CA2868391A1 - Polynucleotides comprising n1-methyl-pseudouridine and methods for preparing the same - Google Patents

Abstract

The invention relates to compositions and methods for the preparation, manufacture and therapeutic use of polynucleotides, primary transcripts and mmRNA molecules.

Description

DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

MODIFIED POLYNUCLEOTIDES FOR THE PRODUCTION OF BIOLOGICS
AND PROTEINS ASSOCIATED WITH HUMAN DISEASE
REFERENCE TO SEQUENCE LISTING
[0001] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing file entitled M300PCTSQLST.txt, was created on March 9, 2013 and is 49,417,315 bytes in size. The information in electronic format of the Sequence Listing is incorporated herein by reference in its entirety.
CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No 61/681,742, filed, August 10, 2012, entitled Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides, U.S. Provisional Patent Application No 61/737,224, filed December 14, 2012, entitled Terminally Optimized Modified RNAs, International Application No PCT/U52012/069610, filed December 14, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions, U.S.
Provisional Patent Application No 61/618,862, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Biologics, U.S. Provisional Patent Application No 61/681,645, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Biologics, U.S.
Provisional Patent Application No 61/737,130, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Biologics, U.S. Provisional Patent Application No 61/618,866, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Antibodies, U.S. Provisional Patent Application No 61/681,647, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Antibodies, U.S. Provisional Patent Application No 61/737,134, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Antibodies, U.S. Provisional Patent Application No 61/618,868, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Vaccines, U.S. Provisional Patent Application No 61/681,648, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Vaccines, U.S.
Provisional Patent Application No 61/737,135, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Vaccines, U.S. Provisional Patent Application No 61/618,870, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides, U.S. Provisional Patent Application No 61/681,649, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides, U.S. Provisional Patent Application No 61/737,139, filed December 14, 2012, Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides, U.S. Provisional Patent Application No 61/618,873, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Secreted Proteins, U.S. Provisional Patent Application No 61/681,650, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Secreted Proteins, U.S.
Provisional Patent Application No 61/737,147, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Secreted Proteins, U.S.
Provisional Patent Application No 61/618,878, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Plasma Membrane Proteins, U.S. Provisional Patent Application No 61/681,654, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Plasma Membrane Proteins, U.S. Provisional Patent Application No 61/737,152, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Plasma Membrane Proteins, U.S. Provisional Patent Application No 61/618,885, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins, U.S. Provisional Patent Application No 61/681,658, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins, U.S. Provisional Patent Application No 61/737,155, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins, U.S. Provisional Patent Application No 61/618,896, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins, U.S. Provisional Patent Application No 61/668,157, filed July 5, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins, U.S. Provisional Patent Application No 61/681,661, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins, U.S. Provisional Patent Application No 61/737,160, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins, U.S. Provisional Patent Application No 61/618,911, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Nuclear Proteins, U.S.
Provisional Patent Application No 61/681,667, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Nuclear Proteins, U.S. Provisional Patent Application No 61/737,168, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Nuclear Proteins, U.S. Provisional Patent Application No 61/618,922, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Proteins, U.S.
Provisional Patent Application No 61/681,675, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Proteins, U.S. Provisional Patent Application No 61/737,174, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Proteins, U.S. Provisional Patent Application No 61/618,935, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S.
Provisional Patent Application No 61/681,687, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S.
Provisional Patent Application No 61/737,184, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No 61/618,945, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S.
Provisional Patent Application No 61/681,696, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S.
Provisional Patent Application No 61/737,191, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No 61/618,953, filed April 2, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S.
Provisional Patent Application No 61/681,704, filed August 10, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S.
Provisional Patent Application No 61/737,203, filed December 14, 2012, entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease, U.S. Provisional Patent Application No 61/618,961, filed April 2, 2012, entitled Dosing Methods for Modified mRNA, U.S. Provisional Patent Application No 61/648,286, filed May 17, 2012, entitled Dosing Methods for Modified mRNA, the contents of each of which are herein incorporated by reference in its entirety.

[0003] This application is also related to International Publication No.
PCT/US2012/58519, filed October 3, 2012, entitled Modified Nucleosides, Nucleotides, and Nucleic Acids, and Uses Thereof and International Publication No.
PCT/US2012/69610, filed December 14, 2012, entitled Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions.

[0004] The instant application is also related to co-pending applications, each filed concurrently herewith on March 9, 2013 and having Attorney Docket Number M301.20, (PCT/US13/ ) entitled Modified Polynucleotides; Attorney Docket Number M304.20 (PCT/US13/ ), entitled Modified Polynucleotides for the Production of Secreted Proteins; Attorney Docket Number M305.20 (PCT/U513/)0000(), entitled Modified Polynucleotides for the Production of Membrane Proteins; Attorney Docket Number M306.20 (PCT/U513/ ), entitled Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins; Attorney Docket Number M308.20 (PCT/U513/ ), entitled Modified Polynucleotides for the Production of Nuclear Proteins; Attorney Docket Number M309.20 (PCT/U513/ ), entitled Modified Polynucleotides for the Production of Proteins; Attorney Docket Number M310.20 (PCT/US13/ ), entitled Modified Polynucleotides for the Production of Proteins Associated with Human Disease; Attorney Docket Number MNC1.20 (PCT/US13/ ), entitled Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides and Attorney Docket Number MNC2.20 (PCT/U513/ ), entitled Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides, the contents of each of which are herein incorporated by reference in its entirety.
FIELD OF THE INVENTION

[0005] The invention relates to compositions, methods, processes, kits and devices for the design, preparation, manufacture and/or formulation of polynucleotides, primary constructs and modified mRNA molecules (mmRNA).
BACKGROUND OF THE INVENTION

[0006] There are multiple problems with prior methodologies of effecting protein expression. For example, introduced DNA can integrate into host cell genomic DNA at some frequency, resulting in alterations and/or damage to the host cell genomic DNA.
Alternatively, the 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. In addition, assuming proper delivery and no damage or integration into the host genome, there are multiple steps which must occur before the encoded 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. Not only do the multiple processing steps from administered DNA to protein create lag times before the generation of the functional protein, each step represents an opportunity for error and damage to the cell. Further, it is known to be difficult to obtain DNA expression in cells as DNA
frequently enters a cell but is not expressed or not expressed at reasonable rates or concentrations. This can be a particular problem when DNA is introduced into primary cells or modified cell lines.

[0007] In the early 1990's Bloom and colleagues successfully rescued vasopressin-deficient rats by injecting in vitro-transcribed vasopressin mRNA into the hypothalamus (Science 255: 996-998; 1992). However, the low levels of translation and the immunogenicity of the molecules hampered the development of mRNA as a therapeutic and efforts have since focused on alternative applications that could instead exploit these pitfalls, i.e. immunization with mRNAs coding for cancer antigens.

[0008] Others have investigated the use of mRNA to deliver a polypeptide of interest and shown that certain chemical modifications of mRNA molecules, particularly pseudouridine and 5-methyl-cytosine, have reduced immunostimulatory effect.

[0009] These studies are disclosed in, for example, Ribostem Limited in United Kingdom patent application serial number 0316089.2 filed on July 9, 2003 now abandoned, PCT application number PCT/GB2004/002981 filed on July 9, 2004 published as W02005005622, United States patent application national phase entry serial number 10/563,897 filed on June 8, 2006 published as US20060247195 now abandoned, and European patent application national phase entry serial number EP2004743322 filed on July 9, 2004 published as EP1646714 now withdrawn; Novozymes, Inc. in PCT
application number PCT/U52007/88060 filed on December 19, 2007 published as W02008140615, United States patent application national phase entry serial number 12/520,072 filed on July 2, 2009 published as US20100028943 and European patent application national phase entry serial number EP2007874376 filed on July 7, published as EP2104739; University of Rochester in PCT application number PCT/US2006/46120 filed on December 4, 2006 published as W02007064952 and United States patent application serial number 11/606,995 filed on December 1, 2006 published as U520070141030; BioNTech AG in European patent application serial number EP2007024312 filed December 14, 2007 now abandoned, PCT application number PCT/EP2008/01059 filed on December 12, 2008 published as W02009077134, European patent application national phase entry serial number EP2008861423 filed on June 2, 2010 published as EP2240572, United States patent application national phase entry serial number 12/,735,060 filed November 24, 2010 published as US20110065103, German patent application serial number DE 10 2005 046 490 filed September 28, 2005, PCT application PCT/EP2006/0448 filed September 28, 2006 published as W02007036366, national phase European patent EP1934345 published March, 21, and national phase US patent application serial number 11/992,638 filed August 14, 2009 published as 20100129877; Immune Disease Institute Inc. in United States patent application serial number 13/088,009 filed April 15, 2011 published as and PCT application PCT/US2011/32679 filed April 15, 2011 published as W020110130624; Shire Human Genetic Therapeutics in United States patent application serial number 12/957,340 filed on November 20, 2010 published as US20110244026;
Sequitur Inc. in PCT application PCT/U51998/019492 filed on September 18, 1998 published as W01999014346; The Scripps Research Institute in PCT application number PCT/U52010/00567 filed on February 24, 2010 published as W02010098861, and United States patent application national phase entry serial number 13/203,229 filed November 3, 2011 published as US20120053333; Ludwig-Maximillians University in PCT application number PCT/EP2010/004681 filed on July 30, 2010 published as W02011012316; Cellscript Inc. in United States patent number 8,039,214 filed June 30, 2008 and granted October 18, 2011, United States patent application serial numbers 12/962,498 filed on December 7, 2010 published as US20110143436, 12/962,468 filed on December 7, 2010 published as US20110143397, 13/237,451 filed on September 20, 2011 published as US20120009649, and PCT applications PCT/U52010/59305 filed December 7, 2010 published as W02011071931 and PCT/U52010/59317 filed on December 7, 2010 published as W02011071936; The Trustees of the University of Pennsylvania in PCT application number PCT/US2006/32372 filed on August 21, published as W02007024708, and United States patent application national phase entry serial number 11/990,646 filed on March 27, 2009 published as U520090286852;
Curevac GMBH in German patent application serial numbers DE10 2001 027 283.9 filed June 5,2001, DE10 2001 062 480.8 filed December 19, 2001, and DE 20 2006 051 filed October 31, 2006 all abandoned, European patent numbers EP1392341 granted March 30, 2005 and EP1458410 granted January 2, 2008, PCT application numbers PCT/EP2002/06180 filed June 5, 2002 published as W02002098443, PCT/EP2002/14577 filed on December 19, 2002 published as W02003051401, PCT/EP2007/09469 filed on December 31, 2007 published as W02008052770, PCT/EP2008/03033 filed on April 16, 2008 published as W02009127230, PCT/EP2006/004784 filed on May 19, 2005 published as W02006122828, PCT/EP2008/00081 filed on January 9, 2007 published as W02008083949, and United States patent application serial numbers 10/729,830 filed on December 5, 2003 published as U520050032730, 10/870,110 filed on June 18, 2004 published as U520050059624, 11/914,945 filed on July 7, 2008 published as U520080267873, 12/446,912 filed on October 27, 2009 published as U52010047261 now abandoned, 12/522,214 filed on January 4,2010 published as U520100189729, 12/787,566 filed on May 26, 2010 published as US20110077287, 12/787,755 filed on May 26, 2010 published as U520100239608, 13/185,119 filed on July 18, 2011 published as US20110269950, and 13/106,548 filed on May 12, 2011 published as US20110311472 all of which are herein incorporated by reference in their entirety.

