immunofluorescence test alexin β 103A
A.HeLa cell transfecting
HeLa cell is coated on Eagles minimum essential medium (EMEM, Life Technologies, Grand Island, NY) in 24 porose discs (Corning Life Sciences, Tewksbury, MA) (7.5x104Cells/well) on simultaneously The overnight incubation under standard cell culture conditions, the culture medium are supplemented with 10% fetal calf serum (FCS, Life Technologies, Grand Island, NY) and 1X glutamax reagent (Life Technologies, Grand Island, NY).By the way that the alexin β 103A (DEFB103A) of 250ng is modified mRNA (shown in SEQ ID NO:1631 MRNA sequence；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) or mCherry modification MRNA (mRNA sequence shown in SEQ ID NO:21439；PolyA tail with about 160 nucleotide does not show in sequence Out；5 ' caps, Cap1) with 50 μ l Opti-MEM reagents (Life Technologies, Grand Island, NY) in the first pipe Merge, and with the L2000 transfection reagent of 1 μ l (Life Technologies, Grand Island, NY) in the Opti- of 50 μ l Merge in MEM in the second pipe to prepare the Transfection solution for each hole to be processed.After preparation, by the first pipe and second Pipe is incubated at room temperature 5 minutes, later merges respective content.Combined Transfection solution is incubated at room temperature 15 points Clock.Then the Transfection solution of 100 μ l is added into each hole.Cell is further cultured for 16 hours, continues to analyze later.
B. pass through Immunofluorescence test DEFB103A
20 points are handled at room temperature by the cells rinsed with PBS of transfection, and with fixed solution (PBS with 4% formaldehyde) Clock.Then it is washed with PBS and uses permeabilization/lock solution (Tris with 5% bovine serum albumin(BSA) together with 0.1%Tween-20 Buffered saline) processing cell.With stirring gently, cell is incubated at room temperature 2 hours, later with containing 0.05% The PBS of Tween-20 is washed 3 times.Then make cell do not have to a process resistant, with anti-DEFB103A primary antibody (rabbit-anti DEFB103A, Abcam, Cambridge, MA) it processing or is handled at room temperature 2 hours with normal IgG control antibodies, with containing 0.05% The PBS of Tween-20 is washed 3 times, and marks (R&D together with red fluorescence with the donkey anti-rabbit IgG for containing 1: 200 dilution Systems, Minneapolis, MN) two corresponding anti-solution processing.Cell is washed simultaneously with the PBS containing 0.05%Tween-20 again It is detected by fluorescent microscopic imaging.Without immunofluorescence dyeing, show the cell of the modification mRNA transfection with coding mCherry.
The confirmation of 164. protein expression of embodiment
Being urinated with 5-methylcytosine and vacation to protein described in coding schedule 205 by fluorescence and/or Western blotting It is that glycosides (5mC/pU) is modified completely, being modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU), wherein use That 5-methylcytosine replaces that 25% uridine (s2U/5mC) modified instead of 25% cytimidine and with 2- thio uridine, Analyzed with 1- methyl pseudouridine (1mpU) modification completely or with the modification mRNA that pseudouridine (pU) is modified completely to confirm that The length of the expression of protein and expressed protein.Table 205, which describes modification mRNA, (has about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) or cDNA (for T7 promoter, the 5 ' non-translational regions of (IVT) to be transcribed in vitro (UTR) and 3 ' UTR, show in the sequence) sequence, the chemical modification and length (in terms of base-pair) evaluated, if If knowing.
205. protein of table and expression
165. vascular endothelial growth factor expression of embodiment and cell viability
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 100,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole of 100ul 24 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, by 62ng, 250ng, 750ng Or 1500ng modified completely with 5-methylcytosine and pseudouridine (5mC/pU), urinated with 5-methylcytosine and 1- methyl vacation It is that glycosides (5mC/1mpU) is modified completely, with 1- methyl pseudouridine (1mpU) modification or (unmodified) blood without containing modification completely Endothelial tube growth factor (VEGF) modifies RNA (mRNA sequence shown in SEQ ID NO:1672；With about 160 nucleosides The polyA tail of acid is not shown in sequence；5 ' caps, Cap1) and as the 2ul Lipofectamine 2000 for transfecting auxiliary agent (LifeTechnologies, Grand Island, NY) mixing, and it is added to cell in duplicate.
A.VEGF protein expression
After being incubated for 24 hours, the cell culture supernatant of the cell of VEGF expression is collected and at 10.000rcf Centrifugation 2 minutes.Then VEGF- specific ELISA kit (R&D Systems, Minneapolis, MN) is used, according to manufacturer Explanation clear supernatant is analyzed.All samples are diluted, until the value measured is in ELISA standard curve The range of linearity in.Also 5-methylcytosine and pseudouridine are used to untreated cell, in duplicate with 62ng or 750ng (5mC/pU) modify completely, it is being modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU), with 1- methyl Pseudouridine (1mpU) modification completely or (unmodified) green fluorescent protein (GFP) without containing modification modify mRNA (SEQ ID MRNA sequence shown in NO:1672；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) Control and 250ng mCherry modify mRNA (mRNA sequence shown in SEQ ID NO:21439；With about 160 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1) control analyzed.The amount (pg/ml) of produced VEGF exists It is shown in table 206 and Figure 48.In Figure 48, " eGFP " refers to that green fluorescent protein modifies mRNA, and " MC " refers to that mCherry is modified MRNA, " UNT-1 " refer to untreated cell, and " G0 " refers to that " G1 " refers to phonetic with 5- methyl born of the same parents without the modification mRNA of modification What pyridine and pseudouridine were modified completely, " G1 " refers to be modified with 5-methylcytosine and 1- methyl pseudouridine completely, and " G5 " Refer to and is modified completely with 1- methyl pseudouridine.
Using being modified with 5-methylcytosine and 1- methyl pseudouridine or modified completely with 1- methyl pseudouridine completely The cell of VEGF modification mRNA transfection shows highest vegf expression.
Table 206.VEGF protein yield (pg/ml)
B.Vascular endothelial growth factor
After being incubated for 24 hours, removes culture medium and measure reagent with the Cell Titer Glo (CTG) in the hole 200ul/ (Promega Corporation, Madison, WI) lytic cell.It is incubated at room temperature after 15 minutes, by two groups of 10ul's Every kind of lysate is transferred in 96 hole plate of White-opalescent polystyrene (Corning, Tewksbury, MA).Again will The Cell Titer Glo measurement reagent of 100ul is added in each aliquot of cell lysate.With general luminous journey The reagent mixture of total volume 110ul is analyzed in the BioTek Synergy H1 plate reader of sequence.It is thin with untreated HeLa Born of the same parents draw the average value measured in duplicate in the graph (1.0=100% vigor) as reference.Also to duplicate Untreated cell and modify mRNA (mRNA sequence shown in SEQ ID NO:1672 with the mCherry of 250ng；Have The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) control of cell of processing evaluated.Normalizing ATP abundance/cell viability of change is shown in table 207 and Figure 49.In Figure 49, " MC " refers to that mCherry modifies mRNA, " UNT-1 " and " UNT-2 " refers to untreated cell, and " G0 " refers to that " G1 " refers to 5- methyl without the modification mRNA of modification What cytimidine and pseudouridine were modified completely, " G1 " refers to be modified with 5-methylcytosine and 1- methyl pseudouridine completely, " G5 " Refer to and modified completely with 1- methyl pseudouridine, and " * " refers to only one data point of acquisition.
Using the cell modified completely with 5-methylcytosine and pseudouridine or unmodified VEGF modification mRNA is transfected In observe indicator cells vigor loss reduced luminescence activity.Using complete with 5-methylcytosine and 1- methyl pseudouridine The cell viability of the cell of the full VEGF modification mRNA transfection modified or modified completely with 1- methyl pseudouridine does not reduce.
The normalized ATP abundance/cell viability of table 207.
Protein output of 166. hematopoietin of embodiment in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 100,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole of 100ul 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng is used into 5- methyl It is that cytimidine and pseudouridine (5mC/pU) are modified completely, repaired completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) Decorations, wherein with the uridine of 2- thio uridine modification 25% and with the cytimidine of 5-methylcytosine modification 25% (s2U and 5mC) Hematopoietin being modified, being modified with pseudouridine (pU) or modified completely with 1- methyl pseudouridine (1mpU) completely (EPO) RNA (mRNA sequence shown in SEQ ID NO:1638 is modified；PolyA tail with about 160 nucleotide, sequence In be not shown；5 ' caps, Cap1) it is diluted in OPTI-MEM (LifeTechnologies, the Grand of 10ul final volume Island, NY) in.Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) as transfection Reagent, and the lipofectamine of 0.2ul 2000 is diluted in the OPTI-MEM of 10ul final volume.It incubates at room temperature It educates after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then solution 20ul merged is added to In the cell culture medium that 100ul contains HeLa cell and it is incubated at room temperature.Also to the thin of the processing of lipofecatmine 2000 The control of born of the same parents (L2000) and untreated cell are analyzed.
After being incubated for 18 to 22 hours, the cell culture supernatant of the cell of expression EPO is collected and in 10.000rcf Lower centrifugation 2 minutes.Then EPO ELISA kit (Stem Cell Technologies, Vancouver Canada) is used, root Clear supernatant is analyzed according to the explanation of manufacturer.All samples are diluted, until the value measured is in ELISA In the range of linearity of standard curve.The amount of generated protein is shown in table 208.
