WO2012045075A1 - Modified nucleosides, nucleotides, and nucleic acids, and uses thereof - Google Patents
Modified nucleosides, nucleotides, and nucleic acids, and uses thereof Download PDFInfo
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- WO2012045075A1 WO2012045075A1 PCT/US2011/054617 US2011054617W WO2012045075A1 WO 2012045075 A1 WO2012045075 A1 WO 2012045075A1 US 2011054617 W US2011054617 W US 2011054617W WO 2012045075 A1 WO2012045075 A1 WO 2012045075A1
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- 0 CCC(C)(CC1OC(*)C(C)C1C)*(C)N Chemical compound CCC(C)(CC1OC(*)C(C)C1C)*(C)N 0.000 description 3
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- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
- C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
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- C07H21/02—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with ribosyl as saccharide radical
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- C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1136—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against growth factors, growth regulators, cytokines, lymphokines or hormones
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
- C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
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- C12N15/09—Recombinant DNA-technology
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- C12N5/0602—Vertebrate cells
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- C12P21/00—Preparation of peptides or proteins
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
- G01N33/559—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody through a gel, e.g. Ouchterlony technique
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- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
- C12N2310/334—Modified C
- C12N2310/3341—5-Methylcytosine
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/30—Chemical structure
- C12N2310/33—Chemical structure of the base
- C12N2310/335—Modified T or U
Definitions
- RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP and GTP, but may contain post-transcriptionally modified nucleotides. Further, approximately one hundred different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). The role of nucleoside modifications on the irnmuno- stimulatory potential, stability, and on the translation efficiency of RNA, and the consequent benefits to this for enhancing protein expression and producing therapeutics however, is unclear.
- heterologous deoxyribonucleic acid (DNA) introduced into a cell can be inherited by daughter cells (whether or not the heterologous DNA has integrated into the chromosome) or by offspring. Introduced DNA can integrate into host cell genomic DNA at some frequency, resulting in alterations and/or damage to the host cell genomic DNA.
- multiple steps must occur before a protein is made. Once inside the cell, DNA must be transported into the nucleus where it is transcribed into RNA. The RNA transcribed from DNA must then enter the cytoplasm where it is translated into protein. This need for multiple processing steps creates lag times before the generation of a protein of interest. Further, it is difficult to obtain DNA expression in cells; frequently DNA enters cells but is not expressed or not expressed at reasonable rates or concentrations. This can be a particular problem when DNA is introduced into cells such as primary cells or modified cell lines.
- modified nucleosides modified nucleotides, and modified nucleic acids which can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo.
- these modified nucleosides, modified nucleotides, and modified nucleic acids described herein can disrupt binding of a major groove interacting partner with the nucleic acid. Because of the reduced immunogenicity and the decrease in major groove interactions, these modified nucleosides, modified nucleotides, and modified nucleic acids can be more efficient during protein production than, e.g., unmodified nucleic acids.
- the present disclosure provides compounds comprising nucleotides that can disrupt binding of a major groove binding partner with a nucleic acid, wherein the nucleotide has decreased binding affinity to the major groove binding partner.
- the present disclosure further provides nucleic acid sequences of at least two nucleotides comprising a compound of Formula I-d:
- compositions comprising at least one compound of Formula I.
- the present disclosure further provides pharmaceutical compositions comprising a compound of Formula I.
- the present disclosure further provides methods of preparing nucleic acid sequences of at least two nucleotides of a compound of Formula I-d.
- the present disclosure further provides methods of amplifying nucleic acid sequences of at least two nucleotides of a compound of Formula I-d.
- kits comprising a compound of Formula I.
- FIGs. 1A and IB depict images of non-denaturing agarose gels of each in vitro- transcribed modified RNA.
- FIGs. 2A and 2B depict images of an Enzyme-linked immunosorbent assay (ELISA) for Human Granulocyte-Colony Stimulating Factor (G-CSF) of in vitro transfected Human
- ELISA Enzyme-linked immunosorbent assay
- G-CSF Human Granulocyte-Colony Stimulating Factor
- Keratinocyte cells with each indicated modRNA encoding human G-CSF and the line indicates a saturating level of maximum detectable limit of secreted G-CSF in the assay.
- FIGs. 3A-N depict line graphs of a series of Enzyme-linked immunosorbent assays (ELISA) for Human Granulocyte-Colony Stimulating Factor (G-CSF) secreted from in vitro- transfected Human Keratinocyte cells at different time points with each indicated human G-CSF- encoding modRNA at the indicated doses.
- the line indicates a saturating level of maximum detectable limit of secreted G-CSF in the assay.
- FIGs. 4A and 4B depict bar graphs of a series of Enzyme-linked immunosorbent assays (ELISA) for endogenous cellular human Tumor Necrosis Factor-a (TNF- a) secreted from in vzYro-transfected Human Keratinocyte cells at 24 hours with each indicated hu-G-CSF-encoding modRNA at increasing doses.
- ELISA Enzyme-linked immunosorbent assays
- FIGs. 4C and 4D depict bar graphs of a series of Enzyme-linked immunosorbent assays (ELISA) for endogenous cellular human Interferon- ⁇ (IFN- ⁇ ) secreted from in vzYro-transfected Human Keratinocyte cells at 24 hours with each indicated hu-G-CSF-encoding modRNA at increasing doses.
- ELISA Enzyme-linked immunosorbent assays
- FIGs. 4E and 4F depict bar graphs of a series of Enzyme-linked immunosorbent assays (ELISA) for human-G-CSF secreted from in vzYro-transfected Human Keratinocyte cells at 24 hours with each indicated hu-G-CSF-encoding modRNA at increasing doses.
- ELISA Enzyme-linked immunosorbent assays
- FIG. 5A is a table showing results from an Enzyme-linked immunosorbent assay
- ELISA for human-G-CSF secreted from in vzYro-transfected Human Keratinocyte cells sampled from individual wells in a co-culture 24-well tissue culture plate 42 hours post-transfection with 750ng of each indicated hu-G-CSF-encoding modRNA.
- FIG. 5B depicts an image of an agarose gel of RT-PCR hu-G-CSF modRNA products from co-culture cell extracts 42 hours post-transfection of the human keratinocyte feeder layer with hu-G-CSG modRNA and the un-transfected Kasumi-1 and KG-1 insert culture cells.
- FIGs. 5C and 5D depict graphs of results from a hu-G-CSF-modRNA-induced cell proliferation assay of Kasumi-1 (FIG. 5C) and KG-1 (FIG. 5D) cells normalized to untransfected cells.
- Kasumi-1 FIG. 5C
- KG-1 FIG. 5D
- FIGs. 6A-L depict graphs of the UV absorbance spectra for exemplary modRNA molecules that incorporate the indicated modified nucleotide.
- the present disclosure provides, inter alia, modified nucleosides, modified nucleotides, and modified nucleic acids that exhibit a reduced innate immune response when introduced into a population of cells.
- the modified nucleosides, modified nucleotides, and modified nucleic acids can be chemically modified on the major groove face, thereby disrupting major groove binding partner interactions, which cause innate immune responses.
- exogenous unmodified nucleic acids particularly viral nucleic acids
- IFN interferon
- RNA ribonucleic acid
- nucleic acids characterized by integration into a target cell are generally imprecise in their expression levels, deleteriously transferable to progeny and neighbor cells, and suffer from the substantial risk of causing mutation.
- nucleic acids encoding useful polypeptides capable of modulating a cell's function and/or activity are provided herein in part, and methods of making and using these nucleic acids and polypeptides. As described herein, these nucleic acids are capable of reducing the innate immune activity of a population of cells into which they are introduced, thus increasing the efficiency of protein production in that cell population. Further, one or more additional advantageous activities and/or properties of the nucleic acids and proteins of the present disclosure are described.
- modified nucleosides, modified nucleotides, and modified nucleic acids described herein can be modified on the major groove face. These major groove modifications can allow for alterations, e.g. a decrease, in the interaction of the modified nucleosides, modified nucleotides, and modified nucleic acids with a binding groove partner.
- the present disclosure provides compounds comprising a nucleotide that can disrupts binding of a major groove interacting, e.g. binding, partner with a nucleic acid, wherein the nucleotide has decreased binding affinity to major groove interacting, e.g. binding, partners.
- the present disclosure provides compounds comprising a nucleotide that contains chemical modifications, wherein the nucleotide can have altered binding to major groove interacting, e.g. binding, partners.
- the chemical modifications are located on the major groove face of the nucleobase, and wherein the chemical modifications can include replacing or substituting an atom of a pyrimidine nucleobase with an amine, an SH, an alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro).
- the chemical modifications can be located on the major groove face of the nucleobase, and wherein the chemical modification can include replacing or substituting an atom of a pyrimidine nucleobase with an amine, an SH, a methyl or ethyl, or a chloro or fluoro.
- the chemical modifications can be located on the sugar moiety of the nucleotide.
- the chemical modifications can be located on the phosphate backbone of the nucleotide.
- the chemical modifications can alter the electrochemistry on the major groove face of the nucleotide.
- the present disclosure provides nucleotides that contain chemical modifications, wherein the nucleotide reduces the cellular innate immune response, as compared to the cellular innate immune induced by a corresponding unmodified nucleic acid.
- the present disclosure provides nucleic acid sequences comprising at least two nucleotides, the nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove interacting partner with the nucleic acid sequence, wherein the nucleotide has decreased binding affinity to the major groove binding partner.
- compositions comprising a compound as described herein.
- the composition is a reaction mixture.
- the composition is a pharmaceutical composition.
- the composition is a cell culture.
- compositions further comprise an RNA polymerase and a cDNA template.
- compositions further comprise a nucleotide selected from the group consisting of adenosine, cytosine, guanosine, and uracil.
- the present disclosure provides for methods of synthesizing a pharmaceutical nucleic acid, comprising providing a complementary deoxyribonucleic acid (cDNA) that encodes a pharmaceutical protein of interest; selecting a nucleotide that is known to disrupt a binding of a major groove binding partner with a nucleic acid, wherein the nucleotide has decreased binding affinity to the major groove binding partner; and contacting the provided cDNA and the selected nucleotide with an RNA polymerase, under conditions such that the pharmaceutical nucleic acid is synthesized.
- cDNA complementary deoxyribonucleic acid
- the pharmaceutical nucleic acid is a ribonucleic acid (RNA).
- the present disclosure provides for methods of making a
- composition comprising a physiologically active secreted protein, comprising transfecting a first population of human cells with a pharmaceutical nucleic acid made by the methods described herein, wherein the secreted protein is active upon a second population of human cells.
- the secreted protein is capable of interacting, e.g. binding, with a receptor on the surface of at least one cell present in the second population.
- the secreted protein is Granulocyte-Colony Stimulating Factor (G-1)
- the second population contains myeloblast cells that express the G-CSF receptor.
- the present disclosure provides for methods of making a
- pharmaceutical formulation comprising human cells comprising a physiologically active secreted protein, comprising transfecting a first population of human cells with a pharmaceutical nucleic acid made by the methods described herein, wherein the secreted protein is active upon a second population of human cells.
- substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges.
- the term "Ci_6 alkyl” is specifically intended to individually disclose methyl, ethyl, C 3 alkyl, C 4 alkyl, C5 alkyl, and C 6 alkyl.
- stable refers to a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
- alkyl is meant to refer to a saturated hydrocarbon group which is straight-chained or branched.
- Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like.
- An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.
- alkenyl refers to an alkyl group having one or more double carbon- carbon bonds.
- Example alkenyl groups include ethenyl, propenyl, and the like.
- alkoxy refers to an -O-alkyl group.
- Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.
- alkynyl refers to an alkyl group having one or more triple carbon- carbon bonds.
- Example alkynyl groups include ethynyl, propynyl, and the like.
- aryl refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl,
- aryl groups have from 6 to about 20 carbon atoms.
- halo or halogen includes fluoro, chloro, bromo, and iodo.
- therapeutic agent refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
- animal refers to any member of the animal kingdom. In some embodiments, “animal” refers to humans at any stage of development. In some embodiments, “animal” refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g. , a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically-engineered animal, or a clone.
- the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
- association with means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.
- biologically active refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when
- nucleic acid administered to an organism, has a biological effect on that organism, is considered to be biologically active.
- a portion of that nucleic acid that shares at least one biological activity of the whole nucleic acid is typically referred to as a "biologically active" portion.
- Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences.
- two or more sequences are said to be "completely conserved” if they are 100% identical to one another.
- two or more sequences are said to be "highly conserved” if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another.
- two or more sequences are said to be "highly conserved” if they are about 70% identical, about 80%> identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another. In some embodiments, two or more sequences are said to be "conserved” if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another.
- two or more sequences are said to be "conserved” if they are about 30% identical, about 40% identical, about 50% identical, about 60%> identical, about 70% identical, about 80%> identical, about 90%> identical, about 95% identical, about 98% identical, or about 99% identical to one another.
- expression of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post- translational modification of a polypeptide or protein.
- a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
- in vitro refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
- an artificial environment e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
- in vivo refers to events that occur within an organism (e.g., animal, plant, or microbe).
- isolated refers to a substance or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some
- isolated agents are more 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 more than about 99% pure.
- a substance is "pure” if it is substantially free of other components.
- subject or patient refers to any organism to which a composition in accordance with the present disclosure may be administered, e.g. , for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
- substantially refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest.
- biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result.
- the term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
- An individual who is "suffering from” a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition.
- an individual who is "susceptible to" a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition.
- an individual who is susceptible to a disease, disorder, and/or condition may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic variant associated with development of the disease, disorder, and/or condition; (2) a genetic
- polymorphism associated with development of the disease, disorder, and/or condition (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition.
- an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition.
- an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
- terapéuticaally effective amount means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
- transcription factor refers to a DNA-binding protein that regulates transcription of DNA into RNA, for example, by activation or repression of transcription. Some transcription factors effect regulation of transcription alone, while others act in concert with other proteins. Some transcription factor can both activate and repress transcription under certain conditions. In general, transcription factors bind a specific target sequence or sequences highly similar to a specific consensus sequence in a regulatory region of a target gene. Transcription factors may regulate transcription of a target gene alone or in a complex with other molecules.
- treating refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition.
- treating cancer may refer to inhibiting survival, growth, and/or spread of a tumor.
- Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
- treatment comprises delivery of a protein associated with a therapeutically active nucleic acid to a subject in need thereof.
- unmodified refers to a nucleic acid prior to being modified, e.g.
- the compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated.
- Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis.
- Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton.
- Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge.
- Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, IH- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, IH- and 2H- isoindole, and IH- and 2H-pyrazole.
- Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
- Compounds of the present disclosure can also include all isotopes of atoms occurring in the intermediates or final compounds.
- Isotopes include those atoms having the same atomic number but different mass numbers.
- isotopes of hydrogen include tritium and deuterium.
- compound as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
- the compounds of the present disclosure are substantially isolated.
- substantially isolated is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure.
- Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%), or at least about 99% by weight of the compound of the present disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art.
- the compounds of the present disclosure, and salts thereof, can also be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
- the present disclosure also includes pharmaceutically acceptable salts of the compounds described herein.
- pharmaceutically acceptable salts refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form.
- examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
- the pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids.
- the pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods.
- such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 17 th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
- phrases "pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
- prodrugs refer to any carriers, typically covalently bonded, which release the active parent drug when administered to a mammalian subject.
- Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds.
- Prodrugs include compounds wherein hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl group respectively.
- prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the present disclosure. Preparation and use of prodrugs is discussed in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference in their entirety.
- nucleoside is defined as a compound containing a five-carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a derivative thereof.
- nucleotide is defined as a nucleoside consisting of a phosphate group.
- the nucleosides and nucleotides described herein are generally chemically modified on the major groove face.
- the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
- Table 1 below identifies the chemical faces of each canonical nucleotide. Circles identify the atoms comprising the respective chemical regions.
- modified nucleosides include pyridin-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 -taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1- methyl- 1 -deaza-pseudouridine, 2-thio-l-
- modified nucleosides include 5-aza-cytidine, pseudoisocytidme, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l- methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-
- modified nucleosides include 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyl
- modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza- guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7- methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2- dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
- the nucleotide can be modified on the major groove face and can include replacement of the hydrogen on C-5 of uracil with a methyl group or a halo group.
- the nucleoside and nucleotide can be a compound of Formula I:
- Z is O or S
- each of Y 1 is independently selected from -OR al , -NR al R bl , and -SR al ;
- each of Y 2 is independently selected from O, NR a , S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
- each of Y 3 is independently selected from O and S;
- Y 4 is selected from H, -OR a , -SR a , and -NHR a ;
- n 0, 1 , 2, or 3;
- n 0, 1 , 2 or 3;
- B is a nucleobase
- R a is H, Ci-20 alkyl, C 2- 2o alkenyl, C 2-2 o alkynyl, or C 6-2 o aryl;
- R al and R bl are each independently H or a counterion
- -Y 3 -R cl is OH or SH at a pH of about 1 or -Y 3 -R cl is O " or S " at physiological pH; or -Y 3 -R cl is Ci-20 alkoxy, C 2-2 o -O-alkenyl, or Ci -2 o -O-alkynyl;
- B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y 1 or Y 2 is not O or OH.
- B is a nucleobase of Formula Il-a, Il-b, or II-c:
- X is O or S
- U and W are each independently C or N;
- V is O, S, C or N;
- R 1 is H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, halo, or -OR c , wherein Ci-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are each optionally substituted with -OH, -NR a R b , -SH, - C(0)R c , -C(0)OR c , -NHC(0)R c , or -NHC(0)OR c ;
- R 2 is H, -OR c , -SR C , -NR a R b , or halo;
- R 1 and R 2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, - OH, -SH, -NR a R b , Ci-20 alkyl, C 2- 2o alkenyl, C 2- 2o alkynyl, C 1-20 alkoxy, or C 1-20 thioalkyl;
- R 3 is H or Ci-20 alkyl
- R 4 is H or C 1-20 alkyl; wherein when ⁇ denotes a double bond then R 4 is absent, or N- R 4 , taken together, forms a positively charged N substituted with C 1-20 alkyl;
- R a and R b are each independently H, C 1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl, or C6-20 aryl;
- R c is H, Ci-20 alkyl, C 2- 2o alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
- B is a nucleobase of Formula Il-al , II-a2, II-a3, II-a4, or II-a5:
- B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil.
- B is a pyrimidine or derivative thereof.
- the nucleotide is selected from the group consisting of:
- the modified nucleotide can be:
- the major groove chemical modification can include replacement of the C-H group at C-5 with an -NH- group or a -NH(CH 3 )- group.
- the modified nucleotide can be:
- the major groove chemical modification can include replacement of the hydrogen at C-5 of cytosine with a halo group or a methyl group.
- the modified nucleotide can be:
- the major groove chemical modification can include a fused ring that is formed by the NH 2 at the C-4 position and the carbon atom at the C-5 position.
- the modified nucleotide can be:
- a modified nucleotide is 5 '-0-(l -Thiophosphate)- Adenosine, 5'- 0-(l-Thiophosphate)-Cytidine, 5'-0-(l-Thiophosphate)-Guanosine, 5'-0-(l-Thiophosphate)- Uridine or 5'-0-(l-Thiophosphate)-Pseudouridine.
- a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages.
- Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment.
- Phosphorothioate linked nucleic acids are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
- modified nucleotides and modified nucleotide combinations are provided below in Table 2.
- N6-methyl-adenosine 25% Nl-Methyl-pseudo-uridine/75%-pseudo-uridine a-thio-adenosine 5-methyl-uridine
- modified nucleosides and nucleotides disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. It is understood that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
- spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
- spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry
- HPLC high performance liquid chromatography
- Preparation of modified nucleosides and nucleotides can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art.
- the chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley
- Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature.
- a given reaction can be carried out in one solvent or a mixture of more than one solvent.
- suitable solvents for a particular reaction step can be selected.
- Resolution of racemic mixtures of modified nucleosides and nucleotides can be carried out by any of numerous methods known in the art.
- An example method includes fractional
- Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids.
- Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent ⁇ e.g., dinitrobenzoylphenylglycine).
- Suitable elution solvent composition can be determined by one skilled in the art.
- Modified nucleosides and nucleotides can also be prepared according to the synthetic methods described in Ogata et al. Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al. Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al. Biochemistry 1(4): 563-568, 1962; and Xu et al. Tetrahedron 48(9): 1729-1740, 1992, each of which are incorporated by reference in their entirety.
- nucleic acids including RNAs such as mRNAs that contain one or more modified nucleosides (termed “modified nucleic acids”) or nucleotides as described herein, which have useful properties including the significant decreast or lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced, or the suppression thereof.
- modified nucleic acids enhance the efficiency of protein production, intracellular retention of nucleic acids, and viability of contacted cells, as well as possess reduced immunogenicity, of these nucleic acids compared to unmodified nucleic acids, having these properties are termed “enhanced nucleic acids” herein.
- nucleic acids which have decreased binding affinity to a major groove interacting, e.g. binding, partner.
- the nucleic acids are comprised of at least one nucleotide that has been chemically modified on the major groove face as described herein.
- nucleic acid in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain.
- exemplary nucleic acids for use in accordance with the present disclosure include, but are not limited to, one or more of DNA, RNA including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc., described in detail herein.
- mRNA messenger mRNA
- modified nucleic acids containing a translatable region and one, two, or more than two different nucleoside modifications.
- the modified nucleic acid exhibits reduced degradation in a cell into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid.
- exemplary nucleic acids include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), locked nucleic acids (LNAs) or a hybrid thereof.
- the modified nucleic acid includes messenger RNAs (mRNAs). As described herein, the nucleic acids of the present disclosure do not substantially induce an innate immune response of a cell into which the mRNA is introduced.
- the present disclosure provides a modified nucleic acid containing a degradation domain, which is capable of being acted on in a directed manner within a cell.
- nucleic acid is optional, and are beneficial in some embodiments.
- a 5' untranslated region (UTR) and/or a 3'UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications.
- nucleoside modifications may also be present in the translatable region. Also provided are nucleic acids containing a Kozak sequence. Additionally, provided are nucleic acids containing one or more intronic nucleotide sequences capable of being excised from the nucleic acid.
- nucleic acids containing an internal ribosome entry site may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA.
- An mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes ("multicistronic mRNA").
- multicistronic mRNA When nucleic acids are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the present disclosure include without limitation, those from picomaviruses (e.g.
- FMDV pest viruses
- CFFV pest viruses
- PV polio viruses
- ECMV encephalomyocarditis viruses
- FMDV foot-and-mouth disease viruses
- HCV hepatitis C viruses
- CSFV classical swine fever viruses
- MLV murine leukemia virus
- SIV simian immune deficiency viruses
- CrPV cricket paralysis viruses
- the nucleic acid sequences comprise a compound of Formula I-d:
- Z is O or S
- each of Y 1 is independently selected from -OR al , -NR al R bl , and -SR al ;
- each of Y 2 is independently selected from O, NR a , S or a linker comprising
- each of Y 3 is independently selected from O and S;
- Y 4 is selected from H, -OR a , -SR a , and -NHR a ;
- B is a nucleobase
- R a is H, C 1-20 alkyl, C 2-2 o alkenyl, C 2-20 alkynyl, or C 6-20 aryl;
- R al and R bl are each independently H or a counterion;
- -Y 3 -R cl is OH or SH at a pH of about 1 or -Y 3 -R cl is O " or S " at physiological pH;
- B is an unmodified nucleobase selected from cytosine, guanine, thymidine, uracil and adenine, then at least one of Z, Y 1 or Y 2 is not O or OH.
- B is a nucleobase of Formula Il-a, Il-b, or II-c:
- ⁇ denotes a single or double bond
- X is O or S
- U and W are each independently C or N;
- V is O, S, C or N;
- R 1 is H, Ci -6 alkyl, Ci -6 alkenyl, Ci -6 alkynyl, halo, or -OR c , wherein Ci-20 alkyl, C 2-2 o alkenyl, C 2-2 o alkynyl are each optionally substituted with -OH, -NR a R b , -SH, - C(0)R c , -C(0)OR c , -NHC(0)R c , or -NHC(0)OR c ;
- R 2 is H, -OR c , -SR C , -NR a R b , or halo;
- R 1 and R 2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, - OH, -SH, -NR a R b , Ci-20 alkyl, C 2-2 o alkenyl, C 2-2 o alkynyl, Ci -2 o alkoxy, or Ci -2 o thioalkyl;
- R 3 is H or Ci-20 alkyl
- R 4 is H or C 1-20 alkyl; wherein when ⁇ denotes a double bond then R 4 is absent, or N- R 4 , taken together, forms a positively charged N substituted with Ci -2 o alkyl;
- R a and R b are each independently H, Ci -2 o alkyl, C 2-2 o alkenyl, C 2-2 o alkynyl, or C6 -2 o aryl;
- R c is H, Ci-20 alkyl, C 2-2 o alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
- B is a nucleobase of Formula Il-al, II-a2, II-a3, II-a4, or II-a5:
- At least 25% of the cytosines are replaced by a compound of Formula I-a (e.g., 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%).
- a compound of Formula I-a e.g., 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%).
- At least 25% of the uracils are replaced by a compound of Formula I-a (e.g., 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%).
- a compound of Formula I-a e.g., 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%).
- At least 25% of the cytosines and 25% of the uracils are replaced by a compound of Formula I-a (e.g., 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%).
- a compound of Formula I-a e.g., 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%).
- the nucleic acid is translatable.
- RNA recognition receptors that detect and respond to RNA ligands through interactions, e.g. binding, with the major groove face of a nucleotide or nucleic acid.
- RNA ligands comprising modified nucleotides or nucleic acids as described herein decrease interactions with major groove binding partners, and therefore decrease an innate immune response, or expression and secretion of pro-inflammatory cytokines, or both.
- Example major groove interacting, e.g. binding, partners include, but are not limited to the following nucleases and helicases. Within membranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single- and double-stranded RNAs.
- helicases within the cytoplasm, members of the superfamily 2 class of DE (D/H) helicases and ATPases can sense RNAs to initiate antiviral responses.
- D/H superfamily 2 class of DE
- helicases include the RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated gene 5).
- RIG-I retinoic acid-inducible gene I
- MDA5 melanoma differentiation-associated gene 5
- Other examples include laboratory of genetics and physiology 2 (LGP2), ⁇ -200 domain containing proteins, or Helicase-domain containing proteins.
- innate immune response includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. Protein synthesis is also reduced during the innate cellular immune response. While it is advantageous to eliminate the innate immune response in a cell, the present disclosure provides modified mRNAs that substantially reduce the immune response, including interferon signaling, without entirely eliminating such a response.
- the immune response is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% as compared to the immune response induced by a corresponding unmodified nucleic acid.
- a reduction can be measured by expression or activity level of Type 1 interferons or the expression of interferon-regulated genes such as the toll-like receptors (e.g., TLR7 and TLR8).
- Reduction of innate immune response can also be measured by decreased cell death following one or more administrations of modified RNAs to a cell population; e.g., cell death is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequency observed with a corresponding unmodified nucleic acid.
- cell death may affect fewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than 0.01% of cells contacted with the modified nucleic acids.
- the present disclosure provides for the repeated introduction (e.g., transfection) of modified nucleic acids into a target cell population, e.g., in vitro, ex vivo, or in vivo.
- the step of contacting the cell population may be repeated one or more times (such as two, three, four, five or more than five times).
- the step of contacting the cell population with the modified nucleic acids is repeated a number of times sufficient such that a predetermined efficiency of protein translation in the cell population is achieved. Given the reduced cytotoxicity of the target cell population provided by the nucleic acid modifications, such repeated transfections are achievable in a diverse array of cell types.
- nucleic acids that encode variant polypeptides, which have a certain identity with a reference polypeptide sequence.
- identity refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, “identity” also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. “Identity” measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related peptides can be readily calculated by known methods.
- Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
- the polypeptide variant has the same or a similar activity as the reference polypeptide.
- the variant has an altered activity (e.g., increased or decreased) relative to a reference polypeptide.
- variants of a particular polynucleotide or polypeptide of the present disclosure will have at least about 40%, 45%, 50%>, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
- protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of this present disclosure.
- a reference protein meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical
- any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%>, about 70%), about 80%>, about 90%>, about 95%, or about 100%.
- identical to any of the sequences described herein can be utilized in accordance with the present disclosure.
- a protein sequence to be utilized in accordance with the present disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
- polynucleotide libraries containing nucleoside modifications wherein the polynucleotides individually contain a first nucleic acid sequence encoding a polypeptide, such as an antibody, protein binding partner, scaffold protein, and other polypeptides known in the art.
- a polypeptide such as an antibody, protein binding partner, scaffold protein, and other polypeptides known in the art.
- the polynucleotides are mRNA in a form suitable for direct introduction into a target cell host, which in turn synthesizes the encoded polypeptide.
- multiple variants of a protein are produced and tested to determine the best variant in terms of
- Such a library may contain 10, 10 , 10 , 10 , 10 , 10 , 10 , 10 , or over 10 9 possible variants (including substitutions, deletions of one or more residues, and insertion of one or more residues).
- Proper protein translation involves the physical aggregation of a number of polypeptides and nucleic acids associated with the mRNA.
- Provided by the present disclosure are protein- nucleic acid complexes, containing a translatable mRNA having one or more nucleoside modifications (e.g., at least two different nucleoside modifications) and one or more polypeptides bound to the mRNA.
- the proteins are provided in an amount effective to prevent or reduce an innate immune response of a cell into which the complex is introduced.
- mRNAs having sequences that are substantially not translatable. Such mRNA is effective as a vaccine when administered to a mammalian subject.
- modified nucleic acids that contain one or more noncoding regions. Such modified nucleic acids are generally not translated, but are capable of binding to and sequestering one or more translational machinery component such as a ribosomal protein or a transfer RNA (tRNA), thereby effectively reducing protein expression in the cell.
- the modified nucleic acid may contain a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).
- Nucleic acids for use in accordance with the present disclosure may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription, enzymatic or chemical cleavage of a longer precursor, etc.
- Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M.J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference).
- modified nucleosides and nucleotides disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. It is understood that where typical or preferred process conditions ⁇ i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
- product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
- spectroscopic means such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13 C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry
- chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
- Preparation of modified nucleosides and nucleotides can involve the protection and deprotection of various chemical groups.
- the need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art.
- the chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
- Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature.
- a given reaction can be carried out in one solvent or a mixture of more than one solvent.
- suitable solvents for a particular reaction step can be selected.
- An example method includes fractional recrystallization using a "chiral resolving acid" which is an optically active, salt-forming organic acid.
- Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids.
- Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine).
- Suitable elution solvent composition can be determined by one skilled in the art.
- Modified nucleic acids need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures may exist at various positions in the nucleic acid.
- nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased.
- a modification may also be a 5 Or 3' terminal modification.
- the nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50%> modified nucleotides, at least 80%> modified nucleotides, or at least 90% modified nucleotides.
- the nucleic acids may contain a modified pyrimidine such as uracil or cytosine.
- at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil.
- the modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid is replaced with a modified cytosine.
- the modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
- the shortest length of a modified mRNA of the present disclosure can be the length of an mRNA sequence that is sufficient to encode for a dipeptide.
- the length of the mRNA sequence is sufficient to encode for a tripeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a tetrapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a pentapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a hexapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a heptapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for an octapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a nonapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a decapeptide.
- dipeptides that the modified nucleic acid sequences can encode for include, but are not limited to, carnosine and anserine.
- the mRNA is greater than 30 nucleotides in length. In another embodiment, the RNA molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least 90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides.
- the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides.
- the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides.
- the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.
- the present disclosure provides methods of preparing a nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove interacting partner with the nucleic acid sequence, wherein the nucleic acid sequence comprises a compound of Formula I-d:
- Z is O or S
- each of Y 1 is independently selected from -OR al , -NR al R bl , and -SR al ;
- each of Y 2 is independently selected from O, NR a , S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
- each of Y 3 is independently selected from O and S;
- Y 4 is selected from H, -OR a , -SR a , and -NHR a ;
- B is a nucleobase
- R a is H, Ci-20 alkyl, C 2- 2o alkenyl, C 2-2 o alkynyl, or C 6-2 o aryl;
- R al and R bl are each independently H or a counterion
- -Y 3 -R cl is OH or SH at a pH of about 1 or -Y 3 -R cl is O " or S " at physiological pH; or -Y 3 -R cl is Ci-20 alkoxy, C 2-2 o -O-alkenyl, or Ci -2 o -O-alkynyl;
- B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y 1 or Y 2 is not O or OH;
- the reaction is repeated from 1 to about 7,000 times.
- B is a nucleobase of Formula Il-a, Il-b, or II-c:
- X is O or S
- U and W are each independently C or N;
- V is O, S, C or N;
- R 1 is H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, halo, or -OR c , wherein Ci-20 alkyl, C 2-2 o alkenyl, C 2-2 o alkynyl are each optionally substituted with -OH, -NR a R b , -SH, - C(0)R c , -C(0)OR c , -NHC(0)R c , or -NHC(0)OR c ;
- R 2 is H, -OR c , -SR C , -NR a R b , or halo;
- R 1 and R 2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, - OH, -SH, -NR a R b , Ci-20 alkyl, C 2-2 o alkenyl, C 2-2 o alkynyl, C 1-20 alkoxy, or C 1-20 thioalkyl;
- R 3 is H or Ci-20 alkyl
- R 4 is H or C 1-20 alkyl; wherein when ⁇ denotes a double bond then R 4 is absent, or N- R 4 , taken together, forms a positively charged N substituted with C 1-20 alkyl;
- R a and R b are each independently H, C 1-20 alkyl, C 2-2 o alkenyl, C 2-2 o alkynyl, or C6 -2 o aryl;
- R c is H, Ci-20 alkyl, C 2-2 o alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
- B is a nucleobase of Formula Il-al , II-a2, II-a3, II-a4, or II-a5:
- the methods further comprise a nucleotide selected from the group consisting of adenosine, cytosine, guanosine, and uracil.
- the nucleobase is a pyrimidine or derivative thereof.
- the present disclosure provides methods of amplifying a nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove binding partner with the nucleic acid sequence, the method comprising:
- Z is O or S
- each of Y 1 is independently selected from -OR al , -NR al R bl , and -SR al ;
- each of Y 2 is independently selected from O, NR a , S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
- each of Y 3 is independently selected from O and S;
- Y 4 is selected from H, -OR a , -SR a , and -NHR a ;
- B is a nucleobase
- R a is H, Ci-20 alkyl, C 2- 2o alkenyl, C 2-2 o alkynyl, or C 6-2 o aryl;
- R al and R bl are each independently H or a counterion
- -Y 3 -R cl is OH or SH at a pH of about 1 or -Y 3 -R cl is O " or S " at physiological pH; or -Y 3 -R cl is Ci-20 alkoxy, C 2-2 o -O-alkenyl, or Ci -2 o -O-alkynyl;
- B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y 1 or Y 2 is not O or OH;
- RNA polymerase a primer, a cDNA template, and an RNA polymerase.
- B is a nucleobase of Formula Il-a, Il-b, or II-c:
- ⁇ denotes a single or double bond
- X is O or S
- U and W are each independently C or N;
- V is O, S, C or N; wherein when V is C then R 1 is H, C 1-6 alkyl, C 1-6 alkenyl, C 1-6 alkynyl, halo, or -OR c , wherein Ci-20 alkyl, C 2- 2o alkenyl, C 2-2 o alkynyl are each optionally substituted with -OH, -NR a R b , -SH, - C(0)R c , -C(0)OR c , -NHC(0)R c , or -NHC(0)OR c ;
- R 2 is H, -OR c , -SR C , -NR a R b , or halo;
- R 1 and R 2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, - OH, -SH, -NR a R b , Ci-20 alkyl, C 2-2 o alkenyl, C 2-2 o alkynyl, C 1-20 alkoxy, or C 1-20 thioalkyl;
- R 3 is H or Ci-20 alkyl
- R 4 is H or C 1-20 alkyl; wherein when ⁇ denotes a double bond then R 4 is absent, or N- R 4 , taken together, forms a positively charged N substituted with C 1-20 alkyl;
- R a and R b are each independently H, C 1-20 alkyl, C 2-2 o alkenyl, C 2-2 o alkynyl, or C6 -2 o aryl;
- R c is H, Ci-20 alkyl, C 2-2 o alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
- B is a nucleobase of Formula Il-al, II-a2, II-a3, II-a4, or II-a5:
- the methods further comprise a nucleotide selected from the group consisting of adenosine, cytosine, guanosine, and uracil.
- the nucleobase is a pyrimidine or derivative thereof.
- modified nucleic acids and the proteins translated from the modified nucleic acids described herein can be used as therapeutic agents.
- a modified nucleic acid described herein can be administered to a subject, wherein the modified nucleic acid is translated in vivo to produce a therapeutic peptide in the subject.
- compositions, methods, kits, and reagents for treatment or prevention of disease or conditions in humans and other mammals include modified nucleic acids, cells containing modified nucleic acids or polypeptides translated from the modified nucleic acids, polypeptides translated from modified nucleic acids, and cells contacted with cells containing modified nucleic acids or polypeptides translated from the modified nucleic acids.
- combination therapeutics containing one or more modified nucleic acids containing translatable regions that encode for a protein or proteins that boost a mammalian subject's immunity along with a protein that induces antibody-dependent cellular toxitity.
- G-CSF granulocyte-colony stimulating factor
- such combination therapeutics are useful in Her2+ breast cancer patients who develop induced resistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy. 2(6):795-8 (2010)).
- Such translation can be in vivo, ex vivo, in culture, or in vitro.
- the cell population is contacted with an effective amount of a composition containing a nucleic acid that has at least one nucleoside modification, and a translatable region encoding the recombinant polypeptide.
- the population is contacted under conditions such that the nucleic acid is localized into one or more cells of the cell population and the recombinant polypeptide is translated in the cell from the nucleic acid.
- an effective amount of the composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the nucleic acid ⁇ e.g., size, and extent of modified nucleosides), and other determinants.
- an effective amount of the composition provides efficient protein production in the cell, preferably more efficient than a composition containing a corresponding unmodified nucleic acid. Increased efficiency may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the nucleic acid), increased protein translation from the nucleic acid, decreased nucleic acid degradation (as demonstrated, e.g. , by increased duration of protein translation from a modified nucleic acid), or reduced innate immune response of the host cell.
- aspects of the present disclosure are directed to methods of inducing in vivo translation of a recombinant polypeptide in a mammalian subject in need thereof.
- an effective amount of a composition containing a nucleic acid that has at least one nucleoside modification and a translatable region encoding the recombinant polypeptide is administered to the subject using the delivery methods described herein.
- the nucleic acid is provided in an amount and under other conditions such that the nucleic acid is localized into a cell of the subject and the recombinant polypeptide is translated in the cell from the nucleic acid.
- the cell in which the nucleic acid is localized, or the tissue in which the cell is present, may be targeted with one or more than one rounds of nucleic acid administration.
- compositions containing modified nucleic acids are formulated for administration intramuscularly,
- the composition is formulated for extended release.
- the subject to whom the therapeutic agent is administered suffers from or is at risk of developing a disease, disorder, or deleterious condition.
- GWAS genome-wide association studies
- the administered modified nucleic acid directs production of one or more recombinant polypeptides that provide a functional activity which is substantially absent in the cell in which the recombinant polypeptide is translated.
- the missing functional activity may be enzymatic, structural, or gene regulatory in nature.
- the administered modified nucleic acid directs production of one or more recombinant polypeptides that replace a polypeptide (or multiple polypeptides) that is substantially absent in the cell in which the recombinant polypeptide is translated. Such absence may be due to genetic mutation of the encoding gene or regulatory pathway thereof.
- the recombinant polypeptide functions to antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell. Usually, the activity of the endogenous protein is deleterious to the subject, for example, do to mutation of the endogenous protein resulting in altered activity or localization.
- the recombinant polypeptide antagonizes, directly or indirectly, the activity of a biological moiety present in, on the surface of, or secreted from the cell.
- antagonized biological moieties include lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), a nucleic acid, a carbohydrate, or a small molecule toxin.
- the recombinant proteins described herein are engineered for localization within the cell, potentially within a specific compartment such as the nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
- a useful feature of the modified nucleic acids of the present disclosure is the capacity to reduce the innate immune response of a cell to an exogenous nucleic acid.
- the cell is contacted with a first composition that contains a first dose of a first exogenous nucleic acid including a translatable region and at least one nucleoside modification, and the level of the innate immune response of the cell to the first exogenous nucleic acid is determined.
- the cell is contacted with a second composition, which includes a second dose of the first exogenous nucleic acid, the second dose containing a lesser amount of the first exogenous nucleic acid as compared to the first dose.
- the cell is contacted with a first dose of a second exogenous nucleic acid.
- the second exogenous nucleic acid may contain one or more modified nucleosides, which may be the same or different from the first exogenous nucleic acid or, alternatively, the second exogenous nucleic acid may not contain modified nucleosides.
- the steps of contacting the cell with the first composition and/or the second composition may be repeated one or more times. Additionally, efficiency of protein production (e.g., protein translation) in the cell is optionally determined, and the cell may be re-transfected with the first and/or second composition repeatedly until a target protein production efficiency is achieved.
- Therapeutics for diseases and conditions Provided are methods for treating or preventing a symptom of diseases characterized by missing or aberrant protein activity, by replacing the missing protein activity or overcoming the aberrant protein activity. Because of the rapid initiation of protein production following introduction of modified mRNAs, as compared to viral DNA vectors, the compounds of the present disclosure are particularly advantageous in treating acute diseases such as sepsis, stroke, and myocardial infarction. Moreover, the lack of transcriptional regulation of the modified mRNAs of the present disclosure is advantageous in that accurate titration of protein production is achievable.
- Diseases characterized by dysfunctional or aberrant protein activity include, but not limited to, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, and metabolic diseases.
- the present disclosure provides a method for treating such conditions or diseases in a subject by introducing nucleic acid or cell-based therapeutics containing the modified nucleic acids provided herein, wherein the modified nucleic acids encode for a protein that antagonizes or otherwise overcomes the aberrant protein activity present in the cell of the subject.
- Specific examples of a dysfunctional protein are the missense mutation variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produce a dysfunctional protein variant of CFTR protein, which causes cystic fibrosis.
- CFTR cystic fibrosis transmembrane conductance regulator
- the present disclosure provides a method for treating such conditions or diseases in a subject by introducing nucleic acid or cell-based therapeutics containing the modified nucleic acids provided herein, wherein the modified nucleic acids encode for a protein that replaces the protein activity missing from the target cells of the subject.
- a dysfunctional protein are the nonsense mutation variants of the cystic fibrosis
- CFTR transmembrane conductance regulator
- RNA molecules are formulated for administration by inhalation.
- the present disclosure provides a method for treating
- hyperlipidemia in a subject by introducing into a cell population of the subject with a modified mRNA molecule encoding Sortilin, a protein recently characterized by genomic studies, thereby ameliorating the hyperlipidemia in a subject.
- the SORTl gene encodes a trans-Golgi network (TGN) transmembrane protein called Sortilin.
- TGN trans-Golgi network
- Genetic studies have shown that one of five individuals has a single nucleotide polymorphism, rsl2740374, in the lpl3 locus of the SORTl gene that predisposes them to having low levels of low-density lipoprotein (LDL) and very-low- density lipoprotein (VLDL).
- LDL low-density lipoprotein
- VLDL very-low- density lipoprotein
- Methods of the present disclosure enhance nucleic acid delivery into a cell population, in vivo, ex vivo, or in culture.
- a cell culture containing a plurality of host cells ⁇ e.g., eukaryotic cells such as yeast or mammalian cells
- the composition also generally contains a transfection reagent or other compound that increases the efficiency of enhanced nucleic acid uptake into the host cells.
- the enhanced nucleic acid exhibits enhanced retention in the cell population, relative to a
- the retention of the enhanced nucleic acid is greater than the retention of the unmodified nucleic acid. In some embodiments, it is at least about 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than 200% greater than the retention of the unmodified nucleic acid. Such retention advantage may be achieved by one round of transfection with the enhanced nucleic acid, or may be obtained following repeated rounds of transfection.
- the enhanced nucleic acid is delivered to a target cell population with one or more additional nucleic acids. Such delivery may be at the same time, or the enhanced nucleic acid is delivered prior to delivery of the one or more additional nucleic acids.
- the additional one or more nucleic acids may be modified nucleic acids or unmodified nucleic acids. It is understood that the initial presence of the enhanced nucleic acids does not substantially induce an innate immune response of the cell population and, moreover, that the innate immune response will not be activated by the later presence of the unmodified nucleic acids. In this regard, the enhanced nucleic acid may not itself contain a translatable region, if the protein desired to be present in the target cell population is translated from the unmodified nucleic acids.
- modified nucleic acids are provided to express a protein-binding partner or a receptor on the surface of the cell, which functions to target the cell to a specific tissue space or to interact with a specific moiety, either in vivo or in vitro.
- Suitable protein- binding partners include antibodies and functional fragments thereof, scaffold proteins, or peptides.
- modified nucleic acids can be employed to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties.
- a method for epigenetically silencing gene expression in a mammalian subject comprising a nucleic acid where the translatable region encodes a polypeptide or polypeptides capable of directing sequence-specific histone H3 methylation to initiate heterochromatin formation and reduce gene transcription around specific genes for the purpose of silencing the gene.
- a gain-of-function mutation in the Janus Kinase 2 gene is responsible for the family of Myeloproliferative Diseases.
- the present disclosure provides proteins generated from modified mRNAs.
- compositions may optionally comprise one or more additional therapeutically active substances.
- a method of administering pharmaceutical compositions comprising one or more proteins to be delivered to a subject in need thereof is provided.
- compositions are administered to humans.
- active ingredient generally refers to a protein or protein-containing complex as described herein.
- compositions are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for
- compositions suitable for administration to humans Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
- Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.
- Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
- a pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses.
- a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
- the amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.
- Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered.
- the composition may comprise between 0.1% and 100% (w/w) active ingredient.
- compositions may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
- a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
- Remington's The Science and Practice of Pharmacy, 21 st Edition, A. R. Gennaro discloses various excipients used in formulating pharmaceutical
- compositions and known techniques for the preparation thereof Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this present disclosure.
- a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure.
- an excipient is approved for use in humans and for veterinary use.
- an excipient is approved by United States Food and Drug Administration.
- an excipient is pharmaceutical grade.
- an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
- compositions used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
- Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
- Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)
- crospovidone sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
- Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers ⁇ e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum ® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g.
- natural emulsifiers ⁇ e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin
- colloidal clays e.g. bentonite [alumin
- stearyl alcohol cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
- polyoxyethylene monostearate [Myrj ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol ® ), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor ® ), polyoxyethylene ethers, (e.g.
- polyoxyethylene lauryl ether [Brij ® 30]), poly(vinyl-pyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic ® F 68, Poloxamer ® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
- Exemplary binding agents include, but are not limited to, starch ⁇ e.g. cornstarch and starch paste); gelatin; sugars ⁇ e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums ⁇ e.g.
- acacia sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum ® ), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
- Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives.
- Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite.
- Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
- EDTA ethylenediaminetetraacetic acid
- citric acid monohydrate disodium edetate
- dipotassium edetate dipotassium edetate
- edetic acid fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate.
- antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal.
- antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
- exemplary alcohol e.g., butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid.
- preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol.
- acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid.
- preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus ® , Phenonip ® , methylparaben, Germall ® 115, Germaben ® II, Neolone TM , Kathon TM , and/or Euxyl ® .
- Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d- gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, iso
- Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
- oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury
- oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
- Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs.
- liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof.
- inert diluents commonly used in the art such as, for example,
- oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
- adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents.
- compositions are mixed with solubilizing agents such as Cremophor ® , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
- Injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents.
- Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol.
- the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution.
- Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
- any bland fixed oil can be employed including synthetic mono- or diglycerides.
- Fatty acids such as oleic acid can be used in the preparation of injectables.
- Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
- sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
- delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle.
- injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
- compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
- suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
- Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules.
- an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g.
- the dosage form may comprise buffering agents.
- solution retarding agents e.g. paraffin
- absorption accelerators e.g. quaternary ammonium compounds
- wetting agents e.g. cetyl alcohol and glycerol monostearate
- absorbents e.g. kaolin and bentonite clay
- lubricants e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate
- the dosage form may comprise buffering agents.
- Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
- Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or
- embedding compositions which can be used include polymeric substances and waxes.
- Solid compositions of a similar type may be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
- Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches.
- an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.
- transdermal patches which often have the added advantage of providing controlled delivery of a compound to the body.
- dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium.
- rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.
- Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patents 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662.
- compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof.
- Jet injection devices which deliver liquid compositions to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Patents 5,480,381; 5,599,302; 5,334,144; 5,993,412; 5,649,912; 5,569,189; 5,704,911;
- Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable.
- conventional syringes may be used in the classical mantoux method of intradermal administration.
- Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
- liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
- Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent.
- Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
- a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity.
- a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm.
- Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container.
- Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm.
- Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
- Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50% to 99.9% (w/w) of the composition, and active ingredient may constitute 0.1 % to 20% (w/w) of the composition.
- a propellant may further comprise additional ingredients such as a liquid non- ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
- compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension.
- Such formulations may be prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device.
- Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as
- Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.
- Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition.
- Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 ⁇ to 500 ⁇ .
- Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
- Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein.
- a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using
- formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient.
- Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
- a pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration.
- Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0%) (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient.
- Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein.
- Other opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this present disclosure.
- the present disclosure provides methods comprising administering proteins or complexes in accordance with the present disclosure to a subject in need thereof.
- Proteins or complexes, or pharmaceutical, imaging, diagnostic, or prophylactic compositions thereof may be administered to a subject using any amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and/or condition ⁇ e.g., a disease, disorder, and/or condition relating to working memory deficits).
- the exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like.
- Compositions in accordance with the present disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage.
- compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment.
- the specific therapeutically effective, prophylactially effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
- Proteins to be delivered and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be administered to animals, such as mammals ⁇ e.g., humans, domesticated animals, cats, dogs, mice, rats, etc.). In some embodiments, pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof are administered to humans. Proteins to be delivered and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof in accordance with the present disclosure may be administered by any route.
- proteins and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g.
- proteins or complexes, and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof are administered by systemic intravenous injection.
- proteins or complexes and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be administered intravenously and/or orally.
- proteins or complexes, and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be administered in a way which allows the protein or complex to cross the blood- brain barrier, vascular barrier, or other epithelial barrier.
- the present disclosure encompasses the delivery of proteins or complexes, and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof, by any appropriate route taking into consideration likely advances in the sciences of drug delivery.
- the most appropriate route of administration will depend upon a variety of factors including the nature of the protein or complex comprising proteins associated with at least one agent to be delivered (e.g., its stability in the environment of the gastrointestinal tract, bloodstream, etc.), the condition of the patient (e.g., whether the patient is able to tolerate particular routes of administration), etc.
- the present disclosure encompasses the delivery of the pharmaceutical, prophylactic, diagnostic, or imaging compositions by any appropriate route taking into consideration likely advances in the sciences of drug delivery.
- compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day, one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect.
- the desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple
- administrations e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations.
- Proteins or complexes may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents.
- combination with it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure.
- Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent.
- the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
- therapeutically, prophylactically, diagnostically, or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions.
- agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
- kits for example, a composition useful for treating cancer in accordance with the present disclosure may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., control of any adverse effects).
- kits for conveniently and/or effectively carrying out methods of the present disclosure.
- kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
- kits for protein production comprising a first isolated nucleic acid comprising a translatable region and a nucleic acid modification, wherein the nucleic acid is capable of evading an innate immune response of a cell into which the first isolated nucleic acid is introduced, and packaging and instructions.
- kits for protein production comprising: a first isolated nucleic acid comprising a translatable region, provided in an amount effective to produce a desired amount of a protein encoded by the translatable region when introduced into a target cell; a second nucleic acid comprising an inhibitory nucleic acid, provided in an amount effective to substantially inhibit the innate immune response of the cell; and packaging and instructions.
- kits for protein production comprising a first isolated nucleic acid comprising a translatable region and a nucleoside modification, wherein the nucleic acid exhibits reduced degradation by a cellular nuclease, and packaging and instructions.
- kits for protein production comprising a first isolated nucleic acid comprising a translatable region and at least two different nucleoside modifications, wherein the nucleic acid exhibits reduced degradation by a cellular nuclease, and packaging and instructions.
- kits for protein production comprising a first isolated nucleic acid comprising a translatable region and at least one nucleoside modification, wherein the nucleic acid exhibits reduced degradation by a cellular nuclease; a second nucleic acid comprising an inhibitory nucleic acid; and packaging and instructions.
- the first isolated nucleic acid comprises messenger RNA
- the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2- thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5- carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl- pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio- uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio- 1- methyl-pseudouridine, 2-thio- 1-methyl-pseudouridine, 1 -methyl- 1-d
- 2- thio-dihydropseudouridine 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, and 4-methoxy-2-thio-pseudouridine.
- the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio
- the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladen
- the mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy- guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
- nucleoside selected from the
- compositions for protein production comprising a first isolated nucleic acid comprising a translatable region and a nucleoside modification, wherein the nucleic acid exhibits reduced degradation by a cellular nuclease, and a mammalian cell suitable for translation of the translatable region of the first nucleic acid.
- Modified mRNAs were made using standard laboratory methods and materials for in vitro transcription with the exception that the nucleotide mix contained modified nucleotides.
- the open reading frame (ORF) of the gene of interest is flanked by a 5' untranslated region (UTR) containing a strong Kozak translational initiation signal and an alpha-globin 3' UTR terminating with an oligo(dT) sequence for templated addition of a polyA tail for modRNAs not incorporating Adenosine analogs.
- Adenosine-containing modRNAs were synthesized without an oligo (dT) sequence to allow for post-transcription poly (A) polymerase poly-(A) tailing.
- the modRNAs were modified by incorporating chemically modified nucleotides indicated in Table 3 (below) during the in vitro transcription with 100% replacement of the corresponding natural nucleotide or partial replacement of the corresponding natural nucleotide at the indicated percentage.
- Table 3 indicates the chemical identity of each chemically-distinct modified nucleotide incorporated into a modified mRNA with the given chemistry designation number.
- Chem 8 Pseudo-iso-cytidine/a-thio-uridine Chem 36 a-thio-uridine Chem 9 Pseudo-iso-cytidine/5-methyl-uridine Chem 37
- Nanodrop modRNA quantification and UV spectral data modRNAs in TE buffer (1 ⁇ ) were used for Nanodrop UV absorbance readings to quantitate the yield of each modRNA from an in vitro transcription reaction (UV absorbance traces are shown in Figures 6A-6L). These data indicate which chemically modified nucleotides were transcribed into chemically- modified mRNAs. These data also demonstrate that nucleotides with chemical modifications on the major groove and minor groove face of the nucleotide were capable of being transcribed into a modRNA. These data further demonstrate that the nucleotides of the present invention are transcription-competent and compatible with incorporation into a modRNA, which may have altered UV spectra due to the presence of a given modified nucleotide.
- Pyrrolo-C containing modRNAs have an increase in UV absorbance at a lower wavelength due to the presence of the pyrrolo ring of the modified C nucleotide.
- 2-amino-adenine nucleotide-containing modRNAs have an increase in UV absorbance at a higher wavelength due to the presence of an exocyclic amine off the purine ring.
- Nucleotides that are not transcription- competent and cannot be incorporated into a modRNA have a scrambled UV spectrum indicating no product from the transcription reaction.
- RNAiMAX RNAiMAX was prepared as described and mixed with the cells in the multi-well plate within a period of time, e.g., 6 hours, of cell seeding before cells had adhered to the tissue culture plate.
- Keratinocytes were seeded at a cell density of 0.7 x 10 5 .