[00010] Notwithstanding these reports which are limited to a selection of chemical modifications including pseudouridine and 5-methyl-cytosine, there remains a need in the art for therapeutic modalities to address the myriad of barriers surrounding the efficacious modulation of intracellular translation and processing of nucleic acids encoding polypeptides or fragments thereof [00011] To this end, the inventors have shown that certain modified mRNA
sequences have the potential as therapeutics with benefits beyond just evading, avoiding or diminishing the immune response. Such studies are detailed in published co-pending applications International Application PCT/U52011/046861 filed August 5, 2011 and PCT/US2011/054636 filed October 3, 2011, International Application number PCT/US2011/054617 filed October 3, 2011, the contents of which are incorporated herein by reference in their entirety.

[00012] The present invention addresses this need by providing nucleic acid based compounds or polynucleotides which encode a polypeptide of interest (e.g., modified mRNA or mmRNA) and which have structural and/or chemical features that avoid one or more of the problems in the art, for example, features which are useful for optimizing formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity, overcoming the threshold of expression, improving expression rates, half life and/or protein concentrations, optimizing protein localization, and avoiding deleterious bio-responses such as the immune response and/or degradation pathways.
SUMMARY OF THE INVENTION

[00013] Described herein are compositions, methods, processes, kits and devices for the design, preparation, manufacture and/or formulation of modified mRNA
(mmRNA) molecules.

[00014] The details of various embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and the drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS

[00015] The foregoing and other objects, features and advantages will be apparent from the following description of particular embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating the principles of various embodiments of the invention.

[00016] FIG. 1 is a schematic of a primary construct of the present invention.

[00017] FIG. 2 illustrates lipid structures in the prior art useful in the present invention. Shown are the structures for 98N12-5 (TETA5-LAP), DLin-DMA, DLin-K-DMA (2,2-Dilinoley1-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA and C12-200.

[00018] FIG. 3 is a representative plasmid useful in the IVT reactions taught herein.
The plasmid contains Insert 64818, designed by the instant inventors.

[00019] FIG. 4 is a gel profile of modified mRNA encapsulated in PLGA
micro spheres.

[00020] FIG. 5 is a histogram of Factor IX protein production PLGA formulation Factor IX modified mRNA.

[00021] FIG. 6 is a histogram showing VEGF protein production in human keratinocyte cells after transfection of modified mRNA at a range of doses.
Figure 6A
shows protein production after transfection of modified mRNA comprising natural nucleoside triphosphate (NTP). Figure 6B shows protein production after transfection of modified mRNA fully modified with pseudouridine (Pseudo-U) and 5-methylcytosine (5mC). Figure 6C shows protein production after transfection of modified mRNA
fully modified with Nl-methyl-pseudouridine (N 1-methyl-Pseudo-U)and 5-methylcytosine (5mC).

[00022] FIG. 7 is a histogram of VEGF protein production in HEK293 cells.

[00023] FIG. 8 is a histogram of VEGF expression and IFN-alpha induction after transfection of VEGF modified mRNA in peripheral blood mononuclear cells (PBMC).
Figure 8A shows VEGF expression. Figure 8B shows IFN-alpha induction.

[00024] FIG. 9 is a histogram of VEGF protein production in HeLa cells from VEGF
modified mRNA.

[00025] FIG. 10 is a histogram of VEGF protein production from lipoplexed VEGF

modified mRNA in mice.

[00026] FIG. 11 is a histogram of G-CSF protein production in HeLa cells from G-CSF modified mRNA.

[00027] FIG. 12 is a histogram of G-CSF protein production in mice from lipoplexedG-CSF modified mRNA.

[00028] FIG. 13 is a histogram of Factor IX protein production in HeLa cell supernatant from Factor IX modified mRNA.

[00029] FIG. 14 is a histogram of AP0A1 protein production in HeLa cells from AP0A1 wild-type modified mRNA, AP0A1 Milano modified mRNA or AP0A1 Paris modified mRNA.

[00030] FIG. 15 is a gel profile of AP0A1 protein from AP0A1 wild-type modified mRNA.

[00031] FIG. 16 is a gel profile of AP0A1 protein from AP0A1 Paris modified mRNA.

[00032] FIG. 17 is a gel profile of AP0A1 protein from AP0A1 Milano modified mRNA.

[00033] FIG. 18 is a gel profile of Fibrinogen alpha (FGA) protein from FGA
modified mRNA.

[00034] FIG. 19 is a histogram of Plasminogen protein production in HeLa cell supernatant from Plasminogen modified mRNA.

[00035] FIG. 20 is a gel profile of Plasminogen protein from Plasminogen modified mRNA.

[00036] FIG. 21 is a gel profile of galactose- 1 -phosphate uridylyltransferase (GALT) protein from GALT modified mRNA.

[00037] FIG. 22 is a gel profile of argininosuccinate lyase (ASL) protein from ASL
modified mRNA.

[00038] FIG. 23 is a gel profile of tyrosine aminotransferase (TAT) protein from TAT
modified mRNA.

[00039] FIG. 24 is a gel profile of glucan (1,4-alpha-), branching enzyme 1 (GBE1) protein from GBE1 modified mRNA.

[00040] FIG. 25 is a histogram of Prothrombin protein production in HeLa cell supernatant from Prothrombin modified mRNA.

[00041] FIG. 26 is a histogram of Prothrombin protein production in HeLa cell supernatant from Prothrombin modified mRNA.

[00042] FIG. 27 is a gel profile of ceruloplasmin (CP or CLP) protein from CP
modified mRNA.

[00043] FIG. 28 is a histogram of transforming growth factor beta 1 (TGF-betal) protein production in HeLa cell supernatant from TGF-betal modified mRNA.

[00044] FIG. 29 is a gel profile of ornithine carbamoyltransferase (OTC) protein from OTC modified mRNA.

[00045] FIG. 30 is a flow cytometry plot of low density lipoprotein receptor (LDLR) modified mRNA.

[00046] FIG. 31 is a gel profile of UDP glucuronosyltransferase 1 family, polypeptide Al (UGT1A1) protein from UGT1A1 modified mRNA.

[00047] FIG. 32 is a histogram of Factor XI protein production in HEK293 cells.

[00048] FIG. 33 is a gel profile of Aquaporin-5 protein from Aquaporin-5 modified mRNA.

[00049] FIG. 34 is a histogram of Factor VII protein production in HeLa cells from Factor VII modified mRNA.

[00050] FIG. 35 is a histogram of Insulin Glargine protein production in HeLa cells from Insulin Glargine modified mRNA.

[00051] FIG. 36 is a histogram of Tissue Factor protein production in HeLa cells from Tissue Factor modified mRNA.

[00052] FIG. 37 is a histogram of Factor XI protein production in HeLa cells from Factor XI modified mRNA.

[00053] FIG. 38 is a histogram of Factor XI protein production in HeLa cell supernatant from Factor XI modified mRNA.

[00054] FIG. 39 is a histogram of Insulin Aspart protein production in HeLa cells from Insulin Aspart modified mRNA.

[00055] FIG. 40 is a histogram of Insulin Lispro protein production in HeLa cells from Insulin Lispro modified mRNA.

[00056] FIG. 41 is a histogram of Insulin Glulisine protein production in HeLa cells from Insulin Glulisine modified mRNA.

[00057] FIG. 42 is a histogram of human growth hormone protein production in HeLa cells from human growth hormone modified mRNA.

[00058] FIG. 43 is a gel profile of tumor protein 53 (p53) protein from p53 modified mRNA. Figure 43A shows the expected size of p53. Figure 43B shows the expected size of p53.

[00059] FIG. 44 is a gel profile of tuftelin (TUFT1) protein from TUFT1 modified mRNA.