208. protein output of table
The vivoexpression of 167. insulin aspart of embodiment modification mRNA
With concentration shown in table 209 with and from Invitrogen (Carlsbad, CA) Compound being modified completely with 5-methylcytosine and pseudouridine (5mC/pU), phonetic with 5- methyl born of the same parents of Lipofectamine2000 It is that pyridine and 1- methyl pseudouridine (5mC/1mpU) are modified completely, wherein with the uridine of 2- thio uridine modification 25% and with 5- methyl Cytimidine modification 25% cytimidine (s2U and 5mC) modified, with pseudouridine (pU) completely modification or with 1- methyl vacation urine The insulin aspart that glycosides (1mpU) is modified completely modifies mRNA (mRNA sequence shown in SEQ ID NO:21468；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1) transfection cell.The total volume of every 96 hole transfection mixture For 50 μ l, it is divided into two equal portions, every part of 25 μ l.The aliquot of one 25 μ l contains the corresponding mmRNA of 1 μ l and 24 μ l Opti- MEM, the aliquot of another 25 μ l contain 0.4 μ l Lipofectamine2000 and 24.6 μ l Opti-MEM.It is needing When, by aliquot serial dilution in Opti-MEM containing modification mRNA and Opti-MEM.Two aliquots are in room temperature It is lower to be individually incubated for 15min.Then, two aliquots are mixed, and is incubated for 20min again at room temperature, it finally will be total Totally 50 μ l transfection mixtures are transferred to 96 holes containing the 100 additional 35000HEK293 cells of μ l cell culture medium.Then by cell In 37 DEG C/5%CO of humidification2It is incubated in cell incubator for 24 hours, harvests cell culture supernatant or preparation cell cracking later Object.Protein expression is detected by ELISA, and protein (pg/ml) is shown in Figure 50 and table 209.In table 209, " > ", which is meant, to be greater than.
209. protein expression of table
The vivoexpression of 168. insulin glargine of embodiment modification mRNA
With concentration shown in table 210 with and from Invitrogen (Carlsbad, CA) Compound being modified completely with 5-methylcytosine and pseudouridine (5mC/pU), phonetic with 5- methyl born of the same parents of Lipofectamine2000 It is that pyridine and 1- methyl pseudouridine (5mC/1mpU) are modified completely, wherein with the uridine of 2- thio uridine modification 25% and with 5- methyl Cytimidine modification 25% cytimidine (s2U and 5mC) modified, with pseudouridine (pU) completely modification or with 1- methyl vacation urine The insulin glargine that glycosides (1mpU) is modified completely modifies mRNA (mRNA sequence shown in SEQ ID NO:21465；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap1) transfection cell.The total volume of every 96 hole transfection mixture For 50 μ l, it is divided into two equal portions, every part of 25 μ l.The aliquot of one 25 μ l contains the corresponding mmRNA of 1 μ l and 24 μ l Opti- MEM, the aliquot of another 25 μ l contain 0.4 μ l Lipofectamine2000 and 24.6 μ l Opti-MEM.It is needing When, by aliquot serial dilution in Opti-MEM containing modification mRNA and Opti-MEM.Two aliquots are in room temperature It is lower to be individually incubated for 15min.Then, two aliquots are mixed, and is incubated for 20min again at room temperature, it finally will be total Totally 50 μ l transfection mixtures are transferred to 96 holes containing the 100 additional 35000HEK293 cells of μ l cell culture medium.Then by cell In 37 DEG C/5%CO of humidification2It is incubated in cell incubator for 24 hours, harvests cell culture supernatant or preparation cell cracking later Object.Protein expression is detected by ELISA, and protein (pg/ml) is shown in Figure 51 and table 210.
210. protein expression of table
169. paddy of embodiment relies the vivoexpression of insulin modification mRNA
To concentration shown in table 211 and from Invitrogen (Carlsbad, CA) Compound being modified completely with 5-methylcytosine and pseudouridine (5mC/pU), phonetic with 5- methyl born of the same parents of Lipofectamine2000 It is that pyridine and 1- methyl pseudouridine (5mC/1mpU) are modified completely, wherein with the uridine of 2- thio uridine modification 25% and with 5- methyl Cytimidine modification 25% cytimidine (s2U and 5mC) modified, with pseudouridine (pU) completely modification or with 1- methyl vacation urine The paddy that glycosides (1mpU) is modified completely relies insulin to modify mRNA (mRNA sequence shown in SEQ ID NO:21469；With about The polyA tail of 160 nucleotide is not shown in sequence；5 ' caps, Cap) transfection cell.The total volume of every 96 hole transfection mixture For 50 μ l, it is divided into two equal portions, every part of 25 μ l.The aliquot of one 25 μ l contains the corresponding mmRNA of 1 μ l and 24 μ l Opti- MEM, the aliquot of another 25 μ l contain 0.4 μ l Lipofectamine2000 and 24.6 μ l Opti-MEM.It is needing When, by aliquot serial dilution in Opti-MEM containing modification mRNA and Opti-MEM.Two aliquots are in room temperature It is lower to be individually incubated for 15min.Then, two aliquots are mixed, and is incubated for 20min again at room temperature, it finally will be total Totally 50 μ l transfection mixtures are transferred to 96 holes containing the 100 additional 35000HEK293 cells of μ l cell culture medium.Then by cell In 37 DEG C/5%CO of humidification2It is incubated in cell incubator for 24 hours, harvests cell culture supernatant or preparation cell cracking later Object.Protein expression is detected by ELISA, and protein (pg/ml) is shown in Figure 52 and table 211.In table 211, " > ", which is meant, to be greater than.
211. protein expression of table
170. dose response of embodiment and injection site selection and timing
In order to measure the influence of dose response trend, injection site and inject the influence of timing, summarized according to embodiment 35 Scheme execute research.In these researchs, 1ug, 5ug, 10ug, 25ug, 50ug and the difference of intervenient value are used Dosage measures dose response result.The divided doses of 100ug accumulated dose include three times or six 1.6ug, 4.2ug, 8.3ug, 16.6ug or equal to applying the selected value of accumulated dose and the dosage of accumulated dose.
Injection site is selected from limbs or shows any body surface of the enough area suitable for injection.This may also include The selection of injection depth, to target corium (intradermal), epidermis (epidermis), subcutaneous tissue (SC) or muscle (IM).Injection angles will Changed based on target site of delivery, wherein target the injection at intradermal position and the plane of skin surface into 10-15 degree angle, for Subcutaneous injection, the plane with skin surface is at the angle between 20 degree to 45 degree, and for being largely injected into muscle, at 60 degree Angle between 90 degree.
Protein output of 171. transforminggrowthfactor-β1 of embodiment in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 100,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole of 100ul 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng is used into 5- methyl It is that cytimidine and pseudouridine (5mC/pU) are modified completely, repaired completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) Decorations, wherein with the uridine of 2- thio uridine modification 25% and with the cytimidine of 5-methylcytosine modification 25% (s2U and 5mC) Transforminggrowthfactor-β1 being modified, being modified with pseudouridine (pU) or modified completely with 1- methyl pseudouridine (1mpU) completely (TGFB1) RNA (mRNA sequence shown in SEQ ID NO:1668 is modified；PolyA tail with about 140 nucleotide, sequence It is not shown in column；5 ' caps, Cap1) it is diluted in OPTI-MEM (LifeTechnologies, the Grand of 10ul final volume Island, NY) in.Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) as transfection Reagent, and the lipofectamine of 0.2ul 2000 is diluted in the OPTI-MEM of 10ul final volume.It incubates at room temperature It educates after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then solution 20ul merged is added to In the cell culture medium that 100ul contains HeLa cell and it is incubated at room temperature.Also to the cell of lipofecatmine2000 processing (L2000) it is analyzed with the control of untreated cell.
Be incubated for 18 to 22 hours after, collect expression TGFB1 cell cell culture supernatant and 10.000rcf lower centrifugation 2 minutes.Then TGFB1 ELISA kit (R&D Systems, Minneapolis, MN) is used, according to The explanation of manufacturer analyzes clear supernatant.All samples are diluted, until the value measured is marked in ELISA In the range of linearity of directrix curve.The amount of generated protein is shown in table 212.
212. protein output of table
Protein output of 172. transforminggrowthfactor-β1 of embodiment in HeLa cell supernatant
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 100,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole of 100ul 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, 250ng is used into 5- methyl It is that cytimidine and pseudouridine (5mC/pU) are modified completely, repaired completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU) Decorations, wherein with the uridine of 2- thio uridine modification 25% and with the cytimidine of 5-methylcytosine modification 25% (s2U and 5mC) Transforminggrowthfactor-β1 being modified, being modified with pseudouridine (pU) or modified completely with 1- methyl pseudouridine (1mpU) completely (TGFB1) RNA (mRNA sequence shown in SEQ ID NO:1668 is modified；PolyA tail with about 140 nucleotide, sequence It is not shown in column；5 ' caps, Cap1) it is diluted in OPTI-MEM (LifeTechnologies, the Grand of 10ul final volume Island, NY) in.Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) as transfection Reagent, and the lipofectamine of 0.2ul 2000 is diluted in the OPTI-MEM of 10ul final volume.It incubates at room temperature It educates after five minutes, two kinds of solution is merged, and is incubated for again at room temperature 15 minutes.Then solution 20ul merged is added to In the cell culture medium that 100ul contains HeLa cell and it is incubated at room temperature.Also to the thin of the processing of lipofecatmine 2000 The control of born of the same parents (L2000) and untreated cell are analyzed.