- Keratinocytes were seeded at a cell density of 0.3 x 10 5 .
- RNAiMAX RNAiMAX was prepared as described and transfected onto the cells in the multi-well plate over 24 hours after cell seeding and adherence to the tissue culture plate.
- FIGs. 2A and 2B show an Enzyme-linked immunosorbent assay (ELISA) for Human Granulocyte-Colony Stimulating Factor (hu-G-CSF) of in vitro transfected Human Keratinocyte cells. Keratinocytes were grown in EpiLife medium with Supplement S7 from Invitrogen at a confluence of >70%.
- FIG. 2A keratinocytes were reverse transfected with 300 ng of the indicated chemically modified mRNA complexed with RNAiMAX from Invitrogen.
- FIG. 2B keratinocytes were forward transfected with 300 ng modRNA complexed with RNAiMAX from Invitrogen.
- RNA:RNAiMAX complex was formed by first incubating the RNA with Supplement- free EpiLife media in a 5X volumetric dilution for 10 minutes at room temperature. In a second vial, RNAiMAX reagent was incubated with Supplement- free EpiLife Media in a 10X volumetric dilution for 10 minutes at room temperature. The RNA vial was then mixed with the RNAiMAX vial and incubated for 20-30 at room temperature before being added to the cells in a drop-wise fashion. Secreted huG-CSF concentration in the culture medium was measured at 18 hours post-transfection for each of the chemically modified mRNAs in triplicate.
- G-CSF Human Granulocyte-Colony Stimulating Factor
- FIGs. 3A-N show Enzyme-linked immunosorbent assays (ELISA) for Human
- G-CSF Granulocyte-Colony Stimulating Factor
- G-CSF Stimulating Factor
- modified RNA containing modified nucleotides with chemical modifications on the major groove face of pyrimidine analogs have the highest levels of secreted hu-G-CSF into the cell culture medium and that 750ng of modRNA elicits the highest level of secreted hu-G-CSF.
- FIGs. 4A-F show an Enzyme-linked immunosorbent assay (ELISA) for Human Tumor Necrosis Factor-a (TNF-a) (FIGs. 4A and 4B); Human Interferon- ⁇ (IFN- ⁇ ) (FIGs. 4C and 4D); and Human Granulocyte-Colony Stimulating Factor (G-CSF) (FIGs. 4E and 4F) secreted from in vzYro-transfected Human Keratinocyte cells. Keratinocytes were grown in EpiLife medium with Human Keratinocyte Growth Supplement in the absence of hydrocortisone from Invitrogen at a confluence of >70%. In FIGs.
- ELISA Enzyme-linked immunosorbent assay
- keratinocytes were reverse transfected with Ong, 93.75ng, 187.5ng, 375ng, 750ng, 1500ng or 3000ng of the indicated chemically modified mRNA complexed with RNAiMAX from Invitrogen as described in triplicate.
- Secreted TNF-a in the culture medium was measured 24 hours post-transfection for each of the chemically modified mRNAs using an ELISA kit from Invitrogen according to the manufacturer protocols.
- G-CSF Human Granulocyte-Colony Stimulating Factor
- modified RNAs containing modified nucleotides were capable of eliciting a reduced cellular innate immune response in comparison to natural and other chemically modified nucleotides by measuring exemplary type I cytokines TNF-a and IFN- ⁇ . These data show that modified RNAs containing modified nucleotides with chemical
- modifications on the major groove face of pyrimidine analogs have the lowest levels of secreted TNF-a and IFN- ⁇ into the cell culture medium while maintaining high levels of modRNA- encoding hu-G-CSF secretion into the cell culture medium.
- FIGs. 5A-D show modRNA-encoding hu-G-CSF produced by a human keratinocyte feeder cell layer induced the proliferation of both human myeloblast cells KG-1 and Kasumi-1 that express the G-CSF-receptor where the cell populations are separated by a semi-permeable membrane.
- 5A is a table showing the results from an Enzyme-linked immunosorbent assay (ELISA) for human-G-CSF secreted from in vzYro-transfected Human Keratinocyte cells sampled from individual wells in a co-culture 24- well tissue culture plate 42 hours post-transfection with 750ng of each indicated hu-G-CSF- encoding modRNA.
- ELISA Enzyme-linked immunosorbent assay
- G-CSF Human Granulocyte-Colony Stimulating Factor
- RNA from sample cells was extracted and lysed using RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer instructions. Extracted total RNA was submitted to RT-PCR for specific amplification of modRNA-G-CSF using ProtoScript® M-MuLV Taq RT-PCR kit (New England BioLabs, Ipswich, MA) according to the manufacturer instructions with hu-G-CSF-specific primers (see below). RT-PCR products were visualized by 1.2% agarose gel electrophoresis (FIG. 5B). Table 6 below shows which modRNAs were run on the agarose gel.
- Keratinocytes are grown in EpiLife medium with Human Keratinocyte Growth Supplement in the absence of hydrocortisone from Invitrogen at a confluence of >70%. Keratinocytes are reverse transfected with Ong, 46.875ng, 93.75ng, 187.5ng, 375ng, 750ng, 1500ng, 3000ng, or 6000ng of modRNA complexed with RNAiMAX from Invitrogen. The modRNA:RNAiMAX complex is formed. Secreted huG-CSF
- G-CSF Human Granulocyte-Colony Stimulating Factor
- the modified mRNA comprised of chemically-distinct modified nucleotides encoding human Granulocyte-Colony Stimulating Factor (G-CSF) may stimulate the cellular proliferation of a transfection incompetent cell in co-culture environment.
- the co-culture includes a highly transfectable cell type such as a human keratinocyte and a transfection incompetent cell type such as a white blood cell (WBC).
- WBC white blood cell
- the modified mRNA encoding G-CSF may be transfected into the highly transfectable cell allowing for the production and secretion of G-CSF protein into the extracellular environment where G-CSF acts in a paracrine-like manner to stimulate the white blood cell expressing the G-CSF receptor to proliferate.
- the expanded WBC population may be used to treat immune-compromised patients or partially reconstitute the WBC population of an immunosuppressed patient and thus reduce the risk of opportunistic infections.
- a highly transfectable cell such as a fibroblast may be transfected with certain growth factors to support and simulate the growth, maintenance, or differentiation of poorly transfectable embryonic stem cells or induced pluripotent stem cells.
- the cloning, gene synthesis and vector sequencing was performed by DNA2.0 Inc. (Menlo Park, CA). Sequence and insert sequence are set forth herein.
- the ORF was restriction digested using Xbal and used for cDNA synthesis using tailed-or tail-less-PCR.
- the tailed-PCR cDNA product was used as the template for the modified mRNA synthesis reaction using 25mM mixture each modified nucleotide (all modified nucleotides were custom synthesized or purchased from TriLink Biotech, San Diego, CA except pyrrolo-C triphosphate purchased from Glen Research, Sterling VA; unmodifed nucleotides were purchased from Epicenter
- modRNAs were post-transcriptionally capped using recombinant Vaccinia Virus Capping Enzyme (New England BioLabs, Ipswich, MA) and a recombinant 2'-0-methyltransferase (Epicenter Biotechnologies, Madison, WI) to generate the 5'-guanosine Capl structure.
- Cap 2 structure and Cap 3 structures may be generated using additional 2'-0-methyltransferases.
- the in vitro transcribed mRNA product was run on an agarose gel and visualized. modRNA was purified with Ambion/ Applied Biosystems (Austin, TX) MEGAClear RNATM purification kit. PCR used PureLinkTM PCR purification kit
- the product was quantified on NanodropTM UV Absorbance (ThermoFisher, Waltham, MA). Quality, UV absorbance quality and visualization of the product was performed on an 1.2% agarose gel. The product was resuspended in TE buffer.
- 5 '-modRNA capping may be completed concomitantly during the in vzYro-transcription reaction using the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3 ' -0-Me-m 7 G(5')ppp(5')G; G(5')ppp(5')A;
- 5'- modRNA capping may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure: m 7 G(5')ppp(5')G (New England BioLabs, Ipswich, MA).
- Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2'-0 methyl-transferase to generate: m7G(5')ppp(5')G-2'-0-methyl.
- Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-0-methylation of the 5 '-antepenultimate nucleotide using a 2'-0 methyl-transferase.
- Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-0-methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase.
- Enzymes are preferably derived from a recombinant source.
- G-CSF protein
- G-CSF modRNA RT-PCR primers
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Abstract
The present disclosure provides modified nucleosides, nucleotides, and nucleic acids, and methods of using thereof.
Description
MODIFIED NUCLEOSIDES, NUCLEOTIDES, AND NUCLEIC ACIDS, AND USES
THEREOF
CLAIM OF PRIORITY
This application claims the benefit of U.S. Provisional Application Serial No.
61/404,413, filed on October 1, 2010, which is incorporated herein by reference in its entirety.
BACKGROUND
Naturally occurring RNAs are synthesized from four basic ribonucleotides: ATP, CTP, UTP and GTP, but may contain post-transcriptionally modified nucleotides. Further, approximately one hundred different nucleoside modifications have been identified in RNA (Rozenski, J, Crain, P, and McCloskey, J. (1999). The RNA Modification Database: 1999 update. Nucl Acids Res 27: 196-197). The role of nucleoside modifications on the irnmuno- stimulatory potential, stability, and on the translation efficiency of RNA, and the consequent benefits to this for enhancing protein expression and producing therapeutics however, is unclear.
There are multiple problems with prior methodologies of effecting protein expression. For example, heterologous deoxyribonucleic acid (DNA) introduced into a cell can be inherited by daughter cells (whether or not the heterologous DNA has integrated into the chromosome) or by offspring. Introduced DNA can integrate into host cell genomic DNA at some frequency, resulting in alterations and/or damage to the host cell genomic DNA. In addition, multiple steps must occur before a protein is made. Once inside the cell, DNA must be transported into the nucleus where it is transcribed into RNA. The RNA transcribed from DNA must then enter the cytoplasm where it is translated into protein. This need for multiple processing steps creates lag times before the generation of a protein of interest. Further, it is difficult to obtain DNA expression in cells; frequently DNA enters cells but is not expressed or not expressed at reasonable rates or concentrations. This can be a particular problem when DNA is introduced into cells such as primary cells or modified cell lines.
There is a need in the art for biological modalities to address the modulation of intracellular translation of nucleic acids.
SUMMARY
The present disclosure provides, inter alia, modified nucleosides, modified nucleotides, and modified nucleic acids which can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. Further, these modified nucleosides, modified nucleotides, and modified nucleic acids described herein can disrupt binding of a major groove interacting partner with the nucleic acid. Because of the reduced immunogenicity and the decrease in major groove interactions, these modified nucleosides, modified nucleotides, and modified nucleic acids can be more efficient during protein production than, e.g., unmodified nucleic acids.
Thus, the present disclosure provides compounds comprising nucleotides that can disrupt binding of a major groove binding partner with a nucleic acid, wherein the nucleotide has decreased binding affinity to the major groove binding partner.
The present disclosure further provides compounds having Formula I:
I
wherein constituent variables are provided herein.
The present disclosure further provides nucleic acid sequences of at least two nucleotides comprising a compound of Formula I-d:
wherein constituent variables are provided herein.
The present disclosure further provides compositions comprising at least one compound of Formula I.
The present disclosure further provides pharmaceutical compositions comprising a compound of Formula I.
The present disclosure further provides methods of preparing nucleic acid sequences of at least two nucleotides of a compound of Formula I-d.
The present disclosure further provides methods of amplifying nucleic acid sequences of at least two nucleotides of a compound of Formula I-d.
The present disclosure further provides kits comprising a compound of Formula I.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
BRIEF DESCRIPTION OF DRAWINGS FIGs. 1A and IB depict images of non-denaturing agarose gels of each in vitro- transcribed modified RNA.
FIGs. 2A and 2B depict images of an Enzyme-linked immunosorbent assay (ELISA) for Human Granulocyte-Colony Stimulating Factor (G-CSF) of in vitro transfected Human
Keratinocyte cells with each indicated modRNA encoding human G-CSF and the line indicates a saturating level of maximum detectable limit of secreted G-CSF in the assay.
FIGs. 3A-N depict line graphs of a series of Enzyme-linked immunosorbent assays (ELISA) for Human Granulocyte-Colony Stimulating Factor (G-CSF) secreted from in vitro-
transfected Human Keratinocyte cells at different time points with each indicated human G-CSF- encoding modRNA at the indicated doses. The line indicates a saturating level of maximum detectable limit of secreted G-CSF in the assay.
FIGs. 4A and 4B depict bar graphs of a series of Enzyme-linked immunosorbent assays (ELISA) for endogenous cellular human Tumor Necrosis Factor-a (TNF- a) secreted from in vzYro-transfected Human Keratinocyte cells at 24 hours with each indicated hu-G-CSF-encoding modRNA at increasing doses.
FIGs. 4C and 4D depict bar graphs of a series of Enzyme-linked immunosorbent assays (ELISA) for endogenous cellular human Interferon-β (IFN-β) secreted from in vzYro-transfected Human Keratinocyte cells at 24 hours with each indicated hu-G-CSF-encoding modRNA at increasing doses.
FIGs. 4E and 4F depict bar graphs of a series of Enzyme-linked immunosorbent assays (ELISA) for human-G-CSF secreted from in vzYro-transfected Human Keratinocyte cells at 24 hours with each indicated hu-G-CSF-encoding modRNA at increasing doses.
FIG. 5A is a table showing results from an Enzyme-linked immunosorbent assay
(ELISA) for human-G-CSF secreted from in vzYro-transfected Human Keratinocyte cells sampled from individual wells in a co-culture 24-well tissue culture plate 42 hours post-transfection with 750ng of each indicated hu-G-CSF-encoding modRNA.
FIG. 5B depicts an image of an agarose gel of RT-PCR hu-G-CSF modRNA products from co-culture cell extracts 42 hours post-transfection of the human keratinocyte feeder layer with hu-G-CSG modRNA and the un-transfected Kasumi-1 and KG-1 insert culture cells.
FIGs. 5C and 5D depict graphs of results from a hu-G-CSF-modRNA-induced cell proliferation assay of Kasumi-1 (FIG. 5C) and KG-1 (FIG. 5D) cells normalized to untransfected cells. Hu-G-CSF modRNA identity transfected into human keratinocyte feeder cells is indicated.
FIGs. 6A-L depict graphs of the UV absorbance spectra for exemplary modRNA molecules that incorporate the indicated modified nucleotide.
DETAILED DESCRIPTION
The present disclosure provides, inter alia, modified nucleosides, modified nucleotides, and modified nucleic acids that exhibit a reduced innate immune response when introduced into a population of cells. The modified nucleosides, modified nucleotides, and modified nucleic
acids can be chemically modified on the major groove face, thereby disrupting major groove binding partner interactions, which cause innate immune responses.
In general, exogenous unmodified nucleic acids, particularly viral nucleic acids, introduced into cells induce an innate immune response, resulting in cytokine and interferon (IFN) production and cell death. However, it is of great interest for therapeutics, diagnostics, reagents and for biological assays to deliver a nucleic acid, e.g., a ribonucleic acid (RNA) inside a cell, either in vivo or ex vivo, such as to cause intracellular translation of the nucleic acid and production of the encoded protein. Of particular importance is the delivery and function of a non-integrative nucleic acid, as nucleic acids characterized by integration into a target cell are generally imprecise in their expression levels, deleteriously transferable to progeny and neighbor cells, and suffer from the substantial risk of causing mutation. Provided herein in part are nucleic acids encoding useful polypeptides capable of modulating a cell's function and/or activity, and methods of making and using these nucleic acids and polypeptides. As described herein, these nucleic acids are capable of reducing the innate immune activity of a population of cells into which they are introduced, thus increasing the efficiency of protein production in that cell population. Further, one or more additional advantageous activities and/or properties of the nucleic acids and proteins of the present disclosure are described.
Further, the modified nucleosides, modified nucleotides, and modified nucleic acids described herein can be modified on the major groove face. These major groove modifications can allow for alterations, e.g. a decrease, in the interaction of the modified nucleosides, modified nucleotides, and modified nucleic acids with a binding groove partner.
Accordingly, in a first aspect, the present disclosure provides compounds comprising a nucleotide that can disrupts binding of a major groove interacting, e.g. binding, partner with a nucleic acid, wherein the nucleotide has decreased binding affinity to major groove interacting, e.g. binding, partners.
In another aspect, the present disclosure provides compounds comprising a nucleotide that contains chemical modifications, wherein the nucleotide can have altered binding to major groove interacting, e.g. binding, partners.
In some embodiments, the chemical modifications are located on the major groove face of the nucleobase, and wherein the chemical modifications can include replacing or substituting
an atom of a pyrimidine nucleobase with an amine, an SH, an alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro).
In some embodiments, the chemical modifications can be located on the major groove face of the nucleobase, and wherein the chemical modification can include replacing or substituting an atom of a pyrimidine nucleobase with an amine, an SH, a methyl or ethyl, or a chloro or fluoro.
In some embodiments, the chemical modifications can be located on the sugar moiety of the nucleotide.
In some embodiments, the chemical modifications can be located on the phosphate backbone of the nucleotide.
In some embodiments, the chemical modifications can alter the electrochemistry on the major groove face of the nucleotide.
In another aspect, the present disclosure provides nucleotides that contain chemical modifications, wherein the nucleotide reduces the cellular innate immune response, as compared to the cellular innate immune induced by a corresponding unmodified nucleic acid.
In another aspect, the present disclosure provides nucleic acid sequences comprising at least two nucleotides, the nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove interacting partner with the nucleic acid sequence, wherein the nucleotide has decreased binding affinity to the major groove binding partner.
In another aspect, the present disclosure provides compositions comprising a compound as described herein.
In some embodiments, the composition is a reaction mixture.
In some embodiments, the composition is a pharmaceutical composition.
In some embodiments, the composition is a cell culture.
In some embodiments, the compositions further comprise an RNA polymerase and a cDNA template.
In some embodiments, the compositions further comprise a nucleotide selected from the group consisting of adenosine, cytosine, guanosine, and uracil.
In a further aspect, the present disclosure provides for methods of synthesizing a pharmaceutical nucleic acid, comprising providing a complementary deoxyribonucleic acid (cDNA) that encodes a pharmaceutical protein of interest; selecting a nucleotide that is known to
disrupt a binding of a major groove binding partner with a nucleic acid, wherein the nucleotide has decreased binding affinity to the major groove binding partner; and contacting the provided cDNA and the selected nucleotide with an RNA polymerase, under conditions such that the pharmaceutical nucleic acid is synthesized.
In some embodiments, the pharmaceutical nucleic acid is a ribonucleic acid (RNA).
In a further aspect, the present disclosure provides for methods of making a
pharmaceutical formulation comprising a physiologically active secreted protein, comprising transfecting a first population of human cells with a pharmaceutical nucleic acid made by the methods described herein, wherein the secreted protein is active upon a second population of human cells.
In some embodiments, the secreted protein is capable of interacting, e.g. binding, with a receptor on the surface of at least one cell present in the second population.
In some embodiments, the secreted protein is Granulocyte-Colony Stimulating Factor (G-
CSF).
In some embodiments, the second population contains myeloblast cells that express the G-CSF receptor.
In a further aspect, the present disclosure provides for methods of making a
pharmaceutical formulation comprising human cells comprising a physiologically active secreted protein, comprising transfecting a first population of human cells with a pharmaceutical nucleic acid made by the methods described herein, wherein the secreted protein is active upon a second population of human cells.
Definitions
At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term "Ci_6 alkyl" is specifically intended to individually disclose methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl, and C6 alkyl.
It is further intended that the compounds of the present disclosure are stable. As used herein "stable" refers to a compound that is sufficiently robust to survive isolation to a useful
degree of purity from a reaction mixture, and preferably capable of formulation into an efficacious therapeutic agent.
It is further appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, can also be provided in combination in a single embodiment. Conversely, various features of the present disclosure which are, for brevity, described in the context of a single embodiment, can also be provided separately or in any suitable subcombination.
As used herein, the term "alkyl" is meant to refer to a saturated hydrocarbon group which is straight-chained or branched. Example alkyl groups include methyl (Me), ethyl (Et), propyl (e.g., n-propyl and isopropyl), butyl (e.g., n-butyl, isobutyl, t-butyl), pentyl (e.g., n-pentyl, isopentyl, neopentyl), and the like. An alkyl group can contain from 1 to about 20, from 2 to about 20, from 1 to about 12, from 1 to about 8, from 1 to about 6, from 1 to about 4, or from 1 to about 3 carbon atoms.
As used herein, "alkenyl" refers to an alkyl group having one or more double carbon- carbon bonds. Example alkenyl groups include ethenyl, propenyl, and the like.
As used herein, "alkoxy" refers to an -O-alkyl group. Example alkoxy groups include methoxy, ethoxy, propoxy (e.g., n-propoxy and isopropoxy), t-butoxy, and the like.
As used herein, "alkynyl" refers to an alkyl group having one or more triple carbon- carbon bonds. Example alkynyl groups include ethynyl, propynyl, and the like.
As used herein, "aryl" refers to monocyclic or polycyclic (e.g., having 2, 3 or 4 fused rings) aromatic hydrocarbons such as, for example, phenyl, naphthyl, anthracenyl,
phenanthrenyl, indanyl, indenyl, and the like. In some embodiments, aryl groups have from 6 to about 20 carbon atoms.
As used herein, "halo" or "halogen" includes fluoro, chloro, bromo, and iodo.
As used herein, "therapeutic agent" refers to any agent that, when administered to a subject, has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.
As used herein, "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans at any stage of development. In some embodiments, "animal" refers to non-human animals at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g. , a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a
sheep, cattle, a primate, or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, genetically-engineered animal, or a clone.
As used herein, "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).
As used herein, "associated with," "conjugated," "linked," "attached," and "tethered," when used with respect to two or more moieties, means that the moieties are physically associated or connected with one another, either directly or via one or more additional moieties that serves as a linking agent, to form a structure that is sufficiently stable so that the moieties remain physically associated under the conditions in which the structure is used, e.g., physiological conditions.
As used herein, "biologically active" refers to a characteristic of any substance that has activity in a biological system and/or organism. For instance, a substance that, when
administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, where a nucleic acid is biologically active, a portion of that nucleic acid that shares at least one biological activity of the whole nucleic acid is typically referred to as a "biologically active" portion.
As used herein, "conserved" refers to nucleotides or amino acid residues of a
polynucleotide sequence or amino acid sequence, respectively, that are those that occur unaltered in the same position of two or more related sequences being compared. Nucleotides or amino acids that are relatively conserved are those that are conserved amongst more related sequences than nucleotides or amino acids appearing elsewhere in the sequences. In some embodiments, two or more sequences are said to be "completely conserved" if they are 100% identical to one another. In some embodiments, two or more sequences are said to be "highly conserved" if they are at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be "highly conserved"
if they are about 70% identical, about 80%> identical, about 90% identical, about 95%, about 98%, or about 99% identical to one another. In some embodiments, two or more sequences are said to be "conserved" if they are at least 30% identical, at least 40% identical, at least 50% identical, at least 60% identical, at least 70% identical, at least 80% identical, at least 90% identical, or at least 95% identical to one another. In some embodiments, two or more sequences are said to be "conserved" if they are about 30% identical, about 40% identical, about 50% identical, about 60%> identical, about 70% identical, about 80%> identical, about 90%> identical, about 95% identical, about 98% identical, or about 99% identical to one another.
As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of an RNA into a polypeptide or protein; and (4) post- translational modification of a polypeptide or protein.
As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.
As used herein, "in vitro'" refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, in a Petri dish, etc., rather than within an organism (e.g., animal, plant, or microbe).
As used herein, "in vivo" refers to events that occur within an organism (e.g., animal, plant, or microbe).
As used herein, "isolated" refers to a substance or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more of the other components with which they were initially associated. In some
embodiments, isolated agents are more 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 more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components.
As used herein, "subject" or "patient" refers to any organism to which a composition in accordance with the present disclosure may be administered, e.g. , for experimental, diagnostic, prophylactic, and/or therapeutic purposes. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans) and/or plants.
As used herein, "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.
An individual who is "suffering from" a disease, disorder, and/or condition has been diagnosed with or displays one or more symptoms of a disease, disorder, and/or condition.
An individual who is "susceptible to" a disease, disorder, and/or condition has not been diagnosed with and/or may not exhibit symptoms of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (for example, cancer) may be characterized by one or more of the following: (1) a genetic mutation associated with development of the disease, disorder, and/or condition; (2) a genetic
polymorphism associated with development of the disease, disorder, and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with the disease, disorder, and/or condition; (4) habits and/or lifestyles associated with development of the disease, disorder, and/or condition; (5) a family history of the disease, disorder, and/or condition; and (6) exposure to and/or infection with a microbe associated with development of the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will develop the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition will not develop the disease, disorder, and/or condition.
As used herein, "therapeutically effective amount" means an amount of an agent to be delivered (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and/or condition, to treat, improve symptoms of, diagnose, prevent, and/or delay the onset of the disease, disorder, and/or condition.
As used herein, "transcription factor" refers to a DNA-binding protein that regulates transcription of DNA into RNA, for example, by activation or repression of transcription. Some transcription factors effect regulation of transcription alone, while others act in concert with other proteins. Some transcription factor can both activate and repress transcription under certain conditions. In general, transcription factors bind a specific target sequence or sequences highly similar to a specific consensus sequence in a regulatory region of a target gene. Transcription factors may regulate transcription of a target gene alone or in a complex with other molecules.
As used herein, "treating" refers to partially or completely alleviating, ameliorating, improving, relieving, delaying onset of, inhibiting progression of, reducing severity of, and/or reducing incidence of one or more symptoms or features of a particular disease, disorder, and/or condition. For example, "treating" cancer may refer to inhibiting survival, growth, and/or spread of a tumor. Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition and/or to a subject who exhibits only early signs of a disease, disorder, and/or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment comprises delivery of a protein associated with a therapeutically active nucleic acid to a subject in need thereof.
As used herein, "unmodified" refers to a nucleic acid prior to being modified, e.g.
adenosine, guanosine, cytosine, thymidine, and uracil, or a naturally occurring amino acid. The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C=N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.
Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond together with the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are
isomeric protonation states having the same empirical formula and total charge. Example prototropic tautomers include ketone - enol pairs, amide - imidic acid pairs, lactam - lactim pairs, amide - imidic acid pairs, enamine - imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, for example, IH- and 3H-imidazole, 1H-, 2H- and 4H- 1,2,4-triazole, IH- and 2H- isoindole, and IH- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.
Compounds of the present disclosure can also include all isotopes of atoms occurring in the intermediates or final compounds. Isotopes include those atoms having the same atomic number but different mass numbers. For example, isotopes of hydrogen include tritium and deuterium.
The term "compound," as used herein, is meant to include all stereoisomers, geometric isomers, tautomers, and isotopes of the structures depicted.
In some embodiments, the compounds of the present disclosure are substantially isolated. By "substantially isolated" is meant that the compound is at least partially or substantially separated from the environment in which it was formed or detected. Partial separation can include, for example, a composition enriched in the compound of the present disclosure.
Substantial separation can include compositions containing at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%), or at least about 99% by weight of the compound of the present disclosure, or salt thereof. Methods for isolating compounds and their salts are routine in the art.
The compounds of the present disclosure, and salts thereof, can also be prepared in combination with solvent or water molecules to form solvates and hydrates by routine methods.
The present disclosure also includes pharmaceutically acceptable salts of the compounds described herein. As used herein, "pharmaceutically acceptable salts" refers to derivatives of the disclosed compounds wherein the parent compound is modified by converting an existing acid or base moiety to its salt form. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like. The pharmaceutically acceptable salts of the present disclosure include the conventional non-toxic salts of the parent compound formed, for example, from non-toxic inorganic or organic acids. The pharmaceutically acceptable salts of the present disclosure can be synthesized from the parent compound which
contains a basic or acidic moiety by conventional chemical methods. Generally, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington 's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418 and Journal of Pharmaceutical Science, 66, 2 (1977), each of which is incorporated herein by reference in its entirety.
The phrase "pharmaceutically acceptable" is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The present disclosure also includes prodrugs of the compounds described herein. As used herein, "prodrugs" refer to any carriers, typically covalently bonded, which release the active parent drug when administered to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vivo, to the parent compounds. Prodrugs include compounds wherein hydroxyl, amino, sulfhydryl, or carboxyl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino, sulfhydryl, or carboxyl group respectively. Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of the present disclosure. Preparation and use of prodrugs is discussed in T. Higuchi and V. Stella, "Pro-drugs as Novel Delivery Systems," Vol. 14 of the A.C.S. Symposium Series, and in Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are hereby incorporated by reference in their entirety.
Modified Nucleosides and Nucleotides
The present disclosure provides for modified nucleosides and nucleotides. As described herein "nucleoside" is defined as a compound containing a five-carbon sugar molecule (a pentose or ribose) or derivative thereof, and an organic base, purine or pyrimidine, or a
derivative thereof. As described herein, "nucleotide" is defined as a nucleoside consisting of a phosphate group. The nucleosides and nucleotides described herein are generally chemically modified on the major groove face. In some embodiments, the major groove chemical modifications can include an amino group, a thiol group, an alkyl group, or a halo group.
Table 1 below identifies the chemical faces of each canonical nucleotide. Circles identify the atoms comprising the respective chemical regions.
Pyrimidines
Purines
In some embodiments, modified nucleosides include pyridin-4-one ribonucleoside, 5-aza- uridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3-methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, 1 -taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio-l-methyl-pseudouridine, 1- methyl- 1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine,
dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxyuridine, 2- methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio-pseudouridine.
In some embodiments, modified nucleosides include 5-aza-cytidine, pseudoisocytidme, 3-methyl-cytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5- hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo- pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio-pseudoisocytidine, 4-thio-l- methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza-pseudoisocytidine, 1 -methyl- 1 -deaza- pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl-zebularine, 5-aza-2-thio-zebularine, 2- thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy- pseudoisocytidine, and 4-methoxy- 1-methyl-pseudoisocytidine.
In other embodiments, modified nucleosides include 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6- methyladenosine, N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2- methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine.
In some embodiments, modified nucleosides include inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza- guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7- methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2- dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
In some embodiments, the nucleotide can be modified on the major groove face and can include replacement of the hydrogen on C-5 of uracil with a methyl group or a halo group.
In some embodiments, the nucleoside and nucleotide can be a compound of Formula I:
I
wherein:
Z is O or S;
each of Y1 is independently selected from -ORal, -NRalRbl, and -SRal;
each of Y2 is independently selected from O, NRa, S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
each of Y3 is independently selected from O and S;
Y4 is selected from H, -ORa, -SRa, and -NHRa;
n is 0, 1 , 2, or 3;
m is 0, 1 , 2 or 3;
B is a nucleobase;
Ra is H, Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, or C6-2o aryl;
Ral and Rbl are each independently H or a counterion; and
-Y3-Rcl is OH or SH at a pH of about 1 or -Y3-Rcl is O" or S" at physiological pH; or -Y3-Rcl is Ci-20 alkoxy, C2-2o -O-alkenyl, or Ci-2o -O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y1 or Y2 is not O or OH.
In some embodiments, B is a nucleobase of Formula Il-a, Il-b, or II-c:
Il-a Il-b II-c
wherein:
denotes a single or double bond;
X is O or S;
U and W are each independently C or N;
V is O, S, C or N;
wherein when V is C then R1 is H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, halo, or -ORc, wherein Ci-20 alkyl, C2-20 alkenyl, C2-20 alkynyl are each optionally substituted with -OH, -NRaRb, -SH, - C(0)Rc, -C(0)ORc, -NHC(0)Rc, or -NHC(0)ORc;
and wherein when V is O, S, or N then R1 is absent;
R2 is H, -ORc, -SRC, -NRaRb, or halo;
or when V is C then R1 and R2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, - OH, -SH, -NRaRb, Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, C1-20 alkoxy, or C1-20 thioalkyl;
R3 is H or Ci-20 alkyl;
R4 is H or C 1-20 alkyl; wherein when ^ denotes a double bond then R4 is absent, or N- R4, taken together, forms a positively charged N substituted with C1-20 alkyl;
Ra and Rb are each independently H, C1-20 alkyl, C2-20 alkenyl, C2-20 alkynyl, or C6-20 aryl; and
Rc is H, Ci-20 alkyl, C2-2o alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
In some embodiments, B is a nucleobase of Formula Il-al , II-a2, II-a3, II-a4, or II-a5:
Il-al II-a2 II-a3 II-a4 II-a5..
In some embodiments, B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil.
In some embodiments, B is a pyrimidine or derivative thereof.
In some embodiments the nucleotide is a compound of Formula I-a:
I-a.
In some embodiments the nucleotide is a compound of Formula I-b:
I-b.
In some embodiments the nucleotide is a compound of Formula I-c:
I-c.
In some embodiments, the nucleotide is selected from the group consisting of:
20
For example, the modified nucleotide can be:
In some embodiments, the major groove chemical modification can include replacement of the C-H group at C-5 with an -NH- group or a -NH(CH3)- group.
For example, the modified nucleotide can be:
In another embodiment, the major groove chemical modification can include replacement of the hydrogen at C-5 of cytosine with a halo group or a methyl group.
For example, the modified nucleotide can be:
In yet a further embodiment, the major groove chemical modification can include a fused ring that is formed by the NH2 at the C-4 position and the carbon atom at the C-5 position.
In some embodiments, a modified nucleotide is 5 '-0-(l -Thiophosphate)- Adenosine, 5'- 0-(l-Thiophosphate)-Cytidine, 5'-0-(l-Thiophosphate)-Guanosine, 5'-0-(l-Thiophosphate)- Uridine or 5'-0-(l-Thiophosphate)-Pseudouridine.
5 '-0-( 1 -Thiophosphate)- Adenosine
5 '-0-( 1 -Thiophosphate)-Cytidine
5'-0-(l-Thiophosphate)-Uridine
5'-0-(l-Thiophosphate)-Pseudouridine
The a-thio substituted phosphate moiety is provided to confer stability to RNA and DNA polymers through the unnatural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently a longer half-life in a cellular environment. Phosphorothioate linked nucleic acids are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
Further examples of modified nucleotides and modified nucleotide combinations are provided below in Table 2.
Table 2
5-hydroxy-uridine Pyrrolo-cytidine/5-iodo-uridine
Deoxy-thymidine Pyrrolo-cytidine/Nl-methyl-pseudo-uridine
Pseudo-uridine Pyrrolo-cytidine/a-thio-uridine
Inosine Pyrrolo-cytidine/5-methyl-uridine
a-thio-guanosine Pyrrolo-cytidine/Pseudo-uridine
8-oxo-guanosine 5-methyl-cytidine/5-iodo-uridine
06-methyl-guanosine 5-methyl-cytidine/Nl-methyl-pseudo-uridine
7-deaza-guanosine 5-methyl-cytidine/a-thio-uridine
No modification 5-methyl-cytidine/5-methyl-uridine
Nl-methyl-adenosine 5-methyl-cytidine/Pseudo-uridine
2-amino-6-Chloro- 5-methyl-cytidine
purine
N6-methyl-2-amino- 25% Pseudo-iso-cytidine
purine
6-Chloro-purine 25% Nl-methyl-pseudo-uridine
N6-methyl-adenosine 25% Nl-Methyl-pseudo-uridine/75%-pseudo-uridine a-thio-adenosine 5-methyl-uridine
8-azido-adenosine 5-iodo-cytidine
7-deaza-adenosine
Synthesis of Modified Nucleotides
The modified nucleosides and nucleotides disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. It is understood that where typical or preferred process conditions (i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means, such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
Preparation of modified nucleosides and nucleotides can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
The reactions of the processes described herein can be carried out in suitable solvents, which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.
Resolution of racemic mixtures of modified nucleosides and nucleotides can be carried out by any of numerous methods known in the art. An example method includes fractional
recrystallization using a "chiral resolving acid" which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent {e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.
Exemplary syntheses of modified nucleotides are provided below in Schemes 1 and 2.
1) POCl3
2) Pyrophosphate
Modified nucleosides and nucleotides can also be prepared according to the synthetic methods described in Ogata et al. Journal of Organic Chemistry 74:2585-2588, 2009; Purmal et al. Nucleic Acids Research 22(1): 72-78, 1994; Fukuhara et al. Biochemistry 1(4): 563-568, 1962; and Xu et al. Tetrahedron 48(9): 1729-1740, 1992, each of which are incorporated by reference in their entirety.
Modified Nucleic Acids
The present disclosure provides nucleic acids, including RNAs such as mRNAs that contain one or more modified nucleosides (termed "modified nucleic acids") or nucleotides as described herein, which have useful properties including the significant decreast or lack of a substantial induction of the innate immune response of a cell into which the mRNA is introduced, or the suppression thereof. Because these modified nucleic acids enhance the efficiency of protein production, intracellular retention of nucleic acids, and viability of
contacted cells, as well as possess reduced immunogenicity, of these nucleic acids compared to unmodified nucleic acids, having these properties are termed "enhanced nucleic acids" herein.
In addition, the present disclosure provides nucleic acids, which have decreased binding affinity to a major groove interacting, e.g. binding, partner. For example, the nucleic acids are comprised of at least one nucleotide that has been chemically modified on the major groove face as described herein.
The term "nucleic acid," in its broadest sense, includes any compound and/or substance that is or can be incorporated into an oligonucleotide chain. Exemplary nucleic acids for use in accordance with the present disclosure include, but are not limited to, one or more of DNA, RNA including messenger mRNA (mRNA), hybrids thereof, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, aptamers, vectors, etc., described in detail herein.
Provided are modified nucleic acids containing a translatable region and one, two, or more than two different nucleoside modifications. In some embodiments, the modified nucleic acid exhibits reduced degradation in a cell into which the nucleic acid is introduced, relative to a corresponding unmodified nucleic acid. Exemplary nucleic acids include ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), locked nucleic acids (LNAs) or a hybrid thereof. In preferred embodiments, the modified nucleic acid includes messenger RNAs (mRNAs). As described herein, the nucleic acids of the present disclosure do not substantially induce an innate immune response of a cell into which the mRNA is introduced.
In certain embodiments, it is desirable to intracellularly degrade a modified nucleic acid introduced into the cell, for example if precise timing of protein production is desired. Thus, the present disclosure provides a modified nucleic acid containing a degradation domain, which is capable of being acted on in a directed manner within a cell.
Other components of nucleic acid are optional, and are beneficial in some embodiments. For example, a 5' untranslated region (UTR) and/or a 3'UTR are provided, wherein either or both may independently contain one or more different nucleoside modifications. In such
embodiments, nucleoside modifications may also be present in the translatable region. Also provided are nucleic acids containing a Kozak sequence.
Additionally, provided are nucleic acids containing one or more intronic nucleotide sequences capable of being excised from the nucleic acid.
Further, provided are nucleic acids containing an internal ribosome entry site (IRES). An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. An mRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes ("multicistronic mRNA"). When nucleic acids are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the present disclosure include without limitation, those from picomaviruses (e.g. FMDV), pest viruses (CFFV), polio viruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia virus (MLV), simian immune deficiency viruses (SIV) or cricket paralysis viruses (CrPV).
In some embodiments, the nucleic acid sequences comprise a compound of Formula I-d:
I-d
wherein:
Z is O or S;
each of Y1 is independently selected from -ORal, -NRalRbl, and -SRal;
each of Y2 is independently selected from O, NRa, S or a linker comprising
selected from the group consisting of C, O, N, and S;
each of Y3 is independently selected from O and S;
Y4 is selected from H, -ORa, -SRa, and -NHRa;
B is a nucleobase;
Ra is H, C1-20 alkyl, C2-2o alkenyl, C2-20 alkynyl, or C6-20 aryl;
Ral and Rbl are each independently H or a counterion; and
-Y3-Rcl is OH or SH at a pH of about 1 or -Y3-Rcl is O" or S" at physiological pH;
or -Y3-Rcl is Ci-20 alkoxy, C2-2o -O-alkenyl, or Ci-2o -O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, thymidine, uracil and adenine, then at least one of Z, Y1 or Y2 is not O or OH.
In some embodiments, B is a nucleobase of Formula Il-a, Il-b, or II-c:
II-a Il-b II-c
wherein:
^ denotes a single or double bond;
X is O or S;
U and W are each independently C or N;
V is O, S, C or N;
wherein when V is C then R1 is H, Ci-6 alkyl, Ci-6 alkenyl, Ci-6 alkynyl, halo, or -ORc, wherein Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl are each optionally substituted with -OH, -NRaRb, -SH, - C(0)Rc, -C(0)ORc, -NHC(0)Rc, or -NHC(0)ORc;
and wherein when V is O, S, or N then R1 is absent;
R2 is H, -ORc, -SRC, -NRaRb, or halo;
or when V is C then R1 and R2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, - OH, -SH, -NRaRb, Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, Ci-2o alkoxy, or Ci-2o thioalkyl;
R3 is H or Ci-20 alkyl;
R4 is H or C 1-20 alkyl; wherein when ^ denotes a double bond then R4 is absent, or N- R4, taken together, forms a positively charged N substituted with Ci-2o alkyl;
Ra and Rb are each independently H, Ci-2o alkyl, C2-2o alkenyl, C2-2o alkynyl, or C6-2o aryl; and
Rc is H, Ci-20 alkyl, C2-2o alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
In some embodiments, B is a nucleobase of Formula Il-al, II-a2, II-a3, II-a4, or II-a5:
Il-al II-a2 II-a3 II-a4 II-a5.
In some embodiments, at least 25% of the cytosines are replaced by a compound of Formula I-a (e.g., 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% of the uracils are replaced by a compound of Formula I-a (e.g., 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% of the cytosines and 25% of the uracils are replaced by a compound of Formula I-a (e.g., 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, the nucleic acid is translatable.
Major Groove Interacting Partners
As described herein, the phrase "major groove interacting partner" refers RNA recognition receptors that detect and respond to RNA ligands through interactions, e.g. binding, with the major groove face of a nucleotide or nucleic acid. As such, RNA ligands comprising modified nucleotides or nucleic acids as described herein decrease interactions with major groove binding partners, and therefore decrease an innate immune response, or expression and secretion of pro-inflammatory cytokines, or both.
Example major groove interacting, e.g. binding, partners include, but are not limited to the following nucleases and helicases. Within membranes, TLRs (Toll-like Receptors) 3, 7, and 8 can respond to single- and double-stranded RNAs. Within the cytoplasm, members of the superfamily 2 class of DE (D/H) helicases and ATPases can sense RNAs to initiate antiviral responses. These helicases include the RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiation-associated gene 5). Other examples include laboratory of genetics and physiology 2 (LGP2), ΗΓΝ-200 domain containing proteins, or Helicase-domain containing proteins.
Prevention or reduction of innate cellular immune response activation using modified nucleic acids
The term "innate immune response" includes a cellular response to exogenous nucleic acids, including single stranded nucleic acids, generally of viral or bacterial origin, which involves the induction of cytokine expression and release, particularly the interferons, and cell death. Protein synthesis is also reduced during the innate cellular immune response. While it is advantageous to eliminate the innate immune response in a cell, the present disclosure provides modified mRNAs that substantially reduce the immune response, including interferon signaling, without entirely eliminating such a response. In some embodiments, the immune response is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or greater than 99.9% as compared to the immune response induced by a corresponding unmodified nucleic acid. Such a reduction can be measured by expression or activity level of Type 1 interferons or the expression of interferon-regulated genes such as the toll-like receptors (e.g., TLR7 and TLR8). Reduction of innate immune response can also be measured by decreased cell death following one or more administrations of modified RNAs to a cell population; e.g., cell death is 10%, 25%, 50%, 75%, 85%, 90%, 95%, or over 95% less than the cell death frequency observed with a corresponding unmodified nucleic acid. Moreover, cell death may affect fewer than 50%, 40%, 30%, 20%, 10%, 5%, 1%, 0.1%, 0.01% or fewer than 0.01% of cells contacted with the modified nucleic acids.
The present disclosure provides for the repeated introduction (e.g., transfection) of modified nucleic acids into a target cell population, e.g., in vitro, ex vivo, or in vivo. The step of contacting the cell population may be repeated one or more times (such as two, three, four, five
or more than five times). In some embodiments, the step of contacting the cell population with the modified nucleic acids is repeated a number of times sufficient such that a predetermined efficiency of protein translation in the cell population is achieved. Given the reduced cytotoxicity of the target cell population provided by the nucleic acid modifications, such repeated transfections are achievable in a diverse array of cell types.
Polypeptide variants
Provided are nucleic acids that encode variant polypeptides, which have a certain identity with a reference polypeptide sequence. The term "identity" as known in the art, refers to a relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between peptides, as determined by the number of matches between strings of two or more amino acid residues. "Identity" measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms"). Identity of related peptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;
Computer Analysis of Sequence Data, Part 1 , Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991 ; and Carillo et al, SIAM J. Applied Math. 48, 1073 (1988).
In some embodiments, the polypeptide variant has the same or a similar activity as the reference polypeptide. Alternatively, the variant has an altered activity (e.g., increased or decreased) relative to a reference polypeptide. Generally, variants of a particular polynucleotide or polypeptide of the present disclosure will have at least about 40%, 45%, 50%>, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular reference polynucleotide or polypeptide as determined by sequence alignment programs and parameters described herein and known to those skilled in the art.
As recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of this present disclosure. For example, provided herein is any protein fragment of a reference protein (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) 10, 20, 30, 40, 50, 60, 70, 80, 90, 100 or greater than 100 amino acids in length In another example, any protein that includes a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids which are about 40%, about 50%, about 60%>, about 70%), about 80%>, about 90%>, about 95%, or about 100%. identical to any of the sequences described herein can be utilized in accordance with the present disclosure. In certain
embodiments, a protein sequence to be utilized in accordance with the present disclosure includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
Polypeptide libraries
Also provided are polynucleotide libraries containing nucleoside modifications, wherein the polynucleotides individually contain a first nucleic acid sequence encoding a polypeptide, such as an antibody, protein binding partner, scaffold protein, and other polypeptides known in the art. Preferably, the polynucleotides are mRNA in a form suitable for direct introduction into a target cell host, which in turn synthesizes the encoded polypeptide.
In certain embodiments, multiple variants of a protein, each with different amino acid modification(s), are produced and tested to determine the best variant in terms of
pharmacokinetics, stability, biocompatibility, and/or biological activity, or a biophysical property
2 3 4 5 6 7 8 9 such as expression level. Such a library may contain 10, 10 , 10 , 10 , 10 , 10 , 10 , 10 , 10 , or over 109 possible variants (including substitutions, deletions of one or more residues, and insertion of one or more residues).
Polypeptide-nucleic acid complexes
Proper protein translation involves the physical aggregation of a number of polypeptides and nucleic acids associated with the mRNA. Provided by the present disclosure are protein- nucleic acid complexes, containing a translatable mRNA having one or more nucleoside modifications (e.g., at least two different nucleoside modifications) and one or more
polypeptides bound to the mRNA. Generally, the proteins are provided in an amount effective to prevent or reduce an innate immune response of a cell into which the complex is introduced.
Untranslatable Modified Nucleic Acids
As described herein, provided are mRNAs having sequences that are substantially not translatable. Such mRNA is effective as a vaccine when administered to a mammalian subject.
Also provided are modified nucleic acids that contain one or more noncoding regions. Such modified nucleic acids are generally not translated, but are capable of binding to and sequestering one or more translational machinery component such as a ribosomal protein or a transfer RNA (tRNA), thereby effectively reducing protein expression in the cell. The modified nucleic acid may contain a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).
Synthesis of Modified Nucleic Acids
Nucleic acids for use in accordance with the present disclosure may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription, enzymatic or chemical cleavage of a longer precursor, etc. Methods of synthesizing RNAs are known in the art (see, e.g., Gait, M.J. (ed.) Oligonucleotide synthesis: a practical approach, Oxford [Oxfordshire], Washington, DC: IRL Press, 1984; and Herdewijn, P. (ed.) Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology, v. 288 (Clifton, N.J.) Totowa, N.J.: Humana Press, 2005; both of which are incorporated herein by reference).
The modified nucleosides and nucleotides disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. It is understood that where typical or preferred process conditions {i.e., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are given; other process conditions can also be used unless otherwise stated. Optimum reaction conditions may vary with the particular reactants or solvent used, but such conditions can be determined by one skilled in the art by routine optimization procedures.
The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by spectroscopic means,
such as nuclear magnetic resonance spectroscopy (e.g., 1H or 13C) infrared spectroscopy, spectrophotometry (e.g., UV-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.
Preparation of modified nucleosides and nucleotides can involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups can be readily determined by one skilled in the art. The chemistry of protecting groups can be found, for example, in Greene, et al., Protective Groups in Organic Synthesis, 2d. Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.
The reactions of the processes described herein can be carried out in suitable solvents, which can be readily selected by one of skill in the art of organic synthesis. Suitable solvents can be substantially nonreactive with the starting materials (reactants), the intermediates, or products at the temperatures at which the reactions are carried out, i.e., temperatures which can range from the solvent's freezing temperature to the solvent's boiling temperature. A given reaction can be carried out in one solvent or a mixture of more than one solvent. Depending on the particular reaction step, suitable solvents for a particular reaction step can be selected.
Resolution of racemic mixtures of modified nucleosides and nucleotides can be carried out by any of numerous methods known in the art. An example method includes fractional recrystallization using a "chiral resolving acid" which is an optically active, salt-forming organic acid. Suitable resolving agents for fractional recrystallization methods are, for example, optically active acids, such as the D and L forms of tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid or the various optically active camphorsulfonic acids. Resolution of racemic mixtures can also be carried out by elution on a column packed with an optically active resolving agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent composition can be determined by one skilled in the art.Modified nucleic acids need not be uniformly modified along the entire length of the molecule. Different nucleotide modifications and/or backbone structures may exist at various positions in the nucleic acid. One of ordinary skill in the art will appreciate that the nucleotide analogs or other modification(s) may be located at any position(s) of a nucleic acid such that the function of the nucleic acid is not substantially decreased. A modification may also be a 5 Or 3' terminal modification. The nucleic acids may contain at a minimum one and at maximum 100% modified nucleotides, or any intervening
percentage, such as at least 5% modified nucleotides, at least 10% modified nucleotides, at least 25% modified nucleotides, at least 50%> modified nucleotides, at least 80%> modified nucleotides, or at least 90% modified nucleotides. For example, the nucleic acids may contain a modified pyrimidine such as uracil or cytosine. In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the uracil in the nucleic acid is replaced with a modified uracil. The modified uracil can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures). In some embodiments, at least 5%, at least 10%, at least 25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine in the nucleic acid is replaced with a modified cytosine. The modified cytosine can be replaced by a compound having a single unique structure, or can be replaced by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures).
Generally, the shortest length of a modified mRNA of the present disclosure can be the length of an mRNA sequence that is sufficient to encode for a dipeptide. In another
embodiment, the length of the mRNA sequence is sufficient to encode for a tripeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a tetrapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a pentapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a hexapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a heptapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for an octapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a nonapeptide. In another embodiment, the length of an mRNA sequence is sufficient to encode for a decapeptide.
Examples of dipeptides that the modified nucleic acid sequences can encode for include, but are not limited to, carnosine and anserine.
In a further embodiment, the mRNA is greater than 30 nucleotides in length. In another embodiment, the RNA molecule is greater than 35 nucleotides in length. In another embodiment, the length is at least 40 nucleotides. In another embodiment, the length is at least 45 nucleotides. In another embodiment, the length is at least 55 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 60 nucleotides. In another embodiment, the length is at least 80 nucleotides. In another embodiment, the length is at least
90 nucleotides. In another embodiment, the length is at least 100 nucleotides. In another embodiment, the length is at least 120 nucleotides. In another embodiment, the length is at least 140 nucleotides. In another embodiment, the length is at least 160 nucleotides. In another embodiment, the length is at least 180 nucleotides. In another embodiment, the length is at least 200 nucleotides. In another embodiment, the length is at least 250 nucleotides. In another embodiment, the length is at least 300 nucleotides. In another embodiment, the length is at least 350 nucleotides. In another embodiment, the length is at least 400 nucleotides. In another embodiment, the length is at least 450 nucleotides. In another embodiment, the length is at least 500 nucleotides. In another embodiment, the length is at least 600 nucleotides. In another embodiment, the length is at least 700 nucleotides. In another embodiment, the length is at least 800 nucleotides. In another embodiment, the length is at least 900 nucleotides. In another embodiment, the length is at least 1000 nucleotides. In another embodiment, the length is at least 1100 nucleotides. In another embodiment, the length is at least 1200 nucleotides. In another embodiment, the length is at least 1300 nucleotides. In another embodiment, the length is at least 1400 nucleotides. In another embodiment, the length is at least 1500 nucleotides. In another embodiment, the length is at least 1600 nucleotides. In another embodiment, the length is at least 1800 nucleotides. In another embodiment, the length is at least 2000 nucleotides. In another embodiment, the length is at least 2500 nucleotides. In another embodiment, the length is at least 3000 nucleotides. In another embodiment, the length is at least 4000 nucleotides. In another embodiment, the length is at least 5000 nucleotides, or greater than 5000 nucleotides.
The present disclosure provides methods of preparing a nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove interacting partner with the nucleic acid sequence, wherein the nucleic acid sequence comprises a compound of Formula I-d:
Z is O or S;
each of Y1 is independently selected from -ORal, -NRalRbl, and -SRal;
each of Y2 is independently selected from O, NRa, S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
each of Y3 is independently selected from O and S;
Y4 is selected from H, -ORa, -SRa, and -NHRa;
B is a nucleobase;
Ra is H, Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, or C6-2o aryl;
Ral and Rbl are each independently H or a counterion; and
-Y3-Rcl is OH or SH at a pH of about 1 or -Y3-Rcl is O" or S" at physiological pH; or -Y3-Rcl is Ci-20 alkoxy, C2-2o -O-alkenyl, or Ci-2o -O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y1 or Y2 is not O or OH;
the method comprising:
reacting a compound of Formula I-c:
I-c
with an RNA polymerase, and a cDNA template.
In some embodiments, the reaction is repeated from 1 to about 7,000 times.
In some embodiments, B is a nucleobase of Formula Il-a, Il-b, or II-c:
Il-a Il-b II-c
wherein:
denotes a single or double bond;
X is O or S;
U and W are each independently C or N;
V is O, S, C or N;
wherein when V is C then R1 is H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, halo, or -ORc, wherein Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl are each optionally substituted with -OH, -NRaRb, -SH, - C(0)Rc, -C(0)ORc, -NHC(0)Rc, or -NHC(0)ORc;
and wherein when V is O, S, or N then R1 is absent;
R2 is H, -ORc, -SRC, -NRaRb, or halo;
or when V is C then R1 and R2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, - OH, -SH, -NRaRb, Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, C1-20 alkoxy, or C1-20 thioalkyl;
R3 is H or Ci-20 alkyl;
R4 is H or C 1-20 alkyl; wherein when ^ denotes a double bond then R4 is absent, or N- R4, taken together, forms a positively charged N substituted with C1-20 alkyl;
Ra and Rb are each independently H, C1-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, or C6-2o aryl; and
Rc is H, Ci-20 alkyl, C2-2o alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
In some embodiments, B is a nucleobase of Formula Il-al , II-a2, II-a3, II-a4, or II-a5:
Il-al II-a2 II-a3 II-a4 II-a5.
In some embodiments, the methods further comprise a nucleotide selected from the group consisting of adenosine, cytosine, guanosine, and uracil.
In some embodiments, the nucleobase is a pyrimidine or derivative thereof.