[00060] FIG. 45 is a gel profile of galactokinase 1 (GALK1) protein from GALK1 modified mRNA. Figure 45A shows the expected size of GALK1. Figure 45B shows the expected size of GALK1.

[00061] FIG. 46 is a gel profile of defensin, beta 103A (DEFB103A) protein from DEFB103A modified mRNA.

[00062] FIG. 47 is a flow cytometry plot of LDLR modified mRNA.

[00063] FIG. 48 is a histogram of vascular endothelial growth factor expression in HeLa.

[00064] FIG. 49 is a histogram of the cell vitality of HeLa cells transfected with vascular endothelial growth factor mRNA.

[00065] FIG. 50 is a histogram of Insulin Aspart protein expression.

[00066] FIG. 51 is a histogram of Insulin Glargine protein expression.

[00067] FIG. 52 is a histogram of Insulin Glulisine protein expression.

[00068] FIG. 53 is a histogram of Interleukin 7 (IL-7) protein expression.

[00069] FIG. 54 is a histogram of Erythropoietin (EPO) protein expression.

[00070] FIG. 55 is a gel profile of Lysosomal Acid Lipase protein from Lysosomal Acid Lipase modified mRNA.

[00071] FIG. 56 is a gel profile of Glucocerebrosidase protein from Glucocerebrosidase modified mRNA.

[00072] FIG. 57 is a gel profile of Iduronate 2-Sulfatase protein from Iduronate 2-Sulfatase modified mRNA.

[00073] FIG. 58 is a gel profile of Luciferase protein from Luciferase modified mRNA.

[00074] FIG. 59 is a histogram of IgG concentration after administration with formulated Herceptin modified mRNA in mammals.

[00075] FIG. 60 is a histogram of IgG concentration (in ng/ml) after transfection with Herceptin modified mRNA.

[00076] FIG. 61 is a gel profile of Herceptin protein from Herceptin modified mRNA.

[00077] FIG. 62 is a histogram of Glucocerebrosidase enzyme activity.

[00078] FIG. 63 is a histogram of Lysosomal Acid Lipase enzyme activity.

[00079] FIG. 64 a is histogram of Factor VIII protein expression.

[00080] FIG. 65 is a histogram of Factor VIII Chromogenic Activity.

[00081] FIG. 66 is a graph of LDLR Expression. Figure 66A shows LDL Receptor Expression of cells compared to LDLR mRNA added. Figure 66B shows LDL Receptor Expression of cells post transfection. Figure 66C shows the saturation of BODIPYO
labeled LRL. Figure 66D shows the binding affinity of BODIPY-LDL to cells.

[00082] FIG. 67 is a graph showing the percent positive cells for UGT1A1 Expression.

[00083] FIG. 68 is a graph showing UGT1A1 protein accumulation.

[00084] FIG. 69 is a gel profile of UGT1A1 protein and OTC from eith UGT1A1 or OTC modified mRNA.

[00085] FIG. 70 is a flow cytometry plot of HEK293 cells transfected with PAh or UGT1A1.

[00086] FIG. 71 is a gel profile of UGT1A1 protein from UGT1A1 modified mRNA.

[00087] FIG. 72 is a gel profile of microsomal extracts of mice treated with LNPs containing UGT1A1.
DETAILED DESCRIPTION

[00088] It is of great interest in the fields of therapeutics, diagnostics, reagents and for biological assays to be able to deliver a nucleic acid, e.g., a ribonucleic acid (RNA) inside a cell, whether in vitro, in vivo, in situ or ex vivo, such as to cause intracellular translation of the nucleic acid and production of an encoded polypeptide of interest. Of particular importance is the delivery and function of a non-integrative polynucleotide.

[00089] Described herein are compositions (including pharmaceutical compositions) and methods for the design, preparation, manufacture and/or formulation of polynucleotides encoding one or more polypeptides of interest. Also provided are systems, processes, devices and kits for the selection, design and/or utilization of the polynucleotides encoding the polypeptides of interest described herein.

[00090] According to the present invention, these polynucleotides are preferably modified as to avoid the deficiencies of other polypeptide-encoding molecules of the art.
Hence these polynucleotides are referred to as modified mRNA or mmRNA.

[00091] The use of modified polynucleotides in the fields of antibodies, viruses, veterinary applications and a variety of in vivo settings has been explored by the inventors and these studies are disclosed in for example, co-pending and co-owned United States provisional patent application serial numbers 61/470,451 filed March 31, 2011 teaching in vivo applications of mmRNA; 61/517,784 filed on April 26, teaching engineered nucleic acids for the production of antibody polypeptides;

61/519,158 filed May 17, 2011 teaching veterinary applications of mmRNA
technology;
61/533, 537 filed on September 12, 2011 teaching antimicrobial applications of mmRNA
technology; 61/533,554 filed on September 12, 2011 teaching viral applications of mmRNA technology, 61/542,533 filed on October 3, 2011 teaching various chemical modifications for use in mmRNA technology; 61/570,690 filed on December 14, teaching mobile devices for use in making or using mmRNA technology;
61/570,708 filed on December 14, 2011 teaching the use of mmRNA in acute care situations;

61/576,651 filed on December 16, 2011 teaching terminal modification architecture for mmRNA; 61/576,705 filed on December 16, 2011 teaching delivery methods using lipidoids for mmRNA; 61/578,271 filed on December 21, 2011 teaching methods to increase the viability of organs or tissues using mmRNA; 61/581,322 filed on December 29, 2011 teaching mmRNA encoding cell penetrating peptides; 61/581,352 filed on December 29, 2011 teaching the incorporation of cytotoxic nucleosides in mmRNA
and 61/631,729 filed on January 10, 2012 teaching methods of using mmRNA for crossing the blood brain barrier; all of which are herein incorporated by reference in their entirety.

[00092] Provided herein, in part, are polynucleotides, primary constructs and/or mmRNA encoding polypeptides of interest which have been designed to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretion efficiency (when applicable), accessibility to circulation, protein half-life and/or modulation of a cell's status, function and/or activity.
I. Compositions of the Invention (mmRNA) [00093] The present invention provides nucleic acid molecules, specifically polynucleotides, primary constructs and/or mmRNA which encode one or more polypeptides of interest. The term "nucleic acid," in its broadest sense, includes any compound and/or substance that comprise a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a 0- D-ribo configuration, a-LNA having an a-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having a 2'-amino functionalization, and 2'-amino- a-LNA having a 2'-amino functionalization) or hybrids thereof [00094] In preferred embodiments, the nucleic acid molecule is a messenger RNA

(mRNA). As used herein, the term "messenger RNA" (mRNA) refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo.

[00095] Traditionally, the basic components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap and a poly-A tail. Building on this wild type modular structure, the present invention expands the scope of functionality of traditional mRNA molecules by providing polynucleotides or primary RNA constructs which maintain a modular organization, but which comprise one or more structural and/or chemical modifications or alterations which impart useful properties to the polynucleotide including, in some embodiments, the lack of a substantial induction of the innate immune response of a cell into which the polynucleotide is introduced.
As such, modified mRNA molecules of the present invention are termed "mmRNA." As used herein, a "structural" feature or modification is one in which two or more linked nucleotides are inserted, deleted, duplicated, inverted or randomized in a polynucleotide, primary construct or mmRNA without significant chemical modification to the nucleotides themselves. Because chemical bonds will necessarily be broken and reformed to effect a structural modification, structural modifications are of a chemical nature and hence are chemical modifications. However, structural modifications will result in a different sequence of nucleotides. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G". The same polynucleotide may be structurally modified from "ATCG" to "ATCCCG". Here, the dinucleotide "CC" has been inserted, resulting in a structural modification to the polynucleotide.

mmRNA Architecture [00096] The mmRNA of the present invention are distinguished from wild type mRNA in their functional and/or structural design features which serve to, as evidenced herein, overcome existing problems of effective polypeptide production using nucleic acid-based therapeutics.

[00097] Figure 1 shows a representative polynucleotide primary construct 100 of the present invention. As used herein, the term "primary construct" or "primary mRNA
construct" refers to a polynucleotide transcript which encodes one or more polypeptides of interest and which retains sufficient structural and/or chemical features to allow the polypeptide of interest encoded therein to be translated. Primary constructs may be polynucleotides of the invention. When structurally or chemically modified, the primary construct may be referred to as an mmRNA.

[00098] Returning to FIG. 1, the primary construct 100 here contains a first region of linked nucleotides 102 that is flanked by a first flanking region 104 and a second flaking region 106. As used herein, the "first region" may be referred to as a "coding region" or "region encoding" or simply the "first region." This first region may include, but is not limited to, the encoded polypeptide of interest. The polypeptide of interest may comprise at its 5' terminus one or more signal sequences encoded by a signal sequence region 103.
The flanking region 104 may comprise a region of linked nucleotides comprising one or more complete or incomplete 5' UTRs sequences. The flanking region 104 may also comprise a 5' terminal cap 108. The second flanking region 106 may comprise a region of linked nucleotides comprising one or more complete or incomplete 3' UTRs.
The flanking region 106 may also comprise a 3' tailing sequence 110.

[00099] Bridging the 5' terminus of the first region 102 and the first flanking region 104 is a first operational region 105. Traditionally this operational region comprises a Start codon. The operational region may alternatively comprise any translation initiation sequence or signal including a Start codon.

[000100] Bridging the 3' terminus of the first region 102 and the second flanking region 106 is a second operational region 107. Traditionally this operational region comprises a Stop codon. The operational region may alternatively comprise any translation initiation sequence or signal including a Stop codon. According to the present invention, multiple serial stop codons may also be used.