After being incubated for 18 to 22 hours, the Cell Culture Lysis object of the cell of expression TGFB1 is collected and in 10.000rcf Lower centrifugation 2 minutes.Then TGFB1ELISA kit (R&D Systems, Minneapolis, MN) is used, according to saying for manufacturer It is bright that cell lysate (7ug/ELISA) is analyzed.All samples are diluted, until the value measured is marked in ELISA In the range of linearity of directrix curve.The amount of generated protein is shown in table 213.
213. protein output of table
The confirmation of peptide identity of the embodiment 173. from modification mRNA
As described in embodiment 157, contained using having the LC-MS/MS of quantitative LC-MRM to evaluate by phonetic with 5- methyl born of the same parents It is that pyridine and pseudouridine (5mC and pU) are modified completely, modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC and 1mpU) , wherein with 2- thio uridine modification 25% uridine and with the cytimidine of 5-methylcytosine modification 25% (s2U and 5mC) into Angiotensin converting enzyme that row is modified, being modified with pseudouridine (pU) or modified completely with 1- methyl pseudouridine (1mpU) completely 2 (ACE2) modify mRNA (mRNA sequence shown in SEQ ID NO:21678；PolyA tail with about 140 nucleotide, It is not shown in sequence；5 ' caps, Cap1), activity-dependent neuroprotective protein (ADNP) modify mRNA (SEQ ID NO:21679 Shown in mRNA sequence；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), born of the same parents split egg White -4 (ARTS) modify mRNA (mRNA sequence shown in SEQ ID NO:21680；PolyA with about 140 nucleotide Tail is not shown in sequence；5 ' caps, Cap1), bone morphogenetic protein 2 (BMP2) modification mRNA (show in SEQ ID NO:21681 MRNA sequence out；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1), blood plasma covellite egg White (CP) modifies mRNA (mRNA sequence shown in SEQ ID NO:1620；PolyA tail with about 160 nucleotide, sequence It is not shown in column；5 ' caps, Cap1), protein 1 (COMMD1) containing COMM structural domain modify mRNA (SEQ ID NO:21682 Shown in mRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1), diablo IAP- combination mitochondrial protein (DIABLO or SMAC) modifies mRNA (mRNA sequence shown in SEQ ID NO:21683；Have The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1), factor XIII α (F13A1) modify mRNA (mRNA sequence shown in SEQ ID NO:21684；PolyA tail with about 160 nucleotide is not shown in sequence；5' Cap, Cap1), hepcidin (HEPC) modify mRNA (mRNA sequence shown in SEQ ID NO:21471；With about 160 The polyA tail of nucleotide is not shown in sequence；5 ' caps, Cap1), IgM heavy chain modification mRNA (shown in SEQ ID NO:21685 MRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1), IL-21 (IL21) mRNA (mRNA sequence shown in SEQ ID NO:21686 is modified；PolyA tail with about 160 nucleotide, It is not shown in sequence；5 ' caps, Cap1), N-acetylglutamate synthetase (NAGS) modification mRNA (show in SEQ ID NO:21687 MRNA sequence out；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) or E3 ubiquitin-egg White ligase (XIAP) modifies mRNA (mRNA sequence shown in SEQ ID NO:21688；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) generate protein cell lysate.Evaluated protein is identified Peptide fragment shown in table 214.
214. protein and peptide fragment sequence of table
The confirmation of 174. protein expression of embodiment
Being urinated with 5-methylcytosine and vacation to protein described in coding schedule 215 by fluorescence and/or Western blotting It is that glycosides (5mC/pU) is modified completely, being modified completely with 5-methylcytosine and 1- methyl pseudouridine (5mC/1mpU), wherein use That 5-methylcytosine replaces that 25% uridine (s2U/5mC) modified instead of 25% cytimidine and with 2- thio uridine, Analyzed with 1- methyl pseudouridine (1mpU) modification completely or with the modification mRNA that pseudouridine (pU) is modified completely to confirm that The length of the expression of protein and expressed protein.Table 215, which describes modification mRNA, (has about 140 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1) sequence, the chemical modification and length (in terms of base-pair) evaluated, such as If fruit is known.
215. protein of table and expression
The 1- methyl pseudouridine or 5-methylcytosine and 1- that embodiment 175. is prepared in cation lipid nano particle The intranasal pulmonary delivery of the luciferase mRNA of methyl pseudouridine modification
It will be with 1- methyl pseudouridine (Luc-G5-LNP-KC2) modification completely or with 1- methyl pseudouridine as described in table 216 The luciferase modification mRNA modified completely with 5-methylcytosine (Luc-G2-LNP-KC2) (shows in SEQ ID NO:21446 MRNA sequence out；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) it prepares in cation In lipidic nanoparticles (LNP-KC2).
216. preparation of table
Preparation is applied to Balb-C mouse intranasal (I.N.) with the dosage of 0.3mg/kg.20 minutes before imaging, with 150mg/kg injects D- luciferin solution into mouse peritoneum.Then it is by Animal Anesthesia and using IVIS Lumina II imaging System (Perkin Elmer) acquires image.Bioluminescence is measured as the total flow (photons/second) of whole mouse.2 is small upon administration When, 8 hours, 48 hours and 72 hours mouse is imaged, average overall flow rate (photons/second) is measured and is shown in table 217 Out.Background traffic is about 6.3+05p/s.Imaging results of the Luc-G5-LNP-KC2 or Luc-G5-LNP-KC2 relative to medium It is shown in table 217, " NT ", which refers to, not to be tested.
Luciferase expression after 217. intranasal administration of table
The lipid in Lipofectamine 2000 of embodiment 176. compound 1- methyl pseudouridine or 5-methylcytosine With the intranasal pulmonary delivery of the luciferase mRNA of 1- methyl pseudouridine modification
It will be urinated in two steps with 1- methyl pseudouridine (Luc-G5- lipid complex) modification completely or with 1- methyl vacation The luciferase that glycosides and 5-methylcytosine (Luc-G2- lipid complex) are modified completely modifies mRNA (SEQ ID NO:21446 Shown in mRNA sequence；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1) lipid is multiple It closes, first step is that the luciferase mRNA of the G5 or G2 modification by 16.6ul from 3mg/ml are diluted in 108.5ul DMEM In, and second step is that 100ul LF2000 is diluted in 25ul DMEM, then by the two be gently mixed together, with The total mixture of 250ul is prepared, before injection, the mixture is incubated at room temperature 5-10min to form lipid-mRNA Compound.It is applied with the dosage of 0.5mg/kg to Balb-C mouse intranasal (I.N.) come 1- methyl pseudouridine or the 1- methyl vacation of using by oneself The lipid complex for the luciferase mRNA that uridine and 5-methylcytosine are modified completely.
20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by animal fiber crops Liquor-saturated and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as whole mouse Total flow (photons/second).2 hours upon administration, 8 hours and 24 hours to mouse be imaged, to average overall flow rate (photons/second) into Row is measured and is shown in table 218.Background signal is about 4.8+05p/s.Background traffic is about 6.3+05p/s.Luc-G5- lipid Compound or Luc-G2- lipid complex are shown in table 218 relative to the imaging results of medium.
Luciferase expression after 218. intranasal administration of table
1- methyl pseudouridine or 5-methylcytosine and 1- the methyl pseudouridine modification that embodiment 177. is prepared in PBS The intranasal pulmonary delivery of luciferase mRNA
It is formulated in PBS (pH 7.4) with the dosage of 7.5mg/kg to Balb-C mouse intranasal (I.N.) application and uses 1- first Base pseudouridine (Luc-G5- buffer (PBS)) modification completely or with 1- methyl pseudouridine and 5-methylcytosine (Luc-G2- (PBS)) the luciferase modification mRNA (mRNA sequence shown in SEQ ID NO:21446 modified completely；With about 140 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1).
20 minutes before imaging, D- luciferin solution is injected into mouse peritoneum with 150mg/kg.Then by animal fiber crops Liquor-saturated and use IVIS Lumina II imaging system (Perkin Elmer) acquires image.Bioluminescence is measured as whole mouse Total flow (photons/second).2 hours upon administration, 8 hours and 24 hours to mouse be imaged, to average overall flow rate (photons/second) into Row is measured and is shown in table 219.Background traffic is about 4.8+05p/s.Background traffic is about 6.3+05p/s.In buffer Luc-G5 shown in table 219 relative to the imaging results of medium.