In a further aspect, the present disclosure provides methods of amplifying a nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove binding partner with the nucleic acid sequence, the method comprising:
I-c
Z is O or S;
each of Y1 is independently selected from -ORal, -NRalRbl, and -SRal;
each of Y2 is independently selected from O, NRa, S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
each of Y3 is independently selected from O and S;
Y4 is selected from H, -ORa, -SRa, and -NHRa;
B is a nucleobase;
Ra is H, Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, or C6-2o aryl;
Ral and Rbl are each independently H or a counterion; and
-Y3-Rcl is OH or SH at a pH of about 1 or -Y3-Rcl is O" or S" at physiological pH; or -Y3-Rcl is Ci-20 alkoxy, C2-2o -O-alkenyl, or Ci-2o -O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y1 or Y2 is not O or OH;
with a primer, a cDNA template, and an RNA polymerase.
In some embodiments, B is a nucleobase of Formula Il-a, Il-b, or II-c:
Il-a Il-b II-c
wherein:
^ denotes a single or double bond;
X is O or S;
U and W are each independently C or N;
V is O, S, C or N;
wherein when V is C then R1 is H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, halo, or -ORc, wherein Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl are each optionally substituted with -OH, -NRaRb, -SH, - C(0)Rc, -C(0)ORc, -NHC(0)Rc, or -NHC(0)ORc;
and wherein when V is O, S, or N then R1 is absent;
R2 is H, -ORc, -SRC, -NRaRb, or halo;
or when V is C then R1 and R2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, - OH, -SH, -NRaRb, Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, C1-20 alkoxy, or C1-20 thioalkyl;
R3 is H or Ci-20 alkyl;
R4 is H or C 1-20 alkyl; wherein when ^ denotes a double bond then R4 is absent, or N- R4, taken together, forms a positively charged N substituted with C1-20 alkyl;
Ra and Rb are each independently H, C1-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, or C6-2o aryl; and
Rc is H, Ci-20 alkyl, C2-2o alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
In some embodiments, B is a nucleobase of Formula Il-al, II-a2, II-a3, II-a4, or II-a5:
Il-al II-a2 II-a3 II-a4 II-a5.
In some embodiments, the methods further comprise a nucleotide selected from the group consisting of adenosine, cytosine, guanosine, and uracil.
In some embodiments, the nucleobase is a pyrimidine or derivative thereof.
Uses of Modified Nucleic Acids
Therapeutic Agents
The modified nucleic acids and the proteins translated from the modified nucleic acids described herein can be used as therapeutic agents. For example, a modified nucleic acid described herein can be administered to a subject, wherein the modified nucleic acid is translated in vivo to produce a therapeutic peptide in the subject. Accordingly, provided herein are
compositions, methods, kits, and reagents for treatment or prevention of disease or conditions in humans and other mammals. The active therapeutic agents of the present disclosure include modified nucleic acids, cells containing modified nucleic acids or polypeptides translated from the modified nucleic acids, polypeptides translated from modified nucleic acids, and cells contacted with cells containing modified nucleic acids or polypeptides translated from the modified nucleic acids.
In certain embodiments, provided are combination therapeutics containing one or more modified nucleic acids containing translatable regions that encode for a protein or proteins that boost a mammalian subject's immunity along with a protein that induces antibody-dependent cellular toxitity. For example, provided are therapeutics containing one or more nucleic acids that encode trastuzumab and granulocyte-colony stimulating factor (G-CSF). In particular, such combination therapeutics are useful in Her2+ breast cancer patients who develop induced resistance to trastuzumab. (See, e.g., Albrecht, Immunotherapy. 2(6):795-8 (2010)).
Provided are methods of inducing translation of a recombinant polypeptide in a cell population using the modified nucleic acids described herein. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell population is contacted with an effective amount of a composition containing a nucleic acid that has at least one nucleoside modification, and a translatable region encoding the recombinant polypeptide. The population is contacted under conditions such that the nucleic acid is localized into one or more cells of the cell population and the recombinant polypeptide is translated in the cell from the nucleic acid.
An effective amount of the composition is provided based, at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the nucleic acid {e.g., size, and extent of modified nucleosides), and other determinants. In general, an effective amount of the composition provides efficient protein production in the cell, preferably more efficient than a composition containing a corresponding unmodified nucleic acid. Increased efficiency may be demonstrated by increased cell transfection (i.e., the percentage of cells transfected with the nucleic acid), increased protein translation from the nucleic acid, decreased nucleic acid degradation (as demonstrated, e.g. , by increased duration of protein translation from a modified nucleic acid), or reduced innate immune response of the host cell.
Aspects of the present disclosure are directed to methods of inducing in vivo translation of a recombinant polypeptide in a mammalian subject in need thereof. Therein, an effective amount of a composition containing a nucleic acid that has at least one nucleoside modification and a translatable region encoding the recombinant polypeptide is administered to the subject using the delivery methods described herein. The nucleic acid is provided in an amount and under other conditions such that the nucleic acid is localized into a cell of the subject and the recombinant polypeptide is translated in the cell from the nucleic acid. The cell in which the nucleic acid is localized, or the tissue in which the cell is present, may be targeted with one or more than one rounds of nucleic acid administration.
Other aspects of the present disclosure relate to transplantation of cells containing modified nucleic acids to a mammalian subject. Administration of cells to mammalian subjects is known to those of ordinary skill in the art, such as local implantation {e.g., topical or subcutaneous administration), organ delivery or systemic injection {e.g., intravenous injection or inhalation), as is the formulation of cells in pharmaceutically acceptable carrier. Compositions containing modified nucleic acids are formulated for administration intramuscularly,
transarterially, intraperitoneally, intravenously, intranasally, subcutaneously, endoscopically, transdermally, or intrathecally. In some embodiments, the composition is formulated for extended release.
The subject to whom the therapeutic agent is administered suffers from or is at risk of developing a disease, disorder, or deleterious condition. Provided are methods of identifying, diagnosing, and classifying subjects on these bases, which may include clinical diagnosis, biomarker levels, genome-wide association studies (GWAS), and other methods known in the art.
In certain embodiments, the administered modified nucleic acid directs production of one or more recombinant polypeptides that provide a functional activity which is substantially absent in the cell in which the recombinant polypeptide is translated. For example, the missing functional activity may be enzymatic, structural, or gene regulatory in nature.
In other embodiments, the administered modified nucleic acid directs production of one or more recombinant polypeptides that replace a polypeptide (or multiple polypeptides) that is substantially absent in the cell in which the recombinant polypeptide is translated. Such absence may be due to genetic mutation of the encoding gene or regulatory pathway thereof.
Alternatively, the recombinant polypeptide functions to antagonize the activity of an endogenous protein present in, on the surface of, or secreted from the cell. Usually, the activity of the endogenous protein is deleterious to the subject, for example, do to mutation of the endogenous protein resulting in altered activity or localization. Additionally, the recombinant polypeptide antagonizes, directly or indirectly, the activity of a biological moiety present in, on the surface of, or secreted from the cell. Examples of antagonized biological moieties include lipids (e.g., cholesterol), a lipoprotein (e.g., low density lipoprotein), a nucleic acid, a carbohydrate, or a small molecule toxin.
The recombinant proteins described herein are engineered for localization within the cell, potentially within a specific compartment such as the nucleus, or are engineered for secretion from the cell or translocation to the plasma membrane of the cell.
As described herein, a useful feature of the modified nucleic acids of the present disclosure is the capacity to reduce the innate immune response of a cell to an exogenous nucleic acid. Provided are methods for performing the titration, reduction or elimination of the immune response in a cell or a population of cells. In some embodiments, the cell is contacted with a first composition that contains a first dose of a first exogenous nucleic acid including a translatable region and at least one nucleoside modification, and the level of the innate immune response of the cell to the first exogenous nucleic acid is determined. Subsequently, the cell is contacted with a second composition, which includes a second dose of the first exogenous nucleic acid, the second dose containing a lesser amount of the first exogenous nucleic acid as compared to the first dose. Alternatively, the cell is contacted with a first dose of a second exogenous nucleic acid. The second exogenous nucleic acid may contain one or more modified nucleosides, which may be the same or different from the first exogenous nucleic acid or, alternatively, the second exogenous nucleic acid may not contain modified nucleosides. The steps of contacting the cell with the first composition and/or the second composition may be repeated one or more times. Additionally, efficiency of protein production (e.g., protein translation) in the cell is optionally determined, and the cell may be re-transfected with the first and/or second composition repeatedly until a target protein production efficiency is achieved.
Therapeutics for diseases and conditions
Provided are methods for treating or preventing a symptom of diseases characterized by missing or aberrant protein activity, by replacing the missing protein activity or overcoming the aberrant protein activity. Because of the rapid initiation of protein production following introduction of modified mRNAs, as compared to viral DNA vectors, the compounds of the present disclosure are particularly advantageous in treating acute diseases such as sepsis, stroke, and myocardial infarction. Moreover, the lack of transcriptional regulation of the modified mRNAs of the present disclosure is advantageous in that accurate titration of protein production is achievable.
Diseases characterized by dysfunctional or aberrant protein activity include, but not limited to, cancer and proliferative diseases, genetic diseases (e.g., cystic fibrosis), autoimmune diseases, diabetes, neurodegenerative diseases, cardiovascular diseases, and metabolic diseases. The present disclosure provides a method for treating such conditions or diseases in a subject by introducing nucleic acid or cell-based therapeutics containing the modified nucleic acids provided herein, wherein the modified nucleic acids encode for a protein that antagonizes or otherwise overcomes the aberrant protein activity present in the cell of the subject. Specific examples of a dysfunctional protein are the missense mutation variants of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which produce a dysfunctional protein variant of CFTR protein, which causes cystic fibrosis.
Multiple diseases are characterized by missing (or substantially diminished such that proper protein function does not occur) protein activity. Such proteins may not be present, or are essentially non-functional. The present disclosure provides a method for treating such conditions or diseases in a subject by introducing nucleic acid or cell-based therapeutics containing the modified nucleic acids provided herein, wherein the modified nucleic acids encode for a protein that replaces the protein activity missing from the target cells of the subject. Specific examples of a dysfunctional protein are the nonsense mutation variants of the cystic fibrosis
transmembrane conductance regulator (CFTR) gene, which produce a nonfunctional protein variant of CFTR protein, which causes cystic fibrosis.
Thus, provided are methods of treating cystic fibrosis in a mammalian subject by contacting a cell of the subject with a modified nucleic acid having a translatable region that encodes a functional CFTR polypeptide, under conditions such that an effective amount of the CTFR polypeptide is present in the cell. Preferred target cells are epithelial cells, such as the
lung, and methods of administration are determined in view of the target tissue; i.e., for lung delivery, the RNA molecules are formulated for administration by inhalation.
In another embodiment, the present disclosure provides a method for treating
hyperlipidemia in a subject, by introducing into a cell population of the subject with a modified mRNA molecule encoding Sortilin, a protein recently characterized by genomic studies, thereby ameliorating the hyperlipidemia in a subject. The SORTl gene encodes a trans-Golgi network (TGN) transmembrane protein called Sortilin. Genetic studies have shown that one of five individuals has a single nucleotide polymorphism, rsl2740374, in the lpl3 locus of the SORTl gene that predisposes them to having low levels of low-density lipoprotein (LDL) and very-low- density lipoprotein (VLDL). Each copy of the minor allele, present in about 30% of people, alters LDL cholesterol by 8 mg/dL, while two copies of the minor allele, present in about 5% of the population, lowers LDL cholesterol 16 mg/dL. Carriers of the minor allele have also been shown to have a 40% decreased risk of myocardial infarction. Functional in vivo studies in mice describes that overexpression of SORTl in mouse liver tissue led to significantly lower LDL- cholesterol levels, as much as 80% lower, and that silencing SORTl increased LDL cholesterol approximately 200% (Musunuru K et al. From noncoding variant to phenotype via SORTl at the lpl3 cholesterol locus. Nature 2010; 466: 714-721).
Methods of cellular nucleic acid delivery
Methods of the present disclosure enhance nucleic acid delivery into a cell population, in vivo, ex vivo, or in culture. For example, a cell culture containing a plurality of host cells {e.g., eukaryotic cells such as yeast or mammalian cells) is contacted with a composition that contains an enhanced nucleic acid having at least one nucleoside modification and, optionally, a translatable region. The composition also generally contains a transfection reagent or other compound that increases the efficiency of enhanced nucleic acid uptake into the host cells. The enhanced nucleic acid exhibits enhanced retention in the cell population, relative to a
corresponding unmodified nucleic acid. The retention of the enhanced nucleic acid is greater than the retention of the unmodified nucleic acid. In some embodiments, it is at least about 50%, 75%, 90%, 95%, 100%, 150%, 200% or more than 200% greater than the retention of the unmodified nucleic acid. Such retention advantage may be achieved by one round of
transfection with the enhanced nucleic acid, or may be obtained following repeated rounds of transfection.
In some embodiments, the enhanced nucleic acid is delivered to a target cell population with one or more additional nucleic acids. Such delivery may be at the same time, or the enhanced nucleic acid is delivered prior to delivery of the one or more additional nucleic acids. The additional one or more nucleic acids may be modified nucleic acids or unmodified nucleic acids. It is understood that the initial presence of the enhanced nucleic acids does not substantially induce an innate immune response of the cell population and, moreover, that the innate immune response will not be activated by the later presence of the unmodified nucleic acids. In this regard, the enhanced nucleic acid may not itself contain a translatable region, if the protein desired to be present in the target cell population is translated from the unmodified nucleic acids.
Targeting Moieties
In some embodiments, modified nucleic acids are provided to express a protein-binding partner or a receptor on the surface of the cell, which functions to target the cell to a specific tissue space or to interact with a specific moiety, either in vivo or in vitro. Suitable protein- binding partners include antibodies and functional fragments thereof, scaffold proteins, or peptides. Additionally, modified nucleic acids can be employed to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties.
Permanent Gene Expression Silencing
A method for epigenetically silencing gene expression in a mammalian subject, comprising a nucleic acid where the translatable region encodes a polypeptide or polypeptides capable of directing sequence-specific histone H3 methylation to initiate heterochromatin formation and reduce gene transcription around specific genes for the purpose of silencing the gene. For example, a gain-of-function mutation in the Janus Kinase 2 gene is responsible for the family of Myeloproliferative Diseases.
Pharmaceutical Compositions
The present disclosure provides proteins generated from modified mRNAs.
Pharmaceutical compositions may optionally comprise one or more additional therapeutically active substances. In accordance with some embodiments, a method of administering pharmaceutical compositions comprising one or more proteins to be delivered to a subject in need thereof is provided. In some embodiments, compositions are administered to humans. For the purposes of the present disclosure, the phrase "active ingredient" generally refers to a protein or protein-containing complex as described herein.
Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for
administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions is contemplated include, but are not limited to, humans and/or other primates; mammals, including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, dogs, mice, and/or rats; and/or birds, including commercially relevant birds such as chickens, ducks, geese, and/or turkeys.
Formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with an excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage such as, for example, one-half or one- third of such a dosage.
Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the present disclosure will vary, depending upon the identity, size, and/or condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.
Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical
compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this present disclosure.
In some embodiments, a pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, calcium hydrogen phosphate, sodium phosphate lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, cornstarch, powdered sugar, etc., and/or combinations thereof.
Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponge, cation-exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinyl-pyrrolidone)
(crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methylcellulose, pregelatinized starch (starch 1500), microcrystalline starch, water insoluble starch, calcium carboxymethyl cellulose, magnesium aluminum silicate (Veegum), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.
Exemplary surface active agents and/or emulsifiers include, but are not limited to, natural emulsifiers {e.g. acacia, agar, alginic acid, sodium alginate, tragacanth, chondrux, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, wool fat, cholesterol, wax, and lecithin), colloidal clays (e.g. bentonite [aluminum silicate] and Veegum® [magnesium aluminum silicate]), long chain amino acid derivatives, high molecular weight alcohols (e.g. stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate, and propylene glycol monostearate, polyvinyl alcohol), carbomers (e.g. carboxy polymethylene, polyacrylic acid, acrylic acid polymer, and carboxyvinyl polymer), carrageenan, cellulosic derivatives (e.g. carboxymethylcellulose sodium, powdered cellulose, hydroxymethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methylcellulose), sorbitan fatty acid esters (e.g.
polyoxyethylene sorbitan monolaurate [Tween®20], polyoxyethylene sorbitan [T ween® 60], polyoxyethylene sorbitan monooleate [Tween®80], sorbitan monopalmitate [Span®40], sorbitan monostearate [Span®60], sorbitan tristearate [Span®65], glyceryl monooleate, sorbitan monooleate [Span®80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [Myrj®45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate, and Solutol®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. Cremophor®), polyoxyethylene ethers, (e.g. polyoxyethylene lauryl ether [Brij®30]), poly(vinyl-pyrrolidone),
diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, Pluronic®F 68, Poloxamer®188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.
Exemplary binding agents include, but are not limited to, starch {e.g. cornstarch and starch paste); gelatin; sugars {e.g. sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol,); natural and synthetic gums {e.g. acacia, sodium alginate, extract of Irish moss, panwar gum, ghatti gum, mucilage of isapol husks, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, microcrystalline cellulose, cellulose acetate, poly(vinyl-pyrrolidone), magnesium aluminum silicate (Veegum®), and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol; etc.; and combinations thereof.
Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acidic preservatives, and/or other preservatives. Exemplary antioxidants include, but are not limited to, alpha tocopherol, ascorbic acid, acorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butyl paraben, methyl paraben, ethyl paraben, propyl paraben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate, and/or sorbic acid. Exemplary alcohol
preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplary
acidic preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta- carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid, and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisol (BHA), butylated hydroxytoluened (BHT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, sodium metabisulfite, potassium sulfite, potassium metabisulfite, Glydant Plus®, Phenonip®, methylparaben, Germall®115, Germaben®II, Neolone™, Kathon™, and/or Euxyl®.
Exemplary buffering agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, d- gluconic acid, calcium glycerophosphate, calcium lactate, propanoic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, potassium phosphate mixtures, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline, Ringer's solution, ethyl alcohol, etc., and/or combinations thereof.
Exemplary lubricating agents include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silica, talc, malt, glyceryl behanate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate, etc., and combinations thereof.
Exemplary oils include, but are not limited to, almond, apricot kernel, avocado, babassu, bergamot, black current seed, borage, cade, camomile, canola, caraway, carnauba, castor, cinnamon, cocoa butter, coconut, cod liver, coffee, corn, cotton seed, emu, eucalyptus, evening primrose, fish, flaxseed, geraniol, gourd, grape seed, hazel nut, hyssop, isopropyl myristate, jojoba, kukui nut, lavandin, lavender, lemon, litsea cubeba, macademia nut, mallow, mango seed, meadowfoam seed, mink, nutmeg, olive, orange, orange roughy, palm, palm kernel, peach kernel, peanut, poppy seed, pumpkin seed, rapeseed, rice bran, rosemary, safflower, sandalwood, sasquana, savoury, sea buckthorn, sesame, shea butter, silicone, soybean, sunflower, tea tree,
thistle, tsubaki, vetiver, walnut, and wheat germ oils. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanol, oleyl alcohol, silicone oil, and/or combinations thereof.
Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and/or elixirs. In addition to active ingredients, liquid dosage forms may comprise inert diluents commonly used in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed, groundnut, corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid esters of sorbitan, and mixtures thereof. Besides inert diluents, oral compositions can include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and/or perfuming agents. In certain embodiments for parenteral administration, compositions are mixed with solubilizing agents such as Cremophor®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing agents, wetting agents, and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions, and/or emulsions in nontoxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution, U.S.P., and isotonic sodium chloride solution. Sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used in the preparation of injectables.
Injectable formulations can be sterilized, for example, by filtration through a bacterial- retaining filter, and/or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved or dispersed in sterile water or other sterile injectable medium prior to use.
In order to prolong the effect of an active ingredient, it is often desirable to slow the absorption of the active ingredient from subcutaneous or intramuscular injection. This may be accomplished by the use of a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug then depends upon its rate of dissolution which, in turn, may depend upon crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is accomplished by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the drug in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of drug to polymer and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). Depot injectable formulations are prepared by entrapping the drug in liposomes or microemulsions which are compatible with body tissues.
Compositions for rectal or vaginal administration are typically suppositories which can be prepared by mixing compositions with suitable non-irritating excipients such as cocoa butter, polyethylene glycol or a suppository wax which are solid at ambient temperature but liquid at body temperature and therefore melt in the rectum or vaginal cavity and release the active ingredient.
Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In such solid dosage forms, an active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate or dicalcium phosphate and/or fillers or extenders (e.g. starches, lactose, sucrose, glucose, mannitol, and silicic acid), binders (e.g. carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose, and acacia), humectants (e.g. glycerol), disintegrating agents (e.g. agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate), solution retarding agents (e.g. paraffin), absorption accelerators (e.g. quaternary ammonium compounds), wetting agents (e.g. cetyl alcohol and glycerol monostearate), absorbents (e.g. kaolin and bentonite clay), and lubricants (e.g. talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may comprise buffering agents.
Solid compositions of a similar type may be employed as fillers in soft and hard-filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight
polyethylene glycols and the like. Solid dosage forms of tablets, dragees, capsules, pills, and granules can be prepared with coatings and shells such as enteric coatings and other coatings well known in the pharmaceutical formulating art. They may optionally comprise opacifying agents and can be of a composition that they release the active ingredient(s) only, or
preferentially, in a certain part of the intestinal tract, optionally, in a delayed manner. Examples of embedding compositions which can be used include polymeric substances and waxes. Solid compositions of a similar type may be employed as fillers in soft and hard- filled gelatin capsules using such excipients as lactose or milk sugar as well as high molecular weight polyethylene glycols and the like.
Dosage forms for topical and/or transdermal administration of a composition may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, inhalants and/or patches. Generally, an active ingredient is admixed under sterile conditions with a pharmaceutically acceptable excipient and/or any needed preservatives and/or buffers as may be required.
Additionally, the present disclosure contemplates the use of transdermal patches, which often have the added advantage of providing controlled delivery of a compound to the body. Such dosage forms may be prepared, for example, by dissolving and/or dispensing the compound in the proper medium. Alternatively or additionally, rate may be controlled by either providing a rate controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.
Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Patents 4,886,499; 5,190,521; 5,328,483; 5,527,288; 4,270,537; 5,015,235; 5,141,496; and 5,417,662. Intradermal
compositions may be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in PCT publication WO 99/34850 and functional equivalents thereof. Jet injection devices which deliver liquid compositions to the dermis via a liquid jet injector and/or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis are suitable. Jet injection devices are described, for example, in U.S. Patents 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 publications WO 97/37705 and WO 97/13537. Ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis are suitable.
Alternatively or additionally, conventional syringes may be used in the classical mantoux method of intradermal administration.
Formulations suitable for topical administration include, but are not limited to, liquid and/or semi liquid preparations such as liniments, lotions, oil in water and/or water in oil emulsions such as creams, ointments and/or pastes, and/or solutions and/or suspensions.
Topically-administrable formulations may, for example, comprise from about 1% to about 10% (w/w) active ingredient, although the concentration of active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more of the additional ingredients described herein.
A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for pulmonary administration via the buccal cavity. Such a formulation may comprise dry particles which comprise the active ingredient and which have a diameter in the range from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. Such compositions are conveniently in the form of dry powders for administration using a device comprising a dry powder reservoir to which a stream of propellant may be directed to disperse the powder and/or using a self propelling solvent/powder dispensing container such as a device comprising the active ingredient dissolved and/or suspended in a low-boiling propellant in a sealed container. Such powders comprise particles wherein at least 98% of the particles by weight have a diameter greater than 0.5 nm and at least 95% of the particles by number have a diameter less than 7 nm. Alternatively, at least 95% of the particles by weight have a diameter greater than 1 nm and at least 90% of the particles by number have a diameter less than 6 nm. Dry powder compositions may include a solid fine powder diluent such as sugar and are conveniently provided in a unit dose form.
Low boiling propellants generally include liquid propellants having a boiling point of below 65 °F at atmospheric pressure. Generally the propellant may constitute 50% to 99.9% (w/w) of the composition, and active ingredient may constitute 0.1 % to 20% (w/w) of the composition. A propellant may further comprise additional ingredients such as a liquid non- ionic and/or solid anionic surfactant and/or a solid diluent (which may have a particle size of the same order as particles comprising the active ingredient).
Pharmaceutical compositions formulated for pulmonary delivery may provide an active ingredient in the form of droplets of a solution and/or suspension. Such formulations may be
prepared, packaged, and/or sold as aqueous and/or dilute alcoholic solutions and/or suspensions, optionally sterile, comprising active ingredient, and may conveniently be administered using any nebulization and/or atomization device. Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, and/or a preservative such as
methylhydroxybenzoate. Droplets provided by this route of administration may have an average diameter in the range from about 0.1 nm to about 200 nm.
Formulations described herein as being useful for pulmonary delivery are useful for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 μιη to 500 μιη. Such a formulation is administered in the manner in which snuff is taken, i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nose.
Formulations suitable for nasal administration may, for example, comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) of active ingredient, and may comprise one or more of the additional ingredients described herein. A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for buccal administration. Such formulations may, for example, be in the form of tablets and/or lozenges made using
conventional methods, and may, for example, 0.1% to 20% (w/w) active ingredient, the balance comprising an orally dissolvable and/or degradable composition and, optionally, one or more of the additional ingredients described herein. Alternately, formulations suitable for buccal administration may comprise a powder and/or an aerosolized and/or atomized solution and/or suspension comprising active ingredient. Such powdered, aerosolized, and/or aerosolized formulations, when dispersed, may have an average particle and/or droplet size in the range from about 0.1 nm to about 200 nm, and may further comprise one or more of any additional ingredients described herein.
A pharmaceutical composition may be prepared, packaged, and/or sold in a formulation suitable for ophthalmic administration. Such formulations may, for example, be in the form of eye drops including, for example, a 0.1/1.0%) (w/w) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further comprise buffering agents, salts, and/or one or more other of any additional ingredients described herein. Other
opthalmically-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form and/or in a liposomal preparation. Ear drops and/or eye drops are contemplated as being within the scope of this present disclosure.
General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
Administration
The present disclosure provides methods comprising administering proteins or complexes in accordance with the present disclosure to a subject in need thereof. Proteins or complexes, or pharmaceutical, imaging, diagnostic, or prophylactic compositions thereof, may be administered to a subject using any amount and any route of administration effective for preventing, treating, diagnosing, or imaging a disease, disorder, and/or condition {e.g., a disease, disorder, and/or condition relating to working memory deficits). The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the disease, the particular composition, its mode of administration, its mode of activity, and the like. Compositions in accordance with the present disclosure are typically formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present disclosure will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactially effective, or appropriate imaging dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts.
Proteins to be delivered and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be administered to animals, such as mammals {e.g., humans, domesticated animals, cats, dogs, mice, rats, etc.). In some embodiments, pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof are administered to humans.
Proteins to be delivered and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof in accordance with the present disclosure may be administered by any route. In some embodiments, proteins and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof, are administered by one or more of a variety of routes, including oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subcutaneous, intraventricular, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (e.g. by powders, ointments, creams, gels, lotions, and/or drops), mucosal, nasal, buccal, enteral, vitreal, intratumoral, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or through a portal vein catheter. In some embodiments, proteins or complexes, and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof, are administered by systemic intravenous injection. In specific embodiments, proteins or complexes and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof may be administered intravenously and/or orally. In specific embodiments, proteins or complexes, and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof, may be administered in a way which allows the protein or complex to cross the blood- brain barrier, vascular barrier, or other epithelial barrier.
However, the present disclosure encompasses the delivery of proteins or complexes, and/or pharmaceutical, prophylactic, diagnostic, or imaging compositions thereof, by any appropriate route taking into consideration likely advances in the sciences of drug delivery.
In general the most appropriate route of administration will depend upon a variety of factors including the nature of the protein or complex comprising proteins associated with at least one agent to be delivered (e.g., its stability in the environment of the gastrointestinal tract, bloodstream, etc.), the condition of the patient (e.g., whether the patient is able to tolerate particular routes of administration), etc. The present disclosure encompasses the delivery of the pharmaceutical, prophylactic, diagnostic, or imaging compositions by any appropriate route taking into consideration likely advances in the sciences of drug delivery.
In certain embodiments, compositions in accordance with the present disclosure may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg, of subject body weight per day,
one or more times a day, to obtain the desired therapeutic, diagnostic, prophylactic, or imaging effect. The desired dosage may be delivered three times a day, two times a day, once a day, every other day, every third day, every week, every two weeks, every three weeks, or every four weeks. In certain embodiments, the desired dosage may be delivered using multiple
administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).
Proteins or complexes may be used in combination with one or more other therapeutic, prophylactic, diagnostic, or imaging agents. By "in combination with," it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the present disclosure. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. In some embodiments, the present disclosure encompasses the delivery of pharmaceutical, prophylactic, diagnostic, or imaging compositions in combination with agents that improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.
It will further be appreciated that therapeutically, prophylactically, diagnostically, or imaging active agents utilized in combination may be administered together in a single composition or administered separately in different compositions. In general, it is expected that agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually.
The particular combination of therapies (therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies employed may achieve a desired effect for the same disorder (for example, a composition useful for treating cancer in accordance with the present disclosure may be administered concurrently with a chemotherapeutic agent), or they may achieve different effects (e.g., control of any adverse effects).
Kits
The present disclosure provides a variety of kits for conveniently and/or effectively carrying out methods of the present disclosure. Typically kits will comprise sufficient amounts and/or numbers of components to allow a user to perform multiple treatments of a subject(s) and/or to perform multiple experiments.
In one aspect, the disclosure provides kits for protein production, comprising a first isolated nucleic acid comprising a translatable region and a nucleic acid modification, wherein the nucleic acid is capable of evading an innate immune response of a cell into which the first isolated nucleic acid is introduced, and packaging and instructions.
In one aspect, the disclosure provides kits for protein production, comprising: a first isolated nucleic acid comprising a translatable region, provided in an amount effective to produce a desired amount of a protein encoded by the translatable region when introduced into a target cell; a second nucleic acid comprising an inhibitory nucleic acid, provided in an amount effective to substantially inhibit the innate immune response of the cell; and packaging and instructions.
In one aspect, the disclosure provides kits for protein production, comprising a first isolated nucleic acid comprising a translatable region and a nucleoside modification, wherein the nucleic acid exhibits reduced degradation by a cellular nuclease, and packaging and instructions.
In one aspect, the disclosure provides kits for protein production, comprising a first isolated nucleic acid comprising a translatable region and at least two different nucleoside modifications, wherein the nucleic acid exhibits reduced degradation by a cellular nuclease, and packaging and instructions.
In one aspect, the disclosure provides kits for protein production, comprising a first isolated nucleic acid comprising a translatable region and at least one nucleoside modification, wherein the nucleic acid exhibits reduced degradation by a cellular nuclease; a second nucleic acid comprising an inhibitory nucleic acid; and packaging and instructions.
In some embodiments, the first isolated nucleic acid comprises messenger RNA
(mRNA). In some embodiments the mRNA comprises at least one nucleoside selected from the group consisting of pyridin-4-one ribonucleoside, 5-aza-uridine, 2-thio-5-aza-uridine, 2- thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxyuridine, 3-methyluridine, 5- carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-
pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio- uridine, l-taurinomethyl-4-thio-uridine, 5-methyl-uridine, 1-methyl-pseudouridine, 4-thio- 1- methyl-pseudouridine, 2-thio- 1-methyl-pseudouridine, 1 -methyl- 1-deaza-pseudouridine, 2-thio-
1 - methyl- 1-deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine,
2- thio-dihydropseudouridine, 2-methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy- pseudouridine, and 4-methoxy-2-thio-pseudouridine.
In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocytidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1 -methyl- 1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5-methyl- zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2-methoxy-5-methyl- cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy- 1-methyl-pseudoisocytidine.
In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8- aza-2, 6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6-isopentenyladenosine, N6- (cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl) adenosine, N6- glycinylcarbamoyladenosine, N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6-dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2- methoxy-adenine.
In some embodiments, the mRNA comprises at least one nucleoside selected from the group consisting of inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7- deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza- guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy- guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo- guanosine, 7-methyl-8-oxo-guanosine, l-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.
In another aspect, the disclosure provides compositions for protein production, comprising a first isolated nucleic acid comprising a translatable region and a nucleoside
modification, wherein the nucleic acid exhibits reduced degradation by a cellular nuclease, and a mammalian cell suitable for translation of the translatable region of the first nucleic acid.
EXAMPLES
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Example 1. Modified mRNA In Vitro Transcription
Materials and Methods
Modified mRNAs (modRNAs) were made using standard laboratory methods and materials for in vitro transcription with the exception that the nucleotide mix contained modified nucleotides. The open reading frame (ORF) of the gene of interest is flanked by a 5' untranslated region (UTR) containing a strong Kozak translational initiation signal and an alpha-globin 3' UTR terminating with an oligo(dT) sequence for templated addition of a polyA tail for modRNAs not incorporating Adenosine analogs. Adenosine-containing modRNAs were synthesized without an oligo (dT) sequence to allow for post-transcription poly (A) polymerase poly-(A) tailing. The modRNAs were modified by incorporating chemically modified nucleotides indicated in Table 3 (below) during the in vitro transcription with 100% replacement of the corresponding natural nucleotide or partial replacement of the corresponding natural nucleotide at the indicated percentage.
Table 3 indicates the chemical identity of each chemically-distinct modified nucleotide incorporated into a modified mRNA with the given chemistry designation number.
Table 3
N l-methyl-pseudouridine Chem 7 Pseudo-iso-cytidine/N l-methyl-pseudo-uridine Chem 35
5,6-dihydrouridine Chem 8 Pseudo-iso-cytidine/a-thio-uridine Chem 36 a-thio-uridine Chem 9 Pseudo-iso-cytidine/5-methyl-uridine Chem 37
4-thio-uridine Chem 10 Pseudo-iso-cytidine/Pseudo-uridine Chem 38
6-aza-uridine Chem 11 Pyrrolo-cytidine Chem 39
5-hydroxy-uridine Chem 12 Pyrrolo-cytidine/5-iodo-uridine Chem 40
Deoxy-thymidine Chem 13 Pyrrolo-cytidine/N l-methyl-pseudo-uridine Chem 41
Pseudo-uridine Chem 14 Pyrrolo-cytidine/a-thio-uridine Chem 42
Inosine Chem 15 Pyrrolo-cytidine/5-methyl-uridine Chem 43 a-thio-guanosine Chem 16 Pyrrolo-cytidine/Pseudo-uridine Chem 44
8-oxo-guanosine Chem 17 5-methyl-cytidine/5-iodo-uridine Chem 45
06-methyl-guanosine Chem 18 5-methyl-cytidine/N l-methyl-pseudo-uridine Chem 46
7-deaza-guanosine Chem 19 5-methyl-cytidine/a-thio-uridine Chem 47
No modification Chem 20 5-methyl-cytidine/5-methyl-uridine Chem 48
N 1-methyl-adenosine Chem 21 5-methyl-cytidine/Pseudo-uridine Chem 49
2-amino-6-Chloro-purine Chem 22 5-methyl-cytidine Chem 50
N6-methyl-2-amino- Chem 23 25% Pseudo-iso-cytidine Chem 51 purine
6-Chloro-purine Chem 24 25% N l-methyl-pseudo-uridine Chem 52
N6-methyl-adenosine Chem 25 25% N l-Methyl-pseudo-uridine/75%-pseudo- Chem 53 uridine
a-thio-adenosine Chem 26 5-methyl-uridine Chem 54
8-azido-adenosine Chem 27 5-iodo-cytidine Chem 55
7-deaza-adenosine Chem 28
Agarose Gel Electrophoresis of modRNA: Individual modRNAs (200-400 ng in a 20 μΐ volume) were loaded into a well on a non-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, CA) and run for 12-15 minutes according to the manufacturer protocol (FIG. 1A). Tables 4 and 5 below indicate the modified nucleotide (Table 4) or nucleic acid (Table 5) loaded in each lane. These data indicate which chemically modified nucleotides were transcribed into chemically-modified mRNAs and the quality of each individual modRNA. These data demonstrate that nucleotides with chemical modifications on the major groove and minor groove face of the nucleotide were capable of being transcribed into a modRNA.
Table 4
Lane Modified NTP
1 a-thio-cytidine
2 Pseudo-iso-cytidine
3 5-aminoallyl-uridine
4 5-iodo-uridine
5 Nl-methyl-pseudo-uridine
6 a-thio-uridine
7 4-t io-uridine
8 5-hydroxy-uridine
9 Deoxy-thymidine
10 Pseudo-uridine
11 Inosine
12 a-thio-guanosine
13 8-oxo-guanosine
14 Nl-methyl-guanosine
15 06-methyl-guanosine
16 No modification
17 Nl-methyl-adenosine
18 2-amino-6-Chloro-purine
19 N6-methyl-2-amino-purine
20 6-Chloro-purine
21 ct-thio-adenosine
22 8-azido-adenosine
23 7-deaza-adenosine
24 6-aza-cytidine
25 2-thio-cytidine
26 5,6-dihydro-uridine
27 6-aza-uridine
28 7-deaza-guanosine
29 N6-methyl-adenosine
Table 5
Lane Modified NTP combination
1 a-thio-cytidine/5-iodo-uridine
2 a-thio-cytidine/Nl-methyl-pseudouridine
3 a-thio-cytidine/a-thio-uridine
4 a-thio-cytidine/5-methyl-uridine
5 a-thio-cytidine/pseudouridine
6 5-iodo-cytidine/5-iodo-uridine
7 5-iodo-cytidine/Nl-methyl-pseudouridine
8 5-iodo-cytidine/a-thio-uridine
9 5-iodo-cytidine/5-methyl-uridine
10 5-iodo-cytidine/pseudouridine
11 Pseudo-iso-cytidine/5-iodo-uridine
12 Pyrrolo-cytidine
13 Pyrrolo-cytidine/5-iodo-uridine
14 Pyrrolo-cytidine/Nl-methyl-pseudouridine
15 Py rro 1 o-cyti d i n e/a-t hio- uri d i n e
16 Pyrrolo-cytidine/5-methyl-uridine
17 Py rro 1 o-cyti d i n e/pseudo uri d i n e
18 5-methyl-cytidine/5-iodo-uridine
19 5-methyl-cytidine/Nl-methyl-uridine
20 5-methyl-cytidine/a-thio-uridine
21 5-methyl-cytidine/5-methyl-uridine
22 5-methyl-cytidine/pseudouridine
23 Pseudo-iso-cytidine/Nl-methyl-pseudouridine
24 Pseudo-iso-cytidine/a-thio-uridine
25 Pseudo-iso-cytidine/5-methyl-uridine
26 Pseudo-iso-cytidine/pseudouridine
27 5-methyl-cytidine
28 25% pseudo-iso-cytidine
29 25% Nl-methyl-pseudouridine
30 25% Nl-methyl-pseudouridine/75% pseudouridine
Agarose Gel Electrophoresis of RT-PCR products: Individual reverse transcribed-PCR products (200-400ng) were loaded into a well of a non-denaturing 1.2% Agarose E-Gel
(Invitrogen, Carlsbad, CA) and run for 12-15 minutes according to the manufacturer protocol (FIG. IB). Table 5 below indicates the modified nucleotide loaded in each lane.
Nanodrop modRNA quantification and UV spectral data: modRNAs in TE buffer (1 μΐ) were used for Nanodrop UV absorbance readings to quantitate the yield of each modRNA
from an in vitro transcription reaction (UV absorbance traces are shown in Figures 6A-6L). These data indicate which chemically modified nucleotides were transcribed into chemically- modified mRNAs. These data also demonstrate that nucleotides with chemical modifications on the major groove and minor groove face of the nucleotide were capable of being transcribed into a modRNA. These data further demonstrate that the nucleotides of the present invention are transcription-competent and compatible with incorporation into a modRNA, which may have altered UV spectra due to the presence of a given modified nucleotide. For example, Pyrrolo-C containing modRNAs have an increase in UV absorbance at a lower wavelength due to the presence of the pyrrolo ring of the modified C nucleotide. In another example, 2-amino-adenine nucleotide-containing modRNAs have an increase in UV absorbance at a higher wavelength due to the presence of an exocyclic amine off the purine ring. Nucleotides that are not transcription- competent and cannot be incorporated into a modRNA have a scrambled UV spectrum indicating no product from the transcription reaction.
Example 2. Modified RNA Transfection
Reverse Transfection: For experiments performed in a 24-well collagen-coated tissue culture plate, Keratinocytes were seeded at a cell density of 1 x 105. For experiments performed in a 96-well collagen-coated tissue culture plate, Keratinocytes were seeded at a cell density of 0.5 x 105. For each modRNA to be transfected, modRNA: RNAiMAX was prepared as described and mixed with the cells in the multi-well plate within a period of time, e.g., 6 hours, of cell seeding before cells had adhered to the tissue culture plate.
Forward Transfection: In a 24-well collagen-coated tissue culture plate, Keratinocytes were seeded at a cell density of 0.7 x 105. For experiments performed in a 96-well collagen- coated tissue culture plate, Keratinocytes were seeded at a cell density of 0.3 x 105.
Keratinocytes were then grown to a confluency of >70% for over 24 hours. For each modRNA to be transfected, modRNA: RNAiMAX was prepared as described and transfected onto the cells in the multi-well plate over 24 hours after cell seeding and adherence to the tissue culture plate.
modRNA Translation Screen: G-CSF ELISA
FIGs. 2A and 2B show an Enzyme-linked immunosorbent assay (ELISA) for Human Granulocyte-Colony Stimulating Factor (hu-G-CSF) of in vitro transfected Human Keratinocyte
cells. Keratinocytes were grown in EpiLife medium with Supplement S7 from Invitrogen at a confluence of >70%. FIG. 2A keratinocytes were reverse transfected with 300 ng of the indicated chemically modified mRNA complexed with RNAiMAX from Invitrogen. FIG. 2B keratinocytes were forward transfected with 300 ng modRNA complexed with RNAiMAX from Invitrogen. The RNA:RNAiMAX complex was formed by first incubating the RNA with Supplement- free EpiLife media in a 5X volumetric dilution for 10 minutes at room temperature. In a second vial, RNAiMAX reagent was incubated with Supplement- free EpiLife Media in a 10X volumetric dilution for 10 minutes at room temperature. The RNA vial was then mixed with the RNAiMAX vial and incubated for 20-30 at room temperature before being added to the cells in a drop-wise fashion. Secreted huG-CSF concentration in the culture medium was measured at 18 hours post-transfection for each of the chemically modified mRNAs in triplicate. Secretion of Human Granulocyte-Colony Stimulating Factor (G-CSF) from transfected human keratinocytes was quantified using an ELISA kit from Invitrogen or R&D Systems (Minneapolis, MN) following the manufacturers recommended instructions. These data show that huG-CSF modRNAs comprised of chemically distinct nucleotide analogs (SEQ ID NO: 2) is capable of being translated in Human Keratinocyte cells and that huG-CSF is transported out of the cells and released into the extracellular environment. These data indicate which modified nucleotides were translated into protein when incorporated into a chemically modified mRNA. These data show that modified RNA containing nucleotides with chemical modifications on the major groove face of pyrimidine analogs have the highest levels of secreted hu-G-CSF into the cell culture medium.
modRNA Dose and Duration: G-CSF ELISA
FIGs. 3A-N show Enzyme-linked immunosorbent assays (ELISA) for Human
Granulocyte-Colony Stimulating Factor (G-CSF) of in vitro transfected Human Keratinocyte cells. Keratinocytes were grown in EpiLife medium with Supplement S7 from Invitrogen at a confluence of >70%. Keratinocytes were reverse transfected with Ong, 46.875ng, 93.75ng, 187.5ng, 375ng, 750ng, or 1500ng modRNA complexed with RNAiMAX from Invitrogen. The modRNA:RNAiMAX complex was formed as described. Secreted huG-CSF concentration in the culture medium was measured at 0, 6, 12, 24, and 48 hours post-transfection for each concentration of each modRNA in triplicate. Secretion of Human Granulocyte-Colony
Stimulating Factor (G-CSF) from transfected human keratinocytes was quantified using an
ELISA kit from Invitrogen or R&D Systems following the manufacturers recommended instructions. These data show that huG-CSF modRNAs comprised of chemically distinct nucleotide analogs (SEQ ID NO: X and Table 6) secreted hu-G-CSF protein in a modRNA dose- dependent manner from Human Keratinocyte cells and that huG-CSF is transported out of the cells and released into the extracellular environment. These data indicate which modified RNAs containing modified nucleotide analogs sustain hu-G-CSF expression for the longest and at the highest levels. These data show that modified RNA containing modified nucleotides with chemical modifications on the major groove face of pyrimidine analogs have the highest levels of secreted hu-G-CSF into the cell culture medium and that 750ng of modRNA elicits the highest level of secreted hu-G-CSF.
Example 3. Cellular Innate Immune Response to modRNA
IFN-β ELISA and TNF-a ELISA:
FIGs. 4A-F show an Enzyme-linked immunosorbent assay (ELISA) for Human Tumor Necrosis Factor-a (TNF-a) (FIGs. 4A and 4B); Human Interferon-β (IFN-β) (FIGs. 4C and 4D); and Human Granulocyte-Colony Stimulating Factor (G-CSF) (FIGs. 4E and 4F) secreted from in vzYro-transfected Human Keratinocyte cells. Keratinocytes were grown in EpiLife medium with Human Keratinocyte Growth Supplement in the absence of hydrocortisone from Invitrogen at a confluence of >70%. In FIGs. 4A and 4B, keratinocytes were reverse transfected with Ong, 93.75ng, 187.5ng, 375ng, 750ng, 1500ng or 3000ng of the indicated chemically modified mRNA complexed with RNAiMAX from Invitrogen as described in triplicate. Secreted TNF-a in the culture medium was measured 24 hours post-transfection for each of the chemically modified mRNAs using an ELISA kit from Invitrogen according to the manufacturer protocols.
In FIGs. 4C and 4D, secreted IFN-β in the same culture medium was measured 24 hours post-transfection for each of the chemically modified mRNAs using an ELISA kit from
Invitrogen according to the manufacturer protocols. In FIGs. 4E and 4F, secreted hu-G-CSF concentration in the same culture medium was measured at 24 hours post-transfection for each of the chemically modified mRNAs. Secretion of Human Granulocyte-Colony Stimulating Factor (G-CSF) from transfected human keratinocytes was quantified using an ELISA kit from
Invitrogen or R&D Systems (Minneapolis, MN) following the manufacturers recommended instructions. These data indicate which modified RNAs containing modified nucleotides were
capable of eliciting a reduced cellular innate immune response in comparison to natural and other chemically modified nucleotides by measuring exemplary type I cytokines TNF-a and IFN-β. These data show that modified RNAs containing modified nucleotides with chemical
modifications on the major groove face of pyrimidine analogs have the lowest levels of secreted TNF-a and IFN-β into the cell culture medium while maintaining high levels of modRNA- encoding hu-G-CSF secretion into the cell culture medium.
Example 4. Human Granulocyte-Colony Stimulating Factor-modified RNA-induced Cell Proliferation Assay
FIGs. 5A-D show modRNA-encoding hu-G-CSF produced by a human keratinocyte feeder cell layer induced the proliferation of both human myeloblast cells KG-1 and Kasumi-1 that express the G-CSF-receptor where the cell populations are separated by a semi-permeable membrane.
Human keratinocytes were grown in EpiLife medium with Supplement S7 from
Invitrogen at a confluence of >70% in a 24-well collagen-coated Transwell® (Corning, Lowell, MA) co-culture tissue culture plate. Keratinocytes were reverse transfected with 750ng of the indicated chemically modified mRNA complexed with RNAiMAX from Invitrogen as described in triplicate. The modRNA:RNAiMAX complex was formed as described. Keratinocyte media was exchanged 6-8 hours post-transfection. 42-hours post-transfection, the 24-well Transwell® plate insert with a Ο μιη-ροΓε semi-permeable polyester membrane was placed into the hu-G- CSF modRNA-transfected keratinocyte containing culture plate. FIG. 5A is a table showing the results from an Enzyme-linked immunosorbent assay (ELISA) for human-G-CSF secreted from in vzYro-transfected Human Keratinocyte cells sampled from individual wells in a co-culture 24- well tissue culture plate 42 hours post-transfection with 750ng of each indicated hu-G-CSF- encoding modRNA.
Human myeloblast cells, Kasumi-1 cells (FIG. 5C) or KG-1 (FIG. 5D) (0.2 x 105 cells), were seeded into the insert well and cell proliferation was quantified 42 hours post-co-culture initiation using the CyQuant Direct Cell Proliferation Assay (Invitrogen) in a 100-120 μΐ volume in a 96-well plate. modRNA-encoding hu-G-CSF-induced myeloblast cell proliferation was expressed as a percent cell proliferation normalized to untransfected keratinocyte/myeloblast co- culture control wells. Secreted hu-G-CSF concentration in both the keratinocyte and myeloblast insert co-culture wells was measured at 42 hours post-co-culture initiation for each modRNA in
duplicate. Secretion of Human Granulocyte-Colony Stimulating Factor (G-CSF) was quantified using an ELISA kit from Invitrogen following the manufacturers recommended instructions.
Transfected hu-G-CSF modRNA in human keratinocyte feeder cells and untransfected human myeloblast cells were detected by RT-PCR. Total RNA from sample cells was extracted and lysed using RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer instructions. Extracted total RNA was submitted to RT-PCR for specific amplification of modRNA-G-CSF using ProtoScript® M-MuLV Taq RT-PCR kit (New England BioLabs, Ipswich, MA) according to the manufacturer instructions with hu-G-CSF-specific primers (see below). RT-PCR products were visualized by 1.2% agarose gel electrophoresis (FIG. 5B). Table 6 below shows which modRNAs were run on the agarose gel.
Table 6
Lane Cell tvDe RT-PCR hu-G-CSF modRNA Tareet
1 Keratinocyte KG-1 Feeder Vehicle
2 Keratinocyte KG-1 Feeder Scramble RNA
3 Keratinocyte KG-1 Feeder No Modification
4 Keratinocyte KG-1 Feeder Chem 7
5 Keratinocyte KG-1 Feeder Ch m 6
6 Keratinocyte KG-1 Feeder Chem 37
7 Keratinocyte Kasumi-1 Feeder Vehicle
8 Keratinocyte Kasumi-1 Feeder Scramble RNA
9 Keratinocyte Kasumi-1 Feeder No Modification
10 Keratinocyte Kasumi-1 Feeder Chem 7
11 Keratinocyte Kasumi-1 Feeder Chem 6
12 Keratinocyte Kasumi-1 Feeder Chem 37
13 Keratinocyte KG-1 Feeder Chem 46
14 Keratinocyte KG-1 Feeder Chem 48
15 Keratinocyte KG-1 Feeder Chem 49
16 Keratinocyte KG-1 Feeder Chem 53
17 Keratinocyte Kasumi-1 Feeder Chem 46
18 Keratinocyte Kasumi-1 Feeder Chem 48
19 Keratinocyte Kasumi-1 Feeder Chem 49
20 Keratinocyte Kasumi-1 Feeder Chem 53
21 Kasumi-1 Vehicle
22 KG-1 Vehicle
23 Kasumi-1 Vehicle
24 Kasumi-1 Scramble RNA
25 Kasumi-1 No Modification
26 Kasumi-1 Chem 7
27 Kasumi-1 Chem 6
28 Kasumi-1 Chem 37
29 Kasumi-1 Chem 46
30 Kasumi-1 Chem 48
3i Kasumi-1 Chem 49
32 Kasumi-1 Chem 53
33 KG-1 Vehicle
34 KG-1 Scramble RNA
35 KG-1 No Modification
36 KG-1 Chem 7
37 Empty Empty
38 Empty Empty
39 Empty Empty
40 Empty Empty
41 Empty Empty
42 Empty Empty
Empty Empty
44 Empty Empty
These data show that human keratinocyte cells containing hu-G-CSF modRNAs comprised of chemically distinct nucleotide analogs secreted hu-G-CSF protein and that the secreted hu-G-CSF was physiologically-active in inducing the proliferation of human myeloblast cells expressing the G-CSF receptor. These data also show the secreted hu-G-CSF protein was permeable across a semi-permeable membrane and acted on a different non-G-CSF-producing cell population. Additionally, these data show that hu-G-CSF modRNA-transfected into human keratinocyte cells in a co-culture environment was present in only the transfected keratinocyte cells and not the un-transfected myeloblast cells. Further, these data show that the modified nucleotide chemical composition of hu-G-CSF modRNA did not affect resultant protein activity.
Example 5. The Effect of modRNA on Cellular Viability
Cytotoxicity and Apoptosis:
This experiment demonstrates cellular viability, cytotoxity and apoptosis for distinct modRNA- in vitro transfected Human Keratinocyte cells. Keratinocytes are grown in EpiLife medium with Human Keratinocyte Growth Supplement in the absence of hydrocortisone from Invitrogen at a confluence of >70%. Keratinocytes are reverse transfected with Ong, 46.875ng, 93.75ng, 187.5ng, 375ng, 750ng, 1500ng, 3000ng, or 6000ng of modRNA complexed with RNAiMAX from Invitrogen. The modRNA:RNAiMAX complex is formed. Secreted huG-CSF
concentration in the culture medium is measured at 0, 6, 12, 24, and 48 hours post-transfection for each concentration of each modRNA in triplicate. Secretion of Human Granulocyte-Colony
Stimulating Factor (G-CSF) from transfected human keratinocytes is quantified using an ELISA kit from Invitrogen or R&D Systems following the manufacturers recommended instructions. Cellular viability, cytotoxicity and apoptosis is measured at 0, 12, 48, 96, and 192 hours post- transfection using the ApoToxGlo kit from Promega (Madison, WI) according to manufacturer instructions.
Example 6. Co-culture
The modified mRNA comprised of chemically-distinct modified nucleotides encoding human Granulocyte-Colony Stimulating Factor (G-CSF) may stimulate the cellular proliferation of a transfection incompetent cell in co-culture environment. The co-culture includes a highly transfectable cell type such as a human keratinocyte and a transfection incompetent cell type such as a white blood cell (WBC). The modified mRNA encoding G-CSF may be transfected into the highly transfectable cell allowing for the production and secretion of G-CSF protein into the extracellular environment where G-CSF acts in a paracrine-like manner to stimulate the white blood cell expressing the G-CSF receptor to proliferate. The expanded WBC population may be used to treat immune-compromised patients or partially reconstitute the WBC population of an immunosuppressed patient and thus reduce the risk of opportunistic infections.
Another example, a highly transfectable cell such as a fibroblast may be transfected with certain growth factors to support and simulate the growth, maintenance, or differentiation of poorly transfectable embryonic stem cells or induced pluripotent stem cells.
Example 7. 5'-Guanosine Capping on Modified Nucleic Acids (modRNAs)
The cloning, gene synthesis and vector sequencing was performed by DNA2.0 Inc. (Menlo Park, CA). Sequence and insert sequence are set forth herein. The ORF was restriction digested using Xbal and used for cDNA synthesis using tailed-or tail-less-PCR. The tailed-PCR cDNA product was used as the template for the modified mRNA synthesis reaction using 25mM mixture each modified nucleotide (all modified nucleotides were custom synthesized or purchased from TriLink Biotech, San Diego, CA except pyrrolo-C triphosphate purchased from Glen Research, Sterling VA; unmodifed nucleotides were purchased from Epicenter
Biotechnologies, Madison, WI) and CellScript MegaScript™ (Epicenter Biotechnologies, Madison, WI) complete mRNA synthesis kit. The in vitro transcription reaction was run for 4
hours at 37°C. modRNAs incorporating adenosine analogs were poly (A) tailed using yeast Poly (A) Polymerase (Affymetrix, Santa Clara, CA). PCR reaction used HiFi PCR 2X Master Mix™ (Kapa Biosystems, Woburn, MA). modRNAs were post-transcriptionally capped using recombinant Vaccinia Virus Capping Enzyme (New England BioLabs, Ipswich, MA) and a recombinant 2'-0-methyltransferase (Epicenter Biotechnologies, Madison, WI) to generate the 5'-guanosine Capl structure. Cap 2 structure and Cap 3 structures may be generated using additional 2'-0-methyltransferases. The in vitro transcribed mRNA product was run on an agarose gel and visualized. modRNA was purified with Ambion/ Applied Biosystems (Austin, TX) MEGAClear RNA™ purification kit. PCR used PureLink™ PCR purification kit
(Invitrogen, Carlsbad, CA). The product was quantified on Nanodrop™ UV Absorbance (ThermoFisher, Waltham, MA). Quality, UV absorbance quality and visualization of the product was performed on an 1.2% agarose gel. The product was resuspended in TE buffer.