[000101] Generally, the shortest length of the first region of the primary construct of the present invention can be the length of a nucleic acid sequence that is sufficient to encode for a dipeptide, a tripeptide, a tetrapeptide, a pentapeptide, a hexapeptide, a heptapeptide, an octapeptide, a nonapeptide, or a decapeptide. In another embodiment, the length may be sufficient to encode a peptide of 2-30 amino acids, e.g. 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may be sufficient to encode for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25 or 30 amino acids, or a peptide that is no longer than 40 amino acids, e.g. no longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11 or 10 amino acids.
Examples of dipeptides that the polynucleotide sequences can encode or include, but are not limited to, carnosine and anserine.

[000102] Generally, the length of the first region encoding the polypeptide of interest of the present invention is greater than about 30 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000, 10,000, 20,000, 30,000, 40,000, 50,000, 60,000, 70,000, 80,000, 90,000 or up to and including 100,000 nucleotides). As used herein, the "first region" may be referred to as a "coding region" or "region encoding" or simply the "first region."

[000103] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes from about 30 to about 100,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 1,000, from 30 to 1,500, from 30 to 3,000, from 30 to 5,000, from 30 to 7,000, from 30 to 10,000, from 30 to 25,000, from 30 to 50,000, from 30 to 70,000, from 100 to 250, from 100 to 500, from 100 to 1,000, from 100 to 1,500, from 100 to 3,000, from 100 to 5,000, from 100 to 7,000, from 100 to 10,000, from 100 to 25,000, from 100 to 50,000, from 100 to 70,000, from 100 to 100,000, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 3,000, from 500 to 5,000, from 500 to 7,000, from 500 to 10,000, from 500 to 25,000, from 500 to 50,000, from 500 to 70,000, from 500 to 100,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 3,000, from 1,000 to 5,000, from 1,000 to 7,000, from 1,000 to 10,000, from 1,000 to 25,000, from 1,000 to 50,000, from 1,000 to 70,000, from 1,000 to 100,000, from 1,500 to 3,000, from 1,500 to 5,000, from 1,500 to 7,000, from 1,500 to 10,000, from 1,500 to 25,000, from 1,500 to 50,000, from 1,500 to 70,000, from 1,500 to 100,000, from 2,000 to 3,000, from 2,000 to 5,000, from 2,000 to 7,000, from 2,000 to 10,000, from 2,000 to 25,000, from 2,000 to 50,000, from 2,000 to 70,000, and from 2,000 to 100,000).

[000104] According to the present invention, the first and second flanking regions may range independently from 15-1,000 nucleotides in length (e.g., greater than 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotides or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotides).

[000105] According to the present invention, the tailing sequence may range from absent to 500 nucleotides in length (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotides). Where the tailing region is a polyA tail, the length may be determined in units of or as a function of polyA Binding Protein binding.
In this embodiment, the polyA tail is long enough to bind at least 4 monomers of PolyA
Binding Protein. PolyA Binding Protein monomers bind to stretches of approximately 38 nucleotides. As such, it has been observed that polyA tails of about 80 nucleotides and 160 nucleotides are functional.

[000106] According to the present invention, the capping region may comprise a single cap or a series of nucleotides forming the cap. In this embodiment the capping region may be from 1 to 10, e.g. 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or 10 or fewer nucleotides in length. In some embodiments, the cap is absent.

[000107] According to the present invention, the first and second operational regions may range from 3 to 40, e.g., 5-30, 10-20, 15, or at least 4, or 30 or fewer nucleotides in length and may comprise, in addition to a Start and/or Stop codon, one or more signal and/or restriction sequences.
Cyclic mmRNA

[000108] According to the present invention, a primary construct or mmRNA may be cyclized, or concatemerized, to generate a translation competent molecule to assist interactions between poly-A binding proteins and 5'-end binding proteins. The mechanism of cyclization or concatemerization may occur through at least 3 different routes: 1) chemical, 2) enzymatic, and 3) ribozyme catalyzed. The newly formed 5'-/3'-linkage may be intramolecular or intermolecular.

[000109] In the first route, the 5'-end and the 3'-end of the nucleic acid contain chemically reactive groups that, when close together, form a new covalent linkage between the 5'-end and the 3'-end of the molecule. The 5'-end may contain an NHS-ester reactive group and the 3'-end may contain a 3'-amino-terminated nucleotide such that in an organic solvent the 3'-amino-terminated nucleotide on the 3'-end of a synthetic mRNA
molecule will undergo a nucleophilic attack on the 5'-NHS-ester moiety forming a new 5'-/3'-amide bond.

[000110] In the second route, T4 RNA ligase may be used to enzymatically link a 5'-phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic acid forming a new phosphorodiester linkage. In an example reaction, liug of a nucleic acid molecule is incubated at 37 C for 1 hour with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. The ligation reaction may occur in the presence of a split oligonucleotide capable of base-pairing with both the 5'- and 3'-region in juxtaposition to assist the enzymatic ligation reaction.

[000111] In the third route, either the 5'-or 3'-end of the cDNA template encodes a ligase ribozyme sequence such that during in vitro transcription, the resultant nucleic acid molecule can contain an active ribozyme sequence capable of ligating the 5'-end of a nucleic acid molecule to the 3'-end of a nucleic acid molecule. The ligase ribozyme may be derived from the Group I Intron, Group I Intron, Hepatitis Delta Virus, Hairpin ribozyme or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37 C.
mmRNA Multimers [000112] According to the present invention, multiple distinct polynucleotides, primary constructs or mmRNA may be linked together through the 3'-end using nucleotides which are modified at the 3'-terminus. Chemical conjugation may be used to control the stoichiometry of delivery into cells. For example, the glyoxylate cycle enzymes, isocitrate lyase and malate synthase, may be supplied into HepG2 cells at a 1:1 ratio to alter cellular fatty acid metabolism. This ratio may be controlled by chemically linking polynucleotides, primary constructs or mmRNA using a 3'-azido terminated nucleotide on one polynucleotide, primary construct or mmRNA species and a C5-ethynyl or alkynyl-containing nucleotide on the opposite polynucleotide, primary construct or mmRNA species. The modified nucleotide is added post-transcriptionally using terminal transferase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. After the addition of the 3'-modified nucleotide, the two polynucleotide, primary construct or mmRNA species may be combined in an aqueous solution, in the presence or absence of copper, to form a new covalent linkage via a click chemistry mechanism as described in the literature.

[000113] In another example, more than two polynucleotides may be linked together using a functionalized linker molecule. For example, a functionalized saccharide molecule may be chemically modified to contain multiple chemical reactive groups (SH-, NH2-, N3, etc...) to react with the cognate moiety on a 3'-functionalized mRNA
molecule (i.e., a 3'-maleimide ester, 3'-NHS-ester, alkynyl). The number of reactive groups on the modified saccharide can be controlled in a stoichiometric fashion to directly control the stoichiometric ratio of conjugated polynucleotide, primary construct or mmRNA.

mmRNA Conjugates and Combinations [000114] In order to further enhance protein production, primary constructs or mmRNA
of the present invention can be designed to be conjugated to other polynucleotides, dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell, hormones and hormone receptors, non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, or a drug.

[000115] Conjugation may result in increased stability and/or half life and may be particularly useful in targeting the polynucleotides, primary constructs or mmRNA to specific sites in the cell, tissue or organism.

[000116] According to the present invention, the mmRNA or primary constructs may be administered with, or further encode one or more of RNAi agents, siRNAs, shRNAs, miRNAs, miRNA binding sites, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers or vectors, and the like.
Bifunctional mmRNA

[000117] In one embodiment of the invention are bifunctional polynucleotides (e.g., bifunctional primary constructs or bifunctional mmRNA). As the name implies, bifunctional polynucleotides are those having or capable of at least two functions. These molecules may also by convention be referred to as multi-functional.

[000118] The multiple functionalities of bifunctional polynucleotides may be encoded by the RNA (the function may not manifest until the encoded product is translated) or may be a property of the polynucleotide itself. It may be structural or chemical.
Bifunctional modified polynucleotides may comprise a function that is covalently or electrostatically associated with the polynucleotides. Further, the two functions may be provided in the context of a complex of a mmRNA and another molecule.

[000119] Bifunctional polynucleotides may encode peptides which are anti-proliferative. These peptides may be linear, cyclic, constrained or random coil. They may function as aptamers, signaling molecules, ligands or mimics or mimetics thereof Anti-proliferative peptides may, as translated, be from 3 to 50 amino acids in length.
They may be 5-40, 10-30, or approximately 15 amino acids long. They may be single chain, multichain or branched and may form complexes, aggregates or any multi-unit structure once translated.
Noncoding polynucleotides and primary constructs [000120] As described herein, provided are polynucleotides and primary constructs having sequences that are partially or substantially not translatable, e.g., having a noncoding region. Such noncoding region may be the "first region" of the primary construct. Alternatively, the noncoding region may be a region other than the first region.
Such molecules are generally not translated, but can exert an effect on protein production by one or more of binding to and sequestering one or more translational machinery components such as a ribosomal protein or a transfer RNA (tRNA), thereby effectively reducing protein expression in the cell or modulating one or more pathways or cascades in a cell which in turn alters protein levels. The polynucleotide or primary construct may contain or encode one or more long noncoding RNA (lncRNA, or lincRNA) or portion thereof, a small nucleolar RNA (sno-RNA), micro RNA (miRNA), small interfering RNA (siRNA) or Piwi-interacting RNA (piRNA).
Polypeptides of interest [000121] According to the present invention, the primary construct is designed to encode one or more polypeptides of interest or fragments thereof. A
polypeptide of interest may include, but is not limited to, whole polypeptides, a plurality of polypeptides or fragments of polypeptides, which independently may be encoded by one or more nucleic acids, a plurality of nucleic acids, fragments of nucleic acids or variants of any of the aforementioned. As used herein, the term "polypeptides of interest" refer to any polypeptide which is selected to be encoded in the primary construct of the present invention. As used herein, "polypeptide" means a polymer of amino acid residues (natural or unnatural) linked together most often by peptide bonds. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances the polypeptide encoded is smaller than about 50 amino acids and the polypeptide is then termed a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments and other equivalents, variants, and analogs of the foregoing. A
polypeptide may be a single molecule or may be a multi-molecular complex such as a dimer, trimer or tetramer. They may also comprise single chain or multichain polypeptides such as antibodies or insulin and may be associated or linked.
Most commonly disulfide linkages are found in multichain polypeptides. The term polypeptide may also apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid.