Luciferase expression after 219. intranasal administration of table
The in-vitro transfection of 178. interleukin-17 of embodiment
Interleukin-17 (IL7) is modified into mRNA (mRNA sequence shown in SEQ ID NO:1656 by reverse transfection Column；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is transfected into the people in 24 porous flat plates In keratinocyte.Human keratinocytes are made to have replenishers S7's from Invitrogen (Carlsbad, CA)It is grown in culture medium, until it reaches converging for 50%-70%.With 0ng, 46.875ng, 93.75ng, 187.5ng, 375ng and 750ng with come from Invitrogen (Carlsbad, CA) RNAIMAXTMCompound coding The modification mRNA (mmRNA) of IL-7 transfects cell.By the way that RNA is used with 5X volume dilution degree without replenishers firstCulture medium is incubated at room temperature 10 minutes to form RNA:RNAIMAXTMCompound.In second bottle, with 10X volume dilution degree is by RNAIMAXTMReagent with without replenishersCulture medium is incubated at room temperature 10 together Minute.Then by RNA bottle and RNAIMAXTMBottle mixing, and be incubated at room temperature 20-30 minutes, later in a manner of dropwise It is added in cell.
MRNA (the mRNA sequence shown in SEQ ID NO:1656 of IL-7 optimized completely will be encoded；With about 160 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1) it is modified completely with 5-methylcytosine and pseudouridine.With volume The mmRNA of code IL-7 transfects cell, for each concentration, 6 and 12 hours after transfection, and using coming from Invitrogen The explanation that the ELISA kit of (Carlsbad, CA) is recommended according to manufacturer carries out the IL-7 concentration (ρ g/ml) in culture medium Measurement.The modification mRNA of these data code displayings IL-7 shown in Figure 53 can be under the dosage of 375ng and 750ng It is translated in Human keratinocytes.
The in-vitro transfection of 179. hematopoietin of embodiment
Hematopoietin (EPO) is modified into mRNA (mRNA shown in SEQ ID NO:1638 by reverse transfection Sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is transfected into 24 porous flat plates In Human keratinocytes.Make Human keratinocytes that there is replenishers S7 from Invitrogen (Carlsbad, CA) 'sIt is grown in culture medium, until it reaches converging for 50%-70%.With 46.875ng, 93.75ng, 187.5ng, 375ng and 750ng with come from Invitrogen (Carlsbad, CA) RNAIMAXTMCompound coding The modification mRNA (mmRNA) of EPO transfects cell.By the way that RNA is used with 5X volume dilution degree without replenishers firstCulture medium is incubated at room temperature 10 minutes to form RNA:RNAIMAXTMCompound.In second bottle, with 10X volume dilution degree is by RNAIMAXTMReagent with without replenishersCulture medium is incubated at room temperature 10 together Minute.Then by RNA bottle and RNAIMAXTMBottle mixing, and be incubated at room temperature 20-30 minutes, later in a manner of dropwise It is added in cell.
MRNA (the mRNA sequence shown in SEQ ID NO:1638 of EPO optimized completely will be encoded；With about 160 The polyA tail of a nucleotide is not shown in sequence；5 ' caps, Cap1) it is modified completely with 5-methylcytosine and pseudouridine.With volume The mmRNA of code EPO transfects cell, for each concentration, 6 and 12 hours after transfection, and using coming from Invitrogen The explanation that the ELISA kit of (Carlsbad, CA) is recommended according to manufacturer carries out the EPO concentration (ρ g/ml) in culture medium Measurement.The modification mRNA of these data code displayings EPO shown in Figure 54 can be translated in Human keratinocytes.
The detection of 180. lysosomal acid lipase albumen of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by the lysosomal acid lipase of 750ng modified completely with 5-methylcytosine and pseudouridine (LAL) mRNA (mRNA sequence shown in SEQ ID NO:1657；PolyA tail with about 160 nucleotide, in sequence It is not shown；5 ' caps, Cap1) it is diluted in 250ul final volume(LifeTechnologies, Grand Island, NY) in.Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) as transfection Reagent, and 5.5ul is diluted in 250ul final volumeIn.It is incubated at room temperature after five minutes, by two Kind solution merges, and is incubated for again at room temperature 15 minutes.Then solution 500ul merged is added to 3ml, and to contain HeLa thin In the cell culture medium of born of the same parents.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 The anti-lysosomal acid lipase antibody of primary antibody of anti-target proteins under dilution in the 1X TBS solution of 3ml 5%BSA； Abcam Cat No.ab89771), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.Adjoint stirring gently, Film is washed 3 times, every time five minutes with 1X TBS/0.1%Tween.((Western Breeze, Invitrogen) sews secondary antibody It is bonded to horseradish peroxidase and is bound to primary antibody.Secondary antibody is diluted 1: 1000 to 1: 5000 in the 1X TBS solution of 5%BSA And it is incubated at room temperature 3 hours.As shown in Figure 55, the Western blotting of LAL is detected close to 45.4kDa.In Figure 55, swimming Road 1 and 2 shows modification mRNA, and swimming lane 3 and 4 only shows administration medium.
The detection of 181. glucocerebroside zymoprotein of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by the glucocerebrosidase mRNA of 750ng modified completely with 5-methylcytosine and pseudouridine (mRNA sequence shown in SEQ ID NO:1645；PolyA tail with about 160 nucleotide is not shown in sequence；5' Cap, Cap1) it is diluted in 250ul final volume(LifeTechnologies, Grand Island, NY) In.It is used as transfection reagent using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY), and 5.5ul is diluted in 250ul final volumeIn.It is incubated at room temperature after five minutes, two kinds of solution is closed And it and is incubated for again at room temperature 15 minutes.Then solution 500ul merged is added to the cell that 3ml contains HeLa cell In culture medium.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 Primary antibody (the anti-GBA antibody of anti-target proteins under dilution in the 1X TBS solution of 3ml 5%BSA；Abcam catalog number (Cat.No.) Ab55080 and Sigma catalog number (Cat.No.) G4046), it is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With gently Stirring, is washed film 3 times, every time five minutes with 1X TBS/0.1%Tween.Secondary antibody (Western Breeze, Invitrogen it) is conjugated to horseradish peroxidase and is bound to primary antibody.Secondary antibody is diluted 1 in the 1X TBS solution of 5%BSA : it 1000 to 1: 5000 and is incubated at room temperature 3 hours.As shown in Figure 56, the Western blotting detection of glucocerebrosidase Close to 59.7kDa.In Figure 56, swimming lane 1 and 2 shows modification mRNA, and swimming lane 3 and 4 only shows administration medium.
The detection of 182. iduronic acid 2- sulfatase protein of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by the iduronic acid 2- sulfuric acid of 750ng modified completely with 5-methylcytosine and pseudouridine Esterase mRNA (mRNA sequence shown in SEQ ID NO:1652；PolyA tail with about 160 nucleotide, in sequence not It shows；5 ' caps, Cap1) it is diluted in 250ul final volume(LifeTechnologies, Grand Island, NY) in.Using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) as transfection Reagent, and 5.5ul is diluted in 250ul final volumeIn.It is incubated at room temperature after five minutes, by two Kind solution merges, and is incubated for again at room temperature 15 minutes.Then solution 500ul merged is added to 3ml, and to contain HeLa thin In the cell culture medium of born of the same parents.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 (anti-2 sulfatase of iduronic acid is anti-for the primary antibody of anti-target proteins under dilution in the 1X TBS solution of 3ml 5%BSA Body；Abcam catalog number (Cat.No.) ab 70025 and R&D Systems catalog number (Cat.No.) AF2449), it is kept for 3 hours and is shaken in track at room temperature It is gently mixed on bed.With stirring gently, film is washed 3 times, every time five minutes with 1X TBS/0.1%Tween.Secondary antibody (Western Breeze, Invitrogen) is conjugated to horseradish peroxidase and is bound to primary antibody.By secondary antibody 5%BSA's 1: 1000 to 1: 5000 are diluted in 1X TBS solution and are incubated at room temperature 3 hours.As shown in Figure 57, iduronic acid 2- The Western blotting of sulfatase is detected close to 76kDa.In Figure 57, swimming lane 1 and 2 shows modification mRNA, and swimming lane 3 and 4 only shows Medium is administered out.
The detection of 183. luciferase protein of embodiment: Western blotting
In the day before transfection, by with trypsin-EDTA (EDTA) solution (LifeTechnologies, Grand Island, NY) it handles to harvest 750,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and connect Kind (is supplemented with 10% fetal calf serum (FCS) and 1x in Eagle ' the s minimum essential medium (EMEM) of total volume 3ml Glutamax in 6 hole cel culture plates (Corning, Manassas, VA) of)/hole.Make cell at 37 DEG C in 5%CO2 gas It is grown overnight in atmosphere.Second day, by the luciferase mRNA (SEQ of 750ng modified completely with 5-methylcytosine and pseudouridine MRNA sequence shown in ID NO:21446；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is diluted in 250ul final volumeIn (LifeTechnologies, Grand Island, NY). It is used as transfection reagent using Lipofectamine 2000 (LifeTechnologies, Grand Island, NY), and will 5.5ul is diluted in 250ul final volumeIn.It is incubated at room temperature after five minutes, two kinds of solution is merged, And it is incubated for again at room temperature 15 minutes.Then solution 500ul merged is added to the cell culture that 3ml contains HeLa cell In base.Then it is incubated for plate as described above.
After being incubated for 16-18 hours, removes culture medium and wash cell with 1ml PBS.After completely removing PBS, Add the fresh PBS of 500ul.By harvesting cell with cell scraper scraping.Then by the identical mRNA's of receiving harvested Cell merges in a 1.5ml Eppendorf pipe.