5' Capping Modified Nucleic Acid (mRNA) Structure:
5 '-modRNA capping may be completed concomitantly during the in vzYro-transcription reaction using the following chemical RNA cap analogs to generate the 5'-guanosine cap structure according to manufacturer protocols: 3 '-0-Me-m7G(5')ppp(5')G; 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). 5'- modRNA capping may be completed post-transcriptionally using a Vaccinia Virus Capping Enzyme to generate the "Cap 0" structure: m7G(5')ppp(5')G (New England BioLabs, Ipswich, MA). Cap 1 structure may be generated using both Vaccinia Virus Capping Enzyme and a 2'-0 methyl-transferase to generate: m7G(5')ppp(5')G-2'-0-methyl. Cap 2 structure may be generated from the Cap 1 structure followed by the 2'-0-methylation of the 5 '-antepenultimate nucleotide using a 2'-0 methyl-transferase. Cap 3 structure may be generated from the Cap 2 structure followed by the 2'-0-methylation of the 5'-preantepenultimate nucleotide using a 2'-0 methyl-transferase. Enzymes are preferably derived from a recombinant source.
Sequences:
G-CSF cDNA:
agcttttggaccctcgtacagaagctaatacgactcactatagggaaataagagagaaaagaagagtaagaagaaatataagag ccaccatggccggtcccgcgacccaaagccccatgaaacttatggccctgcagttgctgctttggcactcggccctctggacagtccaaga
agcgactcctctcggacctgcctcatcgttgccgcagtcattccttttgaagtgtctggagcaggtgcgaaagattcagggcgatggagccg cactccaagagaagctctgcgcgacatacaaactttgccatcccgaggagctcgtactgctcgggcacagcttggggattccctgggctcc tctctcgtcctgtccgtcgcaggctttgcagttggcagggtgcctttcccagctccactccggtttgttcttgtatcagggactgctgcaagccc ttgagggaatctcgccagaattgggcccgacgctggacacgttgcagctcgacgtggcggatttcgcaacaaccatctggcagcagatgg aggaactggggatggcacccgcgctgcagcccacgcagggggcaatgccggcctttgcgtccgcgtttcagcgcagggcgggtggagt cctcgtagcgagccaccttcaatcatttttggaagtctcgtaccgggtgctgagacatcttgcgcagccgtgaagcgctgccttctgcggggc ttgccttctggccatgcccttcttctctcccttgcacctgtacctcttggtctttgaataaagcctgagtaggaaggcggccgctcgagcatgca tctagagggcccaattcgccctattcgaagtcg (SEQ ID NO: 1)
G-CSF mRNA:
agcuuuuggacccucguacagaagcuaauacgacucacuauagggaaauaagagagaaaagaagaguaagaagaaau auaagagccaccauggccggucccgcgacccaaagccccaugaaacuuauggcccugcaguugcugcuuuggcacucggcccu cuggacaguccaagaagcgacuccucucggaccugccucaucguugccgcagucauuccuuuugaagugucuggagcaggug cgaaagauucagggcgauggagccgcacuccaagagaagcucugcgcgacauacaaacuuugccaucccgaggagcucguacu gcucgggcacagcuuggggauucccugggcuccucucucguccuguccgucgcaggcuuugcaguuggcagggugccuuuc ccagcuccacuccgguuuguucuuguaucagggacugcugcaagcccuugagggaaucucgccagaauugggcccgacgcug gacacguugcagcucgacguggcggauuucgcaacaaccaucuggcagcagauggaggaacuggggauggcacccgcgcugc agcccacgcagggggcaaugccggccuuugcguccgcguuucagcgcagggcggguggaguccucguagcgagccaccuuca aucauuuuuggaagucucguaccgggugcugagacaucuugcgcagccgugaagcgcugccuucugcggggcuugccuucu ggccaugcccuucuucucucccuugcaccuguaccucuuggucuuugaauaaagccugaguaggaaggcggccgcucgagca ugcaucuagagggcccaauucgcccuauucgaagucg (SEQ ID NO: 2)
G-CSF protein:
MAGPATQSPMKLMALQLLLWHSALWTVQEATPLGPASSLPQSFLLKCLEQVRKI QGDGAALQEKLVSECATYKLCHPEELVLLGHSLGIPWAPLSSCPSQALQLAGCLSQLHS GLFLYQGLLQALEGISPELGPTLDTLQLDVADFATTIWQQMEELGMAPALQPTQGAMPA FASAFQRRAGGVLVASHLQSFLEVSYPvVLRHLAQP (SEQ ID NO: 3) cDNA synthesis primers:
Forward Primer: 5*- TTG GAC CCT CGT ACA GAA GCT AAT ACG (SEQ ID NO: 4)
Reverse Primer for template Poly (A) tailing: 5*- T(i20)CT TCC TAC TCA GGC TTT ATT CAA AGA CCA (SEQ ID NO: 5)
Reverse Primer for post-transcriptional Poly (A) Polymerase tailing: 5'- CTT CCT ACT CAG GCT TTA TTC AAA GAC CA (SEQ ID NO: 6)
G-CSF modRNA RT-PCR primers:
Forward Primer: 5*- TGG CCG GTC CCG CGA CCC AA (SEQ ID NO: 7)
Reverse Primer: 5*- GCT TCA CGG CTG CGC AAG AT (SEQ ID NO: 8)
OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
Claims
WHAT IS CLAIMED IS:
1. A compound comprising a nucleotide that disrupts binding of a major groove binding partner with a nucleic acid comprising the nucleotide, wherein the nucleotide has decreased binding affinity to the major groove binding partner.
2. The compound of claim 1, wherein the nucleotide comprises a chemical modification located on the major groove face of a nucleobase portion of the nucleotide.
3. The compound of claim 2, wherein the nucleobase portion comprises a pyrimidine nucleobase, and wherein the chemical modification comprises replacing or substituting an atom of the major groove face of the pyrimidine nucleobase with an amine, an SH, a methyl, an ethyl, a chloro or a fluoro group.
4. The compound of claim 2, wherein the chemical modification is located on a sugar portion of the nucleotide.
5. The compound of claim 2, wherein the chemical modification is located on a phosphate backbone of the nucleotide.
6. The compound of claim 1, having Formula I:
Z is O or S;
each of Y1 is independently selected from -ORal, -NRalRbl, and -SRal;
each of Y2 is independently selected from O, NRa, S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
each of Y3 is independently selected from O and S;
Y4 is selected from H, -ORa, -SRa, and -NHRa;
n is 0, 1, 2, or 3;
m is 0, 1, 2 or 3;
B is a nucleobase;
Ra is H, Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, or C6-2o aryl;
Ral and Rbl are each independently H or a counterion; and
-Y3-Rcl is OH or SH at a pH of about 1 or -Y3-Rcl is O" or S" at physiological pH; or -Y3-Rcl is Ci-20 alkoxy, C2-2o -O-alkenyl, or Ci-2o -O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y1 or Y2 is not O or OH.
The compound of claim 6, wherein B is a nucleobase of Formula Il-a, Il-b, or II-c:
R
Il-a Il-b II-c
wherein:
^ denotes a single or double bond;
X is O or S;
V, U and W are each independently C or N;
wherein when V is C then R1 is H, Ci_6 alkyl, Ci_6 alkenyl, Ci_6 alkynyl, halo, or -ORc, wherein Ci_6 alkyl, Ci_6 alkenyl, Ci_6 alkynyl are each optionally substituted with -OH, -NRaRb, SH, -C(0)Rc, -C(0)ORc, -NHC(0)Rc, or -NHC(0)ORc;
and wherein when V is N then R1 is absent;
R2 is H, -ORc, -SRC, -NRaRb, or halo;
or when V is C then R1 and R2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, -OH, -SH, -NRaRb, Ci-6 alkyl, Ci-6 alkenyl, Ci-6 alkynyl, Ci-6 alkoxy, or Ci-6 thioalkyl;
R3 is H or Ci-6 alkyl;
R4 is H or Ci-6 alkyl; wherein when ^ denotes a double bond then R4 is absent, or N-R4, taken together, forms a positively charged N substituted with Ci_6 alkyl;
Ra and Rb are each independently H, Ci_6 alkyl, Ci_6 alkenyl, Ci_6 alkynyl, or C6-io aryl; and
Rc is H, Ci-6 alkyl, Ci_6 alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
8. The compound of claim 7, wherein B is a nucleobase of Formula Il-al , II-a2, II-a3, II-a4, or II-a5:
Il-al II-a2 II-a3 II-a4 II-a5.
9. The compound of claim 6, wherein B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil.
The compound of claim 6, having Formula I-
I-a.
I-b.
I-c.
15. A nucleic acid sequence comprising at least two nucleotides, the nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove binding partner with the nucleic acid sequence, wherein the nucleotide has decreased binding affinity to the major groove binding partner.
The nucleic acid sequence of claim 15 comprising a compound of Formula I-d:
wherein:
Z is O or S;
each of Y1 is independently selected from -ORal, -NRalRbl, and -SRal;
each of Y2 is independently selected from O, NRa, S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
B is a nucleobase;
Ra is H, Ci-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, or C6-10 aryl; and
Ral and Rbl are each independently H or a counterion; and
-ORcl is OH at a pH of about 1 or -ORcl is 0~ at physiological pH;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y1 or Y2 is not O or OH.
17. The nucleic acid sequence of claim 16, wherein B is a nucleobase of Formula Il-a, Il-b, or II-c:
Il-a Il-b II-c
wherein:
^ denotes a single or double bond;
X is O or S;
V, U and W are each independently C or N;
wherein when V is C then R1 is H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, halo, or -ORc, wherein C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl are each optionally substituted with -OH, -NRaRb, - SH, -C(0)Rc, -C(0)ORc, -NHC(0)Rc, or -NHC(0)ORc;
and wherein when V is N then R1 is absent;
R2 is H, -ORc, -SRC, -NRaRb, or halo;
or when V is C then R1 and R2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, -OH, -SH, -NRaRb, Ci-6 alkyl, Ci-6 alkenyl, Ci-6 alkynyl, Ci-6 alkoxy, or Ci-6 thioalkyl;
R3 is H or Ci_6 alkyl;
R4 is H or C1-6 alkyl; wherein when ^ denotes a double bond then R4 is absent, or N-R4, taken together, forms a positively charged N substituted with C1-6 alkyl;
Ra and Rb are each independently H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, or C6-10 aryl; and
Rc is H, Ci-6 alkyl, C1-6 alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
18. The nucleic acid sequence of claim 17, wherein B is a nucleobase of Formula Il-al, II- II-a3, II-a4, -a5:
Il-al II-a2 II-a3 II-a4 II-a5.
19. The nucleic acid sequence of claim 16, wherein B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil.
20. The nucleic acid sequence of claim 16, wherein the nucleic acid sequence contains a plurality of structurally unique compounds of Formula I-d.
21. The nucleic acid sequence of claim 16, wherein at least 25% of the cytosines are replaced by a compound of Formula I-d and/or wherein at least 25% of the uracils are replaced by a compound of Formula I-d.
22. The compound of claim 1, wherein the major groove interacting partners are selected from the group consisting of: TLRs (Toll-like Receptors) 3, 7, and 8; RIG-I (retinoic acid- inducible gene I); MDA5 (melanoma differentiation-associated gene 5); laboratory of genetics and physiology 2 (LGP2); ΗΓΝ-200 domain containing proteins; and Helicase-domain containing proteins.
23. A non-naturally occurring nucleotide comprising one or more chemical modifications of a naturally occurring nucleotide, wherein the nucleotide reduces the induction of the cellular
innate immune response of a cell to a modified nucleic acid comprising the non-naturally occurring nucleotide when the modified nucleic acid is introduced into the cell, as compared to the induction of the cellular innate immune in a cell induced by a corresponding unmodified nucleic acid.
24. The compound of claim 23, wherein the nucleotide reduces the innate immune response or the secretion of pro-inflammatory cytokines or both by at least about 10%.
25. The compound of claim 23, wherein the nucleotide reduces the innate immune response or the secretion of pro-inflammatory cytokines or both by about 75%.
26. The compound of claim 23, wherein the nucleotide reduces the innate immune response or the secretion of pro-inflammatory cytokines or both by at least 90%.
27. The compound of claim 23, further comprising a translateable region encoding a protein of interest.
28. A composition comprising the compound of claim 27, in an amount sufficient to increase the production of the protein of interest when introduced into a target cell, as compared to the amount of protein produced in a cell containing a corresponding unmodified nucleic acid encoding the protein of interest.
29. The composition of claim 28, wherein the increase is at least about 10%.
30. The composition of claim 28, wherein the increase is at least about 50%.
31. The composition of claim 28, wherein the increase is at least about 100%.
32. The composition of claim 28, wherein at least 25% of the cytosines in the nucleic acid are replaced by a compound of Formula I-d.
33. The composition of claim 28, wherein at least 90% of the cytosines in the nucleic acid are replaced by a compound of Formula I-d.
34. The composition of claim 28, wherein about 100% of the cytosines in the nucleic acid are replaced by a compound of Formula I-d.
35. The composition of claim 28, wherein at least 25% of the uracils in the nucleic acid are replaced by a compound of Formula I-d.
36. The composition of claim 28, wherein at least 90% of the uracils in the nucleic acid are replaced by a compound of Formula I-d.
37. The composition of claim 28, wherein about 100% of the uracils in the nucleic acid are replaced by a compound of Formula I-d.
38. The composition of claim 28, wherein at least 25% of the cytosines in the nucleic acid are replaced by a first compound of Formula I-d and wherein at least 25% of the uracils in the nucleic acid are replaced by a second compound of Formula I-d.
39. The composition of claim 28, wherein about 100% of the cytosines in the nucleic acid are replaced by a first compound of Formula I-d and wherein about 100% of the uracils in the nucleic acid are replaced by a second compound of Formula I-d.
40. The composition of claim 28, further comprising an RNA polymerase, a cDNA template, or a combination thereof.
41. The composition of claim 40, further comprising a nucleotide selected from the group consisting of adenosine, cytosine, guanosine, and uracil.
42. A method of preparing a nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove binding partner with the nucleic acid sequence, wherein the nucleic acid sequence comprises a compoun f Formula I-d:
I-d
wherein:
the nucleotide has decreased binding affinity to the major groove binding partner;
Z is O or S;
each of Y1 is independently selected from -ORal, -NRalRbl, and -SRal;
each of Y2 is independently selected from O, NRa, S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
B is a nucleobase; and
Ral and Rbl are each independently H or a counterion; and
-ORcl is OH at a pH of about 1 or -ORcl is 0~ at physiological pH;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y1 or Y2 is not O or OH;
the method comprising:
reacting a compound of Formula I-c:
I-c
with an RNA polymerase, and a cDNA template.
43. The method of claim 42, wherein the reaction is repeated from 1 to about 7,000 times.
44. The method of claim 42, wherein B is a nucleobase of Formula Il-a, Il-b, or II-c:
R
Il-a Il-b II-c
wherein:
^ denotes a single or double bond;
X is O or S;
V, U and W are each independently C or N;
wherein when V is C then R1 is H, Ci_6 alkyl, Ci_6 alkenyl, Ci_6 alkynyl, halo, or -ORc, wherein Ci-6 alkyl, Ci-6 alkenyl, Ci-6 alkynyl are each optionally substituted with -OH, -NRaRb, - SH, -C(0)Rc, -C(0)ORc, -NHC(0)Rc, or -NHC(0)ORc;
and wherein when V is N then R1 is absent;
R2 is H, -ORc, -SRC, -NRaRb, or halo;
or when V is C then R1 and R2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, -OH, -SH, -NRaRb, Ci-6 alkyl, Ci-6 alkenyl, Ci-6 alkynyl, Ci-6 alkoxy, or Ci-6 thioalkyl;
R3 is H or Ci-6 alkyl;
R4 is H or Ci-6 alkyl; wherein when ^ denotes a double bond then R4 is absent, or N-R4, taken together, forms a positively charged N substituted with C1-6 alkyl;
Ra and Rb are each independently H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, or C6-10 aryl; and
Rc is H, Ci-6 alkyl, C1-6 alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
45. The method of claim 44, wherein B is a nucleobase of Formula Il-al, II-a2, II-a3, II-a4, or II-a5:
46. A method of amplifying a nucleic acid sequence comprising a nucleotide that disrupts binding of a major groove binding partner with the nucleic acid sequence, the method comprising:
reacting a compound of Formula I-c:
I-c
wherein:
the nucleotide has decreased binding affinity to the major groove binding partner;
Z is O or S;
each of Y1 is independently selected from -ORal, -NRalRbl, and -SRal;
each of Y2 is independently selected from O, NRa, S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
B is a nucleobase; and
Ral and Rbl are each independently H or a counterion; and
-ORcl is OH at a pH of about 1 or -ORcl is 0~ at physiological pH;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y1 or Y2 is not O or OH;
with a primer, a cDNA template, and an RNA polymerase.
47. The method of claim 46, wherein B is a nucleobase of Formula Il-a, Il-b, or II-c:
wherein:
denotes a single or double bond;
X is O or S;
V, U and W are each independently C or N;
wherein when V is C then R1 is H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, halo, or -ORc, wherein C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl are each optionally substituted with -OH, -NRaRb, - SH, -C(0)Rc, -C(0)ORc, -NHC(0)Rc, or -NHC(0)ORc;
and wherein when V is N then R1 is absent;
R2 is H, -ORc, -SRC, -NRaRb, or halo;
or when V is C then R1 and R2 together with the carbon atoms to which they are attached can form a 5- or 6-membered ring optionally substituted with 1-4 substituents selected from halo, -OH, -SH, -NRaRb, Ci-6 alkyl, Ci-6 alkenyl, Ci-6 alkynyl, Ci-6 alkoxy, or Ci-6 thioalkyl;
R3 is H or Ci_6 alkyl;
R4 is H or C1-6 alkyl; wherein when ^ denotes a double bond then R4 is absent, or N-R4, taken together, forms a positively charged N substituted with C1-6 alkyl;
Ra and Rb are each independently H, C1-6 alkyl, C1-6 alkenyl, C1-6 alkynyl, or C6-10 aryl; and
Rc is H, Ci-6 alkyl, C1-6 alkenyl, phenyl, benzyl, a polyethylene glycol group, or an amino-polyethylene glycol group.
48. The method of claim 47, wherein B is a nucleobase of Formula Il-al, II-a2, II-a3, II-a4, or II-a5:
Il-al II-a2 II-a3 II-a4 II-a5.
49. A method of synthesizing a pharmaceutical nucleic acid, comprising the steps of:
a) providing a complementary deoxyribonucleic acid (cDNA) that encodes a pharmaceutical protein of interest;
b) selecting a nucleotide that is known to disrupt a binding of a major groove binding partner with a nucleic acid, wherein the nucleotide has decreased binding affinity to the major groove binding partner; and
c) contacting the provided cDNA and the selected nucleotide with an RNA polymerase, under conditions such that the pharmaceutical nucleic is synthesized.
50. The method of claim 49, wherein the pharmaceutical nucleic acid is a ribonucleic acid (RNA).
51. A method of inducing a physiological change in a target cell population, comprising the steps of:
a) providing a first nucleic acid comprising i) a translatable region encoding a protein of interest and ii) a nucleic acid modification, wherein the first nucleic acid is substantially resistant to cellular degradation; and
b) contacting an effective amount of the first nucleic acid to a producer cell under conditions such that the protein of interest is produced in the producer cell and secreted therefrom, wherein the secreted protein of interest contacts the target cell population and induces a physiological change therein.
52. The method of claim 51 , wherein the protein of interest is capable of binding to a receptor on the surface of at least one cell present in the target cell population.
53. The method of claim 51 , wherein the secreted protein is capable of interacting with a receptor on the surface of at least one cell present in the target cell population.
54. The method of claim 53, wherein the secreted protein is Granulocyte-Colony Stimulating Factor (G-CSF).
55. The method of claim 53, wherein the target cell population comprises one or more cells that express the G-CSF receptor.
56. A compound comprising a nucleic acid comprising one or more nucleotides having Formula I:
I
wherein:
Z is O or S;
each of Y1 is independently selected from -ORal, -NRalRbl, and -SRal;
each of Y2 is independently selected from O, NRa, S or a linker comprising an atom selected from the group consisting of C, O, N, and S;
each of Y3 is independently selected from O and S;
Y4 is selected from H, -ORa, -SRa, and -NHRa;
n is 0, 1 , 2, or 3;
m is 0, 1 , 2 or 3;
B is a nucleobase;
Ra is H, Ci-20 alkyl, C2-2o alkenyl, C2-2o alkynyl, or C6-2o aryl;
Ral and Rbl are each independently H or a counterion; and
-Y3-Rcl is OH or SH at a pH of about 1 or -Y3-Rcl is O" or S" at physiological pH; or -Y3-Rcl is Ci-20 alkoxy, C2-2o -O-alkenyl, or Ci-2o -O-alkynyl;
wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Z, Y1 or Y2 is not O or OH; and
wherein a cell comprising the nucleic acid is characterized by:
i) decreased cellular secretion of s pro-inflammatory cytokine;
ii) decreased activation of a cellular innate immune responder;
iii) decreased suspectibility to a cellular nuclease;
iv) decreased binding to a negative regulator of gene expression;
decreased binding to a nucleic acid; increased protein translation efficiency; increased half-life;
or a combination thereof.
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US8999380B2 (en) | 2012-04-02 | 2015-04-07 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of biologics and proteins associated with human disease |
US9107886B2 (en) | 2012-04-02 | 2015-08-18 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding basic helix-loop-helix family member E41 |
US9181321B2 (en) | 2013-03-14 | 2015-11-10 | Shire Human Genetic Therapies, Inc. | CFTR mRNA compositions and related methods and uses |
US9283287B2 (en) | 2012-04-02 | 2016-03-15 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of nuclear proteins |
US9308281B2 (en) | 2011-06-08 | 2016-04-12 | Shire Human Genetic Therapies, Inc. | MRNA therapy for Fabry disease |
US9334328B2 (en) | 2010-10-01 | 2016-05-10 | Moderna Therapeutics, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US9404127B2 (en) | 2010-06-30 | 2016-08-02 | Protiva Biotherapeutics, Inc. | Non-liposomal systems for nucleic acid delivery |
US9428535B2 (en) | 2011-10-03 | 2016-08-30 | Moderna Therapeutics, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US9464124B2 (en) | 2011-09-12 | 2016-10-11 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
US9512456B2 (en) | 2012-08-14 | 2016-12-06 | Modernatx, Inc. | Enzymes and polymerases for the synthesis of RNA |
US9522176B2 (en) | 2013-10-22 | 2016-12-20 | Shire Human Genetic Therapies, Inc. | MRNA therapy for phenylketonuria |
US9572897B2 (en) | 2012-04-02 | 2017-02-21 | Modernatx, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
EP3052106A4 (en) * | 2013-09-30 | 2017-07-19 | ModernaTX, Inc. | Polynucleotides encoding immune modulating polypeptides |
WO2017127750A1 (en) | 2016-01-22 | 2017-07-27 | Modernatx, Inc. | Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof |
US9751925B2 (en) | 2014-11-10 | 2017-09-05 | Modernatx, Inc. | Alternative nucleic acid molecules containing reduced uracil content and uses thereof |
US9850269B2 (en) | 2014-04-25 | 2017-12-26 | Translate Bio, Inc. | Methods for purification of messenger RNA |
WO2018009838A1 (en) | 2016-07-07 | 2018-01-11 | Rubius Therapeutics, Inc. | Compositions and methods related to therapeutic cell systems expressing exogenous rna |
US9872900B2 (en) | 2014-04-23 | 2018-01-23 | Modernatx, Inc. | Nucleic acid vaccines |
US9957499B2 (en) | 2013-03-14 | 2018-05-01 | Translate Bio, Inc. | Methods for purification of messenger RNA |
WO2018081459A1 (en) | 2016-10-26 | 2018-05-03 | Modernatx, Inc. | Messenger ribonucleic acids for enhancing immune responses and methods of use thereof |
WO2018144775A1 (en) | 2017-02-01 | 2018-08-09 | Modernatx, Inc. | Immunomodulatory therapeutic mrna compositions encoding activating oncogene mutation peptides |
US10093706B2 (en) | 2017-01-30 | 2018-10-09 | Indiana University Research And Technology Corporation | Dominant positive hnRNP-E1 polypeptide compositions and methods |
US10106800B2 (en) | 2005-09-28 | 2018-10-23 | Biontech Ag | Modification of RNA, producing an increased transcript stability and translation efficiency |
WO2018213789A1 (en) | 2017-05-18 | 2018-11-22 | Modernatx, Inc. | Modified messenger rna comprising functional rna elements |
US10143758B2 (en) | 2009-12-01 | 2018-12-04 | Translate Bio, Inc. | Liver specific delivery of messenger RNA |
US10155031B2 (en) | 2012-11-28 | 2018-12-18 | Biontech Rna Pharmaceuticals Gmbh | Individualized vaccines for cancer |
US10258698B2 (en) | 2013-03-14 | 2019-04-16 | Modernatx, Inc. | Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions |
US10323076B2 (en) | 2013-10-03 | 2019-06-18 | Modernatx, Inc. | Polynucleotides encoding low density lipoprotein receptor |
WO2019152557A1 (en) | 2018-01-30 | 2019-08-08 | Modernatx, Inc. | Compositions and methods for delivery of agents to immune cells |
WO2019200171A1 (en) | 2018-04-11 | 2019-10-17 | Modernatx, Inc. | Messenger rna comprising functional rna elements |
WO2019204743A1 (en) | 2018-04-19 | 2019-10-24 | Checkmate Pharmaceuticals, Inc. | Synthetic rig-i-like receptor agonists |
US10485884B2 (en) | 2012-03-26 | 2019-11-26 | Biontech Rna Pharmaceuticals Gmbh | RNA formulation for immunotherapy |
WO2020056304A1 (en) | 2018-09-14 | 2020-03-19 | Modernatx, Inc. | Methods and compositions for treating cancer using mrna therapeutics |
WO2020097409A2 (en) | 2018-11-08 | 2020-05-14 | Modernatx, Inc. | Use of mrna encoding ox40l to treat cancer in human patients |
US10738355B2 (en) | 2011-05-24 | 2020-08-11 | Tron-Translationale Onkologie An Der Universitätsmedizin Der Johannes Gutenberg-Universität Mainz Ggmbh | Individualized vaccines for cancer |
WO2020227510A1 (en) | 2019-05-07 | 2020-11-12 | Modernatx, Inc. | Polynucleotides for disrupting immune cell activity and methods of use thereof |
WO2020227537A1 (en) | 2019-05-07 | 2020-11-12 | Modernatx, Inc | Differentially expressed immune cell micrornas for regulation of protein expression |
US10849920B2 (en) | 2015-10-05 | 2020-12-01 | Modernatx, Inc. | Methods for therapeutic administration of messenger ribonucleic acid drugs |
WO2020263883A1 (en) | 2019-06-24 | 2020-12-30 | Modernatx, Inc. | Endonuclease-resistant messenger rna and uses thereof |
WO2020263985A1 (en) | 2019-06-24 | 2020-12-30 | Modernatx, Inc. | Messenger rna comprising functional rna elements and uses thereof |
WO2021007515A1 (en) | 2019-07-11 | 2021-01-14 | Tenaya Therapeutics, Inc. | Cardiac cell reprogramming with micrornas and other factors |
WO2021050986A1 (en) | 2019-09-11 | 2021-03-18 | Modernatx, Inc. | Lnp-formulated mrna therapeutics and use thereof for treating human subjects |
WO2021081353A1 (en) | 2019-10-23 | 2021-04-29 | Checkmate Pharmaceuticals, Inc. | Synthetic rig-i-like receptor agonists |
US11015211B2 (en) | 2018-08-30 | 2021-05-25 | Tenaya Therapeutics, Inc. | Cardiac cell reprogramming with myocardin and ASCL1 |
WO2021178246A1 (en) | 2020-03-02 | 2021-09-10 | Tenaya Therapeutics, Inc. | Gene vector control by cardiomyocyte-expressed micrornas |
US11156617B2 (en) | 2015-02-12 | 2021-10-26 | BioNTech RNA Pharmaceuticals GbmH | Predicting T cell epitopes useful for vaccination |
US11173120B2 (en) | 2014-09-25 | 2021-11-16 | Biontech Rna Pharmaceuticals Gmbh | Stable formulations of lipids and liposomes |
US11174500B2 (en) | 2018-08-24 | 2021-11-16 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11173190B2 (en) | 2017-05-16 | 2021-11-16 | Translate Bio, Inc. | Treatment of cystic fibrosis by delivery of codon-optimized mRNA encoding CFTR |
WO2021243207A1 (en) | 2020-05-28 | 2021-12-02 | Modernatx, Inc. | Use of mrnas encoding ox40l, il-23 and il-36gamma for treating cancer |
US11222711B2 (en) | 2013-05-10 | 2022-01-11 | BioNTech SE | Predicting immunogenicity of T cell epitopes |
US11224642B2 (en) | 2013-10-22 | 2022-01-18 | Translate Bio, Inc. | MRNA therapy for argininosuccinate synthetase deficiency |
WO2022032154A2 (en) | 2020-08-06 | 2022-02-10 | Modernatx, Inc. | Compositions for the delivery of payload molecules to airway epithelium |
US11253605B2 (en) | 2017-02-27 | 2022-02-22 | Translate Bio, Inc. | Codon-optimized CFTR MRNA |
US11254936B2 (en) | 2012-06-08 | 2022-02-22 | Translate Bio, Inc. | Nuclease resistant polynucleotides and uses thereof |
EP3971287A1 (en) | 2013-07-11 | 2022-03-23 | ModernaTX, Inc. | Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use |
US11298426B2 (en) | 2003-10-14 | 2022-04-12 | BioNTech SE | Recombinant vaccines and use thereof |
EP3718565B1 (en) | 2015-10-22 | 2022-04-27 | ModernaTX, Inc. | Respiratory virus vaccines |
US20220125723A1 (en) | 2010-07-06 | 2022-04-28 | Glaxosmithkline Biologicals Sa | Lipid formulations with viral immunogens |
US11492628B2 (en) | 2015-10-07 | 2022-11-08 | BioNTech SE | 3′-UTR sequences for stabilization of RNA |
US11547673B1 (en) | 2020-04-22 | 2023-01-10 | BioNTech SE | Coronavirus vaccine |
WO2023009421A1 (en) | 2021-07-26 | 2023-02-02 | Modernatx, Inc. | Processes for preparing lipid nanoparticle compositions |
WO2023009422A1 (en) | 2021-07-26 | 2023-02-02 | Modernatx, Inc. | Processes for preparing lipid nanoparticle compositions for the delivery of payload molecules to airway epithelium |
US11596645B2 (en) | 2010-07-06 | 2023-03-07 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11597744B2 (en) | 2017-06-30 | 2023-03-07 | Sirius Therapeutics, Inc. | Chiral phosphoramidite auxiliaries and methods of their use |
WO2023064469A1 (en) | 2021-10-13 | 2023-04-20 | Modernatx, Inc. | Compositions of mrna-encoded il15 fusion proteins and methods of use thereof |
WO2023069498A1 (en) | 2021-10-22 | 2023-04-27 | Senda Biosciences, Inc. | Mrna vaccine composition |
US11639370B2 (en) | 2010-10-11 | 2023-05-02 | Glaxosmithkline Biologicals Sa | Antigen delivery platforms |
WO2023086465A1 (en) | 2021-11-12 | 2023-05-19 | Modernatx, Inc. | Compositions for the delivery of payload molecules to airway epithelium |
US11655475B2 (en) | 2010-07-06 | 2023-05-23 | Glaxosmithkline Biologicals Sa | Immunisation of large mammals with low doses of RNA |
WO2023096858A1 (en) | 2021-11-23 | 2023-06-01 | Senda Biosciences, Inc. | A bacteria-derived lipid composition and use thereof |
WO2023122080A1 (en) | 2021-12-20 | 2023-06-29 | Senda Biosciences, Inc. | Compositions comprising mrna and lipid reconstructed plant messenger packs |
WO2023154818A1 (en) | 2022-02-09 | 2023-08-17 | Modernatx, Inc. | Mucosal administration methods and formulations |
US11744801B2 (en) | 2017-08-31 | 2023-09-05 | Modernatx, Inc. | Methods of making lipid nanoparticles |
US11759422B2 (en) | 2010-08-31 | 2023-09-19 | Glaxosmithkline Biologicals Sa | Pegylated liposomes for delivery of immunogen-encoding RNA |
WO2023196988A1 (en) | 2022-04-07 | 2023-10-12 | Modernatx, Inc. | Methods of use of mrnas encoding il-12 |
US11786607B2 (en) | 2017-06-15 | 2023-10-17 | Modernatx, Inc. | RNA formulations |
WO2023199113A1 (en) | 2022-04-15 | 2023-10-19 | Smartcella Solutions Ab | COMPOSITIONS AND METHODS FOR EXOSOME-MEDIATED DELIVERY OF mRNA AGENTS |
US11865159B2 (en) | 2017-02-28 | 2024-01-09 | Sanofi | Therapeutic RNA |
US11878055B1 (en) | 2022-06-26 | 2024-01-23 | BioNTech SE | Coronavirus vaccine |
US11896636B2 (en) | 2011-07-06 | 2024-02-13 | Glaxosmithkline Biologicals Sa | Immunogenic combination compositions and uses thereof |
US11958891B2 (en) | 2017-01-26 | 2024-04-16 | Surrozen Operating, Inc. | Tissue-specific Wnt signal enhancing molecules and uses thereof |
WO2024044741A3 (en) * | 2022-08-26 | 2024-04-18 | Trilink Biotechnologies, Llc | Efficient method for making highly purified 5'-capped oligonucleotides |
US11981703B2 (en) | 2016-08-17 | 2024-05-14 | Sirius Therapeutics, Inc. | Polynucleotide constructs |
WO2024102434A1 (en) | 2022-11-10 | 2024-05-16 | Senda Biosciences, Inc. | Rna compositions comprising lipid nanoparticles or lipid reconstructed natural messenger packs |
WO2024107827A1 (en) | 2022-11-16 | 2024-05-23 | The Broad Institute, Inc. | Therapeutic exploitation of sting channel activity |
US11993645B2 (en) | 2017-01-11 | 2024-05-28 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions comprising R-Spondin (RSPO) surrogate molecules |
Families Citing this family (92)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9012219B2 (en) | 2005-08-23 | 2015-04-21 | The Trustees Of The University Of Pennsylvania | RNA preparations comprising purified modified RNA for reprogramming cells |
SI3112467T1 (en) | 2009-12-07 | 2018-06-29 | The Trustees Of The University Of Pennsylvania | Rna preparations comprising purified modified rna for reprogramming cells |
EP2686014A1 (en) * | 2011-03-16 | 2014-01-22 | Sanofi | Uses of a dual v region antibody-like protein |
KR102196339B1 (en) | 2011-12-05 | 2020-12-29 | 팩터 바이오사이언스 인크. | Methods and products for transfecting cells |
WO2013090294A1 (en) | 2011-12-12 | 2013-06-20 | The Trustees Of The University Of Pennsylvania | Proteins comprising mrsa pbp2a and fragments thereof, nucleic acids encoding the same, and compositions and their use to prevent and treat mrsa infections |
EA201492055A1 (en) | 2012-06-08 | 2015-11-30 | Шир Хьюман Дженетик Терапис, Инк. | INHALATIVE DELIVERY OF mRNA IN TIGHTNESS CELL TARGETS |
JP6510416B2 (en) | 2012-11-01 | 2019-05-08 | ファクター バイオサイエンス インコーポレイテッド | Methods and products for expressing proteins in cells |
WO2014124457A1 (en) * | 2013-02-11 | 2014-08-14 | University Of Louisville Research Foundation, Inc. | Methods for producing antibodies |
CN105209490A (en) | 2013-03-14 | 2015-12-30 | 夏尔人类遗传性治疗公司 | Methods and compositions for delivering mRNA coded antibodies |
ES2795249T3 (en) | 2013-03-15 | 2020-11-23 | Translate Bio Inc | Synergistic enhancement of nucleic acid delivery through mixed formulations |
EP4279610A3 (en) | 2013-03-15 | 2024-01-03 | ModernaTX, Inc. | Ribonucleic acid purification |
WO2014152030A1 (en) | 2013-03-15 | 2014-09-25 | Moderna Therapeutics, Inc. | Removal of dna fragments in mrna production process |
EP2971033B8 (en) | 2013-03-15 | 2019-07-10 | ModernaTX, Inc. | Manufacturing methods for production of rna transcripts |
WO2014144767A1 (en) | 2013-03-15 | 2014-09-18 | Moderna Therapeutics, Inc. | Ion exchange purification of mrna |
EP3052511A4 (en) | 2013-10-02 | 2017-05-31 | Moderna Therapeutics, Inc. | Polynucleotide molecules and uses thereof |
CN105658800A (en) | 2013-10-22 | 2016-06-08 | 夏尔人类遗传性治疗公司 | CNS delivery of MRNA and uses thereof |
KR102096796B1 (en) | 2013-10-22 | 2020-05-27 | 샤이어 휴먼 지네틱 테라피즈 인크. | Lipid formulations for delivery of messenger rna |
JP2017500865A (en) | 2013-12-19 | 2017-01-12 | ノバルティス アーゲー | Compositions and formulations of leptin mRNA |
FI3690056T3 (en) | 2014-01-31 | 2023-03-19 | Factor Bioscience Inc | Methods and products for nucleic acid production and delivery |
CA2949106C (en) | 2014-05-30 | 2023-10-24 | Shire Human Genetic Therapies, Inc. | Biodegradable lipids for delivery of nucleic acids |
US10286086B2 (en) | 2014-06-19 | 2019-05-14 | Modernatx, Inc. | Alternative nucleic acid molecules and uses thereof |
PE20171238A1 (en) | 2014-06-24 | 2017-08-24 | Shire Human Genetic Therapies | STEREOCHEMICALLY ENRICHED COMPOSITIONS FOR NUCLEIC ACIDS ADMINISTRATION |
CA2953265C (en) | 2014-07-02 | 2023-09-26 | Shire Human Genetic Therapies, Inc. | Encapsulation of messenger rna |
US10407683B2 (en) | 2014-07-16 | 2019-09-10 | Modernatx, Inc. | Circular polynucleotides |
GB201418965D0 (en) * | 2014-10-24 | 2014-12-10 | Ospedale San Raffaele And Fond Telethon | |
US9943595B2 (en) | 2014-12-05 | 2018-04-17 | Translate Bio, Inc. | Messenger RNA therapy for treatment of articular disease |
US10758558B2 (en) | 2015-02-13 | 2020-09-01 | Translate Bio Ma, Inc. | Hybrid oligonucleotides and uses thereof |
AU2016218977C1 (en) | 2015-02-13 | 2023-03-23 | Factor Bioscience Inc. | Nucleic acid products and methods of administration thereof |
JP6895892B2 (en) | 2015-03-19 | 2021-06-30 | トランスレイト バイオ, インコーポレイテッド | MRNA treatment for Pompe disease |
WO2016180430A1 (en) | 2015-05-08 | 2016-11-17 | Curevac Ag | Method for producing rna |
ES2798271T3 (en) | 2015-05-29 | 2020-12-10 | Curevac Real Estate Gmbh | Method for producing and purifying RNA, comprising at least one tangential flow filtration step |
US11364292B2 (en) | 2015-07-21 | 2022-06-21 | Modernatx, Inc. | CHIKV RNA vaccines |
TW201718638A (en) | 2015-07-21 | 2017-06-01 | 現代治療公司 | Infectious disease vaccines |
US11564893B2 (en) | 2015-08-17 | 2023-01-31 | Modernatx, Inc. | Methods for preparing particles and related compositions |
WO2017049286A1 (en) | 2015-09-17 | 2017-03-23 | Moderna Therapeutics, Inc. | Polynucleotides containing a morpholino linker |
CN114686548A (en) | 2015-10-14 | 2022-07-01 | 川斯勒佰尔公司 | Modification of RNA-associated enzymes for enhanced production |
AU2016342045A1 (en) | 2015-10-22 | 2018-06-07 | Modernatx, Inc. | Human cytomegalovirus vaccine |
EP3364982A4 (en) | 2015-10-22 | 2019-04-17 | ModernaTX, Inc. | Sexually transmitted disease vaccines |
EP3364950A4 (en) | 2015-10-22 | 2019-10-23 | ModernaTX, Inc. | Tropical disease vaccines |
AU2016342371B2 (en) | 2015-10-22 | 2023-05-11 | Modernatx, Inc. | Nucleic acid vaccines for varicella zoster virus (VZV) |
ES2763822T3 (en) | 2015-12-09 | 2020-06-01 | Novartis Ag | Mark-free analysis of the efficiency of adding caps to the RNA using rnasa h, probes and liquid chromatography / mass spectrometry |
EP3386484B1 (en) | 2015-12-10 | 2022-03-30 | ModernaTX, Inc. | Compositions and methods for delivery of therapeutic agents |
US10465190B1 (en) | 2015-12-23 | 2019-11-05 | Modernatx, Inc. | In vitro transcription methods and constructs |
TW201738256A (en) * | 2016-04-04 | 2017-11-01 | 日產化學工業股份有限公司 | Production method of protein |
KR102369898B1 (en) | 2016-04-08 | 2022-03-03 | 트랜슬레이트 바이오 인코포레이티드 | Multimeric Encoding Nucleic Acids and Uses Thereof |
US20180126003A1 (en) * | 2016-05-04 | 2018-05-10 | Curevac Ag | New targets for rna therapeutics |
WO2017201340A2 (en) | 2016-05-18 | 2017-11-23 | Modernatx, Inc. | Polynucleotides encoding relaxin |
EP3842530A1 (en) | 2016-06-13 | 2021-06-30 | Translate Bio, Inc. | Messenger rna therapy for the treatment of ornithine transcarbamylase deficiency |
IL264439B1 (en) | 2016-08-17 | 2024-04-01 | Factor Bioscience Inc | non-viral, cell-free composition comprising a synthetic messenger RNA (MRNA) encoding a gene-editing protein for use in treating cancer, and a synthetic RNA encoding a gene-editing protein for use in treatment |
CA3036831A1 (en) | 2016-09-14 | 2018-03-22 | Modernatx, Inc. | High purity rna compositions and methods for preparation thereof |
WO2018058088A1 (en) * | 2016-09-26 | 2018-03-29 | SOLA Biosciences, LLC | Cell-associated secretion-enhancing fusion proteins |
EP3528821A4 (en) | 2016-10-21 | 2020-07-01 | ModernaTX, Inc. | Human cytomegalovirus vaccine |
WO2018089851A2 (en) | 2016-11-11 | 2018-05-17 | Modernatx, Inc. | Influenza vaccine |
US11103578B2 (en) | 2016-12-08 | 2021-08-31 | Modernatx, Inc. | Respiratory virus nucleic acid vaccines |
US11384352B2 (en) | 2016-12-13 | 2022-07-12 | Modernatx, Inc. | RNA affinity purification |
MA47515A (en) | 2017-02-16 | 2019-12-25 | Modernatx Inc | VERY POWERFUL IMMUNOGENIC COMPOSITIONS |
WO2018170256A1 (en) | 2017-03-15 | 2018-09-20 | Modernatx, Inc. | Herpes simplex virus vaccine |
US11464848B2 (en) | 2017-03-15 | 2022-10-11 | Modernatx, Inc. | Respiratory syncytial virus vaccine |
US11576961B2 (en) | 2017-03-15 | 2023-02-14 | Modernatx, Inc. | Broad spectrum influenza virus vaccine |
WO2018170270A1 (en) | 2017-03-15 | 2018-09-20 | Modernatx, Inc. | Varicella zoster virus (vzv) vaccine |
MA47790A (en) | 2017-03-17 | 2021-05-05 | Modernatx Inc | RNA-BASED VACCINES AGAINST ZOONOTIC DISEASES |
WO2018187590A1 (en) | 2017-04-05 | 2018-10-11 | Modernatx, Inc. | Reduction or elimination of immune responses to non-intravenous, e.g., subcutaneously administered therapeutic proteins |
CN106929513A (en) * | 2017-04-07 | 2017-07-07 | 东南大学 | The nano antibody of mRNA codings and its application |
WO2018217897A1 (en) * | 2017-05-23 | 2018-11-29 | David Weiner | Compositions and method for inducing an immune response |
GB2580228B (en) | 2017-07-03 | 2022-11-02 | Torque Therapeutics Inc | Immunostimulatory fusion molecules and uses thereof |
WO2019036685A1 (en) | 2017-08-18 | 2019-02-21 | Modernatx, Inc. | Methods for hplc analysis |
MA49913A (en) | 2017-08-18 | 2021-05-05 | Modernatx Inc | RNA POLYMERASE VARIANTS |
US11866696B2 (en) | 2017-08-18 | 2024-01-09 | Modernatx, Inc. | Analytical HPLC methods |
MA50253A (en) | 2017-09-14 | 2020-07-22 | Modernatx Inc | ZIKA VIRUS RNA VACCINES |
US20210180053A1 (en) | 2017-11-01 | 2021-06-17 | Novartis Ag | Synthetic rnas and methods of use |
AU2018392716A1 (en) | 2017-12-20 | 2020-06-18 | Translate Bio, Inc. | Improved composition and methods for treatment of ornithine transcarbamylase deficiency |
MA51523A (en) | 2018-01-05 | 2020-11-11 | Modernatx Inc | POLYNUCLEOTIDES CODING ANTI-BODY ANTI-CHIKUNGUNYA VIRUS |
MA54676A (en) | 2018-01-29 | 2021-11-17 | Modernatx Inc | RSV RNA VACCINES |
US11351242B1 (en) | 2019-02-12 | 2022-06-07 | Modernatx, Inc. | HMPV/hPIV3 mRNA vaccine composition |
MX2021010075A (en) | 2019-02-20 | 2021-12-10 | Modernatx Inc | Rna polymerase variants for co-transcriptional capping. |
US11851694B1 (en) | 2019-02-20 | 2023-12-26 | Modernatx, Inc. | High fidelity in vitro transcription |
US10501404B1 (en) | 2019-07-30 | 2019-12-10 | Factor Bioscience Inc. | Cationic lipids and transfection methods |
US11576966B2 (en) | 2020-02-04 | 2023-02-14 | CureVac SE | Coronavirus vaccine |
US11241493B2 (en) | 2020-02-04 | 2022-02-08 | Curevac Ag | Coronavirus vaccine |
FI20215508A1 (en) | 2020-04-09 | 2021-10-10 | Niemelae Erik Johan | Mimetic nanoparticles for preventing the spreading and lowering the infection rate of novel coronaviruses |
US11406703B2 (en) | 2020-08-25 | 2022-08-09 | Modernatx, Inc. | Human cytomegalovirus vaccine |
CA3200234A1 (en) | 2020-11-25 | 2022-06-02 | Daryl C. Drummond | Lipid nanoparticles for delivery of nucleic acids, and related methods of use |
MX2023006126A (en) | 2020-12-09 | 2023-07-28 | BioNTech SE | Rna manufacturing. |
KR20230164648A (en) | 2020-12-22 | 2023-12-04 | 큐어백 에스이 | RNA vaccines against SARS-CoV-2 variants |
CN116981692A (en) * | 2021-01-14 | 2023-10-31 | 翻译生物公司 | Methods and compositions for delivering mRNA-encoded antibodies |
AU2021421391A1 (en) | 2021-01-24 | 2023-07-20 | Michael David FORREST | Inhibitors of atp synthase - cosmetic and therapeutic uses |
US11524023B2 (en) | 2021-02-19 | 2022-12-13 | Modernatx, Inc. | Lipid nanoparticle compositions and methods of formulating the same |
CN113736768B (en) * | 2021-08-18 | 2023-06-23 | 新发药业有限公司 | Pseudo uridine synthetase mutant, mutant gene and application thereof in preparation of vitamin B2 |
AU2021461416A1 (en) | 2021-08-24 | 2024-02-22 | BioNTech SE | In vitro transcription technologies |
WO2023068931A1 (en) | 2021-10-21 | 2023-04-27 | Curevac Netherlands B.V. | Cancer neoantigens |
WO2023196898A1 (en) | 2022-04-07 | 2023-10-12 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Beta globin mimetic peptides and their use |
WO2024083345A1 (en) | 2022-10-21 | 2024-04-25 | BioNTech SE | Methods and uses associated with liquid compositions |
Citations (30)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4270537A (en) | 1979-11-19 | 1981-06-02 | Romaine Richard A | Automatic hypodermic syringe |
US4596556A (en) | 1985-03-25 | 1986-06-24 | Bioject, Inc. | Hypodermic injection apparatus |
US4790824A (en) | 1987-06-19 | 1988-12-13 | Bioject, Inc. | Non-invasive hypodermic injection device |
US4886499A (en) | 1986-12-18 | 1989-12-12 | Hoffmann-La Roche Inc. | Portable injection appliance |
US4940460A (en) | 1987-06-19 | 1990-07-10 | Bioject, Inc. | Patient-fillable and non-invasive hypodermic injection device assembly |
US4941880A (en) | 1987-06-19 | 1990-07-17 | Bioject, Inc. | Pre-filled ampule and non-invasive hypodermic injection device assembly |
US5015235A (en) | 1987-02-20 | 1991-05-14 | National Carpet Equipment, Inc. | Syringe needle combination |
US5064413A (en) | 1989-11-09 | 1991-11-12 | Bioject, Inc. | Needleless hypodermic injection device |
US5141496A (en) | 1988-11-03 | 1992-08-25 | Tino Dalto | Spring impelled syringe guide with skin penetration depth adjustment |
US5190521A (en) | 1990-08-22 | 1993-03-02 | Tecnol Medical Products, Inc. | Apparatus and method for raising a skin wheal and anesthetizing skin |
US5312335A (en) | 1989-11-09 | 1994-05-17 | Bioject Inc. | Needleless hypodermic injection device |
US5328483A (en) | 1992-02-27 | 1994-07-12 | Jacoby Richard M | Intradermal injection device with medication and needle guard |
US5334144A (en) | 1992-10-30 | 1994-08-02 | Becton, Dickinson And Company | Single use disposable needleless injector |
US5339163A (en) | 1988-03-16 | 1994-08-16 | Canon Kabushiki Kaisha | Automatic exposure control device using plural image plane detection areas |
US5383851A (en) | 1992-07-24 | 1995-01-24 | Bioject Inc. | Needleless hypodermic injection device |
US5417662A (en) | 1991-09-13 | 1995-05-23 | Pharmacia Ab | Injection needle arrangement |
US5466220A (en) | 1994-03-08 | 1995-11-14 | Bioject, Inc. | Drug vial mixing and transfer device |
US5480381A (en) | 1991-08-23 | 1996-01-02 | Weston Medical Limited | Needle-less injector |
US5527288A (en) | 1990-12-13 | 1996-06-18 | Elan Medical Technologies Limited | Intradermal drug delivery device and method for intradermal delivery of drugs |
US5569189A (en) | 1992-09-28 | 1996-10-29 | Equidyne Systems, Inc. | hypodermic jet injector |
US5599302A (en) | 1995-01-09 | 1997-02-04 | Medi-Ject Corporation | Medical injection system and method, gas spring thereof and launching device using gas spring |
WO1997013537A1 (en) | 1995-10-10 | 1997-04-17 | Visionary Medical Products Corporation | Gas pressured needle-less injection device |
US5649912A (en) | 1994-03-07 | 1997-07-22 | Bioject, Inc. | Ampule filling device |
WO1997037705A1 (en) | 1996-04-11 | 1997-10-16 | Weston Medical Limited | Spring-powered dispensing device for medical purposes |
US5893397A (en) | 1996-01-12 | 1999-04-13 | Bioject Inc. | Medication vial/syringe liquid-transfer apparatus |
WO1999034850A1 (en) | 1998-01-08 | 1999-07-15 | Fiderm S.R.L. | Device for controlling the penetration depth of a needle, for application to an injection syringe |
US5993412A (en) | 1997-05-19 | 1999-11-30 | Bioject, Inc. | Injection apparatus |
WO2007024708A2 (en) | 2005-08-23 | 2007-03-01 | The Trustees Of The University Of Pennsylvania | Rna containing modified nucleosides and methods of use thereof |
WO2009127230A1 (en) * | 2008-04-16 | 2009-10-22 | Curevac Gmbh | MODIFIED (m)RNA FOR SUPPRESSING OR AVOIDING AN IMMUNOSTIMULATORY RESPONSE AND IMMUNOSUPPRESSIVE COMPOSITION |
WO2010111290A1 (en) * | 2009-03-23 | 2010-09-30 | University Of Utah Research Foundation | Methods and compositions related to modified guanine bases for controlling off-target effects in rna interference |
Family Cites Families (1131)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US2008526A (en) | 1932-11-03 | 1935-07-16 | Wappler Frederick Charles | Method and means for treating living tissue |
US3467096A (en) | 1966-04-12 | 1969-09-16 | Ferrell S Horn | Multiple hypodermic syringe arrangement |
BE757653A (en) | 1969-10-21 | 1971-04-16 | Ugine Kuhlmann | NEW DRUGS DERIVED FROM NUCLEIC ACIDS AND METHODS FOR THEIR PREPARATION |
BE786542A (en) | 1971-07-22 | 1973-01-22 | Dow Corning | SUCTION DEVICE ALLOWING TO OBTAIN CELL SAMPLES |
US3906092A (en) | 1971-11-26 | 1975-09-16 | Merck & Co Inc | Stimulation of antibody response |
US4399216A (en) | 1980-02-25 | 1983-08-16 | The Trustees Of Columbia University | Processes for inserting DNA into eucaryotic cells and for producing proteinaceous materials |
US4500707A (en) | 1980-02-29 | 1985-02-19 | University Patents, Inc. | Nucleosides useful in the preparation of polynucleotides |
US5132418A (en) | 1980-02-29 | 1992-07-21 | University Patents, Inc. | Process for preparing polynucleotides |
US4458066A (en) | 1980-02-29 | 1984-07-03 | University Patents, Inc. | Process for preparing polynucleotides |
US4411657A (en) | 1980-05-19 | 1983-10-25 | Anibal Galindo | Hypodermic needle |