[000122] The term "polypeptide variant" refers to molecules which differ in their amino acid sequence from a native or reference sequence. The amino acid sequence variants may possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence, as compared to a native or reference sequence.
Ordinarily, variants will possess at least about 50% identity (homology) to a native or reference sequence, and preferably, they will be at least about 80%, more preferably at least about 90%
identical (homologous) to a native or reference sequence.

[000123] In some embodiments "variant mimics" are provided. As used herein, the term "variant mimic" is one which contains one or more amino acids which would mimic an activated sequence. For example, glutamate may serve as a mimic for phosphoro-threonine and/or phosphoro-serine. Alternatively, variant mimics may result in deactivation or in an inactivated product containing the mimic, e.g., phenylalanine may act as an inactivating substitution for tyrosine; or alanine may act as an inactivating substitution for serine.

[000124] "Homology" as it applies to amino acid sequences is defined as the percentage of residues in the candidate amino acid sequence that are identical with the residues in the amino acid sequence of a second sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent homology. Methods and computer programs for the alignment are well known in the art. It is understood that homology depends on a calculation of percent identity but may differ in value due to gaps and penalties introduced in the calculation.

[000125] By "homologs" as it applies to polypeptide sequences means the corresponding sequence of other species having substantial identity to a second sequence of a second species.

[000126] "Analogs" is meant to include polypeptide variants which differ by one or more amino acid alterations, e.g., substitutions, additions or deletions of amino acid residues that still maintain one or more of the properties of the parent or starting polypeptide.

[000127] The present invention contemplates several types of compositions which are polypeptide based including variants and derivatives. These include substitutional, insertional, deletion and covalent variants and derivatives. The term "derivative" is used synonymously with the term "variant" but generally refers to a molecule that has been modified and/or changed in any way relative to a reference molecule or starting molecule.

[000128] As such, mmRNA encoding polypeptides containing substitutions, insertions and/or additions, deletions and covalent modifications with respect to reference sequences, in particular the polypeptide sequences disclosed herein, are included within the scope of this invention. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the invention (e.g., at the N-terminal or C-terminal ends). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or to allow for biotinylation. Alternatively, amino acid residues located at the carboxy and amino terminal regions of the amino acid sequence of a peptide or protein may optionally be deleted providing for truncated sequences. Certain amino acids (e.g., C-terminal or N-terminal residues) may alternatively be deleted depending on the use of the sequence, as for example, expression of the sequence as part of a larger sequence which is soluble, or linked to a solid support.

[000129] "Substitutional variants" when referring to polypeptides are those that have at least one amino acid residue in a native or starting sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule.

[000130] As used herein the term "conservative amino acid substitution" refers to the substitution of an amino acid that is normally present in the sequence with a different amino acid of similar size, charge, or polarity. Examples of conservative substitutions include the substitution of a non-polar (hydrophobic) residue such as isoleucine, valine and leucine for another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, and between glycine and serine. Additionally, the substitution of a basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue such as aspartic acid or glutamic acid for another acidic residue are additional examples of conservative substitutions. Examples of non-conservative substitutions include the substitution of a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine and/or a polar residue for a non-polar residue.

[000131] "Insertional variants" when referring to polypeptides are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. "Immediately adjacent" to an amino acid means connected to either the alpha-carboxy or alpha-amino functional group of the amino acid.

[000132] "Deletional variants" when referring to polypeptides are those with one or more amino acids in the native or starting amino acid sequence removed.
Ordinarily, deletional variants will have one or more amino acids deleted in a particular region of the molecule.

[000133] "Covalent derivatives" when referring to polypeptides include modifications of a native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent, and/or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent that is capable of reacting with selected side-chains or terminal residues, or by harnessing mechanisms of post-translational modifications that function in selected recombinant host cells. The resultant covalent derivatives are useful in programs directed at identifying residues important for biological activity, for immunoassays, or for the preparation of anti-protein antibodies for immunoaffinity purification of the recombinant glycoprotein. Such modifications are within the ordinary skill in the art and are performed without undue experimentation.

[000134] Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues may be present in the polypeptides produced in accordance with the present invention.

[000135] Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (T. E.
Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp.
79-86 (1983)).

[000136] "Features" when referring to polypeptides are defined as distinct amino acid sequence-based components of a molecule. Features of the polypeptides encoded by the mmRNA of the present invention include surface manifestations, local conformational shape, folds, loops, half-loops, domains, half-domains, sites, termini or any combination thereof [000137] As used herein when referring to polypeptides the term "surface manifestation" refers to a polypeptide based component of a protein appearing on an outermost surface.

[000138] As used herein when referring to polypeptides the term "local conformational shape" means a polypeptide based structural manifestation of a protein which is located within a definable space of the protein.

[000139] As used herein when referring to polypeptides the term "fold" refers to the resultant conformation of an amino acid sequence upon energy minimization. A
fold may occur at the secondary or tertiary level of the folding process. Examples of secondary level folds include beta sheets and alpha helices. Examples of tertiary folds include domains and regions formed due to aggregation or separation of energetic forces.
Regions formed in this way include hydrophobic and hydrophilic pockets, and the like.

[000140] As used herein the term "turn" as it relates to protein conformation means a bend which alters the direction of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.

[000141] As used herein when referring to polypeptides the term "loop" refers to a structural feature of a polypeptide which may serve to reverse the direction of the backbone of a peptide or polypeptide. Where the loop is found in a polypeptide and only alters the direction of the backbone, it may comprise four or more amino acid residues.
Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997). Loops may be open or closed. Closed loops or "cyclic" loops may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids between the bridging moieties.
Such bridging moieties may comprise a cysteine-cysteine bridge (Cys-Cys) typical in polypeptides having disulfide bridges or alternatively bridging moieties may be non-protein based such as the dibromozylyl agents used herein.

[000142] As used herein when referring to polypeptides the term "half-loop"
refers to a portion of an identified loop having at least half the number of amino acid resides as the loop from which it is derived. It is understood that loops may not always contain an even number of amino acid residues. Therefore, in those cases where a loop contains or is identified to comprise an odd number of amino acids, a half-loop of the odd-numbered loop will comprise the whole number portion or next whole number portion of the loop (number of amino acids of the loop/2+/-0.5 amino acids). For example, a loop identified as a 7 amino acid loop could produce half-loops of 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 being 3 or 4).

[000143] As used herein when referring to polypeptides the term "domain"
refers to a motif of a polypeptide having one or more identifiable structural or functional characteristics or properties (e.g., binding capacity, serving as a site for protein-protein interactions).

[000144] As used herein when referring to polypeptides the term "half-domain"
means a portion of an identified domain having at least half the number of amino acid resides as the domain from which it is derived. It is understood that domains may not always contain an even number of amino acid residues. Therefore, in those cases where a domain contains or is identified to comprise an odd number of amino acids, a half-domain of the odd-numbered domain will comprise the whole number portion or next whole number portion of the domain (number of amino acids of the domain/2+/-0.5 amino acids). For example, a domain identified as a 7 amino acid domain could produce half-domains of 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 being 3 or 4). It is also understood that sub-domains may be identified within domains or half-domains, these subdomains possessing less than all of the structural or functional properties identified in the domains or half domains from which they were derived. It is also understood that the amino acids that comprise any of the domain types herein need not be contiguous along the backbone of the polypeptide (i.e., nonadjacent amino acids may fold structurally to produce a domain, half-domain or subdomain).

[000145] As used herein when referring to polypeptides the terms "site" as it pertains to amino acid based embodiments is used synonymously with "amino acid residue"
and "amino acid side chain." A site represents a position within a peptide or polypeptide that may be modified, manipulated, altered, derivatized or varied within the polypeptide based molecules of the present invention.

[000146] As used herein the terms "termini" or "terminus" when referring to polypeptides refers to an extremity of a peptide or polypeptide. Such extremity is not limited only to the first or final site of the peptide or polypeptide but may include additional amino acids in the terminal regions. The polypeptide based molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). Proteins of the invention are in some cases made up of multiple polypeptide chains brought together by disulfide bonds or by non-covalent forces (multimers, oligomers). These sorts of proteins will have multiple N- and C-termini. Alternatively, the termini of the polypeptides may be modified such that they begin or end, as the case may be, with a non-polypeptide based moiety such as an organic conjugate.

[000147] Once any of the features have been identified or defined as a desired component of a polypeptide to be encoded by the primary construct or mmRNA of the invention, any of several manipulations and/or modifications of these features may be performed by moving, swapping, inverting, deleting, randomizing or duplicating.
Furthermore, it is understood that manipulation of features may result in the same outcome as a modification to the molecules of the invention. For example, a manipulation which involved deleting a domain would result in the alteration of the length of a molecule just as modification of a nucleic acid to encode less than a full length molecule would.