Make cell precipitation by being centrifuged 2 minutes at 3,000rpm.Remove PBS, and by carefully drawn with pipette by Radioimmuno-precipitation assay (RIPA) buffer of cell in 250ul (is mixed containing PMSF and eukaryotic protein enzyme inhibitor Object) (being all from BostonBioProducts, Ashland, MA) middle cracking.By being centrifuged 10 at 4 DEG C at 10,000rpm Minute clarifies lysate.Clear lysate is transferred to the Amicon filter with 10,000kd molecular cut value It is rotated 20 minutes in (Waters, Milford, MA) and at 12,000rpm and 4 DEG C.It is placed on newly by the way that filter will be inverted The protein cracking for rotating 1 minute in fresh 1.5ml Eppendorf pipe and at 3,000rpm to recycle concentration.Lysate Final volume in 25ul between 40ul.
For coming from the microtiter plate of Pierce (Thermo Fisher, Rockford, IL), surveyed using BCA kit Determine protein concentration.The standard protein of titration curve is dissolved in RIPA buffer (as described by cell lysate preparation) Rather than dilution buffer.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied to 1: 500 to 1: 2000 Primary antibody (the anti-luciferase of anti-target proteins under dilution in the 1X TBS solution of 3ml 5%BSA；Abcam catalog number (Cat.No.) Ab21176), kept for 3 hours and be gently mixed on orbital shaker at room temperature.With stirring gently, with 1X TBS/ 0.1%Tween washs film 3 times, every time five minutes.Secondary antibody (Western Breeze, Invitrogen) is conjugated to horseradish mistake Oxide enzyme is simultaneously bound to primary antibody.Secondary antibody is diluted 1: 1000 to 1: 5000 in the 1X TBS solution of 5%BSA and in room temperature It is lower to be incubated for 3 hours.As shown in Figure 58, the Western blotting of luciferase is detected close to 60.7kDa.In Figure 58,1 He of swimming lane 2 show modification mRNA, and swimming lane 3 and 4 only shows administration medium.
184. Trastuzumab In vivo study of embodiment
Trastuzumab heavy chain (HC) is modified into mRNA (SEQ ID NO:21451；PolyA with about 140 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and 1- methyl-pseudouridine) and Trastuzumab light chain (LC) mRNA (SEQ ID NO:21452 is modified；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and 1- methyl-pseudouridine) it is formulated in DLin-MC3-DMA and DLin-KC2-DMA In, as described in table 220.
Table 220.DLin-MC3-DMA and DLin-KC2-DMA preparation
Trastuzumab preparation described in Table X 1 is intravenously applied to mouse (CD1) with the dosage of 0.5mg/kg.Also to fluorescence (Luc in Figure 59) is evaluated in the control of plain enzyme mRNA.8 hours after applying Trastuzumab, 24 hours, 48 hours, 96 hours, 168 hours (7 days), 336 hours (14 days), 504 hours (21 days) by mouse bloodletting, with by ELISA (It is total Human IgG detection kit, Cambridge MA) measurement serum IgG concentration.Injection is only compareed into fluorescence the 8th hour time point The animal bloodletting of plain enzyme.As a result it is shown in table 222 and Figure 59.
222. Trastuzumab preparation of table
185. Trastuzumab in vitro study of embodiment
In the day before transfection, by with trypsin-EDTA solutions (LifeTechnologies, Grand Island, NY) processing is to harvest 15,000HeLa cell (ATCC the CCL-2nd；Manassas, VA) and it is inoculated in total volume 100ul EMEM culture medium (being supplemented with 10%FCS and 1x Glutamax)/hole 96 hole cel culture plates (Corning, Manassas, VA) in.Make cell at 37 DEG C in 5%CO2It is grown overnight in atmosphere.Second day, by the Trastuzumab weight of 150ng Chain (HC) mRNA (SEQ ID NO:21451；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and 1- methyl pseudouridine) or 75ng Trastuzumab light chain (LC) (SEQ ID NO: 21452；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1；With 5-methylcytosine and 1- Methyl pseudouridine is modified completely) or the Trastuzumab LC of the Trastuzumab HC and 250ng of 500ng is diluted in 10ul final volume together OPTI-MEM (LifeTechnologies, Grand Island, NY) in.Use Lipofectamine 2000 (LifeTechnologies, Grand Island, NY) is used as transfection reagent, and by the lipofectamine of 0.2ul 2000 are diluted in the OPTI-MEM of 10ul final volume.It is incubated at room temperature after five minutes, two kinds of solution is merged, and in room It is incubated for again under temperature 15 minutes.Then solution 20ul merged is added to 100ul and contains in the cell culture medium of HeLa cell And it is incubated at room temperature.As control, also to recombination human IgG (Hu IgG ctrl), measurement buffer and in measurement buffer In the presence of the culture medium (measurement buffer+control medium) from untreated cell analyzed.
After being incubated for 18 to 22 hours, the cell culture supernatant of the cell of expression Trastuzumab heavy chain or light chain is collected And with total human IgG ELISA kit (Cambridge MA) illustrate to be analyzed according to manufacturer.Institute The amount for generating IgG is shown in table 223 and Figure 60.In Table X 3, " OTC " refers to outside beyond chart.
The research of 223. Trastuzumab of table
Sample IgG(ng/ml)
Recombined human IgG OTC
Trastuzumab heavy chain 12
Trastuzumab light chain 0
Trastuzumab heavy chain+light chain 348
Measure buffer 9
Measure buffer+control medium 12
186. Trastuzumab in vitro study of embodiment: the external generation of antibody is confirmed by immunoblotting
In the case where serum is not present, Trastuzumab heavy chain described in embodiment 185 and Light chain mRNA (Trastuzumab are used Heavy chain (HC) mRNA (SEQ ID NO:21451；PolyA tail with about 140 nucleotide is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and 1- methyl pseudouridine), Trastuzumab light chain (LC) (SEQ ID NO:21452；Tool There is the polyA tail of about 140 nucleotide, is not shown in sequence；5 ' caps, Cap1；It is urinated with 5-methylcytosine and 1- methyl vacation Glycosides is modified completely) or Trastuzumab HC and LC are together (H&L)) in-vitro transfection HeLa cell.24 hours after transfection, collect supernatant Liquid, concentration, and in the case where there is (the swimming lane 1-5 in Figure 61) and (the swimming lane 6-10 in Figure 61) reducing agent is not present, The protein of 4ug is loaded onto each swimming lane of 4%-12%MOPS NuPAGE gel, for being separated by electrophoresis.Then by egg White matter is transferred on pvdf membrane, and is detected using antibody, for detecting weight Ig chain and light Ig chain simultaneously.
As shown in Figure 61, in the swimming lane of the supernatant electrophoresis for the cell for only transfecting light chain, in reduction and non-reduced item Light chain bands are detected under part.In non reducing conditions, the relatively big band at~50kDa is also detected.Only light chain is point The fact that secreting property, well confirms this.The swimming lane of supernatant containing the cell for only transfecting heavy chain mRNA exists or is not depositing Without signal in the case where reducing agent, it was demonstrated that only heavy chain lacks secretion.The cell transfected with heavy chain and Light chain mRNA it is upper Clear liquid all has two band presence or absence of reducing agent.Under two conditions, there is light chain specificity Band.This represents the light chain under the reducing conditions from denaturation Ig molecule and the free light chain albumen under non reducing conditions. Under the reducing conditions, the heavy chain only secreted in association with light chain at 55kDa as it can be seen that and under non reducing conditions, as institute is pre- Phase, without apparent single heavy chain band.In the swimming lane, total Ig band of 160kDa can be detected clearly.This card Real generated light chain and heavy chain egg when the mRNA for encoding the chemical modification of these protein to be transfected into HeLa cell White molecular identity.
In figure 61, " MWM " represents molecular weight marker, and " LC " represents Trastuzumab light chain, and " HC " represents Trastuzumab heavy chain, And " Media " represents only culture medium.
187. enzyme assay of embodiment
A.Glucocerebrosidase enzymatic activity
It is compound with 0ng, 625ng, 125ng, 250ng, 500ng, 1000ng or 2000ng and RNAiMAX (Invitrogen) Glucocerebroside enzyme modification mRNA (mRNA sequence shown in SEQ ID NO:1645；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) transfected HEK 293. Cell is washed with PBS in 6 hours after transfection, 12 hours, 24 hours and 48 hours.Then containing 0.54% tauroursodeoxycholic acid Sodium is together with lytic cell in the 100mM citrate phosphate buffer (pH 5.4) of 1%Triton X-100.Then 0.5mM's In the presence of 4-methyl umbelliferone acyl β-D- galactopyranoside (4-MUG) substrate, it is small that cell lysate is incubated for 1 at 37 DEG C When.After one hour, stop reaction with isometric 200mM sodium hydroxide.In plate reader (Ex=370nm；Em=460nm) The conversion of middle measurement 4-MU.Figure 62, which is shown, is expressed as enzymatic activity for mRNA pairs compound with RNAiMAX (Invitrogen) According to transfection object (iduronic acid 2- sulfatase (mRNA sequence shown in SEQ ID NO:1652；With about 160 cores The polyA tail of thuja acid is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) it is normalized Percentage.