US4973679A (en) | 1981-03-27 | 1990-11-27 | University Patents, Inc. | Process for oligonucleo tide synthesis using phosphormidite intermediates |
US4415732A (en) | 1981-03-27 | 1983-11-15 | University Patents, Inc. | Phosphoramidite compounds and processes |
US4668777A (en) | 1981-03-27 | 1987-05-26 | University Patents, Inc. | Phosphoramidite nucleoside compounds |
US4401796A (en) | 1981-04-30 | 1983-08-30 | City Of Hope Research Institute | Solid-phase synthesis of polynucleotides |
US4373071A (en) | 1981-04-30 | 1983-02-08 | City Of Hope Research Institute | Solid-phase synthesis of polynucleotides |
US4474569A (en) | 1982-06-28 | 1984-10-02 | Denver Surgical Developments, Inc. | Antenatal shunt |
US4737462A (en) | 1982-10-19 | 1988-04-12 | Cetus Corporation | Structural genes, plasmids and transformed cells for producing cysteine depleted muteins of interferon-β |
US4588585A (en) | 1982-10-19 | 1986-05-13 | Cetus Corporation | Human recombinant cysteine depleted interferon-β muteins |
US4816567A (en) | 1983-04-08 | 1989-03-28 | Genentech, Inc. | Recombinant immunoglobin preparations |
US4579849A (en) | 1984-04-06 | 1986-04-01 | Merck & Co., Inc. | N-alkylguanine acyclonucleosides as antiviral agents |
US4957735A (en) | 1984-06-12 | 1990-09-18 | The University Of Tennessee Research Corporation | Target-sensitive immunoliposomes- preparation and characterization |
US4959314A (en) | 1984-11-09 | 1990-09-25 | Cetus Corporation | Cysteine-depleted muteins of biologically active proteins |
US5036006A (en) | 1984-11-13 | 1991-07-30 | Cornell Research Foundation, Inc. | Method for transporting substances into living cells and tissues and apparatus therefor |
US5116943A (en) | 1985-01-18 | 1992-05-26 | Cetus Corporation | Oxidation-resistant muteins of Il-2 and other protein |
CA1288073C (en) | 1985-03-07 | 1991-08-27 | Paul G. Ahlquist | Rna transformation vector |
EP0204401A1 (en) | 1985-04-09 | 1986-12-10 | Biogen, Inc. | Method of improving the yield of polypeptides produced in a host cell by stabilizing mRNA |
US5017691A (en) | 1986-07-03 | 1991-05-21 | Schering Corporation | Mammalian interleukin-4 |
US5153319A (en) | 1986-03-31 | 1992-10-06 | University Patents, Inc. | Process for preparing polynucleotides |
US4879111A (en) | 1986-04-17 | 1989-11-07 | Cetus Corporation | Treatment of infections with lymphokines |
CA1340843C (en) | 1987-07-31 | 1999-12-07 | J. Lawrence Burg | Selective amplification of target polynucleotide sequences |
US6090591A (en) | 1987-07-31 | 2000-07-18 | The Board Of Trustees Of The Leland Stanford Junior University | Selective amplification of target polynucleotide sequences |
EP0359789B1 (en) | 1988-01-21 | 1993-08-04 | Genentech, Inc. | Amplification and detection of nucleic acid sequences |
JP2650159B2 (en) | 1988-02-24 | 1997-09-03 | アクゾ・ノベル・エヌ・ベー | Nucleic acid amplification method |
CA1340807C (en) | 1988-02-24 | 1999-11-02 | Lawrence T. Malek | Nucleic acid amplification process |
EP0336562B1 (en) | 1988-03-04 | 1995-06-14 | Cancer Research Campaign Technology Ltd. | Improvements relating to antigens |
KR900700134A (en) | 1988-04-15 | 1990-08-11 | 원본미기재 | IL-2 Receptor-Specific Chimeric Antibodies |
US5021335A (en) | 1988-06-17 | 1991-06-04 | The Board Of Trustees Of The Leland Stanford Junior University | In situ transcription in cells and tissues |
US5168038A (en) | 1988-06-17 | 1992-12-01 | The Board Of Trustees Of The Leland Stanford Junior University | In situ transcription in cells and tissues |
US5130238A (en) | 1988-06-24 | 1992-07-14 | Cangene Corporation | Enhanced nucleic acid amplification process |
US5759802A (en) | 1988-10-26 | 1998-06-02 | Tonen Corporation | Production of human serum alubumin A |
US5047524A (en) | 1988-12-21 | 1991-09-10 | Applied Biosystems, Inc. | Automated system for polynucleotide synthesis and purification |
US5262530A (en) | 1988-12-21 | 1993-11-16 | Applied Biosystems, Inc. | Automated system for polynucleotide synthesis and purification |
US5530101A (en) | 1988-12-28 | 1996-06-25 | Protein Design Labs, Inc. | Humanized immunoglobulins |
US5693622A (en) | 1989-03-21 | 1997-12-02 | Vical Incorporated | Expression of exogenous polynucleotide sequences cardiac muscle of a mammal |
US6673776B1 (en) | 1989-03-21 | 2004-01-06 | Vical Incorporated | Expression of exogenous polynucleotide sequences in a vertebrate, mammal, fish, bird or human |
US6214804B1 (en) | 1989-03-21 | 2001-04-10 | Vical Incorporated | Induction of a protective immune response in a mammal by injecting a DNA sequence |
EP0465529B1 (en) | 1989-03-21 | 1998-04-29 | Vical, Inc. | Expression of exogenous polynucleotide sequences in a vertebrate |
US6867195B1 (en) | 1989-03-21 | 2005-03-15 | Vical Incorporated | Lipid-mediated polynucleotide administration to reduce likelihood of subject's becoming infected |
US5703055A (en) | 1989-03-21 | 1997-12-30 | Wisconsin Alumni Research Foundation | Generation of antibodies through lipid mediated DNA delivery |
US5012818A (en) | 1989-05-04 | 1991-05-07 | Joishy Suresh K | Two in one bone marrow surgical needle |
IE66597B1 (en) | 1989-05-10 | 1996-01-24 | Akzo Nv | Method for the synthesis of ribonucleic acid (RNA) |
US5240855A (en) | 1989-05-12 | 1993-08-31 | Pioneer Hi-Bred International, Inc. | Particle gun |
US5332671A (en) | 1989-05-12 | 1994-07-26 | Genetech, Inc. | Production of vascular endothelial cell growth factor and DNA encoding same |
CA2020958C (en) | 1989-07-11 | 2005-01-11 | Daniel L. Kacian | Nucleic acid sequence amplification methods |
US5545522A (en) | 1989-09-22 | 1996-08-13 | Van Gelder; Russell N. | Process for amplifying a target polynucleotide sequence using a single primer-promoter complex |
FR2740360B1 (en) | 1995-10-25 | 1997-12-26 | Rhone Poulenc Chimie | WATER REDISPERSABLE GRANULES COMPRISING AN ACTIVE MATERIAL IN LIQUID FORM |
US5215899A (en) | 1989-11-09 | 1993-06-01 | Miles Inc. | Nucleic acid amplification employing ligatable hairpin probe and transcription |
NO904633L (en) | 1989-11-09 | 1991-05-10 | Molecular Diagnostics Inc | AMPLIFICATION OF NUCLEIC ACIDS BY TRANSCRIPABLE HAIRNEL PROBE. |
US5633076A (en) | 1989-12-01 | 1997-05-27 | Pharming Bv | Method of producing a transgenic bovine or transgenic bovine embryo |
US5697901A (en) | 1989-12-14 | 1997-12-16 | Elof Eriksson | Gene delivery by microneedle injection |
US5194370A (en) | 1990-05-16 | 1993-03-16 | Life Technologies, Inc. | Promoter ligation activated transcription amplification of nucleic acid sequences |
CA2087256A1 (en) | 1990-07-25 | 1992-01-26 | Jerry L. Ruth | Circular extension for generating multiple nucleic acid complements |
US5489677A (en) | 1990-07-27 | 1996-02-06 | Isis Pharmaceuticals, Inc. | Oligonucleoside linkages containing adjacent oxygen and nitrogen atoms |
US6140496A (en) | 1990-10-09 | 2000-10-31 | Benner; Steven Albert | Precursors for deoxyribonucleotides containing non-standard nucleosides |
US6100024A (en) | 1991-02-08 | 2000-08-08 | Promega Corporation | Methods and compositions for nucleic acid detection by target extension and probe amplification |
AU668864B2 (en) | 1991-03-18 | 1996-05-23 | Centocor Inc. | Chimeric antibodies specific for human tumor necrosis factor |
US5426180A (en) | 1991-03-27 | 1995-06-20 | Research Corporation Technologies, Inc. | Methods of making single-stranded circular oligonucleotides |
DE69229482T2 (en) | 1991-04-25 | 1999-11-18 | Chugai Pharmaceutical Co Ltd | RECOMBINED HUMAN ANTIBODIES AGAINST THE HUMAN INTERLEUKIN 6 RECEPTOR |
US5169766A (en) | 1991-06-14 | 1992-12-08 | Life Technologies, Inc. | Amplification of nucleic acid molecules |
US5199441A (en) | 1991-08-20 | 1993-04-06 | Hogle Hugh H | Fine needle aspiration biopsy apparatus and method |
US5298422A (en) | 1991-11-06 | 1994-03-29 | Baylor College Of Medicine | Myogenic vector systems |
US5824307A (en) | 1991-12-23 | 1998-10-20 | Medimmune, Inc. | Human-murine chimeric antibodies against respiratory syncytial virus |
EP0625049A4 (en) | 1992-01-23 | 1995-07-12 | Vical Inc | Ex vivo gene transfer. |
JP3368603B2 (en) | 1992-02-28 | 2003-01-20 | オリンパス光学工業株式会社 | Gene therapy treatment device |
US6174666B1 (en) | 1992-03-27 | 2001-01-16 | The United States Of America As Represented By The Department Of Health And Human Services | Method of eliminating inhibitory/instability regions from mRNA |
US6132419A (en) | 1992-05-22 | 2000-10-17 | Genetronics, Inc. | Electroporetic gene and drug therapy |
US5514545A (en) | 1992-06-11 | 1996-05-07 | Trustees Of The University Of Pennsylvania | Method for characterizing single cells based on RNA amplification for diagnostics and therapeutics |
US6670178B1 (en) | 1992-07-10 | 2003-12-30 | Transkaryotic Therapies, Inc. | In Vivo production and delivery of insulinotropin for gene therapy |
DE69310179T2 (en) | 1992-07-31 | 1997-07-31 | Behringwerke Ag | METHOD FOR INTRODUCING DEFINED SEQUENCES AT THE 3 'END OF POLYNUCLEOTIDES |
US5273525A (en) | 1992-08-13 | 1993-12-28 | Btx Inc. | Injection and electroporation apparatus for drug and gene delivery |
US5240885A (en) | 1992-09-21 | 1993-08-31 | Corning Incorporated | Rare earth-doped, stabilized cadmium halide glasses |
AU5665694A (en) | 1992-11-04 | 1994-05-24 | Denver Biomaterials Inc. | Apparatus for removal of pleural effusion fluid |
US5736137A (en) | 1992-11-13 | 1998-04-07 | Idec Pharmaceuticals Corporation | Therapeutic application of chimeric and radiolabeled antibodies to human B lymphocyte restricted differentiation antigen for treatment of B cell lymphoma |
ATE196606T1 (en) | 1992-11-13 | 2000-10-15 | Idec Pharma Corp | THERAPEUTIC USE OF CHIMERIC AND LABELED ANTIBODIES DIRECTED AGAINST A DIFFERENTIATION ANTIGEN WHICH EXPRESSION IS RESTRICTED TO HUMAN B LYMPHOCYTES, FOR THE TREATMENT OF B-CELL LYMPHOMA |
DK0678122T3 (en) | 1993-01-12 | 2000-03-06 | Biogen Inc | Recombinant anti-VLA4 antibody molecules |
FR2703253B1 (en) | 1993-03-30 | 1995-06-23 | Centre Nat Rech Scient | APPLICATOR OF ELECTRIC PULSES FOR TREATING BIOLOGICAL TISSUES. |
US7135312B2 (en) | 1993-04-15 | 2006-11-14 | University Of Rochester | Circular DNA vectors for synthesis of RNA and DNA |
US5773244A (en) | 1993-05-19 | 1998-06-30 | Regents Of The University Of California | Methods of making circular RNA |
US5851829A (en) | 1993-07-16 | 1998-12-22 | Dana-Farber Cancer Institute | Method of intracellular binding of target molecules |
US5672491A (en) | 1993-09-20 | 1997-09-30 | The Leland Stanford Junior University | Recombinant production of novel polyketides |
US6432711B1 (en) | 1993-11-03 | 2002-08-13 | Diacrin, Inc. | Embryonic stem cells capable of differentiating into desired cell lines |
US6096503A (en) | 1993-11-12 | 2000-08-01 | The Scripps Research Institute | Method for simultaneous identification of differentially expresses mRNAs and measurement of relative concentrations |
US5840299A (en) | 1994-01-25 | 1998-11-24 | Athena Neurosciences, Inc. | Humanized antibodies against leukocyte adhesion molecule VLA-4 |
US7435802B2 (en) | 1994-01-25 | 2008-10-14 | Elan Pharaceuticals, Inc. | Humanized anti-VLA4 immunoglobulins |
DE69533295T3 (en) | 1994-02-16 | 2009-07-16 | The Government Of The United States Of America, As Represented By The Secretary, The Department Of Health And Human Services | Melanoma-associated antigens, epitopes thereof and melanoma-containing vaccines |
IL112820A0 (en) | 1994-03-07 | 1995-05-26 | Merck & Co Inc | Coordinate in vivo gene expression |
EP0754242A4 (en) | 1994-03-18 | 1997-06-11 | Lynx Therapeutics Inc | OLIGONUCLEOTIDE N3'-m(7)P5' PHOSPHORAMIDATES: SYNTHESIS AND COMPOUNDS; HYBRIDIZATION AND NUCLEASE RESISTANCE PROPERTIES |
WO1995026204A1 (en) | 1994-03-25 | 1995-10-05 | Isis Pharmaceuticals, Inc. | Immune stimulation by phosphorothioate oligonucleotide analogs |
US5457041A (en) | 1994-03-25 | 1995-10-10 | Science Applications International Corporation | Needle array and method of introducing biological substances into living cells using the needle array |
US6074642A (en) | 1994-05-02 | 2000-06-13 | Alexion Pharmaceuticals, Inc. | Use of antibodies specific to human complement component C5 for the treatment of glomerulonephritis |
DK0759993T3 (en) | 1994-05-18 | 2007-11-12 | Bayer Bioscience Gmbh | DNA sequences encoding enzymes capable of facilitating the synthesis of linear alpha 1,4-glucans in plants, fungi and microorganisms |
CA2191362A1 (en) | 1994-06-02 | 1995-12-14 | Mark Selby | Nucleic acid immunization using a virus-based infection/transfection system |
GB9412230D0 (en) | 1994-06-17 | 1994-08-10 | Celltech Ltd | Interleukin-5 specific recombiant antibodies |
US6239116B1 (en) | 1994-07-15 | 2001-05-29 | University Of Iowa Research Foundation | Immunostimulatory nucleic acid molecules |
AU3272695A (en) | 1994-08-12 | 1996-03-07 | Immunomedics Inc. | Immunoconjugates and humanized antibodies specific for b-cell lymphoma and leukemia cells |
US5665545A (en) | 1994-11-28 | 1997-09-09 | Akzo Nobel N.V. | Terminal repeat amplification method |
US5641665A (en) | 1994-11-28 | 1997-06-24 | Vical Incorporated | Plasmids suitable for IL-2 expression |
US5588960A (en) | 1994-12-01 | 1996-12-31 | Vidamed, Inc. | Transurethral needle delivery device with cystoscope and method for treatment of urinary incontinence |
US5807718A (en) | 1994-12-02 | 1998-09-15 | The Scripps Research Institute | Enzymatic DNA molecules |
AU4634396A (en) | 1995-01-06 | 1996-07-24 | Centrum Voor Plantenveredelings- En Reproduktieonderzoek (Cpro - Dlo) | Dna sequences encoding carbohydrate polymer synthesizing enzymes and method for producing transgenic plants |
US5795587A (en) | 1995-01-23 | 1998-08-18 | University Of Pittsburgh | Stable lipid-comprising drug delivery complexes and methods for their production |
US5824497A (en) | 1995-02-10 | 1998-10-20 | Mcmaster University | High efficiency translation of mRNA molecules |
EP0727187B1 (en) | 1995-02-15 | 2003-08-06 | Joseph Eldor | Multiple hole spinal needle |
DE69621507T2 (en) | 1995-03-28 | 2003-01-09 | Japan Science & Tech Corp | Method for molecular indexing of genes using restriction enzymes |
US5869230A (en) | 1995-03-30 | 1999-02-09 | Beth Israel Hospital Association | Gene transfer into the kidney |
US5986054A (en) | 1995-04-28 | 1999-11-16 | The Hospital For Sick Children, Hsc Research And Development Limited Partnership | Genetic sequences and proteins related to alzheimer's disease |
FR2733762B1 (en) | 1995-05-02 | 1997-08-01 | Genset Sa | METHOD FOR THE SPECIFIC COUPLING OF THE HAIR OF THE 5 'END OF A RNAM FRAGMENT AND PREPARATION OF RNAM AND COMPLETE DNA |
US5700642A (en) | 1995-05-22 | 1997-12-23 | Sri International | Oligonucleotide sizing using immobilized cleavable primers |
US6111095A (en) | 1995-06-07 | 2000-08-29 | Merck & Co., Inc. | Capped synthetic RNA, analogs, and aptamers |
US6051429A (en) | 1995-06-07 | 2000-04-18 | Life Technologies, Inc. | Peptide-enhanced cationic lipid transfections |
US5889136A (en) | 1995-06-09 | 1999-03-30 | The Regents Of The University Of Colorado | Orthoester protecting groups in RNA synthesis |
US5766903A (en) | 1995-08-23 | 1998-06-16 | University Technology Corporation | Circular RNA and uses thereof |
US6265389B1 (en) | 1995-08-31 | 2001-07-24 | Alkermes Controlled Therapeutics, Inc. | Microencapsulation and sustained release of oligonucleotides |
WO1997011085A1 (en) | 1995-09-19 | 1997-03-27 | University Of Massachusetts | Inhibited biological degradation of oligodeoxynucleotides |
US5830879A (en) | 1995-10-02 | 1998-11-03 | St. Elizabeth's Medical Center Of Boston, Inc. | Treatment of vascular injury using vascular endothelial growth factor |
US6265387B1 (en) | 1995-10-11 | 2001-07-24 | Mirus, Inc. | Process of delivering naked DNA into a hepatocyte via bile duct |
EP0771873A3 (en) | 1995-10-27 | 1998-03-04 | Takeda Chemical Industries, Ltd. | Neuronal cell-specific receptor protein |
CU22584A1 (en) | 1995-11-17 | 1999-11-03 | Centro Inmunologia Molecular | PHARMACEUTICAL COMPOSITIONS CONTAINING A MONOCLONAL ANTIBODY THAT RECOGNIZES THE CD6 HUMAN LEUKOCYTARY DIFFERENTIATION ANTIGEN AND ITS USES FOR THE DIAGNOSIS AND TREATMENT OF PSORIASIS |
US6090382A (en) | 1996-02-09 | 2000-07-18 | Basf Aktiengesellschaft | Human antibodies that bind human TNFα |
US5962271A (en) | 1996-01-03 | 1999-10-05 | Cloutech Laboratories, Inc. | Methods and compositions for generating full-length cDNA having arbitrary nucleotide sequence at the 3'-end |
US6261584B1 (en) | 1996-02-02 | 2001-07-17 | Alza Corporation | Sustained delivery of an active agent using an implantable system |
US6395292B2 (en) | 1996-02-02 | 2002-05-28 | Alza Corporation | Sustained delivery of an active agent using an implantable system |
AU1874397A (en) | 1996-02-16 | 1997-09-02 | Stichting Rega Vzw | Hexitol containing oligonucleotides and their use in antisense strategies |
US6534312B1 (en) | 1996-02-22 | 2003-03-18 | Merck & Co., Inc. | Vaccines comprising synthetic genes |
US6090391A (en) | 1996-02-23 | 2000-07-18 | Aviron | Recombinant tryptophan mutants of influenza |
SE9601245D0 (en) | 1996-03-29 | 1996-03-29 | Pharmacia Ab | Chimeric superantigens and their use |
US6300487B1 (en) | 1996-03-19 | 2001-10-09 | Cell Therapuetics, Inc. | Mammalian lysophosphatidic acid acyltransferase |
TW517061B (en) | 1996-03-29 | 2003-01-11 | Pharmacia & Amp Upjohn Ab | Modified/chimeric superantigens and their use |
US5712127A (en) | 1996-04-29 | 1998-01-27 | Genescape Inc. | Subtractive amplification |
US5853719A (en) | 1996-04-30 | 1998-12-29 | Duke University | Methods for treating cancers and pathogen infections using antigen-presenting cells loaded with RNA |
US7329741B2 (en) | 1996-06-05 | 2008-02-12 | Chiron Corporation | Polynucleotides that hybridize to DP-75 and their use |
DE69739524D1 (en) | 1996-06-05 | 2009-09-17 | Novartis Vaccines & Diagnostic | DP-75 ENCODING DNA AND METHOD OF USE THEREOF |
JP2000516445A (en) | 1996-06-21 | 2000-12-12 | メルク エンド カンパニー インコーポレーテッド | Vaccines containing synthetic genes |
JP2002515786A (en) | 1996-06-28 | 2002-05-28 | ソントラ メディカル,エル.ピー. | Ultrasound enhancement of transdermal delivery |
US5677124A (en) | 1996-07-03 | 1997-10-14 | Ambion, Inc. | Ribonuclease resistant viral RNA standards |
US5939262A (en) | 1996-07-03 | 1999-08-17 | Ambion, Inc. | Ribonuclease resistant RNA preparation and utilization |
US7288266B2 (en) | 1996-08-19 | 2007-10-30 | United States Of America As Represented By The Secretary, Department Of Health And Human Services | Liposome complexes for increased systemic delivery |
US5849546A (en) | 1996-09-13 | 1998-12-15 | Epicentre Technologies Corporation | Methods for using mutant RNA polymerases with reduced discrimination between non-canonical and canonical nucleoside triphosphates |
US6114148C1 (en) | 1996-09-20 | 2012-05-01 | Gen Hospital Corp | High level expression of proteins |
US6433155B1 (en) | 1996-09-24 | 2002-08-13 | Tanox, Inc. | Family of genes encoding apoptosis-related peptides, peptides encoded thereby and methods of use thereof |
US6214966B1 (en) | 1996-09-26 | 2001-04-10 | Shearwater Corporation | Soluble, degradable poly(ethylene glycol) derivatives for controllable release of bound molecules into solution |
AU4992197A (en) | 1996-10-11 | 1998-05-11 | Regents Of The University Of California, The | Immunostimulatory polynucleotide/immunomodulatory molecule conjugates |
EP0839912A1 (en) | 1996-10-30 | 1998-05-06 | Instituut Voor Dierhouderij En Diergezondheid (Id-Dlo) | Infectious clones of RNA viruses and vaccines and diagnostic assays derived thereof |
GB9623051D0 (en) | 1996-11-06 | 1997-01-08 | Schacht Etienne H | Delivery of DNA to target cells in biological systems |
US5980887A (en) | 1996-11-08 | 1999-11-09 | St. Elizabeth's Medical Center Of Boston | Methods for enhancing angiogenesis with endothelial progenitor cells |
US6143559A (en) * | 1996-11-18 | 2000-11-07 | Arch Development Corporation | Methods for the production of chicken monoclonal antibodies |
US5759179A (en) | 1996-12-31 | 1998-06-02 | Johnson & Johnson Medical, Inc. | Needle and valve assembly for use with a catheter |
PT971946E (en) | 1997-01-21 | 2006-11-30 | Gen Hospital Corp | Selection of proteins using rna-protein fusions |
EP0855184A1 (en) | 1997-01-23 | 1998-07-29 | Grayson B. Dr. Lipford | Pharmaceutical composition comprising a polynucleotide and an antigen especially for vaccination |
US6228640B1 (en) | 1997-02-07 | 2001-05-08 | Cem Cezayirli | Programmable antigen presenting cell of CD34 lineage |
DK0969862T3 (en) | 1997-02-07 | 2007-02-12 | Merck & Co Inc | Synthetic HIV gag genes |
US6251665B1 (en) | 1997-02-07 | 2001-06-26 | Cem Cezayirli | Directed maturation of stem cells and production of programmable antigen presenting dentritic cells therefrom |
US6696291B2 (en) | 1997-02-07 | 2004-02-24 | Merck & Co., Inc. | Synthetic HIV gag genes |
US6406705B1 (en) | 1997-03-10 | 2002-06-18 | University Of Iowa Research Foundation | Use of nucleic acids containing unmethylated CpG dinucleotide as an adjuvant |
US6306393B1 (en) | 1997-03-24 | 2001-10-23 | Immunomedics, Inc. | Immunotherapy of B-cell malignancies using anti-CD22 antibodies |
US6261281B1 (en) | 1997-04-03 | 2001-07-17 | Electrofect As | Method for genetic immunization and introduction of molecules into skeletal muscle and immune cells |
US5914269A (en) | 1997-04-04 | 1999-06-22 | Isis Pharmaceuticals, Inc. | Oligonucleotide inhibition of epidermal growth factor receptor expression |
AU6972798A (en) | 1997-04-18 | 1998-11-13 | University Of Medicine And Dentistry Of New Jersey | Inhibition of hiv-1 replication by a tat rna-binding domain peptide analog |
US5958688A (en) | 1997-04-28 | 1999-09-28 | The Trustees Of The University Of Pennsylvania | Characterization of mRNA patterns in neurites and single cells for medical diagnosis and therapeutics |
US6235883B1 (en) | 1997-05-05 | 2001-05-22 | Abgenix, Inc. | Human monoclonal antibodies to epidermal growth factor receptor |
US5989911A (en) | 1997-05-09 | 1999-11-23 | University Of Massachusetts | Site-specific synthesis of pseudouridine in RNA |
US6124091A (en) | 1997-05-30 | 2000-09-26 | Research Corporation Technologies, Inc. | Cell growth-controlling oligonucleotides |
US6589940B1 (en) | 1997-06-06 | 2003-07-08 | Dynavax Technologies Corporation | Immunostimulatory oligonucleotides, compositions thereof and methods of use thereof |
JP4280309B2 (en) | 1997-06-06 | 2009-06-17 | ザ リージェンツ オブ ザ ユニバーシティー オブ カリフォルニア | Inhibitors of DNA immunostimulatory sequence activity |
JP2002510319A (en) | 1997-07-01 | 2002-04-02 | アイシス・ファーマシューティカルス・インコーポレーテッド | Compositions and methods for delivery of oligonucleotides through the gastrointestinal tract |
US5994511A (en) | 1997-07-02 | 1999-11-30 | Genentech, Inc. | Anti-IgE antibodies and methods of improving polypeptides |
DE69837706T2 (en) | 1997-07-21 | 2008-01-10 | Active Biotech Ab | CYTOLYSIS OF APPROPRIATE TARGET CELLS WITH SUPERANTIC CONJUGATES USING THE INDUCTION OF T-CELL ACTIVATION |
EP1009441A1 (en) | 1997-07-31 | 2000-06-21 | St. Elizabeth's Medical Center of Boston, Inc. | Method for the treatment of grafts |
JP4418101B2 (en) | 1997-09-18 | 2010-02-17 | ザ・トラスティーズ・オブ・ザ・ユニバーシティ・オブ・ペンシルバニア | Attenuated vifDNA immunization cassette for genetic vaccines |
WO1999014346A2 (en) * | 1997-09-19 | 1999-03-25 | Sequitur, Inc. | SENSE mRNA THERAPY |
US6004573A (en) | 1997-10-03 | 1999-12-21 | Macromed, Inc. | Biodegradable low molecular weight triblock poly(lactide-co-glycolide) polyethylene glycol copolymers having reverse thermal gelation properties |
WO1999018221A1 (en) | 1997-10-07 | 1999-04-15 | University Of Maryland Biotechnology Institute | Method for introducing and expressing rna in animal cells |
JP2001520041A (en) | 1997-10-20 | 2001-10-30 | ジェンザイム トランスジェニックス コーポレイション | Novel modified nucleic acid sequences and methods for increasing mRNA levels and protein expression in cellular systems |
US6019747A (en) | 1997-10-21 | 2000-02-01 | I-Flow Corporation | Spring-actuated infusion syringe |
WO1999022009A1 (en) | 1997-10-24 | 1999-05-06 | Megabios Corporation | Methods for preparing polynucleotide transfection complexes |
ATE550042T1 (en) | 1997-11-20 | 2012-04-15 | Vical Inc | TREATMENT OF CANCER USING CYTOKINE-EXPRESSING POLYNUCLEOTIDES AND COMPOSITIONS THEREOF |
US7655777B2 (en) | 1997-11-24 | 2010-02-02 | Monsanto Technology Llc | Nucleic acid molecules associated with the tocopherol pathway |
AU1819499A (en) | 1997-12-12 | 1999-06-28 | Samyang Corporation | Positively-charged poly{alpha-(omega-aminoalkyl)glycolic acid} for the delivery of a bioactive agent via tissue and cellular uptake |
US6517869B1 (en) | 1997-12-12 | 2003-02-11 | Expression Genetics, Inc. | Positively charged poly(alpha-(omega-aminoalkyl)lycolic acid) for the delivery of a bioactive agent via tissue and cellular uptake |
WO1999033982A2 (en) | 1997-12-23 | 1999-07-08 | Chiron Corporation | Human genes and gene expression products i |
US6383811B2 (en) | 1997-12-30 | 2002-05-07 | Mirus Corporation | Polyampholytes for delivering polyions to a cell |
JP2002500201A (en) | 1998-01-05 | 2002-01-08 | ユニバーシティ オブ ワシントン | Enhanced transport using membrane disruptors |
JP2002500075A (en) | 1998-01-08 | 2002-01-08 | ソントラ メディカル, インコーポレイテッド | Transdermal transport enhanced by ultrasound transmission |
US8287483B2 (en) | 1998-01-08 | 2012-10-16 | Echo Therapeutics, Inc. | Method and apparatus for enhancement of transdermal transport |
US6365346B1 (en) | 1998-02-18 | 2002-04-02 | Dade Behring Inc. | Quantitative determination of nucleic acid amplification products |
US5955310A (en) | 1998-02-26 | 1999-09-21 | Novo Nordisk Biotech, Inc. | Methods for producing a polypeptide in a bacillus cell |
US6432925B1 (en) | 1998-04-16 | 2002-08-13 | John Wayne Cancer Institute | RNA cancer vaccine and methods for its use |
US6429301B1 (en) | 1998-04-17 | 2002-08-06 | Whitehead Institute For Biomedical Research | Use of a ribozyme to join nucleic acids and peptides |
GB9808327D0 (en) | 1998-04-20 | 1998-06-17 | Chiron Spa | Antidiotypic compounds |
US6395253B2 (en) | 1998-04-23 | 2002-05-28 | The Regents Of The University Of Michigan | Microspheres containing condensed polyanionic bioactive agents and methods for their production |
EP1816197B1 (en) | 1998-04-23 | 2009-09-16 | Takara Bio Inc. | Method for synthesizing DNA |
US20020064517A1 (en) | 1998-04-30 | 2002-05-30 | Stewart A. Cederholm-Williams | Fibrin sealant as a transfection/transformation vehicle for gene therapy |
US20090208418A1 (en) | 2005-04-29 | 2009-08-20 | Innexus Biotechnology Internaltional Ltd. | Superantibody synthesis and use in detection, prevention and treatment of disease |
CA2329147A1 (en) | 1998-05-20 | 1999-11-25 | Feng Liu | A hepatocyte targeting polyethylene glyco-grafted poly-l-lysine polymeric gene carrier |
US6503231B1 (en) | 1998-06-10 | 2003-01-07 | Georgia Tech Research Corporation | Microneedle device for transport of molecules across tissue |
US7091192B1 (en) | 1998-07-01 | 2006-08-15 | California Institute Of Technology | Linear cyclodextrin copolymers |
WO2000002950A1 (en) | 1998-07-13 | 2000-01-20 | Expression Genetics, Inc. | Polyester analogue of poly-l-lysine as a soluble, biodegradable gene delivery carrier |
US6222030B1 (en) | 1998-08-03 | 2001-04-24 | Agilent Technologies, Inc. | Solid phase synthesis of oligonucleotides using carbonate protecting groups and alpha-effect nucleophile deprotection |
CA2340091C (en) | 1998-08-11 | 2013-02-05 | Idec Pharmaceuticals Corporation | Combination therapies for b-cell lymphomas comprising administration of anti-cd20 antibody |
GB9817662D0 (en) | 1998-08-13 | 1998-10-07 | Crocker Peter J | Substance delivery |
US6924365B1 (en) | 1998-09-29 | 2005-08-02 | Transkaryotic Therapies, Inc. | Optimized messenger RNA |
US20090017533A1 (en) | 1998-09-29 | 2009-01-15 | Shire Human Genetic Therapies, Inc., A Delaware Corporation | Optimized messenger rna |
EP1133513A4 (en) | 1998-11-03 | 2002-07-03 | Univ Yale | Multidomain polynucleotide molecular sensors |
KR20010099788A (en) | 1998-11-09 | 2001-11-09 | 케네쓰 제이. 울코트 | Chimeric anti-cd20 antibody treatment of patients receiving bmt or pbsc transplants |
MXPA01004648A (en) | 1998-11-09 | 2002-05-06 | Idec Pharma Corp | Treatment of hematologic malignancies associated with circulating tumor cells using chimeric anti-cd20 antibody. |
WO2000027340A2 (en) | 1998-11-12 | 2000-05-18 | The Children's Medical Center Corporation | USE OF t-RNA AND FRAGMENTS FOR INHIBITING ANGIOGENESIS AND COMPOSITIONS THEREOF |
US6210931B1 (en) | 1998-11-30 | 2001-04-03 | The United States Of America As Represented By The Secretary Of Agriculture | Ribozyme-mediated synthesis of circular RNA |
US20040171980A1 (en) | 1998-12-18 | 2004-09-02 | Sontra Medical, Inc. | Method and apparatus for enhancement of transdermal transport |
US6444790B1 (en) | 1998-12-23 | 2002-09-03 | Human Genome Sciences, Inc. | Peptidoglycan recognition proteins |
WO2000050586A2 (en) | 1999-02-22 | 2000-08-31 | European Molecular Biology Laboratory | In vitro translation system |
US6255476B1 (en) | 1999-02-22 | 2001-07-03 | Pe Corporation (Ny) | Methods and compositions for synthesis of labelled oligonucleotides and analogs on solid-supports |
US7629311B2 (en) | 1999-02-24 | 2009-12-08 | Edward Lewis Tobinick | Methods to facilitate transmission of large molecules across the blood-brain, blood-eye, and blood-nerve barriers |
CA2363141C (en) | 1999-02-26 | 2010-04-06 | Chiron Corporation | Microemulsions with adsorbed macromolecules and microparticles |
EP1165798A2 (en) | 1999-03-29 | 2002-01-02 | Statens Serum Institut | Nucleotide construct with optimised codons for an hiv genetic vaccine based on a primary, early hiv isolate and synthetic envelope |
EP1177231B1 (en) | 1999-04-09 | 2009-08-19 | Invitrogen Dynal AS | Process for the preparation of monodisperse polymer particles |
CN100358577C (en) | 1999-05-07 | 2008-01-02 | 杰南技术公司 | Treatment of auto immune diseases with antagonists which bind to B cell surface markers |
KR20020011985A (en) | 1999-05-07 | 2002-02-09 | 파르마솔 게엠베하 | Lipid particles on the basis of mixtures of liquid and solid lipids and method for producing same |
US6346382B1 (en) | 1999-06-01 | 2002-02-12 | Vanderbilt University | Human carbamyl phosphate synthetase I polymorphism and diagnostic methods related thereto |
AU4332399A (en) | 1999-06-04 | 2000-12-28 | Cheng-Ming Chuong | Rna polymerase chain reaction |
US6611707B1 (en) | 1999-06-04 | 2003-08-26 | Georgia Tech Research Corporation | Microneedle drug delivery device |
US6743211B1 (en) | 1999-11-23 | 2004-06-01 | Georgia Tech Research Corporation | Devices and methods for enhanced microneedle penetration of biological barriers |
US6303573B1 (en) | 1999-06-07 | 2001-10-16 | The Burnham Institute | Heart homing peptides and methods of using same |
EP1196558A1 (en) | 1999-06-08 | 2002-04-17 | Aventis Pasteur | Immunostimulant oligonucleotide |
DK1194167T3 (en) | 1999-06-09 | 2009-11-16 | Immunomedics Inc | Immunotherapy of autoimmune diseases using B-cell specific antibodies |
US6949245B1 (en) | 1999-06-25 | 2005-09-27 | Genentech, Inc. | Humanized anti-ErbB2 antibodies and treatment with anti-ErbB2 antibodies |
MXPA01013450A (en) | 1999-06-30 | 2003-09-04 | Advanced Cell Tech Inc | Cytoplasmic transfer to de-differentiate recipient cells. |
US6514948B1 (en) | 1999-07-02 | 2003-02-04 | The Regents Of The University Of California | Method for enhancing an immune response |
CN1360631A (en) | 1999-07-09 | 2002-07-24 | 美国家用产品公司 | Methods and compositions for preventing formation of aberrant RNA during transcription of plasmid sequence |
US8557244B1 (en) | 1999-08-11 | 2013-10-15 | Biogen Idec Inc. | Treatment of aggressive non-Hodgkins lymphoma with anti-CD20 antibody |
NZ517202A (en) | 1999-08-24 | 2004-05-28 | Medarex Inc | Human CTLA-4 antibodies and their uses |
US20050112141A1 (en) | 2000-08-30 | 2005-05-26 | Terman David S. | Compositions and methods for treatment of neoplastic disease |
US20040106567A1 (en) | 1999-09-07 | 2004-06-03 | Hagstrom James E. | Intravascular delivery of non-viral nucleic acid |
EP1619254B1 (en) | 1999-09-09 | 2010-12-22 | CureVac GmbH | Transfer of mRNA using polycationic compounds |
WO2001021810A1 (en) | 1999-09-17 | 2001-03-29 | Aventis Pasteur Limited | Chlamydia antigens and corresponding dna fragments and uses thereof |
US6623457B1 (en) | 1999-09-22 | 2003-09-23 | Becton, Dickinson And Company | Method and apparatus for the transdermal administration of a substance |
WO2002064799A2 (en) | 1999-09-28 | 2002-08-22 | Transkaryotic Therapies, Inc. | Optimized messenger rna |
IL148922A0 (en) | 1999-10-06 | 2002-09-12 | Quark Biotech Inc | Method for enrichment of natural antisense messenger rna |
US7060291B1 (en) | 1999-11-24 | 2006-06-13 | Transave, Inc. | Modular targeted liposomal delivery system |
US6613026B1 (en) | 1999-12-08 | 2003-09-02 | Scimed Life Systems, Inc. | Lateral needle-less injection apparatus and method |
US6277974B1 (en) | 1999-12-14 | 2001-08-21 | Cogent Neuroscience, Inc. | Compositions and methods for diagnosing and treating conditions, disorders, or diseases involving cell death |
US6245929B1 (en) | 1999-12-20 | 2001-06-12 | General Electric Company | Catalyst composition and method for producing diaryl carbonates, using bisphosphines |
AU2019201A (en) | 1999-12-22 | 2001-07-03 | Basell Technology Company B.V. | Alpha-olefin polymerization catalyst system which contains an aromatic silane compound |
AU2764801A (en) | 2000-01-07 | 2001-07-24 | University Of Washington | Enhanced transport of agents using membrane disruptive agents |
EP1276901A2 (en) | 2000-01-13 | 2003-01-22 | Amsterdam Support Diagnostics B.V. | A universal nucleic acid amplification system for nucleic acids in a sample |
CA2395811A1 (en) | 2000-01-31 | 2001-08-02 | Human Genome Sciences, Inc. | Nucleic acids, proteins, and antibodies |
CA2398756A1 (en) | 2000-01-31 | 2001-08-02 | Eyal Raz | Immunomodulatory polynucleotides in treatment of an infection by an intracellular pathogen |
US6602498B2 (en) | 2000-02-22 | 2003-08-05 | Shearwater Corporation | N-maleimidyl polymer derivatives |
CZ2008595A3 (en) | 2000-02-24 | 2017-05-03 | Washington University | A medication for the prevention or treatment of preclinical or clinical Alzheimer's disease |
BR0108962A (en) | 2000-03-03 | 2002-12-24 | Valentis Inc | Nucleic acid formulations for gene distribution and methods of use |
US6896885B2 (en) | 2000-03-31 | 2005-05-24 | Biogen Idec Inc. | Combined use of anti-cytokine antibodies or antagonists and anti-CD20 for treatment of B cell lymphoma |
AU2001249727A1 (en) | 2000-03-31 | 2001-10-15 | Genentech, Inc. | Compositions and methods for detecting and quantifying gene expression |
US6565572B2 (en) | 2000-04-10 | 2003-05-20 | Sdgi Holdings, Inc. | Fenestrated surgical screw and method |
JP2003530847A (en) | 2000-04-12 | 2003-10-21 | ヒューマン ゲノム サイエンシズ インコーポレイテッド | Albumin fusion protein |
US6368801B1 (en) | 2000-04-12 | 2002-04-09 | Molecular Staging, Inc. | Detection and amplification of RNA using target-mediated ligation of DNA by RNA ligase |
US20010046496A1 (en) | 2000-04-14 | 2001-11-29 | Brettman Lee R. | Method of administering an antibody |
US6375972B1 (en) | 2000-04-26 | 2002-04-23 | Control Delivery Systems, Inc. | Sustained release drug delivery devices, methods of use, and methods of manufacturing thereof |
US7871598B1 (en) | 2000-05-10 | 2011-01-18 | Novartis Ag | Stable metal ion-lipid powdered pharmaceutical compositions for drug delivery and methods of use |
US20040229271A1 (en) | 2000-05-19 | 2004-11-18 | Williams Richard B. | Compositions and methods for the identification and selection of nucleic acids and polypeptides |
WO2001092523A2 (en) | 2000-05-30 | 2001-12-06 | Curagen Corporation | Human polynucleotides and polypeptides encoded thereby |
JP2004530629A (en) | 2000-06-07 | 2004-10-07 | バイオシネクサス インコーポレーテッド | Immunostimulatory RNA / DNA hybrid molecule |
IL153004A0 (en) | 2000-06-23 | 2003-06-24 | American Cyanamid Co | Modified morbillivirus v proteins |
CA2414884A1 (en) | 2000-07-03 | 2002-01-10 | Chiron S.P.A. | Immunisation against chlamydia pneumoniae |
US6440096B1 (en) | 2000-07-14 | 2002-08-27 | Becton, Dickinson And Co. | Microdevice and method of manufacturing a microdevice |
CA2416488A1 (en) | 2000-07-21 | 2002-01-31 | Glaxo Group Limited | Codon-optimized papilloma virus sequences |
US6902734B2 (en) | 2000-08-07 | 2005-06-07 | Centocor, Inc. | Anti-IL-12 antibodies and compositions thereof |
US20040142474A1 (en) | 2000-09-14 | 2004-07-22 | Expression Genetics, Inc. | Novel cationic lipopolymer as a biocompatible gene delivery agent |
US6696038B1 (en) | 2000-09-14 | 2004-02-24 | Expression Genetics, Inc. | Cationic lipopolymer as biocompatible gene delivery agent |
AU2001290078A1 (en) | 2000-09-20 | 2002-04-02 | Ruggero Della Bitta | Stem cell therapy |
CA2424216A1 (en) | 2000-10-04 | 2002-04-11 | The Trustees Of The University Of Pennsylvania | Compositions and methods of using capsid protein from flaviviruses and pestiviruses |
US6998115B2 (en) | 2000-10-10 | 2006-02-14 | Massachusetts Institute Of Technology | Biodegradable poly(β-amino esters) and uses thereof |
US7202226B2 (en) | 2000-10-23 | 2007-04-10 | Detroit R & D | Augmentation of wound healing by elF-4E mRNA and EGF mRNA |
US20030077604A1 (en) | 2000-10-27 | 2003-04-24 | Yongming Sun | Compositions and methods relating to breast specific genes and proteins |
US20020132788A1 (en) | 2000-11-06 | 2002-09-19 | David Lewis | Inhibition of gene expression by delivery of small interfering RNA to post-embryonic animal cells in vivo |
AU2002226930A1 (en) | 2000-11-17 | 2002-05-27 | The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Reduction of the nonspecific animal toxicity of immunotoxins by mutating the framework regions of the fv to lower the isoelectric point |
EP1415005B1 (en) | 2000-12-07 | 2012-11-21 | Novartis Vaccines and Diagnostics, Inc. | Endogenous retroviruses up-regulated in prostate cancer |
US20020130430A1 (en) | 2000-12-29 | 2002-09-19 | Castor Trevor Percival | Methods for making polymer microspheres/nanospheres and encapsulating therapeutic proteins and other products |
US7708915B2 (en) | 2004-05-06 | 2010-05-04 | Castor Trevor P | Polymer microspheres/nanospheres and encapsulating therapeutic proteins therein |
EP1224943A1 (en) | 2001-01-19 | 2002-07-24 | Crucell Holland B.V. | Fibronectin as a tumor marker detected by phage antibodies |
HUP0500244A3 (en) | 2001-01-19 | 2011-03-28 | Vironovative Bv | A virus causing respiratory tract illness in suspectible mammals |
US20040110191A1 (en) | 2001-01-31 | 2004-06-10 | Winkler Matthew M. | Comparative analysis of nucleic acids using population tagging |
CA2437737A1 (en) | 2001-02-14 | 2002-08-22 | Stephen D. Ginsberg | Methods and compositions of amplifying rna |
US6652886B2 (en) | 2001-02-16 | 2003-11-25 | Expression Genetics | Biodegradable cationic copolymers of poly (alkylenimine) and poly (ethylene glycol) for the delivery of bioactive agents |
DE10109897A1 (en) | 2001-02-21 | 2002-11-07 | Novosom Ag | Optional cationic liposomes and their use |
US7232425B2 (en) | 2001-03-02 | 2007-06-19 | Sorenson Development, Inc. | Apparatus and method for specific interstitial or subcutaneous diffusion and dispersion of medication |
AU2002242474B2 (en) | 2001-03-09 | 2004-05-20 | Gene Stream Pty Ltd | Novel expression vectors |
JP2002262882A (en) | 2001-03-12 | 2002-09-17 | Nisshinbo Ind Inc | Method for amplifying rna |
FR2822164B1 (en) | 2001-03-19 | 2004-06-18 | Centre Nat Rech Scient | POLYPEPTIDES DERIVED FROM POLYMERASE RNAS, AND USES THEREOF |
US6520949B2 (en) | 2001-04-02 | 2003-02-18 | Martin St. Germain | Method and apparatus for administering fluid to animals subcutaneously |
DE10119005A1 (en) | 2001-04-18 | 2002-10-24 | Roche Diagnostics Gmbh | Process for protein expression starting from stabilized linear short DNA in cell-free in vitro transcription / translation systems with exonuclease-containing lysates or in a cellular system containing exonucleases |
US20030171253A1 (en) | 2001-04-19 | 2003-09-11 | Averil Ma | Methods and compositions relating to modulation of A20 |
WO2002086134A2 (en) | 2001-04-23 | 2002-10-31 | Amaxa Gmbh | Buffer solution for electroporation and a method comprising the use of the same |
US7560424B2 (en) | 2001-04-30 | 2009-07-14 | Zystor Therapeutics, Inc. | Targeted therapeutic proteins |
US6777187B2 (en) | 2001-05-02 | 2004-08-17 | Rubicon Genomics, Inc. | Genome walking by selective amplification of nick-translate DNA library and amplification from complex mixtures of templates |
EP1392587A4 (en) | 2001-05-08 | 2009-03-18 | Magnatech International L P | Electronic length control wire pay-off system and method |
US20050137155A1 (en) | 2001-05-18 | 2005-06-23 | Sirna Therapeutics, Inc. | RNA interference mediated treatment of Parkinson disease using short interfering nucleic acid (siNA) |
US8137911B2 (en) | 2001-05-22 | 2012-03-20 | Cellscript, Inc. | Preparation and use of single-stranded transcription substrates for synthesis of transcription products corresponding to target sequences |
CN100384480C (en) | 2001-05-30 | 2008-04-30 | 斯克里普斯研究学院 | Delivery system for nucleic acids |
EP2305699B1 (en) | 2001-06-05 | 2014-08-13 | CureVac GmbH | Stabilised mRNA with increased G/C content which is optimised for translation in its coded areas for the vaccination against sleeping sickness, leishmaniosis and toxoplasmosis |
US20040175787A1 (en) | 2001-06-18 | 2004-09-09 | Klemens Kaupmann | Novel g-protein coupled receptors and dna sequences thereof |
US7785610B2 (en) | 2001-06-21 | 2010-08-31 | Dynavax Technologies Corporation | Chimeric immunomodulatory compounds and methods of using the same—III |
US7547551B2 (en) | 2001-06-21 | 2009-06-16 | University Of Antwerp. | Transfection of eukaryontic cells with linear polynucleotides by electroporation |
EP1404716A2 (en) | 2001-06-26 | 2004-04-07 | Novartis AG | Novel g protein-coupled receptors and dna sequences thereof |
SE0102327D0 (en) | 2001-06-28 | 2001-06-28 | Active Biotech Ab | A novel engineered superantigen for human therapy |
US20040236092A1 (en) | 2001-07-13 | 2004-11-25 | Roman Dziarski | Peptidologlycan recognition protein encoding nucleic acids and methods of use thereof |
US6586524B2 (en) | 2001-07-19 | 2003-07-01 | Expression Genetics, Inc. | Cellular targeting poly(ethylene glycol)-grafted polymeric gene carrier |
EP1430140B1 (en) | 2001-08-01 | 2010-09-15 | University of Utah | N-terminally truncated isoforms of pde3a cyclic phosphodiesterases |
JP2005502344A (en) | 2001-08-27 | 2005-01-27 | ノバルティス アクチエンゲゼルシャフト | Novel G protein coupled receptor and DNA sequence thereof |
US20040142325A1 (en) | 2001-09-14 | 2004-07-22 | Liat Mintz | Methods and systems for annotating biomolecular sequences |
AR045702A1 (en) | 2001-10-03 | 2005-11-09 | Chiron Corp | COMPOSITIONS OF ASSISTANTS. |
DE10148886A1 (en) | 2001-10-04 | 2003-04-30 | Avontec Gmbh | Inhibition of STAT-1 |
US7276489B2 (en) | 2002-10-24 | 2007-10-02 | Idera Pharmaceuticals, Inc. | Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5′ ends |
WO2003042383A1 (en) | 2001-11-14 | 2003-05-22 | Toyo Boseki Kabushiki Kaisha | Dna synthesis promoters, dna polymerase-associated factors and utilization thereof |
AU2002361642A1 (en) | 2001-11-16 | 2003-06-10 | The University Of Tennessee Research Corporation | Recombinant antibody fusion proteins and methods for detection of apoptotic cells |
US20050032062A1 (en) | 2001-11-29 | 2005-02-10 | Salah-Dine Chibout | Methods for the assessment and prognosis of sarcoidosis |
CA2409775C (en) | 2001-12-03 | 2010-07-13 | F. Hoffmann-La Roche Ag | Reversibly modified thermostable enzymes for dna synthesis and amplification in vitro |
US20060275747A1 (en) | 2001-12-07 | 2006-12-07 | Hardy Stephen F | Endogenous retrovirus up-regulated in prostate cancer |
AU2002368276A1 (en) | 2001-12-07 | 2004-05-13 | Chiron Corporation | Endogenous retrovirus up-regulated in prostate cancer |
WO2003050258A2 (en) | 2001-12-07 | 2003-06-19 | Chiron Corporation | Endogenous retrovirus polypeptides linked to oncogenic transformation |
WO2003051923A2 (en) | 2001-12-17 | 2003-06-26 | Novartis Ag | Novel g-protein coupled receptors and dna sequences thereof |
DE10162480A1 (en) | 2001-12-19 | 2003-08-07 | Ingmar Hoerr | The application of mRNA for use as a therapeutic agent against tumor diseases |
MXPA04004919A (en) | 2001-12-21 | 2004-08-11 | Alcon Inc | Use of synthetic inorganic nanoparticles as carriers for ophthalmic and otic drugs. |
AU2003235707A1 (en) | 2002-01-18 | 2003-07-30 | Curevac Gmbh | Immunogenic preparations and vaccines on the basis of mrna |
US7741297B2 (en) | 2002-02-04 | 2010-06-22 | Oncothyreon Inc. | Immunostimulatory, covalently lipidated oligonucleotides |
EP1472275B1 (en) | 2002-02-05 | 2008-12-17 | Genentech, Inc. | Protein purification |
FR2835749B1 (en) | 2002-02-08 | 2006-04-14 | Inst Nat Sante Rech Med | PHARMACEUTICAL COMPOSITION IMPROVING IN VIVO GENE TRANSFER |
DE10207178A1 (en) | 2002-02-19 | 2003-09-04 | Novosom Ag | Components for the production of amphoteric liposomes |
AR038568A1 (en) | 2002-02-20 | 2005-01-19 | Hoffmann La Roche | ANTI-A BETA ANTIBODIES AND ITS USE |
US7354742B2 (en) | 2002-02-22 | 2008-04-08 | Ortho-Mcneil Pharmaceutical, Inc. | Method for generating amplified RNA |
WO2003102166A2 (en) | 2002-02-26 | 2003-12-11 | Maxygen, Inc. | Novel flavivirus antigens |
DK1487856T3 (en) | 2002-03-04 | 2010-10-18 | Imclone Llc | KDR-specific human antibodies and their use |
EP1485070A1 (en) | 2002-03-13 | 2004-12-15 | Novartis AG | Pharmaceutical microparticles |
US7074596B2 (en) | 2002-03-25 | 2006-07-11 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Synthesis and use of anti-reverse mRNA cap analogues |
NZ535952A (en) | 2002-04-04 | 2009-01-31 | Coley Pharm Gmbh | Immunostimulatory G,U-containing oligoribonucleotides |
JP2005529590A (en) | 2002-04-17 | 2005-10-06 | ノバルティス アクチエンゲゼルシャフト | Method for identifying inhibitor of binding of ARE-containing mRNA and HuR protein |
GB0209539D0 (en) | 2002-04-26 | 2002-06-05 | Avecia Ltd | Monomer Polymer and process |
EP1361277A1 (en) | 2002-04-30 | 2003-11-12 | Centre National De La Recherche Scientifique (Cnrs) | Optimization of transgene expression in mammalian cells |
HUE027590T2 (en) | 2002-05-02 | 2016-11-28 | Wyeth Holdings Llc | Calicheamicin derivative-carrier conjugates |
US7374930B2 (en) | 2002-05-21 | 2008-05-20 | Expression Genetics, Inc. | GLP-1 gene delivery for the treatment of type 2 diabetes |
US20040018525A1 (en) | 2002-05-21 | 2004-01-29 | Bayer Aktiengesellschaft | Methods and compositions for the prediction, diagnosis, prognosis, prevention and treatment of malignant neoplasma |
DE10224200C1 (en) | 2002-05-31 | 2003-08-21 | Artus Ges Fuer Molekularbiolog | Replicating RNA, useful, after reverse transcription, for analysis on microarrays, comprises conversion to cDNA then reverse transcription of this to form antisense sequences |
US7198899B2 (en) | 2002-06-03 | 2007-04-03 | Chiron Corporation | Use of NRG4, or inhibitors thereof, in the treatment of colon and pancreatic cancers |
SE0201907D0 (en) | 2002-06-19 | 2002-06-19 | Atos Medical Ab | Patches for tracheostoma valves |