[000148] Modifications and manipulations can be accomplished by methods known in the art such as, but not limited to, site directed mutagenesis. The resulting modified molecules may then be tested for activity using in vitro or in vivo assays such as those described herein or any other suitable screening assay known in the art.

[000149] According to the present invention, the polypeptides may comprise a consensus sequence which is discovered through rounds of experimentation. As used herein a "consensus" sequence is a single sequence which represents a collective population of sequences allowing for variability at one or more sites.

[000150] 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 polypeptides of interest of this invention. For example, provided herein is any protein fragment (meaning a polypeptide sequence at least one amino acid residue shorter than a reference polypeptide sequence but otherwise identical) of a reference protein 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 invention. In certain embodiments, a polypeptide to be utilized in accordance with the invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.
Encoded Polypeptides [000151] The primary constructs or mmRNA of the present invention may be designed to encode polypeptides of interest selected from any of several target categories including, but not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, cell penetrating peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane bound proteins, nuclear proteins, proteins associated with human disease, targeting moieties or those proteins encoded by the human genome for which no therapeutic indication has been identified but which nonetheless have utility in areas of research and discovery.

[000152] In one embodiment primary constructs or mmRNA may encode variant polypeptides which have a certain identity with a reference polypeptide sequence. As used herein, a "reference polypeptide sequence" refers to a starting polypeptide sequence.
Reference sequences may be wild type sequences or any sequence to which reference is made in the design of another sequence. A "reference polypeptide sequence"
may, e.g., be any one of SEQ ID NOs: 769-1392as disclosed herein, e.g., any of SEQ ID NOs 769, 770, 771, 772, 773, 774, 775, 776, 777, 778, 779, 780, 781, 782, 783, 784, 785, 786, 787, 788, 789, 790, 791, 792, 793, 794, 795, 796, 797, 798, 799, 800, 801, 802, 803, 804, 805, 806, 807, 808, 809, 810, 811, 812, 813, 814, 815, 816, 817, 818, 819, 820, 821, 822, 823, 824, 825, 826, 827, 828, 829, 830, 831, 832, 833, 834, 835, 836, 837, 838, 839, 840, 841, 842, 843, 844, 845, 846, 847, 848, 849, 850, 851, 852, 853, 854, 855, 856, 857, 858, 859, 860, 861, 862, 863, 864, 865, 866, 867, 868, 869, 870, 871, 872, 873, 874, 875, 876, 877, 878, 879, 880, 881, 882, 883, 884, 885, 886, 887, 888, 889, 890, 891, 892, 893, 894, 895, 896, 897, 898, 899, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 919, 920, 921, 922, 923, 924, 925, 926, 927, 928, 929, 930, 931, 932, 933, 934, 935, 936, 937, 938, 939, 940, 941, 942, 943, 944, 945, 946, 947, 948, 949, 950, 951, 952, 953, 954, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 1000, 1001, 1002, 1003, 1004, 1005, 1006, 1007, 1008, 1009, 1010, 1011, 1012, 1013, 1014, 1015, 1016, 1017, 1018, 1019, 1020, 1021, 1022, 1023, 1024, 1025, 1026, 1027, 1028, 1029, 1030, 1031, 1032, 1033, 1034, 1035, 1036, 1037, 1038, 1039, 1040, 1041, 1042, 1043, 1044, 1045, 1046, 1047, 1048, 1049, 1050, 1051, 1052, 1053, 1054, 1055, 1056, 1057, 1058, 1059, 1060, 1061, 1062, 1063, 1064, 1065, 1066, 1067, 1068, 1069, 1070, 1071, 1072, 1073, 1074, 1075, 1076, 1077, 1078, 1079, 1080, 1081, 1082, 1083, 1084, 1085, 1086, 1087, 1088, 1089, 1090, 1091, 1092, 1093, 1094, 1095, 1096, 1097, 1098, 1099, 1100, 1101, 1102, 1103, 1104, 1105, 1106, 1107, 1108, 1109, 1110, 1111, 1112, 1113, 1114, 1115, 1116, 1117, 1118, 1119, 1120, 1121, 1122, 1123, 1124, 1125, 1126, 1127, 1128, 1129, 1130, 1131, 1132, 1133, 1134, 1135, 1136, 1137, 1138, 1139, 1140, 1141, 1142, 1143, 1144, 1145, 1146, 1147, 1148, 1149, 1150, 1151, 1152, 1153, 1154, 1155, 1156, 1157, 1158, 1159, 1160, 1161, 1162, 1163, 1164, 1165, 1166, 1167, 1168, 1169, 1170, 1171, 1172, 1173, 1174, 1175, 1176, 1177, 1178, 1179, 1180, 1181, 1182, 1183, 1184, 1185, 1186, 1187, 1188, 1189, 1190, 1191, 1192, 1193, 1194, 1195, 1196, 1197, 1198, 1199, 1200, 1201, 1202, 1203, 1204, 1205, 1206, 1207, 1208, 1209, 1210, 1211, 1212, 1213, 1214, 1215, 1216, 1217, 1218, 1219, 1220, 1221, 1222, 1223, 1224, 1225, 1226, 1227, 1228, 1229, 1230, 1231, 1232, 1233, 1234, 1235, 1236, 1237, 1238, 1239, 1240, 1241, 1242, 1243, 1244, 1245, 1246, 1247, 1248, 1249, 1250, 1251, 1252, 1253, 1254, 1255, 1256, 1257, 1258, 1259, 1260, 1261, 1262, 1263, 1264, 1265, 1266, 1267, 1268, 1269, 1270, 1271, 1272, 1273, 1274, 1275, 1276, 1277, 1278, 1279, 1280, 1281, 1282, 1283, 1284, 1285, 1286, 1287, 1288, 1289, 1290, 1291, 1292, 1293, 1294, 1295, 1296, 1297, 1298, 1299, 1300, 1301, 1302, 1303, 1304, 1305, 1306, 1307, 1308, 1309, 1310, 1311, 1312, 1313, 1314, 1315, 1316, 1317, 1318, 1319, 1320, 1321, 1322, 1323, 1324, 1325, 1326, 1327, 1328, 1329, 1330, 1331, 1332, 1333, 1334, 1335, 1336, 1337, 1338, 1339, 1340, 1341, 1342, 1343, 1344, 1345, 1346, 1347, 1348, 1349, 1350, 1351, 1352, 1353, 1354, 1355, 1356, 1357, 1358, 1359, 1360, 1361, 1362, 1363, 1364, 1365, 1366, 1367, 1368, 1369, 1370, 1371, 1372, 1373, 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392.

[000153] 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).

[000154] In some embodiments, the polypeptide variant may have the same or a similar activity as the reference polypeptide. Alternatively, the variant may have an altered activity (e.g., increased or decreased) relative to a reference polypeptide.
Generally, variants of a particular polynucleotide or polypeptide of the invention 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% but less than 100% 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. Such tools for alignment include those of the BLAST suite (Stephen F. Altschul, Thomas L.
Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J.
Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.) Other tools are described herein, specifically in the definition of "Identity."

[000155] Default parameters in the BLAST algorithm include, for example, an expect threshold of 10, Word size of 28, Match/Mismatch Scores 1, -2, Gap costs Linear. Any filter can be applied as well as a selection for species specific repeats, e.g., Homo sapiens.
Biologics [000156] The polynucleotides, primary constructs or mmRNA disclosed herein, may encode one or more biologics. As used herein, a "biologic" is a polypeptide-based molecule produced by the methods provided herein and which may be used to treat, cure, mitigate, prevent, or diagnose a serious or life-threatening disease or medical condition.
Biologics, according to the present invention include, but are not limited to, allergenic extracts (e.g. for allergy shots and tests), blood components, gene therapy products, human tissue or cellular products used in transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytics, and immunomodulators, among others.

[000157] According to the present invention, one or more biologics currently being marketed or in development may be encoded by the polynucleotides, primary constructs or mmRNA of the present invention. While not wishing to be bound by theory, it is believed that incorporation of the encoding polynucleotides of a known biologic into the primary constructs or mmRNA of the invention will result in improved therapeutic efficacy due at least in part to the specificity, purity and/or selectivity of the construct designs.
Antibodies [000158] The primary constructs or mmRNA disclosed herein, may encode one or more antibodies or fragments thereof The term "antibody" includes monoclonal antibodies (including full length antibodies which have an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), as well as antibody fragments. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations and/or post-translation modifications (e.g., isomerizations, amidations) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site.

[000159] The monoclonal antibodies herein specifically include "chimeric"
antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is(are) identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
Chimeric antibodies of interest herein include, but are not limited to, "primatized"
antibodies comprising variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World Monkey, Ape etc.) and human constant region sequences.

[000160] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen binding and/or the variable region of the intact antibody.
Examples of antibody fragments include Fab, Fab', F(ab')2 and Fv fragments; diabodies;
linear antibodies; nanobodies; single-chain antibody molecules and multispecific antibodies formed from antibody fragments.

[000161] Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, may be encoded by the mmRNA of the invention, including the heavy chains designated alpha, delta, epsilon, gamma and mu, respectively. Also included are polynucleotide sequences encoding the subclasses, gamma and mu. Hence any of the subclasses of antibodies may be encoded in part or in whole and include the following subclasses:
IgGl, IgG2, IgG3, IgG4, IgAl and IgA2.

[000162] According to the present invention, one or more antibodies or fragments currently being marketed or in development may be encoded by the polynucleotides, primary constructs or mmRNA of the present invention. While not wishing to be bound by theory, it is believed that incorporation into the primary constructs of the invention will result in improved therapeutic efficacy due at least in part to the specificity, purity and selectivity of the mmRNA designs.