B.Lysosomal acid lipase enzymatic activity
It is compound with 0ng, 625ng, 125ng, 250ng, 500ng, 1000ng or 2000ng and RNAiMAX (Invitrogen) Lysosomal acid lipase modify mRNA (mRNA sequence shown in SEQ ID NO:1657；With about 160 nucleotide PolyA tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) transfected HEK 293. Cell is washed with PBS in 6 hours after transfection, 12 hours, 24 hours and 48 hours.Then containing 0.54% tauroursodeoxycholic acid Sodium is together with lytic cell in the 100mM citrate phosphate buffer (pH 5.4) of 1%Triton X-100.Then 0.5mM's In the presence of 4-methyl umbelliferone acyl β-D- galactopyranoside (4-MUG) substrate, it is small that cell lysate is incubated for 1 at 37 DEG C When.After one hour, stop reaction with isometric 200mM sodium hydroxide.In plate reader (Ex=370nm；Em=460nm) The conversion of middle measurement 4-MU.Figure 63, which is shown, is expressed as enzymatic activity for mRNA pairs compound with RNAiMAX (Invitrogen) According to transfection object (iduronic acid 2- sulfatase (mRNA sequence shown in SEQ ID NO:1652；With about 160 cores The polyA tail of thuja acid is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) it is normalized Percentage.
The in-vitro transfection of the embodiment 188.VIII factor: protein detection
The VIII factor is modified into mRNA (mRNA sequence shown in SEQ ID NO:1623 by reverse transfection；With big The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is transfected into the HepG2 cell in 24 porous flat plates In.HepG2 cell is set to have replenishers S7's from Invitrogen (Carlsbad, CA)In culture medium Growth, until it reaches converging for 50%-70%.With 0,250ng, 500ng, 750ng, 1000ng with come from The RNAIMAX of Invitrogen (Carlsbad, CA)TMThe modification mRNA (mmRNA) of compound encoding factor VIII transfects cell. By the way that RNA is used with 5X volume dilution degree without replenishers firstCulture medium is incubated at room temperature 10 minutes Form RNA:RNAIMAXTMCompound.In second bottle, with 10X volume dilution degree by RNAIMAXTMReagent with without mend Fill agentCulture medium is incubated at room temperature 10 minutes together.Then by RNA bottle and RNAIMAXTMBottle is mixed It closes, and is incubated at room temperature 20-30 minutes, be added in cell in a manner of dropwise later.
The mRNA of the encoding factor VIII being transfected into HepG2 cell optimized completely (is shown in SEQ ID NO:1623 MRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it include during translating Modification, such as natural nucleus glycoside triphosphoric acid (NTP), the pseudouridine at each uridine site and the 5- first at each cytimidine site Base cytimidine (vacation-U/5mC), and N1- methyl-pseudouridine at each uridine site and at each cytimidine site 5-methylcytosine (N1- methyl-vacation-U/5mC).Cell is transfected with the mmRNA of encoding factor VIII, for each concentration, 18 hours after transfection, the explanation recommended according to manufacturer of ELISA kit from Invitrogen (Carlsbad, CA) is used The VIII factor concentration (ρ g/ml) of secretion in culture medium is measured.These data shown in table 224 and Figure 64 are aobvious Show that the modification mRNA of encoding factor VIII can be translated in HepG2 cell.
Table 224.VIII factor administration and Protein secretion
The in-vitro transfection of the embodiment 189.VIII factor: Chomogenic activity
The VIII factor is modified into mRNA (mRNA sequence shown in SEQ ID NO:1623 by reverse transfection；With big The polyA tail of about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it is transfected into the HepG2 cell in 24 porous flat plates In.HepG2 cell is set to have replenishers S7's from Invitrogen (Carlsbad, CA)In culture medium Growth, until it reaches converging for 50%-70%.With 0,250ng, 500ng, 750ng, 1000ng with come from The RNAIMAX of Invitrogen (Carlsbad, CA)TMThe modification mRNA (mmRNA) of compound encoding factor VIII transfects cell. By the way that RNA is used with 5X volume dilution degree without replenishers firstCulture medium is incubated at room temperature 10 minutes Form RNA:RNAIMAXTMCompound.In second bottle, with 10X volume dilution degree by RNAIMAXTMReagent with without mend Fill agentCulture medium is incubated at room temperature 10 minutes together.Then by RNA bottle and RNAIMAXTMBottle is mixed It closes, and is incubated at room temperature 20-30 minutes, be added in cell in a manner of dropwise later.
The mRNA of the encoding factor VIII being transfected into HepG2 cell optimized completely (is shown in SEQ ID NO:1623 MRNA sequence；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1) it include during translating Modification, such as pseudouridine at each uridine site and the 5-methylcytosine (vacation-U/5mC) at each cytimidine site, And N1- methyl-pseudouridine at each uridine site and 5-methylcytosine (the N1- first at each cytimidine site Base-vacation-U/5mC).Cell is transfected with the mmRNA of encoding factor VIII.When by thrombin activation, the VIII factor and IXa because Son, phosphatide and calcium form multienzyme complex, when the X factor in measuring liquid with constant density and excess supply when, the multienzyme complex It is Xa factor by the X factor activator.This activity is directly related with the amount of the VIII factor.
Generated Xa factor passes through it for 18 hours on specific Xa factor chromogenic substrate (SXa-11) after transfection Activity is measured, is measured by the amount of the pNA discharged, is measured by the colour developing at 405nm, to measure The activity of the VIII factor.
25%-30%VIII factor concentration reference standard (% activity) is normalized VIII factor active.In 225 He of table The modification mRNA of these data code displaying VIII factors shown in Figure 65 can be translated in HepG2 cell.
Table 225.VIII factor administration and Protein secretion
190. LDL receptor of embodiment (LDLR) expression
A.External LDLR expression
Human embryo kidney epithelium (HEK293) cell (LGC standards GmbH, Wesel, Germany) is seeded in 6 holes On plate (BD Biosciences, San Jose, USA).HEK293 is seeded in 3ml with the density of about 500,000 cells/wells In cell culture medium.With the amount inoculating cell in the hole 4000ng/, the hole 800ng/, the hole 400ng/, the hole 40ng/ and the hole 4ng/ simultaneously After incubation, directly addition contains LDLR mRNA (mRNA shown in SEQ ID NO:21463；With 5-methylcytosine and 1- Methyl pseudouridine is modified completely；5 ' caps, cap 1, the polyA tail (being not shown in sequence) with about 160 nucleotide) or G- CSF mRNA (mRNA shown in SEQ ID NO:21438；It is modified completely with 5-methylcytosine and pseudouridine；5 ' caps, cap 1； PolyA tail with about 160 nucleotide is not shown in sequence) control preparation.G-CSF is handled with anti-LDLR antibody The cell of mRNA transfection, and use the cell of normal goats IgG processing one group of LDLR transfection as control.It is examined by facs analysis Combined primary antibody is surveyed, two process resistant marked later with PE.
As shown in Figure 66 A, facs analysis as the result is shown under the dosage of the LDLR mRNA of 800ng, detect all doors 74.8% expression LDLR in the living cells of control.Under the LDLR mRNA dosage of 40ng, in the living cells that detects all gates 11.6% expression LDLR.Dyeing is not being observed in the cell for the LDLR mRNA processing for compareing nonimmune IgG dyeing.? LDLR positive cell is not detected in the cell of G-CSF mRNA transfection.
B.Protein accumulation
Human embryo kidney epithelium (HEK293) cell (LGC standards GmbH, Wesel, Germany) is seeded in 6 holes On plate (BD Biosciences, San Jose, USA).HEK293 is seeded in 3ml with the density of about 500,000 cells/wells In cell culture medium.In every hole inoculating cell and after being incubated for, directly addition is containing LDLR mRNA (in SEQ ID NO:21463 The mRNA shown；It is modified completely with 5-methylcytosine and 1- methyl pseudouridine；5 ' caps, cap 1 have about 160 nucleosides The polyA tail (being not shown in sequence) of acid) or G-CSF mRNA (mRNA shown in SEQ ID NO:21438；With 5- methyl born of the same parents Pyrimidine and pseudouridine are modified completely；5 ' caps, cap 1；PolyA tail with about 160 nucleotide is not shown in sequence) pair According to preparation.After 15 hours, transfection media is replaced with complete medium.0,2,4,8,24,48 and 72 after culture medium replacement The cell of hour harvest transfection.Be conjugated to phycoerythrin (PE) anti-LDLR antibody handle transfection cell, and be conjugated Normal goat IgG to PE handles the cell of one group of LDLR transfection as control.Institute is detected by facs analysis as described above In conjunction with primary antibody.
As shown in Figure 66 B, facs analysis is shown in wash away transfection media after 0.0-h time point (15.0-h after transfection) examine Measure it is all gate living cells in~65% expression LDLR.Positive cell percentage drops over time at 37 DEG C It is low, so that cannot detect LDLR when after removing transfection media for 24 hours.