WO2004002453A1 (en) | 2002-06-28 | 2004-01-08 | Protiva Biotherapeutics Ltd. | Method and apparatus for producing liposomes |
CA2491567A1 (en) | 2002-07-01 | 2004-01-08 | The Kenneth S. Warren Institute, Inc. | Recombinant tissue protective cytokines and encoding nucleic acids thereof for protection, restoration, and enhancement of responsive cells, tissues, and organs |
DE10229872A1 (en) | 2002-07-03 | 2004-01-29 | Curevac Gmbh | Immune stimulation through chemically modified RNA |
GB0215509D0 (en) | 2002-07-04 | 2002-08-14 | Novartis Ag | Marker genes |
DE60336736D1 (en) | 2002-07-16 | 2011-05-26 | VGX Pharmaceuticals LLC | CODON-OPTIMIZED SYNTHETIC PLASMIDE |
CA2493808A1 (en) | 2002-07-24 | 2004-01-29 | Ptc Therapeutics, Inc. | Methods for identifying small molecules that modulate premature translation termination and nonsense mediated mrna decay |
EP1393745A1 (en) | 2002-07-29 | 2004-03-03 | Hybridon, Inc. | Modulation of immunostimulatory properties of oligonucleotide-based compounds by optimal presentation of 5'ends |
EP1386925A1 (en) | 2002-07-31 | 2004-02-04 | Girindus AG | Method for preparing oligonucleotides |
US6653468B1 (en) | 2002-07-31 | 2003-11-25 | Isis Pharmaceuticals, Inc. | Universal support media for synthesis of oligomeric compounds |
EP1873180B1 (en) | 2002-08-14 | 2014-05-07 | Novartis AG | Ophthalmic device made from a radiation-curable prepolymer |
BR122012021252B8 (en) | 2002-09-06 | 2021-05-25 | Cerulean Pharma Inc | cyclodextrin-based polymers for the delivery of covalently linked therapeutic agents |
MXPA05002632A (en) | 2002-09-09 | 2005-09-20 | Nektar Therapeutics Al Corp | Method for preparing water-soluble polymer derivatives bearing a terminal carboxylic acid. |
US7534872B2 (en) | 2002-09-27 | 2009-05-19 | Syngen, Inc. | Compositions and methods for the use of FMOC derivatives in DNA/RNA synthesis |
KR100932340B1 (en) | 2002-10-17 | 2009-12-16 | 젠맵 에이/에스 | Human monoclonal antibodies against CD20 |
WO2004038018A1 (en) | 2002-10-22 | 2004-05-06 | Eisai Co., Ltd. | Gene expressed specifically in dopamine-producing neuron precursor cells after termination of division |
EP1567675A4 (en) | 2002-11-21 | 2006-05-10 | Epict Technologies | Methods for using primers that encode one strand of a double-stranded promoter |
US7491234B2 (en) | 2002-12-03 | 2009-02-17 | Boston Scientific Scimed, Inc. | Medical devices for delivery of therapeutic agents |
EP3263596A1 (en) | 2002-12-16 | 2018-01-03 | Genentech, Inc. | Immunoglobulin variants and uses thereof |
US7625872B2 (en) | 2002-12-23 | 2009-12-01 | Dynavax Technologies Corporation | Branched immunomodulatory compounds and methods of using the same |
US7169892B2 (en) | 2003-01-10 | 2007-01-30 | Astellas Pharma Inc. | Lipid-peptide-polymer conjugates for long blood circulation and tumor specific drug delivery systems |
WO2004067728A2 (en) | 2003-01-17 | 2004-08-12 | Ptc Therapeutics | Methods and systems for the identification of rna regulatory sequences and compounds that modulate their function |
EP1604011A4 (en) | 2003-01-21 | 2009-12-09 | Ptc Therapeutics Inc | Methods for identifying compounds that modulate untranslated region-dependent gene expression and methods of using same |
US8426194B2 (en) | 2003-01-21 | 2013-04-23 | Ptc Therapeutics, Inc. | Methods and agents for screening for compounds capable of modulating VEGF expression |
US9068234B2 (en) | 2003-01-21 | 2015-06-30 | Ptc Therapeutics, Inc. | Methods and agents for screening for compounds capable of modulating gene expression |
US20040147027A1 (en) | 2003-01-28 | 2004-07-29 | Troy Carol M. | Complex for facilitating delivery of dsRNA into a cell and uses thereof |
EP1592440A4 (en) | 2003-02-10 | 2007-07-11 | Elan Pharm Inc | Immunoglobulin formulation and method of preparation thereof |
US20040167090A1 (en) | 2003-02-21 | 2004-08-26 | Monahan Sean D. | Covalent modification of RNA for in vitro and in vivo delivery |
CA2450289A1 (en) | 2003-03-20 | 2005-05-19 | Imclone Systems Incorporated | Method of producing an antibody to epidermal growth factor receptor |
US7320961B2 (en) | 2003-03-24 | 2008-01-22 | Abbott Laboratories | Method for treating a disease, disorder or adverse effect caused by an elevated serum concentration of an UGT1A1 substrate |
WO2004087868A2 (en) | 2003-03-25 | 2004-10-14 | Stratagene | Dna polymerase fusions and uses thereof |
WO2004092329A2 (en) | 2003-04-08 | 2004-10-28 | Galenica Pharmaceuticals, Inc. | Semi-synthetic saponin analogs with carrier and immune stimulatory activities for dna and rna vaccines |
ZA200507805B (en) | 2003-04-09 | 2006-12-27 | Genentech Inc | Therapy of autoimmune disease in a patient with an inadequate response to a TNF-alpha inhibitor |
KR101180896B1 (en) | 2003-05-05 | 2012-09-07 | 벤-구리온 유니버시티 오브 더 네게브 리서치 앤드 디벨럽먼트 어쏘러티 | Injectable cross-linked polymeric preparations and uses thereof |
TWI353991B (en) | 2003-05-06 | 2011-12-11 | Syntonix Pharmaceuticals Inc | Immunoglobulin chimeric monomer-dimer hybrids |
ES2333598T5 (en) | 2003-05-06 | 2013-09-04 | Biogen Idec Hemophilia Inc | CHEMICAL PROTEINS OF FC COAGULATION FACTOR TO TREAT HEMOPHILIA. |
US7348004B2 (en) | 2003-05-06 | 2008-03-25 | Syntonix Pharmaceuticals, Inc. | Immunoglobulin chimeric monomer-dimer hybrids |
US9567591B2 (en) | 2003-05-15 | 2017-02-14 | Mello Biotechnology, Inc. | Generation of human embryonic stem-like cells using intronic RNA |
GB0313132D0 (en) | 2003-06-06 | 2003-07-09 | Ich Productions Ltd | Peptide ligands |
EP1636385A4 (en) | 2003-06-24 | 2010-06-02 | Mirus Bio Corp | Inhibition of gene function by delivery of polynucleotide-based gene expression inhibitors to mammalian cells in vivo |
GB0316089D0 (en) | 2003-07-09 | 2003-08-13 | Xo Bioscience Ltd | Differentiation method |
US8592197B2 (en) | 2003-07-11 | 2013-11-26 | Novavax, Inc. | Functional influenza virus-like particles (VLPs) |
US7575572B2 (en) | 2003-07-15 | 2009-08-18 | Spinal Generations, Llc | Method and device for delivering medicine to bone |
US20050013870A1 (en) | 2003-07-17 | 2005-01-20 | Toby Freyman | Decellularized extracellular matrix of conditioned body tissues and uses thereof |
ES2523837T3 (en) | 2003-07-18 | 2014-12-01 | Amgen Inc. | Specific binding agents to hepatocyte growth factor |
DE10335833A1 (en) | 2003-08-05 | 2005-03-03 | Curevac Gmbh | Transfection of blood cells with mRNA for immune stimulation and gene therapy |
US8668926B1 (en) | 2003-09-15 | 2014-03-11 | Shaker A. Mousa | Nanoparticle and polymer formulations for thyroid hormone analogs, antagonists, and formulations thereof |
US7135010B2 (en) | 2003-09-30 | 2006-11-14 | Damage Control Surgical Technologies, Inc. | Method and apparatus for rapid deployment chest drainage |
WO2005040416A1 (en) | 2003-10-06 | 2005-05-06 | Novartis Ag | Use of genetic polymorphisms that associate with efficacy of treatment of inflammatory disease |
DE10347710B4 (en) | 2003-10-14 | 2006-03-30 | Johannes-Gutenberg-Universität Mainz | Recombinant vaccines and their use |
US20050130201A1 (en) | 2003-10-14 | 2005-06-16 | Dharmacon, Inc. | Splint-assisted enzymatic synthesis of polyribounucleotides |
EA025962B1 (en) | 2003-11-05 | 2017-02-28 | Роше Гликарт Аг | ANTIBODIES HAVING INCREASED Fc RECEPTOR BINDING AFFINITY AND EFFECTOR FUNCTION |
WO2005047536A2 (en) | 2003-11-13 | 2005-05-26 | Novartis Ag | Detection of genomic amplification and deletion in cancer |
US20070054278A1 (en) | 2003-11-18 | 2007-03-08 | Applera Corporation | Polymorphisms in nucleic acid molecules encoding human enzyme proteins, methods of detection and uses thereof |
US7699852B2 (en) | 2003-11-19 | 2010-04-20 | Zimmer Spine, Inc. | Fenestrated bone tap and method |
US20050153333A1 (en) | 2003-12-02 | 2005-07-14 | Sooknanan Roy R. | Selective terminal tagging of nucleic acids |
CA2548822C (en) | 2003-12-08 | 2015-08-11 | Gel-Del Technologies, Inc. | Mucoadhesive drug delivery devices and methods of making and using thereof |
US7674884B2 (en) | 2003-12-10 | 2010-03-09 | Novimmune S.A. | Neutralizing antibodies and methods of use thereof |
US7927873B2 (en) | 2003-12-19 | 2011-04-19 | University Of Cincinnati | Polyamides for nucleic acid delivery |
ATE552850T1 (en) | 2003-12-23 | 2012-04-15 | Genentech Inc | NEW ANTI-IL 13 ANTIBODIES AND USES THEREOF |
US8957034B2 (en) | 2004-01-28 | 2015-02-17 | Johns Hopkins University | Drugs and gene carrier particles that rapidly move through mucous barriers |
WO2005073375A1 (en) | 2004-01-30 | 2005-08-11 | Maxygen Holdings Ltd. | Regulated stop codon readthrough |
US7309487B2 (en) | 2004-02-09 | 2007-12-18 | George Inana | Methods and compositions for detecting and treating retinal diseases |
WO2005080431A2 (en) | 2004-02-12 | 2005-09-01 | Morphotek, Inc. | Monoclonal antibodies that specifically bind to folate receptor alpha |
US20070265220A1 (en) * | 2004-03-15 | 2007-11-15 | City Of Hope | Methods and compositions for the specific inhibition of gene expression by double-stranded RNA |
EP3736295A1 (en) | 2004-03-24 | 2020-11-11 | Chugai Seiyaku Kabushiki Kaisha | Subtypes of humanized antibody against interleukin-6 receptor |
WO2005098433A2 (en) | 2004-04-01 | 2005-10-20 | Novartis Ag | Diagnostic assays for alzheimer’s disease |
JP5848861B2 (en) | 2004-04-20 | 2016-01-27 | ジェンマブ エー/エスGenmab A/S | Human monoclonal antibody against CD20 |
ES2246694B1 (en) | 2004-04-29 | 2007-05-01 | Instituto Cientifico Y Tecnologico De Navarra, S.A. | PEGILATED NANOPARTICLES. |
WO2005108411A2 (en) | 2004-05-05 | 2005-11-17 | Isis Pharmaceuticals, Inc. | Substituted pixyl protecting groups for oligonucleotide synthesis |
EP2335689A1 (en) | 2004-05-12 | 2011-06-22 | Baxter International Inc. | Method of manufacturing nucleic acid micropheres |
US8012747B2 (en) | 2004-06-01 | 2011-09-06 | San Diego State University Foundation | Expression system |
US7799565B2 (en) | 2004-06-07 | 2010-09-21 | Protiva Biotherapeutics, Inc. | Lipid encapsulated interfering RNA |
ATE537263T1 (en) | 2004-06-07 | 2011-12-15 | Protiva Biotherapeutics Inc | CATIONIC LIPIDS AND METHODS OF USE |
CA2608862C (en) | 2004-06-11 | 2020-05-05 | Trustees Of Tufts College | Silk-based drug delivery system |
WO2006046978A2 (en) | 2004-06-28 | 2006-05-04 | Argos Therapeutics, Inc. | Cationic peptide-mediated transformation |
AU2005260763B2 (en) | 2004-06-30 | 2011-12-22 | Nektar Therapeutics | Polymer-factor IX moiety conjugates |
DE102004035227A1 (en) | 2004-07-21 | 2006-02-16 | Curevac Gmbh | mRNA mixture for vaccination against tumor diseases |
AU2005328382C1 (en) | 2004-07-21 | 2013-01-24 | Alnylam Pharmaceuticals, Inc. | Oligonucleotides comprising a modified or non-natural nucleobase |
US7603349B1 (en) | 2004-07-29 | 2009-10-13 | Yahoo! Inc. | User interfaces for search systems using in-line contextual queries |
GB0417487D0 (en) | 2004-08-05 | 2004-09-08 | Novartis Ag | Organic compound |
SE0402025D0 (en) | 2004-08-13 | 2004-08-13 | Active Biotech Ab | Treatment of hyperproliferative disease with superantigens in combination with another anticancer agent |
US7291208B2 (en) | 2004-08-13 | 2007-11-06 | Gore Enterprise Holdings, Inc. | Grooved active and passive adsorbent filters |
CA2478458A1 (en) | 2004-08-20 | 2006-02-20 | Michael Panzara | Treatment of pediatric multiple sclerosis |
SI2386640T1 (en) | 2004-08-26 | 2015-06-30 | Engeneic Molecular Delivery Pty Ltd | Delivering functional nucleic acids to mammalian cells via bacterially-derived, intact minicells |
DE102004042546A1 (en) | 2004-09-02 | 2006-03-09 | Curevac Gmbh | Combination therapy for immune stimulation |
US7501486B2 (en) | 2004-09-07 | 2009-03-10 | Burnham Institute For Medical Research | Peptides that selectively home to heart vasculature and related conjugates and methods |
US8663599B1 (en) | 2004-10-05 | 2014-03-04 | Gp Medical, Inc. | Pharmaceutical composition of nanoparticles |
JPWO2006041088A1 (en) | 2004-10-12 | 2008-05-15 | 株式会社ティッシュターゲティングジャパン | Brain transitional bone marrow progenitor cells |
AU2005295727A1 (en) | 2004-10-13 | 2006-04-27 | Ptc Therapeutics, Inc. | Pyrazole or triazole compounds and their use for the manufacture of a medicament for treating somatic mutation-related diseases |
US8057821B2 (en) | 2004-11-03 | 2011-11-15 | Egen, Inc. | Biodegradable cross-linked cationic multi-block copolymers for gene delivery and methods of making thereof |
WO2006047842A2 (en) | 2004-11-08 | 2006-05-11 | K.U. Leuven Research And Development | Modified nucleosides for rna interference |
WO2006065480A2 (en) | 2004-11-23 | 2006-06-22 | Ptc Therapeutics, Inc. | Tetrahydrocarbazoles as active agents for inhibiting vegf production by translational control |
US7964571B2 (en) | 2004-12-09 | 2011-06-21 | Egen, Inc. | Combination of immuno gene therapy and chemotherapy for treatment of cancer and hyperproliferative diseases |
EP1856179B1 (en) | 2004-12-10 | 2013-05-15 | Kala Pharmaceuticals, Inc. | Functionalized poly (ether-anhydride) block copolymers |
WO2006071903A2 (en) | 2004-12-28 | 2006-07-06 | Ptc Therapeutics, Inc. | Cell based methods and systems for the identification of rna regulatory sequences and compounds that modulate their functions |
US8535702B2 (en) | 2005-02-01 | 2013-09-17 | Boston Scientific Scimed, Inc. | Medical devices having porous polymeric regions for controlled drug delivery and regulated biocompatibility |
EP2287608B1 (en) | 2005-03-11 | 2014-01-08 | Firalis SAS | Biomarkers for cardiovascular side-effects induced by cox-2 inhibitory compounds |
US8415325B2 (en) | 2005-03-31 | 2013-04-09 | University Of Delaware | Cell-mediated delivery and targeted erosion of noncovalently crosslinked hydrogels |
AU2006235276A1 (en) | 2005-04-07 | 2006-10-19 | Novartis Vaccines And Diagnostics Inc. | CACNA1E in cancer diagnosis, detection and treatment |
EP1910557A2 (en) | 2005-04-07 | 2008-04-16 | Chiron Corporation | Cancer-related genes |
WO2006110776A2 (en) | 2005-04-12 | 2006-10-19 | Nektar Therapeutics Al, Corporation | Polyethylene glycol cojugates of antimicrobial agents |
EP2295466A3 (en) | 2005-04-25 | 2011-08-17 | Pfizer Inc. | Antibodies to myostatin |
US20060241076A1 (en) | 2005-04-26 | 2006-10-26 | Coley Pharmaceutical Gmbh | Modified oligoribonucleotide analogs with enhanced immunostimulatory activity |
RU2406760C3 (en) | 2005-05-09 | 2017-11-28 | Оно Фармасьютикал Ко., Лтд. | HUMAN MONOCLONAL ANTIBODIES TO PROGRAMMABLE DEATH 1 PROTECTION (PD-1) AND METHODS OF CANCER TREATMENT USING ANTI-PD-1-ANTI-BODY, INDEPENDENTLY OR IN COMBINATION WITH OTHER IMMUNETURAH AND I And I And I And I, In The Combine, I And I Do Not Allocate To Them, Combined With Other Overarching |
US20070072175A1 (en) | 2005-05-13 | 2007-03-29 | Biogen Idec Ma Inc. | Nucleotide array containing polynucleotide probes complementary to, or fragments of, cynomolgus monkey genes and the use thereof |
US20060265771A1 (en) | 2005-05-17 | 2006-11-23 | Lewis David L | Monitoring microrna expression and function |
DE102005023170A1 (en) | 2005-05-19 | 2006-11-23 | Curevac Gmbh | Optimized formulation for mRNA |
MX2007015107A (en) | 2005-06-03 | 2008-02-15 | Genentech Inc | Method of producing antibodies with modified fucosylation level. |
US7550264B2 (en) | 2005-06-10 | 2009-06-23 | Datascope Investment Corporation | Methods and kits for sense RNA synthesis |
KR101304157B1 (en) | 2005-06-16 | 2013-09-06 | 넥타르 테라퓨틱스 | Conjugates having a degradable linkage and polymeric reagents useful in preparing such conjugates |
US8202835B2 (en) | 2005-06-17 | 2012-06-19 | Yitzchak Hillman | Disease treatment via antimicrobial peptides or their inhibitors |
CA2611985C (en) | 2005-06-17 | 2016-08-16 | The University Of North Carolina At Chapel Hill | Nanoparticle fabrication methods, systems, and materials |
WO2007005645A2 (en) | 2005-06-30 | 2007-01-11 | Archemix Corp. | Materials and methods for the generation of fully 2'-modified nucleic acid transcripts |
US8101385B2 (en) | 2005-06-30 | 2012-01-24 | Archemix Corp. | Materials and methods for the generation of transcripts comprising modified nucleotides |
WO2007014363A2 (en) | 2005-07-27 | 2007-02-01 | Genentech, Inc. | Vectors for inducible expression of hairpin rna and use thereof |
US7612181B2 (en) | 2005-08-19 | 2009-11-03 | Abbott Laboratories | Dual variable domain immunoglobulin and uses thereof |
US9012219B2 (en) | 2005-08-23 | 2015-04-21 | The Trustees Of The University Of Pennsylvania | RNA preparations comprising purified modified RNA for reprogramming cells |
US20070048741A1 (en) | 2005-08-24 | 2007-03-01 | Getts Robert C | Methods and kits for sense RNA synthesis |
EP2308505A3 (en) | 2005-09-01 | 2011-11-30 | Novartis Vaccines and Diagnostics GmbH | Multiple vaccines including serogroup C meningococcus |
EP1928492B1 (en) | 2005-09-01 | 2011-02-23 | Celgene Corporation | Immunological uses of immunodulatory compounds for vaccine and anti-infections disease therapy |
US8420605B2 (en) | 2005-09-07 | 2013-04-16 | The University Of Strathclyde | Hydrogel compositions |
US20120021042A1 (en) | 2005-09-15 | 2012-01-26 | Steffen Panzner | Efficient Method For Loading Amphoteric Liposomes With Nucleic Acid Active Substances |
DE102005046490A1 (en) | 2005-09-28 | 2007-03-29 | Johannes-Gutenberg-Universität Mainz | New nucleic acid molecule comprising promoter, a transcriptable nucleic acid sequence, a first and second nucleic acid sequence for producing modified RNA with transcriptional stability and translational efficiency |
US20070087437A1 (en) | 2005-10-14 | 2007-04-19 | Jifan Hu | Methods for rejuvenating cells in vitro and in vivo |
PT1951299E (en) | 2005-11-04 | 2012-02-28 | Novartis Vaccines & Diagnostic | Influenza vaccines including combinations of particulate adjuvants and immunopotentiators |
US20070105124A1 (en) | 2005-11-08 | 2007-05-10 | Getts Robert C | Methods and kits for nucleic acid amplification |
EP1960538A2 (en) | 2005-11-18 | 2008-08-27 | Bioline Limited | A method for enhancing enzymatic dna polymerase reactions |
BRPI0619056A2 (en) | 2005-11-28 | 2011-09-20 | Genmab As | monovalent antibody, method for preparing and producing a monovalent antibody, nucleic acid construct, host cell, immunoconjugate, use of a monovalent antibody, and pharmaceutical composition |
AU2005338632B2 (en) | 2005-11-30 | 2010-05-20 | Epicentre Technologies Corporation | Selective terminal tagging of nucleic acids |
TWI389709B (en) | 2005-12-01 | 2013-03-21 | Novartis Ag | Transdermal therapeutic system |
US8603457B2 (en) | 2005-12-02 | 2013-12-10 | University Of Rochester | Nonsense suppression and genetic codon alteration by targeted modification |
JP5806444B2 (en) | 2005-12-02 | 2015-11-10 | ノバルティス アーゲー | Nanoparticles for use in immunogenic compositions |
US7579318B2 (en) | 2005-12-06 | 2009-08-25 | Centre De La Recherche De La Scientifique | Cell penetrating peptides for intracellular delivery of molecules |
EP1969000A2 (en) | 2005-12-06 | 2008-09-17 | Centre National de la Recherche Scientifique | Cell penetrating peptides for intracellular delivery of molecules |
WO2007067968A2 (en) | 2005-12-08 | 2007-06-14 | Novartis Ag | Effects of inhibitors of fgfr3 on gene transcription |
PT1970446E (en) | 2005-12-13 | 2011-09-01 | Univ Kyoto | Nuclear reprogramming factor |
AU2006325030B2 (en) | 2005-12-16 | 2012-07-26 | Cellectis | Cell penetrating peptide conjugates for delivering nucleic acids into cells |
WO2007077042A1 (en) | 2006-01-06 | 2007-07-12 | Topotarget Switzerland Sa | New method for the treatment of gout or pseudogout |
ATE533782T1 (en) | 2006-01-13 | 2011-12-15 | Univ Pennsylvania | VACCINES AND IMMUNOTHERAPEUTICS WITH CODONE-OPTIMIZED IL-15 AND METHODS OF USE THEREOF |
US20070178103A1 (en) | 2006-01-30 | 2007-08-02 | Fey Georg H | CD19-specific immunotoxin and treatment method |
US8946155B2 (en) | 2006-02-03 | 2015-02-03 | Opko Biologics Ltd. | Long-acting polypeptides and methods of producing and administering same |
US9458444B2 (en) | 2006-02-03 | 2016-10-04 | Opko Biologics Ltd. | Long-acting coagulation factors and methods of producing same |
US8476234B2 (en) | 2006-02-03 | 2013-07-02 | Prolor Biotech Inc. | Long-acting coagulation factors and methods of producing same |
DE102006007433A1 (en) | 2006-02-17 | 2007-08-23 | Curevac Gmbh | Immunostimulant adjuvant useful in vaccines against cancer or infectious diseases comprises a lipid-modified nucleic acid |
EP1991560B1 (en) | 2006-02-20 | 2018-04-04 | Ewha University-Industry Collaboration Foundation | Peptide having cell membrane penetrating activity |
EP2319542B1 (en) | 2006-02-21 | 2018-03-21 | Nektar Therapeutics | Segmented degradable polymers and conjugates made therefrom |
EP1991204A2 (en) | 2006-02-24 | 2008-11-19 | Novartis AG | Microparticles containing biodegradable polymer and cationic polysaccharide for use in immunogenic compositions |
WO2007100789A2 (en) | 2006-02-24 | 2007-09-07 | Wyeth | Gpat3 encodes a mammalian, microsomal acyl-coa:glycerol 3-phosphate acyltransferase |
LT2676967T (en) | 2006-02-28 | 2019-09-10 | Biogen Ma Inc. | Methods of treating inflammatory and autoimmune diseases with natalizumab |
US7910152B2 (en) | 2006-02-28 | 2011-03-22 | Advanced Cardiovascular Systems, Inc. | Poly(ester amide)-based drug delivery systems with controlled release rate and morphology |
GB0605217D0 (en) | 2006-03-15 | 2006-04-26 | Novartis Ag | Method and compositions for assessing acute rejection |
US8231907B2 (en) | 2006-03-21 | 2012-07-31 | Morehouse School Of Medicine | Nanoparticles for delivery of active agents |
CA2648099C (en) | 2006-03-31 | 2012-05-29 | The Brigham And Women's Hospital, Inc | System for targeted delivery of therapeutic agents |
CA2648291A1 (en) | 2006-04-04 | 2007-11-01 | Stc.Unm | Swellable particles for drug delivery |
AU2007238624B2 (en) | 2006-04-14 | 2012-05-31 | Cellscript, Llc | Kits and methods for generating 5' capped RNA |
EP1852127A1 (en) | 2006-05-02 | 2007-11-07 | Charité - Universitätsmedizin Berlin | Use of a B-cell-depleting antibody for treatment of polyoma virus infections |
JP5630998B2 (en) | 2006-05-15 | 2014-11-26 | マサチューセッツ インスティテュート オブ テクノロジー | Polymers for functional particles |
EA020805B1 (en) | 2006-05-24 | 2015-01-30 | Мерк Сероно С.А. | Use of combination of cladribine and beta interferon for treating multiple sclerosis |
CN104356230A (en) | 2006-06-02 | 2015-02-18 | 哈佛大学校长及研究员协会 | Protein surface remodeling |
EP2046383B1 (en) | 2006-07-04 | 2014-11-19 | Genmab A/S | Cd20 binding molecules for the treatment of copd |
EP2037899B1 (en) | 2006-07-07 | 2011-03-30 | Aarhus Universitet | Nanoparticles for nucleic acid delivery |
DE602007007082D1 (en) | 2006-07-12 | 2010-07-22 | Novartis Ag | ACTINIC-NETWORKABLE COPOLYMERS FOR THE MANUFACTURE OF CONTACT LENSES |
JP2009544287A (en) | 2006-07-20 | 2009-12-17 | ノバルティス アーゲー | AMIGO-2 inhibitor for treating, diagnosing, or detecting cancer |
US8728527B2 (en) | 2006-07-24 | 2014-05-20 | Luminus Biosciences, Inc. | Solid nanoparticle formulation of water insoluble pharmaceutical substances with reduced ostwald ripening |
US8304529B2 (en) | 2006-07-28 | 2012-11-06 | Life Technologies Corporation | Dinucleotide MRNA cap analogs |
DE102006035618A1 (en) | 2006-07-31 | 2008-02-07 | Curevac Gmbh | New nucleic acid useful as immuno-stimulating adjuvant for manufacture of a composition for treatment of cancer diseases e.g. colon carcinomas and infectious diseases e.g. influenza and malaria |
AU2007280690C1 (en) | 2006-07-31 | 2012-08-23 | Curevac Gmbh | Nucleic acid of formula (I): GIXmGn, or (II): CIXmCn, in particular as an immune-stimulating agent/adjuvant |
JP2010507567A (en) | 2006-08-07 | 2010-03-11 | ジェンザイム・コーポレーション | Combination therapy |
US8658211B2 (en) | 2006-08-18 | 2014-02-25 | Arrowhead Madison Inc. | Polyconjugates for in vivo delivery of polynucleotides |
EP2051965A2 (en) | 2006-08-18 | 2009-04-29 | Nastech Pharmaceutical Company Inc. | Dicer substrate rna peptide conjugates and methods for rna therapeutics |
AU2007292221B2 (en) | 2006-09-06 | 2013-08-29 | The Regents Of The University Of California | Selectively targeted antimicrobial peptides and the use thereof |
US8192927B2 (en) | 2006-09-07 | 2012-06-05 | Crucell Holland B.V. | Human bind molecules capable of neutralizing influenza virus h5n1 and uses thereof |
US20100215580A1 (en) | 2006-09-08 | 2010-08-26 | The Johns Hopkins University | Compositions and methods for enhancing transport through mucus |
US8454948B2 (en) | 2006-09-14 | 2013-06-04 | Medgenics Medical Israel Ltd. | Long lasting drug formulations |
GB0619182D0 (en) | 2006-09-29 | 2006-11-08 | Leuven K U Res & Dev | Oligonucleotide arrays |
EP2068886B1 (en) | 2006-10-03 | 2013-09-18 | Tekmira Pharmaceuticals Corporation | Lipid containing formulations |
AU2007333528B2 (en) | 2006-10-05 | 2013-10-17 | The Johns Hopkins University | Water-dispersible oral, parenteral, and topical formulations for poorly water soluble drugs using smart polymeric nanoparticles |
DE102006051516A1 (en) * | 2006-10-31 | 2008-05-08 | Curevac Gmbh | (Base) modified RNA to increase the expression of a protein |
US8414927B2 (en) | 2006-11-03 | 2013-04-09 | Boston Scientific Scimed, Inc. | Cross-linked polymer particles |
US7999087B2 (en) | 2006-11-15 | 2011-08-16 | Agilent Technologies, Inc. | 2′-silyl containing thiocarbonate protecting groups for RNA synthesis |
US8242258B2 (en) | 2006-12-03 | 2012-08-14 | Agilent Technologies, Inc. | Protecting groups for RNA synthesis |
US8399007B2 (en) | 2006-12-05 | 2013-03-19 | Landec Corporation | Method for formulating a controlled-release pharmaceutical formulation |
EP2532362A1 (en) | 2006-12-06 | 2012-12-12 | Novartis AG | Vaccines including antigen from four strains of influenza virus |
US9034348B2 (en) | 2006-12-11 | 2015-05-19 | Chi2Gel Ltd. | Injectable chitosan mixtures forming hydrogels |
EA202091230A3 (en) | 2006-12-18 | 2021-01-29 | Акселерон Фарма Инк. | ANTAGONISTS OF ACTIVIN-ACTRII AND THEIR APPLICATION FOR INCREASING THE LEVELS OF ERYTHROCYTES |
JP5452230B2 (en) | 2006-12-21 | 2014-03-26 | ストライカー コーポレイション | Sustained release formulations comprising biological agent crystals, polymer gels and particle suspensions |
EP2104739B1 (en) | 2006-12-21 | 2013-06-19 | Novozymes Inc. | Modified messenger rna stabilizing sequences for expressing genes in bacterial cells |
DE102006061015A1 (en) | 2006-12-22 | 2008-06-26 | Curevac Gmbh | Process for the purification of RNA on a preparative scale by HPLC |
CA2673029C (en) | 2006-12-22 | 2017-03-28 | Archemix Corp. | Materials and methods for the generation of transcripts comprising modified nucleotides |
US8338166B2 (en) | 2007-01-04 | 2012-12-25 | Lawrence Livermore National Security, Llc | Sorting, amplification, detection, and identification of nucleic acid subsequences in a complex mixture |
DE102007001370A1 (en) | 2007-01-09 | 2008-07-10 | Curevac Gmbh | RNA-encoded antibodies |
WO2008091799A2 (en) | 2007-01-22 | 2008-07-31 | The Trustees Of Columbia University In The City Of New York | Cell-based methods for identifying inhibitors of parkinson's disease-associated lrrk2 mutants |
KR20170037677A (en) | 2007-01-30 | 2017-04-04 | 에피백스, 인크. | Regulatory t cell epitopes, compositions and uses thereof |
TW201940502A (en) | 2007-02-02 | 2019-10-16 | 美商艾瑟勒朗法瑪公司 | Variants derived from ActRIIB and uses therefor |
WO2008097926A2 (en) | 2007-02-02 | 2008-08-14 | Yale University | Transient transfection with rna |
WO2008096370A2 (en) | 2007-02-05 | 2008-08-14 | Natco Pharma Limited | An efficient and novel purification method of recombinant hg-csf |
US8333799B2 (en) | 2007-02-12 | 2012-12-18 | C. R. Bard, Inc. | Highly flexible stent and method of manufacture |
US8242087B2 (en) | 2007-02-27 | 2012-08-14 | K.U.Leuven Research & Development | Phosphate modified nucleosides useful as substrates for polymerases and as antiviral agents |
AR065573A1 (en) * | 2007-03-02 | 2009-06-17 | Boehringer Ingelheim Int | METHOD OF PRODUCTION OF HETEROLOGICAL PROTEINS BY EXPRESSION OF A PROTEIN WITH DOMAIN OF LIPID TRANSFER RELATED TO REGULATORY PROTEIN OF THE START ACUTE STEROIDOGENESIS START. |
EP1964922A1 (en) * | 2007-03-02 | 2008-09-03 | Boehringer Ingelheim Pharma GmbH & Co. KG | Improvement of protein production |
CA2680206C (en) | 2007-03-05 | 2015-07-07 | Washington University | Nanoparticle delivery systems for membrane-integrating peptides |
US20100297699A1 (en) | 2007-03-20 | 2010-11-25 | Millennium Pharmaceuticals, Inc. | Nucleic Acids Encoding Humanized Immunoglobulin That Binds Alpha4Beta7 Integrin |
WO2014064687A1 (en) | 2012-10-22 | 2014-05-01 | Deliversir Ltd | A system for delivering therapeutic agents into living cells and cells nuclei |
US8386027B2 (en) | 2007-04-27 | 2013-02-26 | Echo Therapeutics, Inc. | Skin permeation device for analyte sensing or transdermal drug delivery |
ES2492943T3 (en) | 2007-04-30 | 2014-09-10 | Glaxosmithkline Llc | Administration procedures for anti-IL5 antibodies |
US8703204B2 (en) | 2007-05-03 | 2014-04-22 | Bend Research, Inc. | Nanoparticles comprising a cholesteryl ester transfer protein inhibitor and anon-ionizable polymer |
US7682789B2 (en) | 2007-05-04 | 2010-03-23 | Ventana Medical Systems, Inc. | Method for quantifying biomolecules conjugated to a nanoparticle |
WO2008137758A2 (en) | 2007-05-04 | 2008-11-13 | Mdrna, Inc. | Amino acid lipids and uses thereof |
WO2009023311A2 (en) | 2007-05-07 | 2009-02-19 | Alba Therapeutics Corporation | Transcutaneous delivery of therapeutic agents |
JP5296328B2 (en) | 2007-05-09 | 2013-09-25 | 独立行政法人理化学研究所 | Single-stranded circular RNA and method for producing the same |
EP2476689B1 (en) | 2007-05-10 | 2015-10-21 | Agilent Technologies, Inc. | Thiocarbon-protecting groups for RNA synthesis |
KR101588061B1 (en) | 2007-05-14 | 2016-01-25 | 메디뮨 엘엘씨 | Methods of reducing eosinophil levels |
WO2008144365A2 (en) | 2007-05-17 | 2008-11-27 | Novartis Ag | Method for making dry powder compositions containing ds-rna based on supercritical fluid technology |
CA2687746A1 (en) | 2007-05-22 | 2008-12-18 | Novartis Ag | Methods of treating, diagnosing and detecting fgf21-associated disorders |
WO2008151058A2 (en) | 2007-05-30 | 2008-12-11 | The General Hospital Corporation | Methods of generating pluripotent cells from somatic cells |
EP2160464B1 (en) | 2007-05-30 | 2014-05-21 | Northwestern University | Nucleic acid functionalized nanoparticles for therapeutic applications |
US8153773B2 (en) | 2007-06-19 | 2012-04-10 | Board Of Supervisors Of Louisiana State University And Agricultural And Mechanical College | Synthesis and use of anti-reverse phosphorothioate analogs of the messenger RNA cap |
EP2173872B1 (en) | 2007-06-29 | 2014-04-02 | CellScript, Inc. | Copy dna and sense rna |
WO2009015071A1 (en) | 2007-07-23 | 2009-01-29 | Dharmacon, Inc. | Screening of micro-rna cluster inhibitor pools |
US9144546B2 (en) | 2007-08-06 | 2015-09-29 | Clsn Laboratories, Inc. | Nucleic acid-lipopolymer compositions |
US20090042825A1 (en) | 2007-08-06 | 2009-02-12 | Majed Matar | Composition, method of preparation & application of concentrated formulations of condensed nucleic acids with a cationic lipopolymer |
CN101835903A (en) | 2007-08-23 | 2010-09-15 | 诺瓦提斯公司 | Methods for detecting oligonucleotides |
WO2009030254A1 (en) | 2007-09-04 | 2009-03-12 | Curevac Gmbh | Complexes of rna and cationic peptides for transfection and for immunostimulation |
EP2197918B1 (en) | 2007-09-05 | 2013-12-18 | F. Hoffmann-La Roche AG | Combination therapy with type i and type ii anti-cd20 antibodies |
US8506928B2 (en) | 2007-09-07 | 2013-08-13 | The Regents Of The University Of California | Methods and compounds for targeting tissues |
US20110086904A1 (en) | 2007-09-17 | 2011-04-14 | The Trustees Of The University Of Pennsylvania | GENERATION OF HYPERSTABLE mRNAs |
EP2195009B1 (en) | 2007-09-26 | 2014-07-30 | Oregon Health and Science University | Cyclic undecapeptides and derivatives as multiple sclerosis therapies |
JP5514727B2 (en) | 2007-09-26 | 2014-06-04 | イントレキソン コーポレーション | Synthetic 5'UTR, expression vector, and method for increasing expression of a transgene |
SI2644192T1 (en) | 2007-09-28 | 2017-08-31 | Pfizer Inc. | Cancer Cell Targeting Using Nanoparticles |
EP2042193A1 (en) | 2007-09-28 | 2009-04-01 | Biomay AG | RNA Vaccines |
WO2009046388A1 (en) | 2007-10-03 | 2009-04-09 | United States Medical Research & Material Command | Cr-2 binding peptide p28 as molecular adjuvant for dna vaccines |
WO2009046738A1 (en) | 2007-10-09 | 2009-04-16 | Curevac Gmbh | Composition for treating lung cancer, particularly of non-small lung cancers (nsclc) |
WO2009046739A1 (en) | 2007-10-09 | 2009-04-16 | Curevac Gmbh | Composition for treating prostate cancer (pca) |
EP2630967A1 (en) | 2007-10-12 | 2013-08-28 | Massachusetts Institute of Technology | Vaccine nanotechnology |
US20090098118A1 (en) | 2007-10-15 | 2009-04-16 | Thomas Friess | Combination therapy of a type ii anti-cd20 antibody with an anti-bcl-2 active agent |
WO2009052830A1 (en) | 2007-10-22 | 2009-04-30 | Genmab A/S | Novel antibody therapies |
AU2008319183B2 (en) | 2007-11-01 | 2014-09-04 | University Of Rochester | Recombinant factor VIII having increased stability |
CA2705263A1 (en) | 2007-11-09 | 2009-05-14 | Novartis Ag | Combination therapy with an antagonist anti-cd 40 antibody and cyclophosphamide, doxorubicin, vincristine and prednisone (chop) for treatment of b-cell malignancies |
EP2223934B1 (en) | 2007-11-14 | 2012-10-03 | Institute Of Microbiology, Chinese Academy Of Sciences | Polypeptides for inhibiting influenza virus infection |
US20090148905A1 (en) | 2007-11-30 | 2009-06-11 | Claire Ashman | Antigen-binding constructs |
CA2707042A1 (en) | 2007-12-10 | 2009-06-18 | Alnylam Pharmaceuticals, Inc. | Compositions and methods for inhibiting expression of factor vii gene |
CN103911378A (en) | 2007-12-11 | 2014-07-09 | 斯克利普斯研究所 | Compositions and methods related to mRNA translational enhancer elements |
US20090238772A1 (en) | 2007-12-13 | 2009-09-24 | Alnylam Pharmaceuticals, Inc. | Methods and compositions for prevention or treatment of rsv infection |
EP2072618A1 (en) | 2007-12-14 | 2009-06-24 | Johannes Gutenberg-Universität Mainz | Use of RNA for reprogramming somatic cells |
EP2231183A2 (en) | 2007-12-21 | 2010-09-29 | Genentech, Inc. | Therapy of rituximab-refractory rheumatoid arthritis patients |
KR20100120663A (en) | 2008-01-23 | 2010-11-16 | 아지노모토 가부시키가이샤 | Method of producing l-amino acid |
JPWO2009093384A1 (en) | 2008-01-24 | 2011-05-26 | 独立行政法人産業技術総合研究所 | Polynucleotide, polynucleotide analogue and gene expression control method using the same |
BRPI0907087B1 (en) | 2008-01-31 | 2021-08-17 | Curevac Ag | RNA MOLECULE, PHARMACEUTICAL COMPOSITION AND ITS USES IN THE TREATMENT OF DISEASES |
WO2009101407A2 (en) | 2008-02-11 | 2009-08-20 | Cambridge Enterprise Limited | Improved reprogramming of mammalian cells, and the cells obtained |
DK2240155T3 (en) | 2008-02-13 | 2012-09-17 | Intarcia Therapeutics Inc | Devices, formulations and methods for the delivery of several beneficial agents |
DE102008009920A1 (en) | 2008-02-15 | 2009-08-20 | Aj Innuscreen Gmbh | Mobile device for nucleic acid isolation |
US8506966B2 (en) | 2008-02-22 | 2013-08-13 | Novartis Ag | Adjuvanted influenza vaccines for pediatric use |
US20120027813A1 (en) | 2008-02-22 | 2012-02-02 | Novartis Vaccines And Diagnostics Srl | Adjuvanted influenza vaccines for pediatric use |
WO2009108891A2 (en) | 2008-02-29 | 2009-09-03 | Egen, Inc. | Modified poloxamers for gene expression and associated methods |
NZ587632A (en) | 2008-03-14 | 2012-06-29 | Biocon Ltd | An anti-cd6 monoclonal antibody and a method thereof |
CN102027043A (en) | 2008-03-14 | 2011-04-20 | 艾根股份有限公司 | Biodegradable cross-linked branched poly (alkylene imines) |
EA201071137A1 (en) | 2008-03-28 | 2011-04-29 | ГЛЭКСОСМИТКЛАЙН ЭлЭлСи | METHODS OF TREATMENT |
WO2009127060A1 (en) | 2008-04-15 | 2009-10-22 | Protiva Biotherapeutics, Inc. | Novel lipid formulations for nucleic acid delivery |
JP5539962B2 (en) | 2008-04-25 | 2014-07-02 | ノースウェスタン、ユニバーシティ | Nanostructure suitable for sequestering cholesterol |
CA2725601A1 (en) | 2008-04-28 | 2009-11-05 | President And Fellows Of Harvard College | Supercharged proteins for cell penetration |
WO2009134717A1 (en) | 2008-04-30 | 2009-11-05 | The Government Of The United States Of America As Represented By The Secretary Of The Department Of Health And Human Services, Centers For Disease Control And Prevention | Chimeric west nile/dengue viruses |
US9394538B2 (en) | 2008-05-07 | 2016-07-19 | Shi-Lung Lin | Development of universal cancer drugs and vaccines |
EP2891501B1 (en) | 2008-05-08 | 2016-09-21 | MiniPumps, LLC | Methods of manufacture of drug-delivery pumps |
US8697098B2 (en) | 2011-02-25 | 2014-04-15 | South Dakota State University | Polymer conjugated protein micelles |
JP5911723B2 (en) | 2008-05-13 | 2016-04-27 | ユニヴァーシティ オブ ワシントン | Diblock copolymer and polynucleotide complex thereof for delivery into cells |
EP2297323A1 (en) | 2008-05-21 | 2011-03-23 | Hartmann, Gunther | 5' triphosphate oligonucleotide with blunt end and uses thereof |
KR101275950B1 (en) | 2008-05-29 | 2013-06-25 | 한올바이오파마주식회사 | Modified erythropoietin (epo) polypeptides that exhibit increased protease resistance and pharmaceutical compositions thereof |
FR2931824B1 (en) | 2008-05-29 | 2014-11-28 | Centre Nat Rech Scient | PROCESS FOR RNA SYNTHESIS THROUGH CHEMICAL. |
WO2009148528A2 (en) | 2008-05-30 | 2009-12-10 | Millennium Pharmaceuticals, Inc. | Assessment of chromosomal alterations to predict clinical outcome of bortezomib treatment |
PL215513B1 (en) | 2008-06-06 | 2013-12-31 | Univ Warszawski | New borane phosphate analogs of dinucleotides, their application, RNA particle, method of obtaining RNA and method of obtaining peptides or protein |
TWI451876B (en) | 2008-06-13 | 2014-09-11 | Lilly Co Eli | Pegylated insulin lispro compounds |
WO2010005723A2 (en) | 2008-06-16 | 2010-01-14 | Bind Biosciences, Inc. | Drug loaded polymeric nanoparticles and methods of making and using same |
WO2010005726A2 (en) | 2008-06-16 | 2010-01-14 | Bind Biosciences Inc. | Therapeutic polymeric nanoparticles with mtor inhibitors and methods of making and using same |
DK2285350T3 (en) | 2008-06-16 | 2017-12-11 | Pfizer | PROCEDURES FOR THE PREPARATION OF DIBLOCK COPOLYMERS FUNCTIONALIZED WITH TARGETING AGENT FOR USE IN THE MANUFACTURE OF THERAPEUTIC NANOPARTICLES |
US20100009424A1 (en) | 2008-07-14 | 2010-01-14 | Natasha Forde | Sonoporation systems and methods |
EP2313085A2 (en) | 2008-07-15 | 2011-04-27 | Novartis AG | Immunogenic amphipathic peptide compositions |
WO2010009065A2 (en) | 2008-07-15 | 2010-01-21 | Novartis Ag | Amphipathic peptide compositions |
EP2331561A4 (en) | 2008-09-03 | 2013-02-27 | Xenome Ltd | Libraries of peptide conjugates and methods for making them |
AU2009288632B2 (en) | 2008-09-06 | 2015-08-13 | Chemgenes Corporation | RNA synthesis - phosphoramidites for synthetic RNA in the reverse direction, and application in convenient introduction of ligands, chromophores and modifications of synthetic RNA at the 3' - end |
WO2010027903A2 (en) | 2008-09-08 | 2010-03-11 | Fred Hutchinson Cancer Research Center | Lung cancer diagnosis |
US20100087337A1 (en) | 2008-09-10 | 2010-04-08 | Bind Biosciences, Inc. | High Throughput Fabrication of Nanoparticles |
TW201438738A (en) | 2008-09-16 | 2014-10-16 | Genentech Inc | Methods for treating progressive multiple sclerosis |
WO2010033906A2 (en) | 2008-09-19 | 2010-03-25 | President And Fellows Of Harvard College | Efficient induction of pluripotent stem cells using small molecule compounds |
WO2010037408A1 (en) | 2008-09-30 | 2010-04-08 | Curevac Gmbh | Composition comprising a complexed (m)rna and a naked mrna for providing or enhancing an immunostimulatory response in a mammal and uses thereof |
WO2010042490A1 (en) | 2008-10-06 | 2010-04-15 | Boston Medical Center Corporation | A single lentiviral vector system for induced pluripotent (ips) stem cells derivation |
EP2350043B9 (en) | 2008-10-09 | 2014-08-20 | TEKMIRA Pharmaceuticals Corporation | Improved amino lipids and methods for the delivery of nucleic acids |
US8535655B2 (en) | 2008-10-10 | 2013-09-17 | Polyactiva Pty Ltd. | Biodegradable polymer—bioactive moiety conjugates |
US8343498B2 (en) | 2008-10-12 | 2013-01-01 | Massachusetts Institute Of Technology | Adjuvant incorporation in immunonanotherapeutics |
WO2010047765A2 (en) | 2008-10-20 | 2010-04-29 | Massachussetts Institute Of Technology | Nanostructures for drug delivery |
WO2010047839A1 (en) | 2008-10-25 | 2010-04-29 | Aura Biosciences | Modified plant virus particles and uses therefor |
CN102245559B (en) | 2008-11-07 | 2015-05-27 | 麻省理工学院 | Aminoalcohol lipidoids and uses thereof |
CN105709229B (en) | 2008-11-10 | 2020-07-28 | 阿布特斯生物制药公司 | Novel lipids and compositions for delivery of therapeutic agents |
WO2010057203A2 (en) | 2008-11-17 | 2010-05-20 | The Board Of Regents Of The University Of Texas System | Hdl particles for delivery of nucleic acids |
EP2191840A1 (en) | 2008-11-28 | 2010-06-02 | Sanofi-Aventis | Antitumor combinations containing antibodies recognizing specifically CD38 and melphalan |
EP2196476A1 (en) | 2008-12-10 | 2010-06-16 | Novartis Ag | Antibody formulation |
US8324368B2 (en) | 2008-12-10 | 2012-12-04 | Alnylam Pharmaceuticals, Inc. | GNAQ targeted dsRNA compositions and methods for inhibiting expression |
WO2010068866A2 (en) | 2008-12-12 | 2010-06-17 | Bind Biosciences | Therapeutic particles suitable for parenteral administration and methods of making and using same |
WO2010068918A2 (en) | 2008-12-12 | 2010-06-17 | The Regents Of The University Of California | Novel targets for treatment of hypercholesterolemia |
EA201100765A1 (en) | 2008-12-15 | 2012-04-30 | Бинд Биосаиэнсис | Long-term circulation nanoparticles |
BRPI1006829A2 (en) | 2009-01-16 | 2016-10-25 | Glaxosmithkline Llc | cancer treatment employing a combination of bendamustine and an anti-cd20 antibody |
WO2010084371A1 (en) | 2009-01-26 | 2010-07-29 | Mitoprod | Novel circular interfering rna molecules |
EP3243504A1 (en) | 2009-01-29 | 2017-11-15 | Arbutus Biopharma Corporation | Improved lipid formulation |
WO2010088927A1 (en) | 2009-02-09 | 2010-08-12 | Curevac Gmbh | Use of pei for the improvement of endosomal release and expression of transfected nucleic acids, complexed with cationic or polycationic compounds |
US20140141089A1 (en) | 2009-02-11 | 2014-05-22 | Colorado School Of Mines | Nanoparticles, Compositions Thereof, and Methods of Use, and Methods of Making the Same |
JP5735927B2 (en) | 2009-02-24 | 2015-06-17 | ザ スクリプス リサーチ インスティテュート | Re-engineering the primary structure of mRNA to enhance protein production |
WO2010141135A2 (en) | 2009-03-05 | 2010-12-09 | Trustees Of Boston University | Bacteriophages expressing antimicrobial peptides and uses thereof |
WO2010102065A1 (en) | 2009-03-05 | 2010-09-10 | Bend Research, Inc. | Pharmaceutical compositions of dextran polymer derivatives |
KR101579771B1 (en) | 2009-03-05 | 2015-12-28 | 애브비 인코포레이티드 | IL-17 binding proteins |
WO2010105277A1 (en) | 2009-03-13 | 2010-09-16 | Egen, Inc. | Compositions and methods for the delivery of biologically active rnas |
AU2010226434A1 (en) | 2009-03-20 | 2011-10-13 | Egen, Inc. | Polyamine derivatives |
JP5622254B2 (en) | 2009-03-31 | 2014-11-12 | 国立大学法人東京大学 | Double-stranded ribonucleic acid polyion complex |
WO2011127032A1 (en) | 2010-04-05 | 2011-10-13 | University Of Chicago | Compositions and methods related to protein a (spa) antibodies as an enhancer of immune response |
JP5789250B2 (en) | 2009-04-03 | 2015-10-07 | ザ・ユニバーシティ・オブ・シカゴThe University Of Chicago | Compositions and methods related to protein A (SpA) variants |
EP2419143B8 (en) | 2009-04-13 | 2018-06-27 | INSERM - Institut National de la Santé et de la Recherche Médicale | Hpv particles and uses thereof |
AU2010236257A1 (en) | 2009-04-17 | 2011-11-03 | Biogen Idec Ma Inc. | Compositions and methods to treat acute myelogenous leukemia |
EP2421563B1 (en) | 2009-04-22 | 2017-04-12 | Massachusetts Institute of Technology | Innate immune suppression enables repeated delivery of long rna molecules |
NZ595235A (en) | 2009-04-27 | 2013-06-28 | Novartis Ag | Compositions and methods for increasing muscle growth |
US20110033389A1 (en) | 2009-04-29 | 2011-02-10 | Zhifeng Chen | Modified antibodies for passive immunotherapy |
WO2010127159A2 (en) | 2009-04-30 | 2010-11-04 | Intezyne Technologies, Incorporated | Polymeric micelles for polynucleotide encapsulation |
US8715736B2 (en) | 2009-04-30 | 2014-05-06 | Florida Agricultural And Mechanical University | Nanoparticle formulations for skin delivery |
NZ711583A (en) | 2009-05-05 | 2017-03-31 | Arbutus Biopharma Corp | Lipid compositions |
DE202009007116U1 (en) | 2009-05-18 | 2010-10-14 | Amoena Medizin-Orthopädie-Technik GmbH | Anti decubitus cushions |
US8574835B2 (en) | 2009-05-29 | 2013-11-05 | Life Technologies Corporation | Scaffolded nucleic acid polymer particles and methods of making and using |
EP3431076B1 (en) | 2009-06-10 | 2021-10-06 | Arbutus Biopharma Corporation | Improved lipid formulation |
EP2440556A1 (en) | 2009-06-10 | 2012-04-18 | Vertex Pharmaceuticals Incorporated | Inhibitors of phosphatidylinositol 3-kinase |
US8273869B2 (en) | 2009-06-15 | 2012-09-25 | Alnylam Pharmaceuticals, Inc. | Lipid formulated dsRNA targeting the PCSK9 gene |
US20110097329A1 (en) | 2009-06-26 | 2011-04-28 | Massachusetts Institute Of Technology | Compositions and methods for treating cancer and modulating stress granule formation |
ES2613498T3 (en) | 2009-07-01 | 2017-05-24 | Protiva Biotherapeutics Inc. | New lipid formulations for the delivery of therapeutic agents to solid tumors |
US8569256B2 (en) | 2009-07-01 | 2013-10-29 | Protiva Biotherapeutics, Inc. | Cationic lipids and methods for the delivery of therapeutic agents |
WO2011005799A2 (en) * | 2009-07-06 | 2011-01-13 | Novartis Ag | Self replicating rna molecules and uses thereof |
AU2010278309B2 (en) | 2009-07-31 | 2013-10-31 | Ethris Gmbh | RNA with a combination of unmodified and modified nucleotides for protein expression |
EP2281579A1 (en) | 2009-08-05 | 2011-02-09 | BioNTech AG | Vaccine composition comprising 5'-Cap modified RNA |
US20110053829A1 (en) | 2009-09-03 | 2011-03-03 | Curevac Gmbh | Disulfide-linked polyethyleneglycol/peptide conjugates for the transfection of nucleic acids |
US20110070227A1 (en) | 2009-09-18 | 2011-03-24 | Anna-Marie Novotney-Barry | Treatment of Autoimmune and Inflammatory Diseases |
US8859284B2 (en) | 2009-10-22 | 2014-10-14 | The United States Of America, As Represented By The Secretary Of The Navy | Delivery of nanoparticles to neurons |
US8449916B1 (en) | 2009-11-06 | 2013-05-28 | Iowa State University Research Foundation, Inc. | Antimicrobial compositions and methods |
WO2011060250A1 (en) | 2009-11-13 | 2011-05-19 | Bend Research, Inc. | Cationic dextran polymer derivatives |
WO2011062965A2 (en) | 2009-11-18 | 2011-05-26 | University Of Washington Through Its Center For Commercialization | Targeting monomers and polymers having targeting blocks |
US8530429B2 (en) | 2009-11-24 | 2013-09-10 | Arch Cancer Therapeutics, Inc. | Brain tumor targeting peptides and methods |
US20110244026A1 (en) | 2009-12-01 | 2011-10-06 | Braydon Charles Guild | Delivery of mrna for the augmentation of proteins and enzymes in human genetic diseases |