[000163] Antibodies encoded in the polynucleotides, primary constructs or mmRNA of the invention may be utilized to treat conditions or diseases in many therapeutic areas such as, but not limited to, blood, cardiovascular, CNS, poisoning (including antivenoms), dermatology, endocrinology, gastrointestinal, medical imaging, musculoskeletal, oncology, immunology, respiratory, sensory and anti-infective.

[000164] In one embodiment, primary constructs or mmRNA disclosed herein may encode monoclonal antibodies and/or variants thereof. Variants of antibodies may also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, deletional variants and/or covalent derivatives. In one embodiment, the primary construct and/or mmRNA disclosed herein may encode an immunoglobulin Fc region. In another embodiment, the primary constructs and/or mmRNA may encode a variant immunoglobulin Fc region. As a non-limiting example, the primary constructs and/or mmRNA may encode an antibody having a variant immunoglobulin Fc region as described in U.S. Pat. No. 8,217,147 herein incorporated by reference in its entirety.
Vaccines [000165] The primary constructs or mmRNA disclosed herein, may encode one or more vaccines. As used herein, a "vaccine" is a biological preparation that improves immunity to a particular disease or infectious agent. According to the present invention, one or more vaccines currently being marketed or in development may be encoded by the polynucleotides, primary constructs or mmRNA of the present invention. While not wishing to be bound by theory, it is believed that incorporation into the primary constructs or mmRNA of the invention will result in improved therapeutic efficacy due at least in part to the specificity, purity and selectivity of the construct designs.

[000166] Vaccines encoded in the polynucleotides, primary constructs or mmRNA
of the invention may be utilized to treat conditions or diseases in many therapeutic areas such as, but not limited to, cardiovascular, CNS, dermatology, endocrinology, oncology, immunology, respiratory, and anti-infective.
Therapeutic proteins or peptides [000167] The primary constructs or mmRNA disclosed herein, may encode one or more validated or "in testing" therapeutic proteins or peptides.

[000168] According to the present invention, one or more therapeutic proteins or peptides currently being marketed or in development may be encoded by the polynucleotides, primary constructs or mmRNA of the present invention. While not wishing to be bound by theory, it is believed that incorporation into the primary constructs or mmRNA of the invention will result in improved therapeutic efficacy due at least in part to the specificity, purity and selectivity of the construct designs.

[000169] Therapeutic proteins and peptides encoded in the polynucleotides, primary constructs or mmRNA of the invention may be utilized to treat conditions or diseases in many therapeutic areas such as, but not limited to, blood, cardiovascular, CNS, poisoning (including antivenoms), dermatology, endocrinology, genetic, genitourinary, gastrointestinal, musculoskeletal, oncology, and immunology, respiratory, sensory and anti-infective.
Cell-Penetrating Polypeptides [000170] The primary constructs or mmRNA disclosed herein, may encode one or more cell-penetrating polypeptides. As used herein, "cell-penetrating polypeptide"
or CPP
refers to a polypeptide which may facilitate the cellular uptake of molecules.
A cell-penetrating polypeptide of the present invention may contain one or more detectable labels. The polypeptides may be partially labeled or completely labeled throughout. The polynucleotide, primary construct or mmRNA may encode the detectable label completely, partially or not at all. The cell-penetrating peptide may also include a signal sequence. As used herein, a "signal sequence" refers to a sequence of amino acid residues bound at the amino terminus of a nascent protein during protein translation. The signal sequence may be used to signal the secretion of the cell-penetrating polypeptide.

[000171] In one embodiment, the polynucleotides, primary constructs or mmRNA
may also encode a fusion protein. The fusion protein may be created by operably linking a charged protein to a therapeutic protein. As used herein, "operably linked"
refers to the therapeutic protein and the charged protein being connected in such a way to permit the expression of the complex when introduced into the cell. As used herein, "charged protein" refers to a protein that carries a positive, negative or overall neutral electrical charge. Preferably, the therapeutic protein may be covalently linked to the charged protein in the formation of the fusion protein. The ratio of surface charge to total or surface amino acids may be approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9.

[000172] The cell-penetrating polypeptide encoded by the polynucleotides, primary constructs or mmRNA may form a complex after being translated. The complex may comprise a charged protein linked, e.g. covalently linked, to the cell-penetrating polypeptide. "Therapeutic protein" refers to a protein that, when administered to a cell has a therapeutic, diagnostic, and/or prophylactic effect and/or elicits a desired biological and/or pharmacological effect.

[000173] In one embodiment, the cell-penetrating polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein, "protein-binding partner" includes, but is not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. The cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner. The cell-penetrating polypeptide may be capable of being secreted from a cell where the polynucleotide, primary construct or mmRNA may be introduced. The cell-penetrating polypeptide may also be capable of penetrating the first cell.

[000174] In a further embodiment, the cell-penetrating polypeptide is capable of penetrating a second cell. The second cell may be from the same area as the first cell, or it may be from a different area. The area may include, but is not limited to, tissues and organs. The second cell may also be proximal or distal to the first cell.

[000175] In one embodiment, the polynucleotides, primary constructs or mmRNA
may encode a cell-penetrating polypeptide which may comprise a protein-binding partner.
The protein binding partner may include, but is not limited to, an antibody, a supercharged antibody or a functional fragment. The polynucleotides, primary constructs or mmRNA may be introduced into the cell where a cell-penetrating polypeptide comprising the protein-binding partner is introduced.
Secreted proteins [000176] Human and other eukaryotic cells are subdivided by membranes into many functionally distinct compartments. Each membrane-bounded compartment, or organelle, contains different proteins essential for the function of the organelle. The cell uses "sorting signals," which are amino acid motifs located within the protein, to target proteins to particular cellular organelles.

[000177] One type of sorting signal, called a signal sequence, a signal peptide, or a leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER).

[000178] Proteins targeted to the ER by a signal sequence can be released into the extracellular space as a secreted protein. Similarly, proteins residing on the cell membrane can also be secreted into the extracellular space by proteolytic cleavage of a "linker" holding the protein to the membrane. While not wishing to be bound by theory, the molecules of the present invention may be used to exploit the cellular trafficking described above. As such, in some embodiments of the invention, polynucleotides, primary constructs or mmRNA are provided to express a secreted protein. The secreted proteins may be selected from those described herein or those in US Patent Publication, 20100255574, the contents of which are incorporated herein by reference in their entirety.

[000179] In one embodiment, these may be used in the manufacture of large quantities of valuable human gene products.
Plasma membrane proteins [000180] In some embodiments of the invention, polynucleotides, primary constructs or mmRNA are provided to express a protein of the plasma membrane.
Cytoplasmic or cytoskeletal proteins [000181] In some embodiments of the invention, polynucleotides, primary constructs or mmRNA are provided to express a cytoplasmic or cytoskeletal protein.
Intracellular membrane bound proteins [000182] In some embodiments of the invention, polynucleotides, primary constructs or mmRNA are provided to express an intracellular membrane bound protein.
Nuclear proteins [000183] In some embodiments of the invention, polynucleotides, primary constructs or mmRNA are provided to express a nuclear protein.
Proteins associated with human disease [000184] In some embodiments of the invention, polynucleotides, primary constructs or mmRNA are provided to express a protein associated with human disease.
Miscellaneous proteins [000185] In some embodiments of the invention, polynucleotides, primary constructs or mmRNA are provided to express a protein with a presently unknown therapeutic function.
Targeting Moieties [000186] In some embodiments of the invention, polynucleotides, primary constructs or mmRNA are provided to express a targeting moiety. These include 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, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. Additionally, polynucleotide, primary construct or mmRNA can be employed to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties or biomolecules.
Polyp eptide Libraries [000187] In one embodiment, the polynucleotides, primary constructs or mmRNA
may be used to produce polypeptide libraries. These libraries may arise from the production of a population of polynucleotides, primary constructs or mmRNA, each containing various structural or chemical modification designs. In this embodiment, a population of polynucleotides, primary constructs or mmRNA may comprise a plurality of encoded polypeptides, including but not limited to, an antibody or antibody fragment, protein binding partner, scaffold protein, and other polypeptides taught herein or known in the art. In a preferred embodiment, the polynucleotides are primary constructs of the present invention, including mmRNA which may be suitable for direct introduction into a target cell or culture which in turn may synthesize the encoded polypeptides.

[000188] In certain embodiments, multiple variants of a protein, each with different amino acid modification(s), may be produced and tested to determine the best variant in terms of pharmacokinetics, stability, biocompatibility, and/or biological activity, or a biophysical property such as expression level. Such a library may contain 10, 102, 103, 104, 105, 106, 107, 108, 109, or over 109 possible variants (including, but not limited to, substitutions, deletions of one or more residues, and insertion of one or more residues).
Anti-Microbial and Anti-viral Polypeptides [000189] The polynucleotides, primary constructs and mmRNA of the present invention may be designed to encode on or more antimicrobial peptides (AMP) or antiviral peptides (AVP). AMPs and AVPs have been isolated and described from a wide range of animals such as, but not limited to, microorganisms, invertebrates, plants, amphibians, birds, fish, and mammals (Wang et al., Nucleic Acids Res. 2009; 37 (Database issue):D933-7). For example, anti-microbial polypeptides are described in Antimicrobial Peptide Database (http://aps.unmc.edu/AP/main.php; Wang et al., Nucleic Acids Res. 2009; 37 (Database issue):D933-7), CAMP: Collection of Anti-Microbial Peptides (http://www.bicnirrh.res.in/antimicrobial/); Thomas et al., Nucleic Acids Res.
2010; 38 (Database issue):D774-80), US 5221732, US 5447914, US 5519115, US 5607914, US
5714577, US 5734015, US 5798336, US 5821224, US 5849490, US 5856127, US
5905187, US 5994308, US 5998374, US 6107460, US 6191254, US 6211148, US
6300489, US 6329504, US 6399370, US 6476189, US 6478825, US 6492328, US
6514701, US 6573361, US 6573361, US 6576755, US 6605698, US 6624140, US
6638531, US 6642203, US 6653280, US 6696238, US 6727066, US 6730659, US
6743598, US 6743769, US 6747007, US 6790833, US 6794490, US 6818407, US
6835536, US 6835713, US 6838435, US 6872705, US 6875907, US 6884776, US
6887847, US 6906035, US 6911524, US 6936432, US 7001924, US 7071293, US
7078380, US 7091185, US 7094759, US 7166769, US 7244710, US 7314858, and US
7582301, the contents of which are incorporated by reference in their entirety.