C. The LDLR of label
In order to which whether the LDLR for evaluating expressed is functional, useThe LDL of label.It is modified with LDLR MRNA (mRNA shown in SEQ ID NO:21463；It is modified completely with 5-methylcytosine and 1- methyl pseudouridine；5 ' caps, cap 1, the polyA tail (being not shown in sequence) with about 16 nucleotide) or G-CSF modification mRNA (in SEQ ID NO:21438 The mRNA shown；It is modified completely with 5-methylcytosine and pseudouridine；5 ' caps, cap 1, the polyA with about 160 nucleotide Tail is not shown in sequence) transfected HEK 293 is overnight, and washing cell is simultaneously incubated with the BODIPY-LDL of increase.? It is incubated for after 1.0-h at 37 DEG C, washs cell and assesses the combination of BODIPY-LDL by FACS.BODIPY-LDL and LDLR The combination of the cell of mRNA transfection is with high-affinity (Kd~60ng/mL) and saturable, as shown in Figure 66 C.
In competition research, the combination (figure of BODIPY-LDL can be reduced by unlabelled LDL with dosage-dependent manner 66D).These data show BODIPY-LDL with expression LDLR mRNA cell combination be it is saturable, specific and With high-affinity.
1 family polypeptides A1 (UGT1A1) of embodiment 191.UDP glucuronyl transferase expression
A.External UGT1A1 expression
Human embryo kidney epithelium (HEK293) cell (LGC standards GmbH, Wesel, Germany) is seeded in 6 holes On plate (BD Biosciences, San Jose, USA).HEK293 is seeded in 3ml with the density of about 500,000 cells/wells In cell culture medium.With the amount inoculating cell in the hole 4000ng/, the hole 800ng/, the hole 400ng/, the hole 40ng/ and the hole 4ng/ simultaneously After incubation, directly addition containing UDP glucuronyl transferase 1 family polypeptides A1 (UGT1A1) mRNA (SEQ ID NO: MRNA shown in 21463；It is modified completely with 5-methylcytosine and 1- methyl pseudouridine；5 ' caps, cap 1 have about 160 The polyA tail (being not shown in sequence) of nucleotide) or G-CSF mRNA (mRNA shown in SEQ ID NO:21438；With 5- first Base cytimidine and pseudouridine are modified completely；5 ' caps, cap 1；PolyA tail with about 160 nucleotide is not shown in sequence) Control preparation.The cell transfected with anti-UGT1A1 antibody processing G-CSF mRNA, and one group is handled with normal goats IgG The cell of UGT1A1 transfection is as control.Combined primary antibody is detected by facs analysis, two process resistant marked later with PE.
As shown in Figure 67, by the positive cell percentage of FACS measurement with the UGT1A1 being transfected into HEK293 cell The amount of mRNA and increase, and be reached under the UGT1A1 mRNA dosage of 800ng the 74.8% of all gate living cells most Big value.Under the maximum dose level (4000ng) tested, further increasing for positive cell percentage is not observed.These data Show mRNA delivering in mammalian cells and/or expression is saturable.What is dyed with the nonimmune IgG of control Dyeing is not observed in the cell of UGT1A1 mRNA processing, and is not detected in the cell transfected with G-CSF mRNA UGT1A1 positive cell.
B.External UGT1A1 expression-protein accumulation
Human embryo kidney epithelium (HEK293) cell (LGC standards GmbH, Wesel, Germany) is seeded in 6 holes On plate (BD Biosciences, San Jose, USA).HEK293 is seeded in 3ml with the density of about 500,000 cells/wells In cell culture medium.In every hole inoculating cell and after being incubated for, directly addition contains UGT1A1 mRNA (SEQ ID NO:2146 Shown in mRNA；It is modified completely with 5-methylcytosine and 1- methyl pseudouridine；5 ' caps, cap 1 have about 160 nucleosides The polyA tail (being not shown in sequence) of acid) or G-CSF mRNA (mRNA shown in SEQ ID NO:21438；With 5- methyl born of the same parents Pyrimidine and pseudouridine are modified completely；5 ' caps, cap 1；PolyA tail with about 160 nucleotide is not shown in sequence) pair According to preparation.After 15 hours, transfection media is replaced with complete medium.0,2,4,8,24,48 and 72 after culture medium replacement The cell of hour harvest transfection.The cell of transfection is handled with the anti-UGT1A1 antibody for being conjugated to phycoerythrin (PE), and with sewing The normal goat IgG for being bonded to PE handles the cell of one group of UGT1A1 transfection as control.It is examined as described above by facs analysis Survey combined primary antibody.
As shown in Figure 68, the UGT1A1 protein accumulation in the cell of UGT1A1 mRNA transfection is incubated after replacing with culture medium The passage for educating the time increased to maximum value at 24 hours and was reduced to baseline value at 72 hours.Under these conditions, The apparent half-life of UGT1A1 mRNA is about 40.0-h.
Embodiment 192. detects UGT1A1 and OTC by Western blotting
HEK293 human embryo kidney epithelium (HEK293) cell (LGC standards GmbH, Wesel, Germany) is connect Kind is on 6 hole plates (BD Biosciences, San Jose, USA).HEK293 is connect with the density of about 500,000 cells/wells Kind is in 2.4ml cell culture medium.With the amount inoculating cell in the hole 800ng/ and after being incubated for 16 hours, directly addition contains UGT1A1 mRNA (mRNA shown in SEQ ID NO:21464；It is modified completely with 5-methylcytosine and 1- methyl pseudouridine； 5 ' caps, cap 1, the polyA tail (being not shown in sequence) with about 160 nucleotide) or OTC mRNA (SEQ ID NO:1659 Shown in mRNA；It is modified completely with 5-methylcytosine and 1- methyl pseudouridine；5 ' caps, cap 1；With about 160 nucleosides The polyA tail of acid is not shown in sequence) preparation.Cell is washed with 500 μ L PBS and by by cell scraping to 500 μ L Cell is harvested in PBS.Cell is centrifuged 5 minutes at 200xg and is resuspended in the 250 μ L for being supplemented with protease inhibitors In RIPA buffer.Pipe is centrifuged 10 minutes at 14,000xg.Supernatant is transferred to centrificon evaporating column, and And 30 minutes are centrifuged at 14,000xg lysate is concentrated.Pass through BCA protein determination (Thermo Fisher Scientific Inc, Rockford, USA) lysate is quantified.
Protein cracking is loaded into the ready-to-use Bis-Tris gel of 1.5mm and 4%-12% acrylamide In the NuPage SDS-PAGE system (chamber and power supply) of gradient, wherein MOPS- buffer (is all from as electrophoresis auxiliary agent Life Technologies, Grand Island, NY).By each lysate sample preparation to 40ul final volume.This sample contains There are the 25ug protein cracking for being in different volumes, RIPA buffer to add to the volume of 26ul, the 10x reducing agent of 4ul And 10ul 4x SDS sample loading buffer (being all from Life Technologies, Grand Island, NY).By sample 95 It heats 5 minutes and is loaded on gel at DEG C.200V, 120mA and maximum value 25W are arranged by manufacturer's selection criteria.Electrophoresis Time is 60 minutes, but is no more than the time for making electrophoretic pigment reach gel lower end.
After running glue and terminating, so that plastic casing is generated crack and the gel of encapsulation is transferred to ready-to-use NC Nitroncellulose Membrane reagent box and power supply (iBLOT；LifeTechnologies, Grand Island, NY).Using default setting, pass through Gao An Protein cracking is transferred on film by training electricity from gel.
After transfer, film is incubated for 15 in 1X tris buffered saline (TBS) solution of 5% bovine serum albumin(BSA) (BSA) Minute, then it is incubated for again 15 minutes in the 1X TBS+0.1%Tween solution of 5%BSA.It is applied under 1: 500 dilution Primary antibody Goat anti-Human UGT1A1 (R&D systems) and rabbit-anti people OTC (NBP1) in the 1X TBS solution of 3ml 5%BSA, It is kept for 3 hours and is gently mixed on orbital shaker at room temperature.With WesternBreeze Chromogenic Kit-Anti- Rabbit detects OTC primary antibody according to fabrication scheme, and with WesternBreeze Chromogenic Kit-Anti-Goat (Invitrogen, Grand Island, NY) detects UGT1A1 primary antibody according to fabrication scheme.
As shown in Figure 69, by anti-UGT1A1 IgG use UGT1A1 mRNA transfect cell lysate in as~ 60kDa band detects UGT1A1 (swimming lane 4), but can't detect (swimming lane 3) in the cell transfected with OTC mRNA.It uses Identical lysate, by anti-OTC IgG as~39kDa band detection in the lysate for the cell for using OTC mRNA to transfect To OTC (swimming lane 7), but it can't detect (swimming lane 8) in the cell transfected with UGT1A1 mRNA.These data determine external source UGT1A1 and OTC mRNA can instruct the synthesis of its homologous protein in mammalian cells.
Embodiment 193.PAH and UGT1A1 expression and detection
A.HEK293, mouse and rat myoblasts transfection
By HEK293 (LGC standards GmbH, Wesel, Germany), C2C12 mouse muscle-forming cell (ATCC) and L6 rat myoblasts (ATCC) cell line is coated on 24 holes in the DMEM (ATCC) for being supplemented with 10% fetal calf serum (ATCC) Disk (BD Biosciences, San Jose, USA) (1.5x105Cells/well)) in.