US20110245756A1 (en) | 2009-12-03 | 2011-10-06 | Rishi Arora | Devices for material delivery, electroporation, sonoporation, and/or monitoring electrophysiological activity |
DE102009056884B4 (en) | 2009-12-03 | 2021-03-18 | Novartis Ag | Vaccine Adjuvants and Improved Methods for Making Same |
ME02964B (en) | 2009-12-06 | 2018-07-20 | Factor viii-fc chimeric and hybrid polypeptides, and methods of use thereof | |
SI3112467T1 (en) | 2009-12-07 | 2018-06-29 | The Trustees Of The University Of Pennsylvania | Rna preparations comprising purified modified rna for reprogramming cells |
US20130189741A1 (en) | 2009-12-07 | 2013-07-25 | Cellscript, Inc. | Compositions and methods for reprogramming mammalian cells |
US9687550B2 (en) | 2009-12-07 | 2017-06-27 | Arbutus Biopharma Corporation | Compositions for nucleic acid delivery |
WO2011069528A1 (en) | 2009-12-09 | 2011-06-16 | Curevac Gmbh | Lyophilization of nucleic acids in lactate-containing solutions |
WO2011069529A1 (en) | 2009-12-09 | 2011-06-16 | Curevac Gmbh | Mannose-containing solution for lyophilization, transfection and/or injection of nucleic acids |
CN102811743B (en) | 2009-12-11 | 2015-11-25 | 佰恩德治疗股份有限公司 | The stabilization formulations of lyophilizing treatment granule |
ES2780156T3 (en) | 2009-12-15 | 2020-08-24 | Pfizer | Therapeutic compositions of polymeric nanoparticles with high glass transition temperature or high molecular weight copolymers |
JP6175237B2 (en) | 2009-12-15 | 2017-08-02 | ファイザー・インク | Therapeutic polymer nanoparticles containing corticosteroids and methods of making and using the same |
EA201290506A1 (en) | 2009-12-16 | 2013-03-29 | Брихэм Энд Уимен'З Хоспитал, Инк. | PARTICLES FOR DELIVERY OF A SET OF AGENTS |
DE102009058769A1 (en) | 2009-12-16 | 2011-06-22 | MagForce Nanotechnologies AG, 10589 | Temperature-dependent activation of catalytic nucleic acids for controlled drug release |
US20130017223A1 (en) | 2009-12-18 | 2013-01-17 | The University Of British Columbia | Methods and compositions for delivery of nucleic acids |
EP2338520A1 (en) | 2009-12-21 | 2011-06-29 | Ludwig Maximilians Universität | Conjugate with targeting ligand and use of same |
KR101762466B1 (en) | 2009-12-23 | 2017-07-27 | 노파르티스 아게 | Lipids, lipid compositions, and methods of using them |
WO2011088309A1 (en) | 2010-01-14 | 2011-07-21 | Regulus Therapeutics Inc. | Microrna compositions and methods |
KR101956623B1 (en) | 2010-02-24 | 2019-03-12 | 애로우헤드 파마슈티컬스 인코포레이티드 | Compositions for Targeted Delivery of siRNA |
CA2791278C (en) | 2010-02-25 | 2015-11-24 | The Johns Hopkins University | Sustained delivery of therapeutic agents to an eye compartment |
US20130133483A1 (en) | 2010-03-08 | 2013-05-30 | University Of Rochester | Synthesis of Nanoparticles Using Reducing Gases |
KR20130006663A (en) | 2010-03-16 | 2013-01-17 | 유니버시티 오브 유타 리서치 파운데이션 | Cleavable modifications to reducible poly(amido ethylenimine)s to enhance nucleotide delivery |
WO2011116072A1 (en) | 2010-03-16 | 2011-09-22 | Escape Therapeutics, Inc. | Hybrid hydrogel scaffold compositions and methods of use |
US20110230816A1 (en) | 2010-03-18 | 2011-09-22 | Tyco Healthcare Group Lp | Gels for Transdermal Delivery |
US9149432B2 (en) | 2010-03-19 | 2015-10-06 | Massachusetts Institute Of Technology | Lipid vesicle compositions and methods of use |
GB201005005D0 (en) | 2010-03-25 | 2010-05-12 | Angeletti P Ist Richerche Bio | New vaccine |
WO2011120053A1 (en) | 2010-03-26 | 2011-09-29 | Mersana Therapeutics, Inc. | Modified polymers for delivery of polynucleotides, method of manufacture, and methods of use thereof |
WO2011119262A1 (en) | 2010-03-26 | 2011-09-29 | Cerulean Pharma Inc. | Methods and systems for generating nanoparticles |
US20110247090A1 (en) | 2010-04-02 | 2011-10-06 | Intrexon Corporation | Synthetic 5'UTRs, Expression Vectors, and Methods for Increasing Transgene Expression |
WO2011127316A1 (en) | 2010-04-07 | 2011-10-13 | Novartis Ag | Method for generating a parvovirus b19 virus-like particle |
EP3391877A1 (en) | 2010-04-08 | 2018-10-24 | The Trustees of Princeton University | Preparation of lipid nanoparticles |
WO2011125469A1 (en) | 2010-04-09 | 2011-10-13 | 国立大学法人東京大学 | Micro-rna-regulated recombinant vaccinia virus and utilization thereof |
ES2745113T3 (en) | 2010-04-09 | 2020-02-27 | Pacira Pharmaceuticals Inc | Method for formulating multivesicular liposomes |
KR101196667B1 (en) | 2010-04-15 | 2012-11-02 | 포항공과대학교 산학협력단 | A DELEVERY SYSTEM OF ANTI-CANCER AGENT USING pH SENSITIVE METAL NANOPARTICLE |
CA2796464C (en) | 2010-04-16 | 2021-08-03 | Immune Disease Institute, Inc. | Sustained polypeptide expression from synthetic, modified rnas and uses thereof |
WO2011127933A1 (en) | 2010-04-16 | 2011-10-20 | Nuevolution A/S | Bi-functional complexes and methods for making and using such complexes |
EP2377938A1 (en) | 2010-04-16 | 2011-10-19 | Eukarys | Capping-prone RNA polymerase enzymes and their applications |
US20130260460A1 (en) | 2010-04-22 | 2013-10-03 | Isis Pharmaceuticals Inc | Conformationally restricted dinucleotide monomers and oligonucleotides |
WO2012046149A1 (en) | 2010-04-28 | 2012-04-12 | Kimberly-Clark Worldwide, Inc. | Method for increasing permeability of an epithelial barrier |
US20130156845A1 (en) | 2010-04-29 | 2013-06-20 | Alnylam Pharmaceuticals, Inc. | Lipid formulated single stranded rna |
JP2013524840A (en) | 2010-04-30 | 2013-06-20 | ノバルティス アーゲー | Predictive markers useful in the treatment of fragile X syndrome (FXS) |
WO2011143230A1 (en) | 2010-05-10 | 2011-11-17 | Alnylam Pharmaceuticals | Methods and compositions for delivery of active agents |
WO2011141704A1 (en) | 2010-05-12 | 2011-11-17 | Protiva Biotherapeutics, Inc | Novel cyclic cationic lipids and methods of use |
JP2013527856A (en) | 2010-05-12 | 2013-07-04 | プロチバ バイオセラピューティクス インコーポレイティッド | Cationic lipids and methods of use |
EP2387999A1 (en) | 2010-05-21 | 2011-11-23 | CureVac GmbH | Histidine-containing solution for transfection and/or injection of nucleic acids and uses thereof |
WO2011149733A2 (en) | 2010-05-24 | 2011-12-01 | Merck Sharp & Dohme Corp. | Novel amino alcohol cationic lipids for oligonucleotide delivery |
JP5957646B2 (en) | 2010-06-04 | 2016-07-27 | サーナ・セラピューティクス・インコーポレイテッドSirna Therapeutics,Inc. | Novel low molecular weight cationic lipids for oligonucleotide delivery |
NZ704192A (en) | 2010-06-14 | 2016-05-27 | Hoffmann La Roche | Cell-penetrating peptides and uses therof |
WO2011163121A1 (en) | 2010-06-21 | 2011-12-29 | Alnylam Pharmaceuticals, Inc. | Multifunctional copolymers for nucleic acid delivery |
EP2585106A1 (en) | 2010-06-25 | 2013-05-01 | Novartis AG | Combinations of meningococcal factor h binding proteins |
CN103079592B (en) | 2010-07-01 | 2015-10-21 | 浦项工科大学校产学协力团 | Use from the microbubble treatment of cell and the method for cancer diagnosis |
EP2588120B1 (en) | 2010-07-02 | 2017-11-15 | The University of Chicago | Compositions and methods related to protein a (spa) variants |
EP4005592B1 (en) | 2010-07-06 | 2022-10-12 | GlaxoSmithKline Biologicals S.A. | Virion-like delivery particles for self-replicating rna molecules |
BR112013000244A2 (en) | 2010-07-06 | 2016-05-17 | Novartis Ag | lipid liposomes having advantageous pka for administration of rna |
NZ606591A (en) | 2010-07-06 | 2015-02-27 | Novartis Ag | Cationic oil-in-water emulsions |
DK2591114T3 (en) | 2010-07-06 | 2016-08-29 | Glaxosmithkline Biologicals Sa | Immunization of large mammals with low doses of RNA |
US9192661B2 (en) | 2010-07-06 | 2015-11-24 | Novartis Ag | Delivery of self-replicating RNA using biodegradable polymer particles |
ES2646669T3 (en) | 2010-07-06 | 2017-12-14 | Glaxosmithkline Biologicals Sa | Procedures for increasing an immune response by providing RNA |
US9770463B2 (en) | 2010-07-06 | 2017-09-26 | Glaxosmithkline Biologicals Sa | Delivery of RNA to different cell types |
EP2591101B1 (en) | 2010-07-09 | 2018-11-07 | Bioverativ Therapeutics Inc. | Systems for factor viii processing and methods thereof |
KR20140017480A (en) | 2010-07-09 | 2014-02-11 | 바이오겐 이데크 헤모필리아 인코포레이티드 | Factor ix polypeptides and methods of use thereof |
WO2012009406A2 (en) | 2010-07-13 | 2012-01-19 | University Of Utah Research Foundation | Gold particles and methods of making and using the same in cancer treatment |
GB201012410D0 (en) | 2010-07-23 | 2010-09-08 | Medical Res Council | Intracellular immunity |
JP5948327B2 (en) | 2010-07-30 | 2016-07-06 | キュアヴァック アーゲー | Nucleic acid complex formation with disulfide-bridged cationic components for transfection and immune stimulation |
WO2012019168A2 (en) | 2010-08-06 | 2012-02-09 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
US9121065B2 (en) | 2010-08-09 | 2015-09-01 | The Trustees Of The University Of Pennsylvania | Nanoparticle-oligonucleotide hybrid structures and methods of use thereof |
WO2012019630A1 (en) | 2010-08-13 | 2012-02-16 | Curevac Gmbh | Nucleic acid comprising or coding for a histone stem-loop and a poly(a) sequence or a polyadenylation signal for increasing the expression of an encoded protein |
US20130142868A1 (en) | 2010-08-20 | 2013-06-06 | University Of Washington | Circumferential Aerosol Device for Delivering Drugs to Olfactory Epithelium and Brain |
CA2808965C (en) | 2010-08-20 | 2020-01-07 | Novartis Ag | Soluble needle arrays for delivery of influenza vaccines |
EA201390145A1 (en) | 2010-08-20 | 2013-11-29 | Серулин Фарма Инк. | CONJUGATES, PARTICLES, COMPOSITIONS AND RELATED METHODS |
HRP20230326T1 (en) | 2010-08-31 | 2023-06-09 | Glaxosmithkline Biologicals Sa | Small liposomes for delivery of immunogen-encoding rna |
SI3556396T1 (en) | 2010-08-31 | 2022-09-30 | Theraclone Sciences, Inc. | Human immunodeficiency virus (hiv)-neutralizing antibodies |
AU2011296268A1 (en) | 2010-08-31 | 2013-02-21 | Merck Sharp & Dohme Corp. | Novel single chemical entities and methods for delivery of oligonucleotides |
DK4066857T3 (en) | 2010-08-31 | 2023-02-20 | Glaxosmithkline Biologicals Sa | Pegylated liposomes for delivery of immunogen-encoding RNA |
EP2611420B1 (en) | 2010-08-31 | 2019-03-27 | GlaxoSmithKline Biologicals SA | Lipids suitable for liposomal delivery of protein-coding rna |
US9549901B2 (en) | 2010-09-03 | 2017-01-24 | The Brigham And Women's Hospital, Inc. | Lipid-polymer hybrid particles |
WO2012034077A2 (en) | 2010-09-09 | 2012-03-15 | The University Of Chicago | Compositions and methods related to attenuated staphylococcal strains |
JP5793194B2 (en) | 2010-09-09 | 2015-10-14 | ザ・ユニバーシティ・オブ・シカゴThe University Of Chicago | Methods and compositions involving protective staphylococcal antigens |
WO2012039979A2 (en) | 2010-09-10 | 2012-03-29 | The Johns Hopkins University | Rapid diffusion of large polymeric nanoparticles in the mammalian brain |
US8466122B2 (en) | 2010-09-17 | 2013-06-18 | Protiva Biotherapeutics, Inc. | Trialkyl cationic lipids and methods of use thereof |
EP3943114A1 (en) | 2010-09-20 | 2022-01-26 | Sirna Therapeutics, Inc. | Novel low molecular weight cationic lipids for oligonucleotide delivery |
US20130183718A1 (en) | 2010-09-21 | 2013-07-18 | RibpxX GmbH | Method for Synthesizing RNA using DNA Template |
EP3412279B1 (en) | 2010-09-24 | 2021-02-17 | The Brigham and Women's Hospital, Inc. | Nanostructured gels capable of controlled release of encapsulated agents |
WO2012050975A2 (en) | 2010-09-29 | 2012-04-19 | The University Of North Carolina At Chapel Hill | Novel circular mammalian rna molecules and uses thereof |
JP2013545723A (en) | 2010-09-30 | 2013-12-26 | メルク・シャープ・エンド・ドーム・コーポレイション | Low molecular weight cationic lipids for oligonucleotide delivery |
LT3590949T (en) | 2010-10-01 | 2022-07-25 | Modernatx, Inc. | Ribonucleic acids containing n1-methyl-pseudouracils and uses thereof |
US10078075B2 (en) | 2011-12-09 | 2018-09-18 | Vanderbilt University | Integrated organ-on-chip systems and applications of the same |
CA2814386C (en) | 2010-10-11 | 2019-08-20 | Novartis Ag | Antigen delivery platforms |
CN103167928B (en) | 2010-10-19 | 2015-01-21 | 三菱电机株式会社 | Laser processing machine control device and laser processing machine control method |
EP3485913A1 (en) | 2010-10-21 | 2019-05-22 | Sirna Therapeutics, Inc. | Low molecular weight cationic lipids for oligonucleotide delivery |
SG10201508700XA (en) | 2010-10-29 | 2015-11-27 | Merck Sharp & Dohme | Recombinant subunit dengue virus vaccine |
AU2011323250B2 (en) | 2010-11-05 | 2015-11-19 | The Johns Hopkins University | Compositions and methods relating to reduced mucoadhesion |
CA2811113A1 (en) | 2010-11-09 | 2012-05-18 | The Regents Of The University Of California | Skin permeating and cell entering (space) peptides and methods of use thereof |
US8927692B2 (en) | 2010-11-12 | 2015-01-06 | The Trustees Of The University Of Pennsylvania | Consensus prostate antigens, nucleic acid molecule encoding the same and vaccine and uses comprising the same |
KR20130116890A (en) | 2010-11-16 | 2013-10-24 | 셀렉타 바이오사이언시즈, 인크. | Immunostimulatory oligonucleotides |
HUE039747T2 (en) | 2010-11-17 | 2019-02-28 | Aduro Biotech Inc | Methods and compositions for inducing an immune response to egfrviii |
WO2012068470A2 (en) | 2010-11-19 | 2012-05-24 | Idera Pharmaceuticals, Inc. | Immune regulatory oligonucleotide (iro) compounds to modulate toll-like receptor based immune response |
WO2012075040A2 (en) | 2010-11-30 | 2012-06-07 | Shire Human Genetic Therapies, Inc. | mRNA FOR USE IN TREATMENT OF HUMAN GENETIC DISEASES |
WO2012072096A1 (en) | 2010-12-03 | 2012-06-07 | Biontech Ag | Method for cellular rna expression |
WO2012103985A2 (en) | 2010-12-16 | 2012-08-09 | Steve Pascolo | Pharmaceutical composition consisting of rna having alkali metal as counter ion and formulated with dications |
US8501930B2 (en) | 2010-12-17 | 2013-08-06 | Arrowhead Madison Inc. | Peptide-based in vivo siRNA delivery system |
JP6088438B2 (en) | 2010-12-22 | 2017-03-01 | プレジデント アンド フェローズ オブ ハーバード カレッジ | Continuous directed evolution |
CN104328121A (en) | 2010-12-29 | 2015-02-04 | 弗·哈夫曼-拉罗切有限公司 | Small molecule conjugates for intracellular delivery of nucleic acids |
WO2012089225A1 (en) | 2010-12-29 | 2012-07-05 | Curevac Gmbh | Combination of vaccination and inhibition of mhc class i restricted antigen presentation |
CA2862765A1 (en) | 2011-01-04 | 2012-07-12 | Brown University | Nanotubes as carriers of nucleic acids into cells |
WO2012094574A2 (en) | 2011-01-06 | 2012-07-12 | The Johns Hopkins University | Stabilized polyribonucleotide nanoparticles |
US20140080766A1 (en) | 2011-01-07 | 2014-03-20 | Massachusetts Institute Of Technology | Compositions and methods for macromolecular drug delivery |
CA2824526C (en) | 2011-01-11 | 2020-07-07 | Alnylam Pharmaceuticals, Inc. | Pegylated lipids and their use for drug delivery |
WO2012099805A2 (en) | 2011-01-19 | 2012-07-26 | Ocean Nanotech, Llc | Nanoparticle based immunological stimulation |
WO2012101235A1 (en) | 2011-01-26 | 2012-08-02 | Cenix Bioscience Gmbh | Delivery system and conjugates for compound delivery via naturally occurring intracellular transport routes |
US10363309B2 (en) | 2011-02-04 | 2019-07-30 | Case Western Reserve University | Targeted nanoparticle conjugates |
WO2012109121A1 (en) | 2011-02-07 | 2012-08-16 | Purdue Research Foundation | Carbohydrate nanoparticles for prolonged efficacy of antimicrobial peptide |
WO2012116715A1 (en) | 2011-03-02 | 2012-09-07 | Curevac Gmbh | Vaccination in newborns and infants |
US20120207840A1 (en) | 2011-02-10 | 2012-08-16 | Aura Biosciences, Inc. | Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For The Treatment Of Non-Melanoma Skin Cancer |
AU2012217788A1 (en) | 2011-02-14 | 2013-08-29 | Swift Biosciences, Inc. | Polynucleotide primers and probes |
CA2827118A1 (en) | 2011-02-15 | 2012-08-23 | Merrimack Pharmaceuticals, Inc. | Compositions and methods for delivering nucleic acid to a cell |
WO2012112689A1 (en) | 2011-02-15 | 2012-08-23 | The University Of North Carolina At Chapel Hill | Nanoparticle, liposomes, polymers, agents and proteins modified with reversible linkers |
EP2489371A1 (en) | 2011-02-18 | 2012-08-22 | Instituto Nacional de Investigacion y Tecnologia Agraria y Alimentaria | Carrier peptides for drug delivery |
WO2012113413A1 (en) | 2011-02-21 | 2012-08-30 | Curevac Gmbh | Vaccine composition comprising complexed immunostimulatory nucleic acids and antigens packaged with disulfide-linked polyethyleneglycol/peptide conjugates |
CN103492574B (en) | 2011-02-22 | 2015-12-09 | 加州理工学院 | Use adeno-associated virus (AAV) vehicle delivery albumen |
US8696637B2 (en) | 2011-02-28 | 2014-04-15 | Kimberly-Clark Worldwide | Transdermal patch containing microneedles |
US20140112950A1 (en) | 2011-03-02 | 2014-04-24 | Manmohan Singh | Combination vaccines with lower doses of antigen and/or adjuvant |
WO2012116714A1 (en) | 2011-03-02 | 2012-09-07 | Curevac Gmbh | Vaccination in elderly patients |
CN103502436A (en) | 2011-03-07 | 2014-01-08 | 麻省理工学院 | Methods for transfecting cells with nucleic acids |
WO2012125680A1 (en) | 2011-03-16 | 2012-09-20 | Novartis Ag | Methods of treating vasculitis using an il-17 binding molecule |
WO2012125987A2 (en) | 2011-03-17 | 2012-09-20 | Massachusetts Institute Of Technology | Delivery system |
KR20140012137A (en) | 2011-03-17 | 2014-01-29 | 노파르티스 아게 | Fgfr and ligands thereof as biomarkers for breast cancer in hr positive subjects |
US10357568B2 (en) | 2011-03-24 | 2019-07-23 | Glaxosmithkline Biologicals S.A. | Adjuvant nanoemulsions with phospholipids |
US9238716B2 (en) | 2011-03-28 | 2016-01-19 | Massachusetts Institute Of Technology | Conjugated lipomers and uses thereof |
CN103476949A (en) | 2011-03-28 | 2013-12-25 | 诺瓦提斯公司 | Markers associated with cyclin-dependent kinase inhibitors |
JP2014511687A (en) | 2011-03-31 | 2014-05-19 | モデルナ セラピューティクス インコーポレイテッド | Engineered nucleic acid delivery and formulation |
WO2012138453A1 (en) | 2011-04-03 | 2012-10-11 | The General Hospital Corporation | Efficient protein expression in vivo using modified rna (mod-rna) |
EP2694524B1 (en) | 2011-04-04 | 2016-05-18 | The U.S.A. As Represented By The Secretary, Department Of Health And Human Services | 2'-o-aminooxymethyl nucleoside derivatives for use in the synthesis and modification of nucleosides, nucleotides and oligonucleotides |
WO2012142132A1 (en) | 2011-04-11 | 2012-10-18 | Life Technologies Corporation | Polymer particles and methods of making and using same |
US11135174B2 (en) | 2011-04-13 | 2021-10-05 | The Trustees Of The University Of Pennsylvania | Coated mesoporous nanoparticles |
WO2013158127A1 (en) | 2012-04-16 | 2013-10-24 | Molecular Transfer, Inc. | Agents for improved delivery of nucleic acids to eukaryotic cells |
US20140178894A1 (en) | 2011-04-20 | 2014-06-26 | Novartis Forschungsstiftung, Zweigniederlassung | Culture medium suitable for the culture of undifferentiated cells |
US20140287022A1 (en) | 2011-04-26 | 2014-09-25 | Molecular Express, Inc. | Liposomal formulations |
AU2012249274A1 (en) | 2011-04-28 | 2013-10-31 | Sloan-Kettering Institute For Cancer Research | Neutralizing antibodies to Nipah and Hendra virus |
CN103687590A (en) | 2011-04-28 | 2014-03-26 | Stc·Unm公司 | Porous nanoparticle-supported lipid bilayers (protocells) for targeted delivery and methods of using same |
US20120301510A1 (en) | 2011-04-29 | 2012-11-29 | Selecta Biosciences, Inc. | Tolerogenic synthetic nanocarriers coupled to cd1d-restricted antigens and methods of use |
US20140056912A1 (en) | 2011-04-29 | 2014-02-27 | Novartis Ag | Methods of treating squamous cell carcinoma |
PL2705143T3 (en) | 2011-05-02 | 2021-07-19 | Wayne State University | A protein-induced pluripotent cell technology uses thereof |
UA116189C2 (en) | 2011-05-02 | 2018-02-26 | Мілленніум Фармасьютікалз, Інк. | FORMULATION FOR ANTI-α4β7 ANTIBODY |
US8945588B2 (en) | 2011-05-06 | 2015-02-03 | The University Of Chicago | Methods and compositions involving protective staphylococcal antigens, such as EBH polypeptides |
WO2012152810A1 (en) | 2011-05-10 | 2012-11-15 | Basf Se | Oil-in-water emulsions |
EP2707033A1 (en) | 2011-05-11 | 2014-03-19 | Ramot at Tel Aviv University, Ltd. | Targeted polymeric conjugates and uses thereof |
CA2835492A1 (en) | 2011-05-12 | 2012-11-15 | Helmut Vockner | Novel pharmaceutical formulation |
DK2706988T3 (en) | 2011-05-12 | 2020-01-20 | Yissum Res Dev Co Of Hebrew Univ Jerusalem Ltd | LIPOSOMES COMPREHENSIVE POLYMER CONJUGATED LIPIDS AND RELATED USE |
RU2013155485A (en) | 2011-05-13 | 2015-06-20 | Новартис Аг | F RSV ANTIGENS IN BEFORE MERGING CONFORMATION |
EP2710136A4 (en) | 2011-05-17 | 2015-01-21 | Moderna Therapeutics Inc | Engineered nucleic acids and methods of use thereof for non-human vertebrates |
US8691750B2 (en) | 2011-05-17 | 2014-04-08 | Axolabs Gmbh | Lipids and compositions for intracellular delivery of biologically active compounds |
WO2012162174A1 (en) | 2011-05-20 | 2012-11-29 | Kohler Co. | Toilet installation system and method |
WO2012159754A2 (en) | 2011-05-24 | 2012-11-29 | Biontech Ag | Individualized vaccines for cancer |
ES2550192T3 (en) | 2011-05-25 | 2015-11-05 | Novartis Ag | Biomarkers for lung cancer |
WO2012166923A2 (en) | 2011-05-31 | 2012-12-06 | Bind Biosciences | Drug loaded polymeric nanoparticles and methods of making and using same |
US20140094461A1 (en) | 2011-06-02 | 2014-04-03 | Novartis Ag | Biomarkers for hedgehog inhibitor therapy |
EP3412282A1 (en) | 2011-06-02 | 2018-12-12 | The Regents of the University of California | Membrane encapsulated nanoparticles and method of use |
US9181553B2 (en) | 2011-06-06 | 2015-11-10 | Novartis Forschungsstiftung Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Method of treatment of breast cancers over-expressing the SHP2 signature genes |
AU2012267531B2 (en) | 2011-06-08 | 2017-06-22 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
EP3354644A1 (en) | 2011-06-08 | 2018-08-01 | Translate Bio, Inc. | Cleavable lipids |
WO2012170607A2 (en) | 2011-06-10 | 2012-12-13 | Novartis Ag | Use of pcsk9 antagonists |
US9364527B2 (en) | 2011-06-10 | 2016-06-14 | Novartis Tiergesundheit Ag | Bovine vaccines and methods |
US8636696B2 (en) | 2011-06-10 | 2014-01-28 | Kimberly-Clark Worldwide, Inc. | Transdermal device containing microneedles |
WO2012168491A1 (en) | 2011-06-10 | 2012-12-13 | Novartis Ag | Pharmaceutical formulations of pcsk9 antagonists |
US8916696B2 (en) | 2011-06-12 | 2014-12-23 | City Of Hope | Aptamer-mRNA conjugates for targeted protein or peptide expression and methods for their use |
WO2012172495A1 (en) | 2011-06-14 | 2012-12-20 | Novartis Ag | Compositions and methods for antibodies targeting tem8 |
CA2839196A1 (en) | 2011-06-15 | 2012-12-20 | Chrontech Pharma Ab | Injection needle and device |
AU2012269929A1 (en) | 2011-06-16 | 2013-12-12 | Novartis Ag | Soluble proteins for use as therapeutics |
US9580475B2 (en) | 2011-06-20 | 2017-02-28 | University of Pittsburgh—of the Commonwealth System of Higher Education | Computationally optimized broadly reactive antigens for H1N1 influenza |
WO2013003475A1 (en) | 2011-06-27 | 2013-01-03 | Cellscript, Inc. | Inhibition of innate immune response |
WO2013066427A1 (en) | 2011-06-28 | 2013-05-10 | Inovio Pharmaceuticals, Inc. | A miniminally invasive dermal electroporation device |
WO2013006437A1 (en) | 2011-07-01 | 2013-01-10 | Novartis Ag | Method for treating metabolic disorders |
WO2013003887A1 (en) | 2011-07-04 | 2013-01-10 | Commonwealth Scientific And Industrial Research Organisation | Nucleic acid complex |
EP2729124B1 (en) | 2011-07-06 | 2018-10-24 | GlaxoSmithKline Biologicals SA | Cationic oil-in-water emulsions |
CA2840989A1 (en) | 2011-07-06 | 2013-01-10 | Novartis Ag | Immunogenic combination compositions and uses thereof |
RU2014104090A (en) | 2011-07-06 | 2015-08-20 | Новартис Аг | LIPOSOMES WITH AN EFFECTIVE N: P RATIO FOR DELIVERY OF PHK MOLECULES |
WO2013006834A1 (en) | 2011-07-06 | 2013-01-10 | Novartis Ag | Oil-in-water emulsions that contain nucleic acids |
WO2013006842A2 (en) | 2011-07-06 | 2013-01-10 | Novartis Ag | Immunogenic compositions and uses thereof |
EP2729501A2 (en) | 2011-07-07 | 2014-05-14 | Life Technologies Corporation | Polymer particles, nucleic acid polymer particles and methods of making and using the same |
US20130012566A1 (en) | 2011-07-10 | 2013-01-10 | Aura Biosciences, Inc. | Virion Derived Protein Nanoparticles For Delivering Diagnostic Or Therapeutic Agents For The Treatment of Alopecia |
WO2013009717A1 (en) | 2011-07-10 | 2013-01-17 | Elisabet De Los Pinos | Virion derived protein nanoparticles for delivering diagnostic or therapeutic agents for the treatment of skin-related diseases |
WO2013009736A2 (en) | 2011-07-10 | 2013-01-17 | President And Fellows Of Harvard College | Compositions and methods for self-assembly of polymers with complementary macroscopic and microscopic scale units |
US20140148503A1 (en) | 2011-07-20 | 2014-05-29 | University Of Iowa Research Foundation | Nucleic acid aptamers |
GB2492999A (en) | 2011-07-20 | 2013-01-23 | Univ Central Lancashire | Neutron detector |
WO2013012476A2 (en) | 2011-07-21 | 2013-01-24 | Arizona Chemical Company, Llc | Branched polyether-polyamide block copolymers and methods of making and using the same |
US10030052B2 (en) | 2011-07-25 | 2018-07-24 | Glaxosmithkline Biologicals Sa | Parvovirus Vp1 unique region polypeptides and compositions thereof |
US9493549B2 (en) | 2011-07-25 | 2016-11-15 | The Rockefeller University | Antibodies directed toward the HIV-1 GP120 CD4 binding site with increased potency and breadth |
US20130039954A1 (en) | 2011-07-29 | 2013-02-14 | Selecta Biosciences, Inc. | Control of antibody responses to synthetic nanocarriers |
CA2845259A1 (en) | 2011-08-15 | 2013-02-21 | The University Of Chicago | Compositions and methods related to antibodies to staphylococcal protein a |
US8932572B2 (en) | 2011-08-26 | 2015-01-13 | Arrowhead Madison Inc. | Poly(vinyl ester) polymers for in vivo nucleic acid delivery |
JP2014527071A (en) | 2011-08-31 | 2014-10-09 | マリンクロッド エルエルシー | Modification of nanoparticle PEG with H-phosphonate |
TR201900264T4 (en) | 2011-08-31 | 2019-02-21 | Glaxosmithkline Biologicals Sa | Pegylated liposomes for delivery of the immunogen encoding RNA. |
US9126966B2 (en) | 2011-08-31 | 2015-09-08 | Protiva Biotherapeutics, Inc. | Cationic lipids and methods of use thereof |
WO2013033620A1 (en) | 2011-09-01 | 2013-03-07 | Irm Llc | Compounds and compositions as pdgfr kinase inhibitors |
JP2014525435A (en) | 2011-09-02 | 2014-09-29 | ノバルティス アーゲー | Organic composition for treating HSF1-related diseases |
US9464124B2 (en) | 2011-09-12 | 2016-10-11 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
WO2013039861A2 (en) | 2011-09-12 | 2013-03-21 | modeRNA Therapeutics | Engineered nucleic acids and methods of use thereof |
EP2755986A4 (en) | 2011-09-12 | 2015-05-20 | Moderna Therapeutics Inc | Engineered nucleic acids and methods of use thereof |
ES2732708T3 (en) | 2011-09-14 | 2019-11-25 | Glaxosmithkline Biologicals Sa | Manufacturing procedures of saccharide-protein glucoconjugates |
BR112014006176A8 (en) | 2011-09-16 | 2017-09-12 | Univ Pennsylvania | IN VITRO TRANSCRIBED RNA OR SYNTHETIC RNA, COMPOSITION, METHOD FOR GENERATING AN RNA GENETICALLY MODIFIED T CELL POPULATION, AND, USE OF A GENETICALLY MODIFIED T CELL |
EP2747761A1 (en) | 2011-09-22 | 2014-07-02 | Bind Therapeutics, Inc. | Methods of treating cancers with therapeutic nanoparticles |
US9375388B2 (en) | 2011-09-23 | 2016-06-28 | Indian Institute Of Technology, Bombay | Nanoparticle based cosmetic composition |
US9458214B2 (en) | 2011-09-26 | 2016-10-04 | Novartis Ag | Dual function fibroblast growth factor 21 proteins |
JO3476B1 (en) | 2011-09-26 | 2020-07-05 | Novartis Ag | Fusion proteins for treating metabolic disorders |
CA2849476A1 (en) | 2011-09-27 | 2013-04-04 | Alnylam Pharmaceuticals, Inc. | Di-aliphatic substituted pegylated lipids |
WO2013045505A1 (en) | 2011-09-28 | 2013-04-04 | Novartis Ag | Biomarkers for raas combination therapy |
MX354267B (en) | 2011-10-03 | 2018-02-21 | Moderna Therapeutics Inc Star | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof. |
JP6305925B2 (en) | 2011-10-11 | 2018-04-18 | ノバルティス アーゲー | Recombinant self-replicating polycistronic RNA molecules |
WO2013055971A1 (en) | 2011-10-11 | 2013-04-18 | Arizona Board Of Regents For And On Behalf Of Arizona State University | Polymers for delivering a substance into a cell |
US20190209690A9 (en) | 2011-10-12 | 2019-07-11 | The Johns Hopkins University | Bioreducible Poly (Beta-Amino Ester)s For siRNA Delivery |
WO2013055331A1 (en) | 2011-10-12 | 2013-04-18 | The Curators Of The University Of Missouri | Pentablock polymers |
WO2013054307A2 (en) | 2011-10-14 | 2013-04-18 | Novartis Ag | Antibodies and methods for wnt pathway-related diseases |
CA2852064A1 (en) | 2011-10-14 | 2013-04-18 | Stc.Unm | Porous nanoparticle-supported lipid bilayers (protocells) for targeted delivery including transdermal delivery of cargo and methods thereof |
CN107266391B (en) | 2011-10-18 | 2020-04-17 | 迪克纳制药公司 | Amine cationic lipids and uses thereof |
CA2852260C (en) | 2011-10-18 | 2020-09-22 | Micell Technologies, Inc. | Drug delivery medical device |
EA034964B1 (en) | 2011-10-20 | 2020-04-13 | Новартис Аг | Biomarkers predictive of responsiveness to alpha 7 nicotinic acetylcholine receptor activator treatment |
US20140248320A1 (en) | 2011-10-20 | 2014-09-04 | Novartis Ag | Adjuvanted influenza b virus vaccines for pediatric priming |
EP2770980A4 (en) | 2011-10-25 | 2015-11-04 | Univ British Columbia | Limit size lipid nanoparticles and related methods |
US20130110043A1 (en) | 2011-10-26 | 2013-05-02 | Nanopass Technologies Ltd. | Microneedle Intradermal Drug Delivery Device with Auto-Disable Functionality |
UA119028C2 (en) | 2011-10-27 | 2019-04-25 | Массачусеттс Інстітьют Оф Текнолоджі | N-terminal functionalized amino acid derivatives capable of forming microspheres encapsulating the drug |
CA2853214C (en) | 2011-10-27 | 2020-03-24 | Kimberly-Clark Worldwide, Inc. | Transdermal delivery of high viscosity bioactive agents |
WO2013063530A2 (en) | 2011-10-28 | 2013-05-02 | Presage Biosciences, Inc. | Methods for drug delivery |
CN108771655A (en) | 2011-10-28 | 2018-11-09 | 诚信生物公司 | Protein formulation containing amino acid |
WO2013062140A1 (en) | 2011-10-28 | 2013-05-02 | Kyoto University | Method for efficiently inducing differentiation of pluripotent stem cells into hepatic lineage cells |
MX360391B (en) | 2011-10-31 | 2018-10-31 | Mallinckrodt Llc | Combinational liposome compositions for cancer therapy. |
CN116077644A (en) | 2011-10-31 | 2023-05-09 | 弗·哈夫曼-拉罗切有限公司 | Antibody formulations |
WO2013067537A1 (en) | 2011-11-04 | 2013-05-10 | Univertiy Of Notre Dame Du Lac | Nanoparticle-based drug delivery |
PL2773326T3 (en) | 2011-11-04 | 2019-06-28 | Nitto Denko Corporation | Method for sterilely producing lipid-nucleic acid particles |
SG11201401717VA (en) | 2011-11-04 | 2014-05-29 | Novartis Ag | Low density lipoprotein-related protein 6 (lrp6) - half life extender constructs |
US9579338B2 (en) | 2011-11-04 | 2017-02-28 | Nitto Denko Corporation | Method of producing lipid nanoparticles for drug delivery |
WO2013066274A1 (en) | 2011-11-04 | 2013-05-10 | Agency For Science, Technology And Research | Self-assembled composite ultrasmall peptide-polymer hydrogels |
US20130115247A1 (en) | 2011-11-05 | 2013-05-09 | Aura Biosciences, Inc. | Virion Derived Protein Nanoparticles For Delivering Radioisotopes For The Diagnosis And Treatment Of Malignant And Systemic Disease And The Monitoring Of Therapy |
US20130116408A1 (en) | 2011-11-05 | 2013-05-09 | Aura Biosciences, Inc. | Virion Derived Protein Nanoparticles For Delivering Radioisotopes For The Diagnosis And Treatment Of Malignant And Systemic Disease And The Monitoring Of Therapy |
EP2776022A1 (en) | 2011-11-08 | 2014-09-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | New treatment for neurodegenerative diseases |
EP2776013B8 (en) | 2011-11-08 | 2023-08-30 | The Board of Trustees of the University of Arkansas | Methods and compositions for x-ray induced release from ph sensitive liposomes |
EP2776459A1 (en) | 2011-11-08 | 2014-09-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Rod cell-specific promoter |
EP2776838A1 (en) | 2011-11-08 | 2014-09-17 | Novartis Forschungsstiftung, Zweigniederlassung Friedrich Miescher Institute For Biomedical Research | Early diagnostic of neurodegenerative diseases |
US10203325B2 (en) | 2011-11-09 | 2019-02-12 | Board Of Trustees Of Michigan State University | Metallic nanoparticle synthesis with carbohydrate capping agent |
CA2889659C (en) | 2011-11-11 | 2023-03-14 | Variation Biotechnologies Inc. | Compositions and methods for treatment of cytomegalovirus |
WO2013071047A1 (en) | 2011-11-11 | 2013-05-16 | Children's Medical Center Corporation | Compositions and methods for in vitro transcription of rna |
RU2014124154A (en) | 2011-11-14 | 2015-12-27 | Новартис Аг | IMMUNOGENOUS COMPLEXES OF POLYANIONAL CARBOMERS AND ENV POLYPEPTIDES AND METHODS FOR PRODUCING AND USING THEM |
MX2014005927A (en) | 2011-11-15 | 2014-06-05 | Novartis Ag | Combination of a phosphoinositide 3-kinase inhibitor and a modulator of the janus kinase 2-signal transducer and activator of transcription 5 pathway. |
RU2768492C2 (en) | 2011-11-18 | 2022-03-24 | Ридженерон Фармасьютикалз, Инк. | Polymer protein microparticles |
EP2783014A1 (en) | 2011-11-21 | 2014-10-01 | Novartis AG | Methods of treating psoriatic arthritis (psa) using il-17 antagonists and psa response or non- response alleles |
WO2013078199A2 (en) | 2011-11-23 | 2013-05-30 | Children's Medical Center Corporation | Methods for enhanced in vivo delivery of synthetic, modified rnas |
WO2013082111A2 (en) | 2011-11-29 | 2013-06-06 | The University Of North Carolina At Chapel Hill | Geometrically engineered particles and methods for modulating macrophage or immune responses |
CA2857502C (en) | 2011-11-30 | 2019-08-13 | 3M Innovative Properties Company | Microneedle device including a peptide therapeutic agent and an amino acid and methods of making and using the same |
US9364549B2 (en) | 2011-11-30 | 2016-06-14 | Andreas Voigt | Hydrophobic drug-delivery material, method for manufacturing thereof and methods for delivery of a drug-delivery composition |
MX351453B (en) | 2011-12-02 | 2017-10-16 | Pegasus Laboratories Inc | Amphipathic lipid-based sustained release compositions. |
WO2013082529A1 (en) | 2011-12-02 | 2013-06-06 | Yale University | Enzymatic synthesis of poly(amine-co-esters) and methods of use thereof for gene delivery |
US20130142781A1 (en) | 2011-12-02 | 2013-06-06 | Invivo Therapeutics Corporation | Peg based hydrogel for peripheral nerve injury applications and compositions and method of use of synthetic hydrogel sealants |
US8497124B2 (en) | 2011-12-05 | 2013-07-30 | Factor Bioscience Inc. | Methods and products for reprogramming cells to a less differentiated state |
WO2013085951A1 (en) | 2011-12-05 | 2013-06-13 | Nano Precision Medical, Inc. | Device having titania nanotube membrane for drug delivery |
KR102196339B1 (en) | 2011-12-05 | 2020-12-29 | 팩터 바이오사이언스 인크. | Methods and products for transfecting cells |
GB201121070D0 (en) | 2011-12-07 | 2012-01-18 | Isis Innovation | composition for delivery of biotherapeutics |
CA2856742A1 (en) | 2011-12-07 | 2013-06-13 | Alnylam Pharmaceuticals, Inc. | Biodegradable lipids for the delivery of active agents |
WO2013086322A1 (en) | 2011-12-07 | 2013-06-13 | Alnylam Pharmaceuticals, Inc. | Branched alkyl and cycloalkyl terminated biodegradable lipids for the delivery of active agents |
WO2013086373A1 (en) | 2011-12-07 | 2013-06-13 | Alnylam Pharmaceuticals, Inc. | Lipids for the delivery of active agents |
US9725687B2 (en) | 2011-12-09 | 2017-08-08 | President And Fellows Of Harvard College | Integrated human organ-on-chip microphysiological systems |
US10087422B2 (en) | 2011-12-09 | 2018-10-02 | President And Fellows Of Harvard College | Organ chips and uses thereof |
EP2787977A4 (en) | 2011-12-09 | 2015-05-06 | Univ California | Liposomal drug encapsulation |
TW201330874A (en) | 2011-12-12 | 2013-08-01 | Kyowa Hakko Kirin Co Ltd | Lipid nano particles comprising combination of cationic lipid |
WO2013090294A1 (en) | 2011-12-12 | 2013-06-20 | The Trustees Of The University Of Pennsylvania | Proteins comprising mrsa pbp2a and fragments thereof, nucleic acids encoding the same, and compositions and their use to prevent and treat mrsa infections |
US9839616B2 (en) | 2011-12-12 | 2017-12-12 | Kyowa Hakko Kirin Co., Ltd. | Lipid nano particles comprising cationic lipid for drug delivery system |
EP2604253A1 (en) | 2011-12-13 | 2013-06-19 | Otto Glatter | Water-in-oil emulsions and methods for their preparation |
KR102046042B1 (en) | 2011-12-13 | 2019-11-18 | 엔진아이씨 몰레큘러 딜리버리 피티와이 리미티드 | Bacterially derived, intact minicells for delivery of therapeutic agents to brain tumors |
US20150000936A1 (en) | 2011-12-13 | 2015-01-01 | Schlumberger Technology Corporation | Energization of an element with a thermally expandable material |
WO2013130161A1 (en) | 2011-12-14 | 2013-09-06 | modeRNA Therapeutics | Methods of responding to a biothreat |
EP2791159A4 (en) | 2011-12-14 | 2015-10-14 | Moderna Therapeutics Inc | Modified nucleic acids, and acute care uses thereof |
US9636414B2 (en) | 2011-12-15 | 2017-05-02 | Biontech Ag | Particles comprising single stranded RNA and double stranded RNA for immunomodulation |
WO2013090897A1 (en) | 2011-12-15 | 2013-06-20 | The Trustees Of The University Of Pennsylvania | Using adaptive immunity to detect drug resistance |
WO2013090601A2 (en) | 2011-12-16 | 2013-06-20 | Massachusetts Institute Of Technology | Compact nanoparticles for biological applications |
BR112014014399A2 (en) | 2011-12-16 | 2017-06-13 | Novartis Ag | medicine delivery aerosol unit regardless of inhalation profile |
WO2013090861A1 (en) | 2011-12-16 | 2013-06-20 | Massachusetts Institute Of Technology | Alpha-aminoamidine polymers and uses thereof |
RU2014129268A (en) | 2011-12-16 | 2016-02-10 | Аллерган, Инк. | OPHTHALMIC COMPOSITIONS THAT CONTAIN GRAVITY POLYVINYL POLYVINYL PROCALT-POLYVINYL ACETATE-POLYETHYLENE Glycol copolymers |
EP2791171A1 (en) | 2011-12-16 | 2014-10-22 | Synthon Biopharmaceuticals B.V. | EXPRESSION OF SECRETORY IgA ANTIBODIES IN DUCKWEED |
US20130156849A1 (en) | 2011-12-16 | 2013-06-20 | modeRNA Therapeutics | Modified nucleoside, nucleotide, and nucleic acid compositions |
ES2627856T3 (en) | 2011-12-19 | 2017-07-31 | The University Of Sydney | Peptide Hydrogel Composite |
US9241829B2 (en) | 2011-12-20 | 2016-01-26 | Abbott Medical Optics Inc. | Implantable intraocular drug delivery apparatus, system and method |
RU2014129863A (en) | 2011-12-21 | 2016-02-10 | Модерна Терапьютикс, Инк. | WAYS TO INCREASE VITALITY OR INCREASE THE LIFE OF A BODY OR EXPLANATE BODY |
WO2013096812A1 (en) | 2011-12-23 | 2013-06-27 | Genentech, Inc. | Articles of manufacture and methods for co-administration of antibodies |
KR101963230B1 (en) | 2011-12-26 | 2019-03-29 | 삼성전자주식회사 | Protein complex comprising multi-specific monoclonal antibodies |
WO2013101908A1 (en) | 2011-12-27 | 2013-07-04 | Massachusetts Institute Of Technology | Microneedle devices and uses thereof |
WO2013098589A1 (en) | 2011-12-29 | 2013-07-04 | Novartis Ag | Adjuvanted combinations of meningococcal factor h binding proteins |
WO2013101690A1 (en) | 2011-12-29 | 2013-07-04 | modeRNA Therapeutics | Modified mrnas encoding cell-penetrating polypeptides |
HUE047947T2 (en) | 2011-12-30 | 2020-05-28 | Cellscript Llc | Making and using in vitro-synthesized ssrna for introducing into mammalian cells to induce a biological or biochemical effect |
EP3735967A1 (en) | 2012-01-06 | 2020-11-11 | NeuroBo Pharmaceuticals, Inc. | Compound for use in methods of reducing risk of cardiovascular disease |
WO2013106496A1 (en) | 2012-01-10 | 2013-07-18 | modeRNA Therapeutics | Methods and compositions for targeting agents into and across the blood-brain barrier |
WO2013112778A1 (en) | 2012-01-26 | 2013-08-01 | Life Technologies Corporation | Methods for increasing the infectivity of viruses |
KR20140129054A (en) | 2012-01-26 | 2014-11-06 | 라이프 테크놀로지스 코포레이션 | Methods for increasing the infectivity of viruses |
WO2013113326A1 (en) | 2012-01-31 | 2013-08-08 | Curevac Gmbh | Pharmaceutical composition comprising a polymeric carrier cargo complex and at least one protein or peptide antigen |
EP2623121A1 (en) | 2012-01-31 | 2013-08-07 | Bayer Innovation GmbH | Pharmaceutical composition comprising a polymeric carrier cargo complex and an antigen |
WO2013113325A1 (en) | 2012-01-31 | 2013-08-08 | Curevac Gmbh | Negatively charged nucleic acid comprising complexes for immunostimulation |
EP2812360B1 (en) | 2012-02-09 | 2022-08-31 | Life Technologies Corporation | Hydrophilic polymeric particles and methods for making same |
US20140037573A1 (en) | 2012-02-22 | 2014-02-06 | Cerulean Pharma Inc. | Conjugates, particles, compositions, and related methods |
US20130243867A1 (en) | 2012-02-23 | 2013-09-19 | University Of South Florida (A Florida Non-Profit Corporation) | Micelle compositions and methods for their use |
WO2013130535A1 (en) | 2012-02-27 | 2013-09-06 | Newgen Biopharma Corporation | Topical delivery of hormonal and non hormonal nano formulations, methods of making and using the same |
US20150037334A1 (en) | 2012-03-01 | 2015-02-05 | Amgen Research (Munich) Gmbh | Long life polypeptide binding molecules |
US20150094259A1 (en) | 2012-03-13 | 2015-04-02 | University Of Kwazulu-Natal | Transdermal Delivery Devices |
US10322089B2 (en) | 2012-03-14 | 2019-06-18 | The Board Of Trustees Of The Leland Stanford Junior University | Nanoparticles, nanoparticle delivery methods, and systems of delivery |
JP5883539B2 (en) | 2012-03-16 | 2016-03-15 | ザ・ジョンズ・ホプキンス・ユニバーシティー | Controlled release formulations for delivery of HIF-1 inhibitors |
NZ631034A (en) | 2012-03-16 | 2016-06-24 | Merck Patent Gmbh | Targeting aminoacid lipids |
AU2013232300B2 (en) | 2012-03-16 | 2015-12-17 | The Johns Hopkins University | Non-linear multiblock copolymer-drug conjugates for the delivery of active agents |
WO2013142349A1 (en) | 2012-03-23 | 2013-09-26 | University Of Chicago | Compositions and methods related to staphylococcal sbi |
US9610346B2 (en) | 2012-03-23 | 2017-04-04 | International Aids Vaccine Initiative | Recombinant viral vectors |
WO2013148186A1 (en) | 2012-03-26 | 2013-10-03 | President And Fellows Of Harvard College | Lipid-coated nucleic acid nanostructures of defined shape |
WO2013143555A1 (en) | 2012-03-26 | 2013-10-03 | Biontech Ag | Rna formulation for immunotherapy |
RU2651498C2 (en) | 2012-03-27 | 2018-04-19 | Кьюрвак Аг | Artificial nucleic acid molecules |
EP2830594B1 (en) | 2012-03-27 | 2018-05-09 | Sirna Therapeutics, Inc. | DIETHER BASED BIODEGRADABLE CATIONIC LIPIDS FOR siRNA DELIVERY |
ES2654205T3 (en) | 2012-03-27 | 2018-02-12 | Curevac Ag | Artificial nucleic acid molecules for enhanced protein or peptide expression |
CN104321432B (en) | 2012-03-27 | 2018-08-10 | 库瑞瓦格股份公司 | Include the artificial nucleic acid molecule of 5 ' TOP UTR |
CA2868030C (en) | 2012-03-29 | 2021-05-25 | Shire Human Genetic Therapies, Inc. | Lipid-derived neutral nanoparticles |
CA2868996A1 (en) | 2012-04-02 | 2013-10-10 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of proteins |
US20140275229A1 (en) | 2012-04-02 | 2014-09-18 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding udp glucuronosyltransferase 1 family, polypeptide a1 |
JP2015518705A (en) | 2012-04-02 | 2015-07-06 | モデルナ セラピューティクス インコーポレイテッドModerna Therapeutics,Inc. | Modified polynucleotides for the production of biologics and proteins associated with human diseases |
US20150050354A1 (en) | 2012-04-02 | 2015-02-19 | Moderna Therapeutics, Inc. | Modified polynucleotides for the treatment of otic diseases and conditions |
US9254311B2 (en) | 2012-04-02 | 2016-02-09 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of proteins |
WO2013151650A1 (en) | 2012-04-05 | 2013-10-10 | University Of Florida Research Foundation, Inc. | Neurophilic nanoparticles |
JP6375289B2 (en) | 2012-04-05 | 2018-08-15 | マサチューセッツ インスティテュート オブ テクノロジー | Immune stimulating composition and method of use thereof |
WO2013152351A2 (en) | 2012-04-06 | 2013-10-10 | The Trustees Of Columbia University In The City Of New York | Fusion polypeptides and methods of use thereof |
ES2821098T3 (en) | 2012-04-08 | 2021-04-23 | Urogen Pharma Ltd | Reversible thermal hydrogel preparations for use in the treatment of urothelial disorders |
WO2013154774A1 (en) | 2012-04-11 | 2013-10-17 | Intezyne Technologies, Inc. | Block copolymers for stable micelles |
US9610250B2 (en) | 2012-04-12 | 2017-04-04 | Yale University | Nanolipogel vehicles for controlled delivery of different pharmaceutical agents |
WO2013154766A1 (en) | 2012-04-13 | 2013-10-17 | New York University | Microrna control of ldl receptor pathway |
US11001797B2 (en) | 2012-04-13 | 2021-05-11 | President And Fellows Of Harvard College | Devices and methods for in vitro aerosol delivery |
AU2012377385A1 (en) | 2012-04-18 | 2014-01-23 | Arrowhead Research Corporation | Poly(acrylate) polymers for in vivo nucleic acid delivery |
CA2870941C (en) | 2012-04-19 | 2021-05-25 | Sirna Therapeutics, Inc. | Diester and triester based low molecular weight, biodegradable cationic lipids for oligonucleotide delivery |
EP2841056A4 (en) | 2012-04-23 | 2015-09-16 | Massachusetts Inst Technology | Stable layer-by-layer coated particles |
CN104254607B (en) | 2012-04-25 | 2020-10-27 | 赛诺菲 | MicroRNA compounds and methods for modulating MIR-21 activity |
EP2844295A1 (en) | 2012-05-03 | 2015-03-11 | Kala Pharmaceuticals, Inc. | Pharmaceutical nanoparticles showing improved mucosal transport |
US9889208B2 (en) | 2012-05-04 | 2018-02-13 | The Johns Hopkins University | Lipid-based drug carriers for rapid penetration through mucus linings |
US20150087671A1 (en) | 2012-05-16 | 2015-03-26 | Micell Technologies, Inc. | Low burst sustained release lipophilic and biologic agent compositions |
WO2013173582A1 (en) | 2012-05-17 | 2013-11-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Hepatitis c virus neutralizing antibody |
WO2013173693A1 (en) | 2012-05-18 | 2013-11-21 | The United States Of America, As Represented By The Secretary, Department Of Health And Human Services | Nanoparticles with enhanced entry into cancer cells |
CN105163721B (en) | 2012-05-23 | 2018-05-22 | 俄亥俄州立大学 | Lipidic nanoparticles composition and preparation and use its method |
ES2719598T3 (en) | 2012-05-25 | 2019-07-11 | Curevac Ag | Reversible immobilization and / or controlled release of nucleic acids contained in nanoparticles by polymeric coatings (biodegradable) |
EP2858708B1 (en) | 2012-06-06 | 2020-07-29 | Loma Vista Medical, Inc. | Inflatable medical devices |
EA201492055A1 (en) | 2012-06-08 | 2015-11-30 | Шир Хьюман Дженетик Терапис, Инк. | INHALATIVE DELIVERY OF mRNA IN TIGHTNESS CELL TARGETS |
PL2858974T3 (en) | 2012-06-08 | 2019-03-29 | Nitto Denko Corporation | Lipids for therapeutic agent delivery formulations |
JP6208750B2 (en) | 2012-06-08 | 2017-10-04 | エスリス ゲーエムベーハーethris GmbH | Pulmonary delivery of messenger RNA |