By by the PAH that is modified completely with 5-methylcytosine and pseudouridine of 250ng modify mRNA (SEQ ID NO: 1660；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1), UGT1A1 modify mRNA (SEQ ID NO:21464；PolyA tail with about 160 nucleotide is not shown in sequence；5 ' caps, Cap1；It is phonetic with 5- methyl born of the same parents Pyridine and pseudouridine are modified completely) or G-CSF modification mRNA (SEQ ID NO:21438；PolyA with about 160 nucleotide Tail is not shown in sequence；5 ' caps, Cap1；Modified completely with 5-methylcytosine and pseudouridine) and 50 μ l Opti-MEM reagents (Life Technologies, Grand Island, NY) merges in the first pipe, and the L2000 transfection reagent with 1 μ l (Life Technologies, Grand Island, NY) merges in the second pipe in the Opti-MEM of 50 μ l to be used for prepare The Transfection solution in each hole to be processed.After preparation, the first pipe and the second pipe are incubated at room temperature 5 minutes, it later will be each From content merge.Combined Transfection solution is incubated at room temperature 15 minutes.Then add 100 μ l's into each hole Transfection solution.Cell is further cultured for 14 hours, continues to analyze later.
B.Pass through Flow cytometry PAH, UGT1A1
After transfection, the 0.25% trypsase (Life of 60 μ l is added from removal culture medium in cell and into each hole Technologies, Grand Island, NY).By cell trypsin digestion 2 minutes, the pancreas egg in 240 holes μ l/ was added later White enzyme inhibitor (Life Technologies, Grand Island, NY).Resulting cell solution is transferred to 96 hole plates (Corning Life Sciences, Tewksbury, MA) makes cell precipitation (800x gravity continues 5 minutes) simultaneously by centrifugation Discard supernatant liquid.Cell precipitation is washed with PBS and is resuspended in Foxp3 and fixes/permeabilization solution (eBioscience, San Diego, CA) in, it is kept for 45 minutes.Cell is precipitated again by centrifugation (800x gravity continues 5 minutes), and is resuspended in Change in buffer (eBiosciences, San Diego, CA), is kept for 10 minutes.Pass through centrifugation (800x gravity continues 5 minutes) It precipitates cell again, and is washed in permeabilization buffer.PAH is handled with the anti-PAH antibody for being conjugated to phycoerythrin (PE) to turn The cell of dye.Use the cell for the normal goat IgG processing one group of PAH transfection for being conjugated to PE as stain control.With being conjugated to The anti-PAH antibody of phycoerythrin (PE) handles the cell of one group of G-CSF transfection as transfection control.Pass through FACS points as described above Analysis detects combined primary antibody.Then cell and FACS buffer solution will be marked (to there is 1% bovine serum albumin(BSA) and 0.1% nitrine Change the PBS of sodium) merge, and it is transferred to gathering pipe, then use BD Accuri (BD Biosciences, San Jose, CA) Label cell is analyzed by flow cytometry.As shown in Figure 70, detect that 11.9% HEK293 with PAH transfection is thin Cellular expression PAH, and detect that the 58.6% HEK293 cell with UGT1A1 transfection expresses UGT1A1.For each mRNA, when When using rat or mouse muscle-forming cell, positive cell percentage is similar with the result obtained with HEK293 cell.
Embodiment 194. captures the expression of UGT1A1 in the HEK293 cell of Capillary Electrophoresis using orientation
With UGT1A1 mRNA transfected HEK 293, and cell lysate is prepared as described above.Use Simple Simon Western system (Protein Simple) executes Western analysis.As shown in Figure 71, by anti-UGT1A1 IgG (swimming lane 1, 4 and 6) rather than by the nonimmune IgG (swimming lane 2 and 5) of same concentrations in the lysate of the cell transfected with UGT1A1 mRNA In detect UGT1A1 (~60kDa band).Similarly, using the cell lysate of the Hep3b cell of same amount of non-transfection (swimming lane 3) or UGT1A1 is not detected in come the lysate (swimming lane 7) of the HEK293 cell for OTC mRNA transfection of using by oneself.
The single dose intravenous of the LNP containing UGT1A1 mRNA is given to normal C57BL6 mouse with the dosage of 0.5mg/kg Interior injection.Mouse is put to death after 24 hours and separates liver from every mouse.By using Polytron homogenizer in frost Homogenate mentions in the phosphate buffered saline (PBS) (pH 7.4) containing protease inhibitors of 5.0mL to prepare microsomal membrane by each liver Take object.The microsome buffer of the frost of tissue homogenate and 40ml is (2.62mM KH2PO4,1.38mM K2HPO4,2% sweet Oil and 0.5mM dithiothreitol (DTT)) mixing, and 20min is centrifuged at 4 DEG C in 12,000xg.Then supernatant fraction is existed 100,000xg is centrifuged 60min at 4 DEG C.Microsomal pellet is resuspended in microsome buffer and is surveyed by Bradford method Determine protein concentration.By utilizing the protein for using anticalcium connexin antibody (Assay Designs, Ann Arbor, MI) Trace, detection calnexin item bring characterization microsome extract.The microsome extract of about 2-5 μ g is applied to capillary Guan Zhong, and execution electrophoresis and protein are fixed as described in manufacturer.As shown in Figure 72, using the antibody to UGT1A1 specificity (described above) (swimming lane 2) rather than with non-specific antibody (swimming lane 4), small with the LNP processing containing UGT1A1 mRNA UGT1A1 is detected in the microsome extract of mouse.In Mouse Liver the molecular weight of UGT1A1 with transfected with UGT1A1 mRNA (swimming lane 3) molecular weight phase observed by the case where the extract of HEK293 cell and being detected with anti-UGT1A1 IgG Together.It can't detect UGT1A1 band (swimming lane 5) in the extract of the HEK293 cell transfected with OTC mRNA.These tables of data UGT1A1 mRNA is illustrated in the intracorporal expression of mouse for having injected the LNP containing UGT1A1 mRNA.
It should be understood that used wording is descriptive wording, rather than restricted wording, and can be in appended claims In the range of make a change, without departing from the true scope and spirit of the invention in broader aspect of the invention.
Although in considerable detail about several described embodiments and quite specifically describe the present invention, simultaneously The not expected present invention should be limited to any details or embodiment or any specific embodiment, but refers to accompanying right and want It asks and is illustrated, to be explained in view of the prior art provides the widest of the claim, and therefore effectively Cover desired extent of the invention in ground.
All announcements, patent application, patent and the other bibliography being mentioned herein are integrally incorporated by reference.If any lance Shield is subject to including this specification defined herein.In addition, chapter title, material, method and embodiment are exemplary only simultaneously And it is not intended to restrictive.

Claims (12)

1. polynucleotides, it includes:
(a) open reading frame of encoding secreted proteins, wherein the open reading frame is by uridine, cytidine, adenosine and the bird modified The nucleotide of glycosides forms, wherein the uridine of the modification is N1- methyl-pseudouridine；
(b) comprising 5 '-UTR of Kozak sequence；
(c) at least one 5 ' cap structure；
(d)3′-UTR；With
(e) 3 ' tailing sequences of nucleosides are connected.
2. composition, it includes: multiple lipidic nanoparticles, wherein the lipidic nanoparticles include described in claim 1 Polynucleotides.
3. composition according to claim 2, wherein when being applied to mammalian cell, relative to comprising by containing Uracil, cytimidine, adenine are compared with the correspondence polynucleotides for the open reading frame that the nucleotide of guanine forms, described more Nucleotide improves the expression of encoded polypeptide.
4. composition according to claim 2 or 3, wherein when being applied to mammalian cell, relative to comprising by containing The correspondence polynucleotides for the open reading frame being made of the nucleotide of uracil, cytimidine, adenine and guanine are compared, described Polynucleotides have area under longer half-life period or higher protein expression profiles.
5. according to the described in any item compositions of claim 2-4, wherein when being applied to peripheral blood mononuclear cells, relative to Correspondence polynucleotides comprising the open reading frame being made of the nucleotide containing uracil, cytimidine, adenine and guanine It compares, INF- α or TNF-α of the polynucleotides induction of detectable reduced levels.
6. according to the described in any item compositions of claim 2-5, wherein the multiple lipidic nanoparticles have 80nm- Average lipid and the polynucleotides ratio (wt/wt) of the average particle size of 160nm, the average PDI of 0.02-0.20 and 10-20.
7. according to the described in any item compositions of claim 2-6, wherein the lipidic nanoparticles include DLin-DMA, DLin-K-DMA、DLin-KC2-DMA、98N12-5、C12-200、DLin-MC3-DMA、DODMA、DSDMA、DLenDMA、 ReLNP, PLGA or PEGylated lipid.
8. according to the described in any item compositions of claim 2-7, wherein the secretory protein is cell factor.
9. according to the described in any item compositions of claim 2-8, wherein 3 ' tailing sequences of the connection nucleotide are selected from about The polyA tail and polyA-G tetrad of 160 nucleotide.
10. according to the described in any item compositions of claim 2-9, wherein at least one described 5 ' cap structure be selected from Cap0, Cap1, ARCA, inosine, 1- methyl-guanosine, 2 ' fluoro- guanosines, 7- denitrogenation-guanosine, 8- oxo-guanosine, 2- amino-guanosine, LNA- Guanosine and 2- azido-guanosine.
11. according to the described in any item compositions of claim 2-10, wherein the open reading frame is through codon optimization with inclined Set G/C content.
12. the method for expressing polypeptide in mammalian cells, the method includes separating cell with described in claim 1 Polynucleotides or claim 2-11 described in any item compositions contact.
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