WO2014014613A2 (en) | 2012-06-20 | 2014-01-23 | President And Fellows Of Harvard College | Self-assembling peptides, peptide nanostructures and uses thereof |
WO2013188979A1 (en) | 2012-06-20 | 2013-12-27 | Frank Gu | Mucoadhesive nanoparticle delivery system |
ES2729603T3 (en) | 2012-06-27 | 2019-11-05 | Merck Sharp & Dohme | IL-23 anti-human crystalline antibodies |
US9150841B2 (en) | 2012-06-29 | 2015-10-06 | Shire Human Genetic Therapies, Inc. | Cells for producing recombinant iduronate-2-sulfatase |
WO2014008334A1 (en) | 2012-07-06 | 2014-01-09 | Alnylam Pharmaceuticals, Inc. | Stable non-aggregating nucleic acid lipid particle formulations |
US9956291B2 (en) | 2012-07-10 | 2018-05-01 | Shaker A. Mousa | Nanoformulation and methods of use of thyroid receptor beta1 agonists for liver targeting |
WO2014014890A1 (en) | 2012-07-16 | 2014-01-23 | Nanoderm Sciences, Inc. | Targeted therapeutic nanoparticles |
EP2687251A1 (en) | 2012-07-17 | 2014-01-22 | Sanofi-Aventis Deutschland GmbH | Drug delivery device |
EP2687252A1 (en) | 2012-07-17 | 2014-01-22 | Sanofi-Aventis Deutschland GmbH | Drug delivery device |
CN112587671A (en) | 2012-07-18 | 2021-04-02 | 博笛生物科技有限公司 | Targeted immunotherapy for cancer |
WO2014015334A1 (en) | 2012-07-20 | 2014-01-23 | Brown University | System and methods for nanostructure protected delivery of treatment agent and selective release thereof |
CN104781416B (en) | 2012-07-24 | 2017-07-04 | 哈佛学院院长及董事 | The self-assembly of nucleic acid nano structure |
WO2014015422A1 (en) | 2012-07-27 | 2014-01-30 | Ontario Institute For Cancer Research | Cellulose-based nanoparticles for drug delivery |
GB201213624D0 (en) | 2012-07-27 | 2012-09-12 | Univ Ulster The | Method and system for production of conjugated nanoparticles |
US9931418B2 (en) | 2012-08-07 | 2018-04-03 | Northeastern University | Compositions for the delivery of RNA and drugs into cells |
WO2014024193A1 (en) | 2012-08-07 | 2014-02-13 | Prodel Pharma Ltd. | Compositions and methods for rapid transmucosal delivery of pharmaceutical ingredients |
WO2014025312A1 (en) | 2012-08-08 | 2014-02-13 | Nanyang Technological University | Methods of manufacturing hydrogel microparticles having living cells, and compositions for manufacturing a scaffold for tissue engineering |
CN104812434A (en) | 2012-08-08 | 2015-07-29 | 普莱萨格生命科学公司 | Extrusion methods and devices for drug delivery |
JP2015525799A (en) | 2012-08-10 | 2015-09-07 | ユニバーシティ・オブ・ノース・テキサス・ヘルス・サイエンス・センターUniversity of North Texas Health Science Center | Drug delivery vehicle comprising conjugate of targeting polyamino acid and fatty acid |
CA2884870C (en) | 2012-08-13 | 2022-03-29 | Massachusetts Institute Of Technology | Amine-containing lipidoids and uses thereof |
WO2014027006A1 (en) | 2012-08-13 | 2014-02-20 | Edko Pazarlama Tanitim Ticaret Limited Sirketi | Bioadhesive formulations for use in drug delivery |
CA2881605A1 (en) | 2012-08-14 | 2014-02-20 | Aaron FROESE | Internal structured self assembling liposomes |
US9512456B2 (en) | 2012-08-14 | 2016-12-06 | Modernatx, Inc. | Enzymes and polymerases for the synthesis of RNA |
WO2014028209A1 (en) | 2012-08-14 | 2014-02-20 | The Trustees Of The University Of Pennsylvania | Stabilizing shear-thinning hydrogels |
AU2013302526B2 (en) | 2012-08-15 | 2018-03-22 | The University Of Chicago | Exosome-based therapeutics against neurodegenerative disorders |
US10179134B2 (en) | 2012-09-05 | 2019-01-15 | Creighton University | Polymeric nanoparticles in a thermosensitive gel for coital-independent vaginal prophylaxis of HIV |
US8703197B2 (en) | 2012-09-13 | 2014-04-22 | International Business Machines Corporation | Branched polyamines for delivery of biologically active materials |
KR20150056618A (en) | 2012-09-17 | 2015-05-26 | 바인드 쎄라퓨틱스, 인크. | Process for preparing therapeutic nanoparticles |
WO2014047649A1 (en) | 2012-09-24 | 2014-03-27 | The Regents Of The University Of California | Methods for arranging and packing nucleic acids for unusual resistance to nucleases and targeted delivery for gene therapy |
US20150246137A1 (en) | 2012-09-27 | 2015-09-03 | The University Of North Carolina At Chapel Hill | Lipid coated nanoparticles containing agents having low aqueous and lipid solubilities and methods thereof |
WO2014093924A1 (en) | 2012-12-13 | 2014-06-19 | Moderna Therapeutics, Inc. | Modified nucleic acid molecules and uses thereof |
US20150202153A1 (en) | 2012-10-04 | 2015-07-23 | University Of The Witwatersrand, Johannesburg | Liposomal drug delivery system |
WO2014053882A1 (en) | 2012-10-04 | 2014-04-10 | Centre National De La Recherche Scientifique | Cell penetrating peptides for intracellular delivery of molecules |
WO2014053879A1 (en) | 2012-10-04 | 2014-04-10 | Centre National De La Recherche Scientifique | Cell penetrating peptides for intracellular delivery of molecules |
WO2014053880A1 (en) | 2012-10-04 | 2014-04-10 | Centre National De La Recherche Scientifique | Cell penetrating peptides for intracellular delivery of molecules |
US20140100178A1 (en) | 2012-10-04 | 2014-04-10 | Aslam Ansari | Composition and methods for site-specific drug delivery to treat malaria and other liver diseases |
EP2716655A1 (en) | 2012-10-04 | 2014-04-09 | Institut Pasteur | Neutralizing antibodies directed against Hepatitis C virus ectodomain glycoprotein E2 |
WO2014053881A1 (en) | 2012-10-04 | 2014-04-10 | Centre National De La Recherche Scientifique | Cell penetrating peptides for intracellular delivery of molecules |
WO2014064534A2 (en) | 2012-10-05 | 2014-05-01 | Chrontech Pharma Ab | Injection needle, device, immunogenic compositions and method of use |
EP2716689A1 (en) | 2012-10-05 | 2014-04-09 | National University of Ireland, Galway | Polymer comprising a plurality of branches having at least one disulfide group and/or at least one vinyl group |
US9931410B2 (en) | 2012-10-09 | 2018-04-03 | The Brigham And Women's Hospital, Inc. | Nanoparticles for targeted delivery of multiple therapeutic agents and methods of use |
US20140106260A1 (en) | 2012-10-11 | 2014-04-17 | The Trustees Of The University Of Pennsylvania | Core-shell nanoparticulate compositions and methods |
BR112015008365A2 (en) | 2012-10-16 | 2017-07-04 | Endocyte Inc | compound of the formula bl (d) x, or a pharmaceutically acceptable salt thereof, pharmaceutical composition, use of a compound, unit dosage form or unit dose composition, composition for treating a cancer in a patient, and method for treating a cancer in a patient |
SI2908912T1 (en) | 2012-10-18 | 2021-03-31 | The Rockefeller University | Broadly-neutralizing anti-hiv antibodies |
WO2014066912A1 (en) | 2012-10-26 | 2014-05-01 | Vanderbilt University | Polymeric nanoparticles |
AU2013336582A1 (en) | 2012-10-26 | 2015-06-11 | Nlife Therapeutics, S.L. | Compositions and methods for selective delivery of oligonucleotide molecules to cell types |
US20150272900A1 (en) | 2012-10-26 | 2015-10-01 | The Johns Hopkins University | Layer-By-Layer Approach to Co-Deliver DNA and siRNA via AuNPs: A Potential Platform for Modifying Release Kinetics |
EP2912180A1 (en) | 2012-10-29 | 2015-09-02 | Technische Universität Dortmund | T7 rna polymerase variants and methods of using the same |
JP6510416B2 (en) | 2012-11-01 | 2019-05-08 | ファクター バイオサイエンス インコーポレイテッド | Methods and products for expressing proteins in cells |
WO2014071072A2 (en) | 2012-11-02 | 2014-05-08 | Pungente Michael D | Novel cationic carotenoid-based lipids for cellular nucleic acid uptake |
WO2014068542A1 (en) | 2012-11-05 | 2014-05-08 | Fondazione Centro San Raffaele | Novel targets in multiple myeloma and other disorders |
US9975916B2 (en) | 2012-11-06 | 2018-05-22 | President And Fellows Of Harvard College | Compositions and methods relating to complex nucleic acid nanostructures |
BR112015010253A2 (en) | 2012-11-06 | 2017-07-11 | Rochal Ind Llc | delivery of biologically active agents using hydrophobic and volatile solvents |
WO2014072999A1 (en) | 2012-11-07 | 2014-05-15 | Council Of Scientific And Industrial Research | Nanocomplex containing cationic peptide for biomolecule delivery |
US9669104B2 (en) | 2012-11-07 | 2017-06-06 | Council Of Scientific And Industrial Research | Nanocomplex containing amphipathic peptide useful for efficient transfection of biomolecules |
US20150258120A1 (en) | 2012-11-08 | 2015-09-17 | Clearside Biomedical, Inc. | Methods and devices for the treatment of ocular diseases in human subjects |
TW201428101A (en) | 2012-11-08 | 2014-07-16 | Inviragen Inc | Compositions, methods and uses for dengue virus serotype-4 constructs |
MX2015005363A (en) | 2012-11-08 | 2015-11-06 | Novozymes Biopharma Dk As | Albumin variants. |
AU2013342163B2 (en) | 2012-11-08 | 2018-08-16 | F. Hoffmann-La Roche Ltd | IL-6 antagonists and uses thereof |
WO2014071963A1 (en) | 2012-11-09 | 2014-05-15 | Biontech Ag | Method for cellular rna expression |
US9200119B2 (en) | 2012-11-09 | 2015-12-01 | Momentive Performance Materials Inc. | Silicon-containing zwitterionic linear copolymer composition |
EP2916853B1 (en) | 2012-11-09 | 2020-05-06 | Velin-Pharma A/S | Compositions for pulmonary delivery |
ES2676470T3 (en) | 2012-11-09 | 2018-07-19 | Biontech Rna Pharmaceuticals Gmbh | Method for RNA expression in cells |
GB201220354D0 (en) | 2012-11-12 | 2012-12-26 | Medpharm Ltd | Dermal compositions |
WO2014075047A2 (en) | 2012-11-12 | 2014-05-15 | Genvec, Inc. | Malaria antigens and methods of use |
CN104884056A (en) | 2012-11-12 | 2015-09-02 | 雷德伍德生物科技股份有限公司 | Compounds and methods for producing a conjugate |
US9943608B2 (en) | 2012-11-13 | 2018-04-17 | Baylor College Of Medicine | Multi-arm biodegradable polymers for nucleic acid delivery |
US9310374B2 (en) | 2012-11-16 | 2016-04-12 | Redwood Bioscience, Inc. | Hydrazinyl-indole compounds and methods for producing a conjugate |
WO2014078636A1 (en) | 2012-11-16 | 2014-05-22 | President And Fellows Of Harvard College | Nucleic acid hydrogel self-assembly |
WO2014076709A1 (en) | 2012-11-19 | 2014-05-22 | Technion Research And Development Foundation Ltd. | Liposomes for in-vivo delivery |
EP2732825B1 (en) | 2012-11-19 | 2015-07-01 | Invivogen | Conjugates of a TLR7 and/or TLR8 agonist and a TLR2 agonist |
US20140141037A1 (en) | 2012-11-20 | 2014-05-22 | Novartis Ag | Rsv f prefusion trimers |
WO2014081849A1 (en) | 2012-11-20 | 2014-05-30 | Phasebio Pharmaceuticals, Inc. | Formulations of active agents for sustained release |
WO2014081299A1 (en) | 2012-11-22 | 2014-05-30 | Tagworks Pharmaceuticals B.V. | Activatable liposomes |
US10927139B2 (en) | 2012-11-22 | 2021-02-23 | Tagworks Pharmaceuticals B.V. | Chemically cleavable group |
WO2014081300A1 (en) | 2012-11-22 | 2014-05-30 | Tagworks Pharmaceuticals B.V. | Channel protein activatable liposomes |
HRP20220607T1 (en) | 2012-11-26 | 2022-06-24 | Modernatx, Inc. | Terminally modified rna |
US20150315541A1 (en) | 2012-12-13 | 2015-11-05 | Moderna Therapeutics, Inc. | Modified polynucleotides for altering cell phenotype |
WO2014108515A1 (en) | 2013-01-10 | 2014-07-17 | Novartis Ag | Influenza virus immunogenic compositions and uses thereof |
EP2946014A2 (en) | 2013-01-17 | 2015-11-25 | Moderna Therapeutics, Inc. | Signal-sensor polynucleotides for the alteration of cellular phenotypes |
US20160022840A1 (en) | 2013-03-09 | 2016-01-28 | Moderna Therapeutics, Inc. | Heterologous untranslated regions for mrna |
EP2968397A4 (en) | 2013-03-12 | 2016-12-28 | Moderna Therapeutics Inc | Diagnosis and treatment of fibrosis |
US20160024181A1 (en) | 2013-03-13 | 2016-01-28 | Moderna Therapeutics, Inc. | Long-lived polynucleotide molecules |
EP2971010B1 (en) | 2013-03-14 | 2020-06-10 | ModernaTX, Inc. | Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions |
EP4279610A3 (en) | 2013-03-15 | 2024-01-03 | ModernaTX, Inc. | Ribonucleic acid purification |
WO2014144767A1 (en) | 2013-03-15 | 2014-09-18 | Moderna Therapeutics, Inc. | Ion exchange purification of mrna |
US20160032273A1 (en) | 2013-03-15 | 2016-02-04 | Moderna Therapeutics, Inc. | Characterization of mrna molecules |
US8980864B2 (en) | 2013-03-15 | 2015-03-17 | Moderna Therapeutics, Inc. | Compositions and methods of altering cholesterol levels |
EP2971033B8 (en) | 2013-03-15 | 2019-07-10 | ModernaTX, Inc. | Manufacturing methods for production of rna transcripts |
WO2014144711A1 (en) | 2013-03-15 | 2014-09-18 | Moderna Therapeutics, Inc. | Analysis of mrna heterogeneity and stability |
WO2014152030A1 (en) | 2013-03-15 | 2014-09-25 | Moderna Therapeutics, Inc. | Removal of dna fragments in mrna production process |
WO2015006747A2 (en) | 2013-07-11 | 2015-01-15 | Moderna Therapeutics, Inc. | Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use. |
US20160194368A1 (en) | 2013-09-03 | 2016-07-07 | Moderna Therapeutics, Inc. | Circular polynucleotides |
CA2923029A1 (en) | 2013-09-03 | 2015-03-12 | Moderna Therapeutics, Inc. | Chimeric polynucleotides |
WO2015038892A1 (en) | 2013-09-13 | 2015-03-19 | Moderna Therapeutics, Inc. | Polynucleotide compositions containing amino acids |
EP3052106A4 (en) | 2013-09-30 | 2017-07-19 | ModernaTX, Inc. | Polynucleotides encoding immune modulating polypeptides |
EA201690675A1 (en) | 2013-10-03 | 2016-08-31 | Модерна Терапьютикс, Инк. | POLYNUCLEOTES ENCODING THE RECEPTOR OF LOW DENSITY LIPOPROTEINS |
EP3058082A4 (en) | 2013-10-18 | 2017-04-26 | ModernaTX, Inc. | Compositions and methods for tolerizing cellular systems |
EP3092250A4 (en) | 2014-01-08 | 2017-05-24 | Moderna Therapeutics, Inc. | Polynucleotides for the in vivo production of antibodies |
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2011
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Patent Citations (33)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4270537A (en) | 1979-11-19 | 1981-06-02 | Romaine Richard A | Automatic hypodermic syringe |
US4596556A (en) | 1985-03-25 | 1986-06-24 | Bioject, Inc. | Hypodermic injection apparatus |
US4886499A (en) | 1986-12-18 | 1989-12-12 | Hoffmann-La Roche Inc. | Portable injection appliance |
US5015235A (en) | 1987-02-20 | 1991-05-14 | National Carpet Equipment, Inc. | Syringe needle combination |
US4790824A (en) | 1987-06-19 | 1988-12-13 | Bioject, Inc. | Non-invasive hypodermic injection device |
US4940460A (en) | 1987-06-19 | 1990-07-10 | Bioject, Inc. | Patient-fillable and non-invasive hypodermic injection device assembly |
US4941880A (en) | 1987-06-19 | 1990-07-17 | Bioject, Inc. | Pre-filled ampule and non-invasive hypodermic injection device assembly |
US5339163A (en) | 1988-03-16 | 1994-08-16 | Canon Kabushiki Kaisha | Automatic exposure control device using plural image plane detection areas |
US5141496A (en) | 1988-11-03 | 1992-08-25 | Tino Dalto | Spring impelled syringe guide with skin penetration depth adjustment |
US5312335A (en) | 1989-11-09 | 1994-05-17 | Bioject Inc. | Needleless hypodermic injection device |
US5064413A (en) | 1989-11-09 | 1991-11-12 | Bioject, Inc. | Needleless hypodermic injection device |
US5503627A (en) | 1989-11-09 | 1996-04-02 | Bioject, Inc. | Ampule for needleless injection |
US5190521A (en) | 1990-08-22 | 1993-03-02 | Tecnol Medical Products, Inc. | Apparatus and method for raising a skin wheal and anesthetizing skin |
US5527288A (en) | 1990-12-13 | 1996-06-18 | Elan Medical Technologies Limited | Intradermal drug delivery device and method for intradermal delivery of drugs |
US5480381A (en) | 1991-08-23 | 1996-01-02 | Weston Medical Limited | Needle-less injector |
US5417662A (en) | 1991-09-13 | 1995-05-23 | Pharmacia Ab | Injection needle arrangement |
US5328483A (en) | 1992-02-27 | 1994-07-12 | Jacoby Richard M | Intradermal injection device with medication and needle guard |
US5383851A (en) | 1992-07-24 | 1995-01-24 | Bioject Inc. | Needleless hypodermic injection device |
US5520639A (en) | 1992-07-24 | 1996-05-28 | Bioject, Inc. | Needleless hypodermic injection methods and device |
US5704911A (en) | 1992-09-28 | 1998-01-06 | Equidyne Systems, Inc. | Needleless hypodermic jet injector |
US5569189A (en) | 1992-09-28 | 1996-10-29 | Equidyne Systems, Inc. | hypodermic jet injector |
US5334144A (en) | 1992-10-30 | 1994-08-02 | Becton, Dickinson And Company | Single use disposable needleless injector |
US5649912A (en) | 1994-03-07 | 1997-07-22 | Bioject, Inc. | Ampule filling device |
US5466220A (en) | 1994-03-08 | 1995-11-14 | Bioject, Inc. | Drug vial mixing and transfer device |
US5599302A (en) | 1995-01-09 | 1997-02-04 | Medi-Ject Corporation | Medical injection system and method, gas spring thereof and launching device using gas spring |
WO1997013537A1 (en) | 1995-10-10 | 1997-04-17 | Visionary Medical Products Corporation | Gas pressured needle-less injection device |
US5893397A (en) | 1996-01-12 | 1999-04-13 | Bioject Inc. | Medication vial/syringe liquid-transfer apparatus |
WO1997037705A1 (en) | 1996-04-11 | 1997-10-16 | Weston Medical Limited | Spring-powered dispensing device for medical purposes |
US5993412A (en) | 1997-05-19 | 1999-11-30 | Bioject, Inc. | Injection apparatus |
WO1999034850A1 (en) | 1998-01-08 | 1999-07-15 | Fiderm S.R.L. | Device for controlling the penetration depth of a needle, for application to an injection syringe |
WO2007024708A2 (en) | 2005-08-23 | 2007-03-01 | The Trustees Of The University Of Pennsylvania | Rna containing modified nucleosides and methods of use thereof |
WO2009127230A1 (en) * | 2008-04-16 | 2009-10-22 | Curevac Gmbh | MODIFIED (m)RNA FOR SUPPRESSING OR AVOIDING AN IMMUNOSTIMULATORY RESPONSE AND IMMUNOSUPPRESSIVE COMPOSITION |
WO2010111290A1 (en) * | 2009-03-23 | 2010-09-30 | University Of Utah Research Foundation | Methods and compositions related to modified guanine bases for controlling off-target effects in rna interference |
Non-Patent Citations (24)
Title |
---|
"Computational Molecular Biology", 1988, OXFORD UNIVERSITY PRESS |
"Remington: The Science and Practice of Pharmacy", 2005, LIPPINCOTT WILLIAMS & WILKINS |
"Remington's Pharmaceutical Sciences", 1985, MACK PUBLISHING COMPANY, pages: 1418 |
A. R. GENNARO: "Remington's The Science and Practice of Pharmacy", 2006, LIPPINCOTT, WILLIAMS & WILKINS |
ALBRECHT, IMMUNOTHERAPY, vol. 2, no. 6, 2010, pages 795 - 798 |
CARILLO ET AL., SIAM J. APPLIED MATH., vol. 48, 1988, pages 1073 |
EDWARD B. ROCHE: "Bioreversible Carriers in Drug Design", 1987, AMERICAN PHARMACEUTICAL ASSOCIATION AND PERGAMON PRESS |
FUKUHARA ET AL., BIOCHEMISTRY, vol. 1, no. 4, 1962, pages 563 - 568 |
GAIT, M.J.: "Oligonucleotide synthesis: a practical approach", 1984, IRL PRESS |
GREENE ET AL.: "Protective Groups in Organic Synthesis", 1991, WILEY & SONS |
GRIBSKOV, M. AND DEVEREUX, J.: "Sequence Analysis Primer", 1991, M. STOCKTON PRESS |
GRIFFIN, A. M., AND GRIFFIN: "Computer Analysis of Sequence Data", 1994, HUMANA PRESS |
HEINJE, G.: "Sequence Analysis in Molecular Biology", 1987, ACADEMIC PRESS |
HERDEWIJN, P.: "Oligonucleotide synthesis: methods and applications, Methods in Molecular Biology", vol. 288, 2005, HUMANA PRESS |
JOURNAL OF PHARMACEUTICAL SCIENCE, vol. 66, no. 2, 1977 |
KAIRO KATALIN ET AL.: "Molecular Therapy", vol. 16, 1 November 2008, NATURE PUBLISHING GROUP, pages: 1833 - 1840 |
LESK, A. M.: "Biocomputing: Informatics and Genome Projects", 1993, ACADEMIC PRESS |
MUSUNURU K ET AL.: "From noncoding variant to phenotype via SORT] at the lpl3 cholesterol locus", NATURE, vol. 466, 2010, pages 714 - 721 |
OGATA ET AL., JOURNAL OF ORGANIC CHEMISTRY, vol. 74, 2009, pages 2585 - 2588 |
PURMAL ET AL., NUCLEIC ACIDS RESEARCH, vol. 22, no. 1, 1994, pages 72 - 78 |
ROZENSKI, J; CRAIN, P; MCCLOSKEY, J: "The RNA Modification Database: 1999 update", NUCL ACIDS RES, vol. 27, 1999, pages 196 - 197 |
T. HIGUCHI; V. STELLA: "Pro-drugs as Novel Delivery Systems", vol. 14, A.C.S. SYMPOSIUM SERIES |
VERMA S. ET AL.: "Modified Oligonucleotides: Synthesis and Strategy for Users", ANNU. REV. BIOCHEM., vol. 67, 1998, pages 99 - 134, XP002412536 * |
XU ET AL., TETRAHEDRON, vol. 48, no. 9, 1992, pages 1729 - 1740 |
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US9518272B2 (en) | 2010-06-30 | 2016-12-13 | Protiva Biotherapeutics, Inc. | Non-liposomal systems for nucleic acid delivery |
US11718852B2 (en) | 2010-06-30 | 2023-08-08 | Arbutus Biopharma Corporation | Non-liposomal systems for nucleic acid delivery |
US11883534B2 (en) | 2010-07-06 | 2024-01-30 | Glaxosmithkline Biologicals Sa | Immunisation with lipid formulations with RNA encoding immunogens |
US11766401B2 (en) | 2010-07-06 | 2023-09-26 | Glaxosmithkline Biologicals Sa | Methods of administering lipid formulations with immunogens |
US11696923B2 (en) | 2010-07-06 | 2023-07-11 | Glaxosmithkline Biologicals, Sa | Delivery of RNA to trigger multiple immune pathways |
US11730754B2 (en) | 2010-07-06 | 2023-08-22 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11690864B2 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11690865B2 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11857681B2 (en) | 2010-07-06 | 2024-01-02 | Glaxosmithkline Biologicals Sa | Lipid formulations with RNA encoding immunogens |
US11865080B2 (en) | 2010-07-06 | 2024-01-09 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11839686B2 (en) | 2010-07-06 | 2023-12-12 | Glaxosmithkline Biologicals Sa | Lipid formulations with viral immunogens |
US11596645B2 (en) | 2010-07-06 | 2023-03-07 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11857562B2 (en) | 2010-07-06 | 2024-01-02 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11690862B1 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11786467B2 (en) | 2010-07-06 | 2023-10-17 | Glaxosmithkline Biologicals Sa | Lipid formulations with immunogens |
US11891608B2 (en) | 2010-07-06 | 2024-02-06 | Glaxosmithkline Biologicals Sa | Immunization of large mammals with low doses of RNA |
US11773395B1 (en) | 2010-07-06 | 2023-10-03 | Glaxosmithkline Biologicals Sa | Immunization of large mammals with low doses of RNA |
US11707482B2 (en) | 2010-07-06 | 2023-07-25 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11690861B2 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11638694B2 (en) | 2010-07-06 | 2023-05-02 | Glaxosmithkline Biologicals Sa | Vaccine for eliciting immune response comprising lipid formulations and RNA encoding multiple immunogens |
US11638693B2 (en) | 2010-07-06 | 2023-05-02 | Glaxosmithkline Biologicals Sa | Vaccine for eliciting immune response comprising RNA encoding an immunogen and lipid formulations comprising mole percentage of lipids |
US11655475B2 (en) | 2010-07-06 | 2023-05-23 | Glaxosmithkline Biologicals Sa | Immunisation of large mammals with low doses of RNA |
US11905514B2 (en) | 2010-07-06 | 2024-02-20 | Glaxosmithkline Biological Sa | Immunisation of large mammals with low doses of RNA |
US11913001B2 (en) | 2010-07-06 | 2024-02-27 | Glaxosmithkline Biologicals Sa | Immunisation of large mammals with low doses of RNA |
US11850305B2 (en) | 2010-07-06 | 2023-12-26 | Glaxosmithkline Biologicals Sa | Method of making lipid formulations with RNA encoding immunogens |
US11759475B2 (en) | 2010-07-06 | 2023-09-19 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11717529B2 (en) | 2010-07-06 | 2023-08-08 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11666534B2 (en) | 2010-07-06 | 2023-06-06 | Glaxosmithkline Biologicals Sa | Methods of administering lipid formulations with viral immunogens |
US11739334B2 (en) | 2010-07-06 | 2023-08-29 | Glaxosmithkline Biologicals Sa | Immunisation of large mammals with low doses of RNA |
US11845925B2 (en) | 2010-07-06 | 2023-12-19 | Glaxosmithkline Biologicals Sa | Immunisation of large mammals with low doses of RNA |
US11690863B2 (en) | 2010-07-06 | 2023-07-04 | Glaxosmithkline Biologicals Sa | Delivery of RNA to trigger multiple immune pathways |
US11851660B2 (en) | 2010-07-06 | 2023-12-26 | Glaxosmithkline Biologicals Sa | Immunisation of large mammals with low doses of RNA |
US20220125723A1 (en) | 2010-07-06 | 2022-04-28 | Glaxosmithkline Biologicals Sa | Lipid formulations with viral immunogens |
US9181319B2 (en) | 2010-08-06 | 2015-11-10 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
US9447164B2 (en) | 2010-08-06 | 2016-09-20 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
US9937233B2 (en) | 2010-08-06 | 2018-04-10 | Modernatx, Inc. | Engineered nucleic acids and methods of use thereof |
US8822663B2 (en) | 2010-08-06 | 2014-09-02 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
US11759422B2 (en) | 2010-08-31 | 2023-09-19 | Glaxosmithkline Biologicals Sa | Pegylated liposomes for delivery of immunogen-encoding RNA |
US9334328B2 (en) | 2010-10-01 | 2016-05-10 | Moderna Therapeutics, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US10064959B2 (en) | 2010-10-01 | 2018-09-04 | Modernatx, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
EP3590949B1 (en) | 2010-10-01 | 2022-05-18 | ModernaTX, Inc. | Ribonucleic acids containing n1-methyl-pseudouracils and uses thereof |
US9657295B2 (en) | 2010-10-01 | 2017-05-23 | Modernatx, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US11639370B2 (en) | 2010-10-11 | 2023-05-02 | Glaxosmithkline Biologicals Sa | Antigen delivery platforms |
US8853377B2 (en) | 2010-11-30 | 2014-10-07 | Shire Human Genetic Therapies, Inc. | mRNA for use in treatment of human genetic diseases |
US11135274B2 (en) | 2010-11-30 | 2021-10-05 | Translate Bio, Inc. | MRNA for use in treatment of human genetic diseases |
US9061021B2 (en) | 2010-11-30 | 2015-06-23 | Shire Human Genetic Therapies, Inc. | mRNA for use in treatment of human genetic diseases |
US9956271B2 (en) | 2010-11-30 | 2018-05-01 | Translate Bio, Inc. | mRNA for use in treatment of human genetic diseases |
US9533047B2 (en) | 2011-03-31 | 2017-01-03 | Modernatx, Inc. | Delivery and formulation of engineered nucleic acids |
US9950068B2 (en) | 2011-03-31 | 2018-04-24 | Modernatx, Inc. | Delivery and formulation of engineered nucleic acids |
US8710200B2 (en) | 2011-03-31 | 2014-04-29 | Moderna Therapeutics, Inc. | Engineered nucleic acids encoding a modified erythropoietin and their expression |
US10738355B2 (en) | 2011-05-24 | 2020-08-11 | Tron-Translationale Onkologie An Der Universitätsmedizin Der Johannes Gutenberg-Universität Mainz Ggmbh | Individualized vaccines for cancer |
US11248264B2 (en) | 2011-05-24 | 2022-02-15 | Tron-Translationale Onkologie An Der Universitätsmedizin Der Johannes Gutenberg-Universität Mainz Ggmbh | Individualized vaccines for cancer |
US10238754B2 (en) | 2011-06-08 | 2019-03-26 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for MRNA delivery |
US11951180B2 (en) | 2011-06-08 | 2024-04-09 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for MRNA delivery |
US10507249B2 (en) | 2011-06-08 | 2019-12-17 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US11291734B2 (en) | 2011-06-08 | 2022-04-05 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US9308281B2 (en) | 2011-06-08 | 2016-04-12 | Shire Human Genetic Therapies, Inc. | MRNA therapy for Fabry disease |
US11951181B2 (en) | 2011-06-08 | 2024-04-09 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US11547764B2 (en) | 2011-06-08 | 2023-01-10 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for MRNA delivery |
US9597413B2 (en) | 2011-06-08 | 2017-03-21 | Shire Human Genetic Therapies, Inc. | Pulmonary delivery of mRNA |
US11730825B2 (en) | 2011-06-08 | 2023-08-22 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US10350303B1 (en) | 2011-06-08 | 2019-07-16 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US11185595B2 (en) | 2011-06-08 | 2021-11-30 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US11951179B2 (en) | 2011-06-08 | 2024-04-09 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for MRNA delivery |
US10413618B2 (en) | 2011-06-08 | 2019-09-17 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US11052159B2 (en) | 2011-06-08 | 2021-07-06 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US11338044B2 (en) | 2011-06-08 | 2022-05-24 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US10888626B2 (en) | 2011-06-08 | 2021-01-12 | Translate Bio, Inc. | Lipid nanoparticle compositions and methods for mRNA delivery |
US11896636B2 (en) | 2011-07-06 | 2024-02-13 | Glaxosmithkline Biologicals Sa | Immunogenic combination compositions and uses thereof |
US10022425B2 (en) | 2011-09-12 | 2018-07-17 | Modernatx, Inc. | Engineered nucleic acids and methods of use thereof |
US10751386B2 (en) | 2011-09-12 | 2020-08-25 | Modernatx, Inc. | Engineered nucleic acids and methods of use thereof |
US9464124B2 (en) | 2011-09-12 | 2016-10-11 | Moderna Therapeutics, Inc. | Engineered nucleic acids and methods of use thereof |
EP4015005A1 (en) | 2011-10-03 | 2022-06-22 | ModernaTX, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
EP3492109B1 (en) | 2011-10-03 | 2020-03-04 | ModernaTX, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US9428535B2 (en) | 2011-10-03 | 2016-08-30 | Moderna Therapeutics, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
EP3682905B1 (en) | 2011-10-03 | 2021-12-01 | ModernaTX, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
CN110511939A (en) * | 2011-10-03 | 2019-11-29 | 现代泰克斯公司 | Nucleosides, nucleotide and nucleic acid of modification and application thereof |
EP3682905A1 (en) | 2011-10-03 | 2020-07-22 | ModernaTX, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
EP3492109A1 (en) | 2011-10-03 | 2019-06-05 | Moderna Therapeutics, Inc. | Modified nucleosides, nucleotides, and nucleic acids, and uses thereof |
US9271996B2 (en) | 2011-12-16 | 2016-03-01 | Moderna Therapeutics, Inc. | Formulation and delivery of PLGA microspheres |
US9186372B2 (en) | 2011-12-16 | 2015-11-17 | Moderna Therapeutics, Inc. | Split dose administration |
US8680069B2 (en) | 2011-12-16 | 2014-03-25 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of G-CSF |
US8664194B2 (en) | 2011-12-16 | 2014-03-04 | Moderna Therapeutics, Inc. | Method for producing a protein of interest in a primate |
US9295689B2 (en) | 2011-12-16 | 2016-03-29 | Moderna Therapeutics, Inc. | Formulation and delivery of PLGA microspheres |
US8754062B2 (en) | 2011-12-16 | 2014-06-17 | Moderna Therapeutics, Inc. | DLIN-KC2-DMA lipid nanoparticle delivery of modified polynucleotides |
EP2791160B1 (en) | 2011-12-16 | 2022-03-02 | ModernaTX, Inc. | Modified mrna compositions |
US11559587B2 (en) | 2012-03-26 | 2023-01-24 | Tron-Translationale Onkologie An Der Universitätsmedizin Der Johannes Gutenberg-Universität Mainz Ggmbh | RNA formulation for immunotherapy |
US10485884B2 (en) | 2012-03-26 | 2019-11-26 | Biontech Rna Pharmaceuticals Gmbh | RNA formulation for immunotherapy |
US9233141B2 (en) | 2012-04-02 | 2016-01-12 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders |
US11564998B2 (en) | 2012-04-02 | 2023-01-31 | Modernatx, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
US9878056B2 (en) | 2012-04-02 | 2018-01-30 | Modernatx, Inc. | Modified polynucleotides for the production of cosmetic proteins and peptides |
US9827332B2 (en) | 2012-04-02 | 2017-11-28 | Modernatx, Inc. | Modified polynucleotides for the production of proteins |
US9828416B2 (en) | 2012-04-02 | 2017-11-28 | Modernatx, Inc. | Modified polynucleotides for the production of secreted proteins |
US9814760B2 (en) | 2012-04-02 | 2017-11-14 | Modernatx, Inc. | Modified polynucleotides for the production of biologics and proteins associated with human disease |
US10501512B2 (en) | 2012-04-02 | 2019-12-10 | Modernatx, Inc. | Modified polynucleotides |
US9782462B2 (en) | 2012-04-02 | 2017-10-10 | Modernatx, Inc. | Modified polynucleotides for the production of proteins associated with human disease |
US9675668B2 (en) | 2012-04-02 | 2017-06-13 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding hepatitis A virus cellular receptor 2 |
US9587003B2 (en) | 2012-04-02 | 2017-03-07 | Modernatx, Inc. | Modified polynucleotides for the production of oncology-related proteins and peptides |
US9572897B2 (en) | 2012-04-02 | 2017-02-21 | Modernatx, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
US9301993B2 (en) | 2012-04-02 | 2016-04-05 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding apoptosis inducing factor 1 |
US9303079B2 (en) | 2012-04-02 | 2016-04-05 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of cytoplasmic and cytoskeletal proteins |
US9283287B2 (en) | 2012-04-02 | 2016-03-15 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of nuclear proteins |
US9254311B2 (en) | 2012-04-02 | 2016-02-09 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of proteins |
US9255129B2 (en) | 2012-04-02 | 2016-02-09 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding SIAH E3 ubiquitin protein ligase 1 |
US9221891B2 (en) | 2012-04-02 | 2015-12-29 | Moderna Therapeutics, Inc. | In vivo production of proteins |
US9220792B2 (en) | 2012-04-02 | 2015-12-29 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding aquaporin-5 |
US9220755B2 (en) | 2012-04-02 | 2015-12-29 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of proteins associated with blood and lymphatic disorders |
US9216205B2 (en) | 2012-04-02 | 2015-12-22 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding granulysin |
US9192651B2 (en) | 2012-04-02 | 2015-11-24 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of secreted proteins |
US9149506B2 (en) | 2012-04-02 | 2015-10-06 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding septin-4 |
US9114113B2 (en) | 2012-04-02 | 2015-08-25 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding citeD4 |
US9107886B2 (en) | 2012-04-02 | 2015-08-18 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding basic helix-loop-helix family member E41 |
US9095552B2 (en) | 2012-04-02 | 2015-08-04 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding copper metabolism (MURR1) domain containing 1 |
US9089604B2 (en) | 2012-04-02 | 2015-07-28 | Moderna Therapeutics, Inc. | Modified polynucleotides for treating galactosylceramidase protein deficiency |
US9061059B2 (en) | 2012-04-02 | 2015-06-23 | Moderna Therapeutics, Inc. | Modified polynucleotides for treating protein deficiency |
US9050297B2 (en) | 2012-04-02 | 2015-06-09 | Moderna Therapeutics, Inc. | Modified polynucleotides encoding aryl hydrocarbon receptor nuclear translocator |
US8999380B2 (en) | 2012-04-02 | 2015-04-07 | Moderna Therapeutics, Inc. | Modified polynucleotides for the production of biologics and proteins associated with human disease |
US11254936B2 (en) | 2012-06-08 | 2022-02-22 | Translate Bio, Inc. | Nuclease resistant polynucleotides and uses thereof |
US9512456B2 (en) | 2012-08-14 | 2016-12-06 | Modernatx, Inc. | Enzymes and polymerases for the synthesis of RNA |
WO2014081507A1 (en) | 2012-11-26 | 2014-05-30 | Moderna Therapeutics, Inc. | Terminally modified rna |
US9597380B2 (en) | 2012-11-26 | 2017-03-21 | Modernatx, Inc. | Terminally modified RNA |
AU2017202228B2 (en) * | 2012-11-26 | 2019-03-14 | Modernatx, Inc. | Terminally modified RNA |
US10925935B2 (en) | 2012-11-26 | 2021-02-23 | Modernatx, Inc. | Terminally Modified RNA |
US10155029B2 (en) | 2012-11-26 | 2018-12-18 | Modernatx, Inc. | Terminally modified RNA |
EP4074834A1 (en) | 2012-11-26 | 2022-10-19 | ModernaTX, Inc. | Terminally modified rna |
US11504419B2 (en) | 2012-11-28 | 2022-11-22 | BioNTech SE | Individualized vaccines for cancer |
US10155031B2 (en) | 2012-11-28 | 2018-12-18 | Biontech Rna Pharmaceuticals Gmbh | Individualized vaccines for cancer |
WO2014093924A1 (en) | 2012-12-13 | 2014-06-19 | Moderna Therapeutics, Inc. | Modified nucleic acid molecules and uses thereof |
US11708396B2 (en) | 2013-01-17 | 2023-07-25 | Modernatx, Inc. | Signal-sensor polynucleotides for the alteration of cellular phenotypes |
WO2014113089A2 (en) | 2013-01-17 | 2014-07-24 | Moderna Therapeutics, Inc. | Signal-sensor polynucleotides for the alteration of cellular phenotypes |
WO2014159813A1 (en) | 2013-03-13 | 2014-10-02 | Moderna Therapeutics, Inc. | Long-lived polynucleotide molecules |
US11692189B2 (en) | 2013-03-14 | 2023-07-04 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US9181321B2 (en) | 2013-03-14 | 2015-11-10 | Shire Human Genetic Therapies, Inc. | CFTR mRNA compositions and related methods and uses |
US9713626B2 (en) | 2013-03-14 | 2017-07-25 | Rana Therapeutics, Inc. | CFTR mRNA compositions and related methods and uses |
US10420791B2 (en) | 2013-03-14 | 2019-09-24 | Translate Bio, Inc. | CFTR MRNA compositions and related methods and uses |
US9957499B2 (en) | 2013-03-14 | 2018-05-01 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11820977B2 (en) | 2013-03-14 | 2023-11-21 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US10876104B2 (en) | 2013-03-14 | 2020-12-29 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US10258698B2 (en) | 2013-03-14 | 2019-04-16 | Modernatx, Inc. | Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions |
US11510937B2 (en) | 2013-03-14 | 2022-11-29 | Translate Bio, Inc. | CFTR MRNA compositions and related methods and uses |
US8980864B2 (en) | 2013-03-15 | 2015-03-17 | Moderna Therapeutics, Inc. | Compositions and methods of altering cholesterol levels |
DE102013005361A1 (en) | 2013-03-28 | 2014-10-02 | Eberhard Karls Universität Tübingen Medizinische Fakultät | polyribonucleotide |
US11222711B2 (en) | 2013-05-10 | 2022-01-11 | BioNTech SE | Predicting immunogenicity of T cell epitopes |
EP3971287A1 (en) | 2013-07-11 | 2022-03-23 | ModernaTX, Inc. | Compositions comprising synthetic polynucleotides encoding crispr related proteins and synthetic sgrnas and methods of use |
EP3041938A1 (en) * | 2013-09-03 | 2016-07-13 | Moderna Therapeutics, Inc. | Circular polynucleotides |
WO2015034925A1 (en) | 2013-09-03 | 2015-03-12 | Moderna Therapeutics, Inc. | Circular polynucleotides |
EP3052106A4 (en) * | 2013-09-30 | 2017-07-19 | ModernaTX, Inc. | Polynucleotides encoding immune modulating polypeptides |
US10023626B2 (en) | 2013-09-30 | 2018-07-17 | Modernatx, Inc. | Polynucleotides encoding immune modulating polypeptides |
US10815291B2 (en) | 2013-09-30 | 2020-10-27 | Modernatx, Inc. | Polynucleotides encoding immune modulating polypeptides |
US10323076B2 (en) | 2013-10-03 | 2019-06-18 | Modernatx, Inc. | Polynucleotides encoding low density lipoprotein receptor |
US11224642B2 (en) | 2013-10-22 | 2022-01-18 | Translate Bio, Inc. | MRNA therapy for argininosuccinate synthetase deficiency |
US9522176B2 (en) | 2013-10-22 | 2016-12-20 | Shire Human Genetic Therapies, Inc. | MRNA therapy for phenylketonuria |
US10208295B2 (en) | 2013-10-22 | 2019-02-19 | Translate Bio, Inc. | MRNA therapy for phenylketonuria |
US11377642B2 (en) | 2013-10-22 | 2022-07-05 | Translate Bio, Inc. | mRNA therapy for phenylketonuria |
EP3134131B1 (en) | 2014-04-23 | 2021-12-22 | ModernaTX, Inc. | Nucleic acid vaccines |
US10022435B2 (en) | 2014-04-23 | 2018-07-17 | Modernatx, Inc. | Nucleic acid vaccines |
US9872900B2 (en) | 2014-04-23 | 2018-01-23 | Modernatx, Inc. | Nucleic acid vaccines |
US10709779B2 (en) | 2014-04-23 | 2020-07-14 | Modernatx, Inc. | Nucleic acid vaccines |
US11059841B2 (en) | 2014-04-25 | 2021-07-13 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11884692B2 (en) | 2014-04-25 | 2024-01-30 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US10155785B2 (en) | 2014-04-25 | 2018-12-18 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US9850269B2 (en) | 2014-04-25 | 2017-12-26 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11173120B2 (en) | 2014-09-25 | 2021-11-16 | Biontech Rna Pharmaceuticals Gmbh | Stable formulations of lipids and liposomes |
US9751925B2 (en) | 2014-11-10 | 2017-09-05 | Modernatx, Inc. | Alternative nucleic acid molecules containing reduced uracil content and uses thereof |
US10072057B2 (en) | 2014-11-10 | 2018-09-11 | Modernatx, Inc. | Alternative nucleic acid molecules containing reduced uracil content and uses thereof |
US11156617B2 (en) | 2015-02-12 | 2021-10-26 | BioNTech RNA Pharmaceuticals GbmH | Predicting T cell epitopes useful for vaccination |
US11590157B2 (en) | 2015-10-05 | 2023-02-28 | Modernatx, Inc. | Methods for therapeutic administration of messenger ribonucleic acid drugs |
US10849920B2 (en) | 2015-10-05 | 2020-12-01 | Modernatx, Inc. | Methods for therapeutic administration of messenger ribonucleic acid drugs |
US11492628B2 (en) | 2015-10-07 | 2022-11-08 | BioNTech SE | 3′-UTR sequences for stabilization of RNA |
EP3718565B1 (en) | 2015-10-22 | 2022-04-27 | ModernaTX, Inc. | Respiratory virus vaccines |
WO2017127750A1 (en) | 2016-01-22 | 2017-07-27 | Modernatx, Inc. | Messenger ribonucleic acids for the production of intracellular binding polypeptides and methods of use thereof |
WO2018009838A1 (en) | 2016-07-07 | 2018-01-11 | Rubius Therapeutics, Inc. | Compositions and methods related to therapeutic cell systems expressing exogenous rna |
US11981703B2 (en) | 2016-08-17 | 2024-05-14 | Sirius Therapeutics, Inc. | Polynucleotide constructs |
WO2018081459A1 (en) | 2016-10-26 | 2018-05-03 | Modernatx, Inc. | Messenger ribonucleic acids for enhancing immune responses and methods of use thereof |
US11993645B2 (en) | 2017-01-11 | 2024-05-28 | The Board Of Trustees Of The Leland Stanford Junior University | Compositions comprising R-Spondin (RSPO) surrogate molecules |
US11958891B2 (en) | 2017-01-26 | 2024-04-16 | Surrozen Operating, Inc. | Tissue-specific Wnt signal enhancing molecules and uses thereof |
US10093706B2 (en) | 2017-01-30 | 2018-10-09 | Indiana University Research And Technology Corporation | Dominant positive hnRNP-E1 polypeptide compositions and methods |
WO2018144775A1 (en) | 2017-02-01 | 2018-08-09 | Modernatx, Inc. | Immunomodulatory therapeutic mrna compositions encoding activating oncogene mutation peptides |
US11253605B2 (en) | 2017-02-27 | 2022-02-22 | Translate Bio, Inc. | Codon-optimized CFTR MRNA |
US11865159B2 (en) | 2017-02-28 | 2024-01-09 | Sanofi | Therapeutic RNA |
US11173190B2 (en) | 2017-05-16 | 2021-11-16 | Translate Bio, Inc. | Treatment of cystic fibrosis by delivery of codon-optimized mRNA encoding CFTR |
EP3625345B1 (en) | 2017-05-18 | 2023-05-24 | ModernaTX, Inc. | Modified messenger rna comprising functional rna elements |
WO2018213789A1 (en) | 2017-05-18 | 2018-11-22 | Modernatx, Inc. | Modified messenger rna comprising functional rna elements |
EP4253544A2 (en) | 2017-05-18 | 2023-10-04 | ModernaTX, Inc. | Modified messenger rna comprising functional rna elements |
US11786607B2 (en) | 2017-06-15 | 2023-10-17 | Modernatx, Inc. | RNA formulations |
US11597744B2 (en) | 2017-06-30 | 2023-03-07 | Sirius Therapeutics, Inc. | Chiral phosphoramidite auxiliaries and methods of their use |
US11744801B2 (en) | 2017-08-31 | 2023-09-05 | Modernatx, Inc. | Methods of making lipid nanoparticles |
WO2019152557A1 (en) | 2018-01-30 | 2019-08-08 | Modernatx, Inc. | Compositions and methods for delivery of agents to immune cells |
WO2019200171A1 (en) | 2018-04-11 | 2019-10-17 | Modernatx, Inc. | Messenger rna comprising functional rna elements |
WO2019204743A1 (en) | 2018-04-19 | 2019-10-24 | Checkmate Pharmaceuticals, Inc. | Synthetic rig-i-like receptor agonists |
US11174500B2 (en) | 2018-08-24 | 2021-11-16 | Translate Bio, Inc. | Methods for purification of messenger RNA |
US11015211B2 (en) | 2018-08-30 | 2021-05-25 | Tenaya Therapeutics, Inc. | Cardiac cell reprogramming with myocardin and ASCL1 |
US11913012B2 (en) | 2018-08-30 | 2024-02-27 | Tenaya Therapeutics, Inc. | Cardiac cell reprogramming with myocardin and ASCL1 |
WO2020056304A1 (en) | 2018-09-14 | 2020-03-19 | Modernatx, Inc. | Methods and compositions for treating cancer using mrna therapeutics |
WO2020097409A2 (en) | 2018-11-08 | 2020-05-14 | Modernatx, Inc. | Use of mrna encoding ox40l to treat cancer in human patients |
WO2020227510A1 (en) | 2019-05-07 | 2020-11-12 | Modernatx, Inc. | Polynucleotides for disrupting immune cell activity and methods of use thereof |
WO2020227537A1 (en) | 2019-05-07 | 2020-11-12 | Modernatx, Inc | Differentially expressed immune cell micrornas for regulation of protein expression |
WO2020263883A1 (en) | 2019-06-24 | 2020-12-30 | Modernatx, Inc. | Endonuclease-resistant messenger rna and uses thereof |
WO2020263985A1 (en) | 2019-06-24 | 2020-12-30 | Modernatx, Inc. | Messenger rna comprising functional rna elements and uses thereof |
WO2021007515A1 (en) | 2019-07-11 | 2021-01-14 | Tenaya Therapeutics, Inc. | Cardiac cell reprogramming with micrornas and other factors |
WO2021050986A1 (en) | 2019-09-11 | 2021-03-18 | Modernatx, Inc. | Lnp-formulated mrna therapeutics and use thereof for treating human subjects |
WO2021081353A1 (en) | 2019-10-23 | 2021-04-29 | Checkmate Pharmaceuticals, Inc. | Synthetic rig-i-like receptor agonists |
WO2021178246A1 (en) | 2020-03-02 | 2021-09-10 | Tenaya Therapeutics, Inc. | Gene vector control by cardiomyocyte-expressed micrornas |
US11951185B2 (en) | 2020-04-22 | 2024-04-09 | BioNTech SE | RNA constructs and uses thereof |
US11547673B1 (en) | 2020-04-22 | 2023-01-10 | BioNTech SE | Coronavirus vaccine |
US11779659B2 (en) | 2020-04-22 | 2023-10-10 | BioNTech SE | RNA constructs and uses thereof |
US11925694B2 (en) | 2020-04-22 | 2024-03-12 | BioNTech SE | Coronavirus vaccine |
WO2021243207A1 (en) | 2020-05-28 | 2021-12-02 | Modernatx, Inc. | Use of mrnas encoding ox40l, il-23 and il-36gamma for treating cancer |
WO2022032154A2 (en) | 2020-08-06 | 2022-02-10 | Modernatx, Inc. | Compositions for the delivery of payload molecules to airway epithelium |
WO2023009421A1 (en) | 2021-07-26 | 2023-02-02 | Modernatx, Inc. | Processes for preparing lipid nanoparticle compositions |
WO2023009422A1 (en) | 2021-07-26 | 2023-02-02 | Modernatx, Inc. | Processes for preparing lipid nanoparticle compositions for the delivery of payload molecules to airway epithelium |
WO2023064469A1 (en) | 2021-10-13 | 2023-04-20 | Modernatx, Inc. | Compositions of mrna-encoded il15 fusion proteins and methods of use thereof |
WO2023069498A1 (en) | 2021-10-22 | 2023-04-27 | Senda Biosciences, Inc. | Mrna vaccine composition |
WO2023086465A1 (en) | 2021-11-12 | 2023-05-19 | Modernatx, Inc. | Compositions for the delivery of payload molecules to airway epithelium |
WO2023096858A1 (en) | 2021-11-23 | 2023-06-01 | Senda Biosciences, Inc. | A bacteria-derived lipid composition and use thereof |
WO2023122080A1 (en) | 2021-12-20 | 2023-06-29 | Senda Biosciences, Inc. | Compositions comprising mrna and lipid reconstructed plant messenger packs |
WO2023154818A1 (en) | 2022-02-09 | 2023-08-17 | Modernatx, Inc. | Mucosal administration methods and formulations |
WO2023196988A1 (en) | 2022-04-07 | 2023-10-12 | Modernatx, Inc. | Methods of use of mrnas encoding il-12 |
WO2023199113A1 (en) | 2022-04-15 | 2023-10-19 | Smartcella Solutions Ab | COMPOSITIONS AND METHODS FOR EXOSOME-MEDIATED DELIVERY OF mRNA AGENTS |
US11878055B1 (en) | 2022-06-26 | 2024-01-23 | BioNTech SE | Coronavirus vaccine |
WO2024044741A3 (en) * | 2022-08-26 | 2024-04-18 | Trilink Biotechnologies, Llc | Efficient method for making highly purified 5'-capped oligonucleotides |
WO2024102434A1 (en) | 2022-11-10 | 2024-05-16 | Senda Biosciences, Inc. | Rna compositions comprising lipid nanoparticles or lipid reconstructed natural messenger packs |
WO2024107827A1 (en) | 2022-11-16 | 2024-05-23 | The Broad Institute, Inc. | Therapeutic exploitation of sting channel activity |
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