[000190] The anti-microbial polypeptides described herein may block cell fusion and/or viral entry by one or more enveloped viruses (e.g., HIV, HCV). For example, the anti-microbial polypeptide can comprise or consist of a synthetic peptide corresponding to a region, e.g., a consecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the transmembrane subunit of a viral envelope protein, e.g., HIV-1 gp120 or gp41. The amino acid and nucleotide sequences of HIV-1 gp120 or gp41 are described in, e.g., Kuiken et at., (2008). "HIV Sequence Compendium,"
Los Alamos National Laboratory.

[000191] In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence. In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding viral protein sequence.

[000192] In other embodiments, the anti-microbial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, e.g., a consecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of a capsid binding protein. In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding sequence of the capsid binding protein.

[000193] The anti-microbial polypeptides described herein may block protease dimerization and inhibit cleavage of viral proproteins (e.g., HIV Gag-pol processing) into functional proteins thereby preventing release of one or more enveloped viruses (e.g., HIV, HCV). In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence.

[000194] In other embodiments, the anti-microbial polypeptide can comprise or consist of a synthetic peptide corresponding to a region, e.g., a consecutive sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of a protease binding protein. In some embodiments, the anti-microbial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding sequence of the protease binding protein.

[000195] The anti-microbial polypeptides described herein can include an in vitro-evolved polypeptide directed against a viral pathogen.

Anti-Microbial Polypeptides [000196] Anti-microbial polypeptides (AMPs) are small peptides of variable length, sequence and structure with broad spectrum activity against a wide range of microorganisms including, but not limited to, bacteria, viruses, fungi, protozoa, parasites, prions, and tumor/cancer cells. (See, e.g., Zaiou, J Mol Med, 2007; 85:317;
herein incorporated by reference in its entirety). It has been shown that AMPs have broad-spectrum of rapid onset of killing activities, with potentially low levels of induced resistance and concomitant broad anti-inflammatory effects.

[000197] In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be under 10kDa, e.g., under 8kDa, 6kDa, 4kDa, 2kDa, or lkDa.
In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) consists of from about 6 to about 100 amino acids, e.g., from about 6 to about 75 amino acids, about 6 to about 50 amino acids, about 6 to about 25 amino acids, about 25 to about 100 amino acids, about 50 to about 100 amino acids, or about 75 to about amino acids. In certain embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may consist of from about 15 to about 45 amino acids.
In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) is substantially cationic.

[000198] In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be substantially amphipathic. In certain embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be substantially cationic and amphipathic. In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be cytostatic to a Gram-positive bacterium. In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be cytotoxic to a Gram-positive bacterium. In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be cytostatic and cytotoxic to a Gram-positive bacterium. In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be cytostatic to a Gram-negative bacterium. In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be cytotoxic to a Gram-negative bacterium. In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be cytostatic and cytotoxic to a Gram-positive bacterium. In some embodiments, the anti-microbial polypeptide may be cytostatic to a virus, fungus, protozoan, parasite, prion, or a combination thereof In some embodiments, the anti-microbial polypeptide may be cytotoxic to a virus, fungus, protozoan, parasite, prion, or a combination thereof. In certain embodiments, the anti-microbial polypeptide may be cytostatic and cytotoxic to a virus, fungus, protozoan, parasite, prion, or a combination thereof In some embodiments, the anti-microbial polypeptide may be cytotoxic to a tumor or cancer cell (e.g., a human tumor and/or cancer cell). In some embodiments, the anti-microbial polypeptide may be cytostatic to a tumor or cancer cell (e.g., a human tumor and/or cancer cell). In certain embodiments, the anti-microbial polypeptide may be cytotoxic and cytostatic to a tumor or cancer cell (e.g., a human tumor or cancer cell). In some embodiments, the anti-microbial polypeptide (e.g., an anti-bacterial polypeptide) may be a secreted polypeptide.

[000199] In some embodiments, the anti-microbial polypeptide comprises or consists of a defensin. Exemplary defensins include, but are not limited to, a-defensins (e.g., neutrophil defensin 1, defensin alpha 1, neutrophil defensin 3, neutrophil defensin 4, defensin 5, defensin 6), 13-defensins (e.g., beta-defensin 1, beta-defensin 2, beta-defensin 103, beta-defensin 107, beta-defensin 110, beta-defensin 136), and 0-defensins. In other embodiments, the anti-microbial polypeptide comprises or consists of a cathelicidin (e.g., hCAP18).
Anti-Viral Polypeptides [000200] Anti-viral polypeptides (AVPs) are small peptides of variable length, sequence and structure with broad spectrum activity against a wide range of viruses. See, e.g., Zaiou, J Mol Med, 2007; 85:317. It has been shown that AVPs have a broad-spectrum of rapid onset of killing activities, with potentially low levels of induced resistance and concomitant broad anti-inflammatory effects. In some embodiments, the anti-viral polypeptide is under 10kDa, e.g., under 8kDa, 6kDa, 4kDa, 2kDa, or lkDa. In some embodiments, the anti-viral polypeptide comprises or consists of from about 6 to about 100 amino acids, e.g., from about 6 to about 75 amino acids, about 6 to about 50 amino acids, about 6 to about 25 amino acids, about 25 to about 100 amino acids, about 50 to about 100 amino acids, or about 75 to about 100 amino acids. In certain embodiments, the anti-viral polypeptide comprises or consists of from about 15 to about 45 amino acids. In some embodiments, the anti-viral polypeptide is substantially cationic. In some embodiments, the anti-viral polypeptide is substantially amphipathic.
In certain embodiments, the anti-viral polypeptide is substantially cationic and amphipathic. In some embodiments, the anti-viral polypeptide is cytostatic to a virus. In some embodiments, the anti-viral polypeptide is cytotoxic to a virus. In some embodiments, the anti-viral polypeptide is cytostatic and cytotoxic to a virus. In some embodiments, the anti-viral polypeptide is cytostatic to a bacterium, fungus, protozoan, parasite, prion, or a combination thereof In some embodiments, the anti-viral polypeptide is cytotoxic to a bacterium, fungus, protozoan, parasite, prion or a combination thereof In certain embodiments, the anti-viral polypeptide is cytostatic and cytotoxic to a bacterium, fungus, protozoan, parasite, prion, or a combination thereof. In some embodiments, the anti-viral polypeptide is cytotoxic to a tumor or cancer cell (e.g., a human cancer cell). In some embodiments, the anti-viral polypeptide is cytostatic to a tumor or cancer cell (e.g., a human cancer cell). In certain embodiments, the anti-viral polypeptide is cytotoxic and cytostatic to a tumor or cancer cell (e.g., a human cancer cell). In some embodiments, the anti-viral polypeptide is a secreted polypeptide.
Cytotoxic Nucleosides [000201] In one embodiment, the polynucleotides, primary constructs or mmRNA
of the present invention may incorporate one or more cytotoxic nucleosides. For example, cytotoxic nucleosides may be incorporated into polynucleotides, primary constructs or mmRNA such as bifunctional modified RNAs or mRNAs. Cytotoxic nucleoside anti-cancer agents include, but are not limited to, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, FTORAFURO (a combination of tegafur and uracil), tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), and 6-mercaptopurine.

[000202] A number of cytotoxic nucleoside analogues are in clinical use, or have been the subject of clinical trials, as anticancer agents. Examples of such analogues include, but are not limited to, cytarabine, gemcitabine, troxacitabine, decitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), cladribine, clofarabine, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine and 1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl)-cytosine. Another example of such a compound is fludarabine phosphate. These compounds may be administered systemically and may have side effects which are typical of cytotoxic agents such as, but not limited to, little or no specificity for tumor cells over proliferating normal cells.

[000203] A number of prodrugs of cytotoxic nucleoside analogues are also reported in the art. Examples include, but are not limited to, N4-behenoy1-1-beta-D-arabinofuranosylcytosine, N4-octadecy1-1-beta-D-arabinofuranosylcytosine, N4-palmitoy1-1-(2-C-cyano-2-deoxy-beta-D-arabino-pentofuranosyl) cytosine, and P-(cytarabine 5'-elaidic acid ester). In general, these prodrugs may be converted into the active drugs mainly in the liver and systemic circulation and display little or no selective release of active drug in the tumor tissue. For example, capecitabine, a prodrug of 5'-deoxy-5-fluorocytidine (and eventually of 5-fluorouracil), is metabolized both in the liver and in the tumor tissue. A series of capecitabine analogues containing "an easily hydrolysable radical under physiological conditions" has been claimed by Fujiu et al.
(U.S. Pat. No. 4,966,891) and is herein incorporated by reference. The series described by Fujiu includes N4 alkyl and aralkyl carbamates of 5'-deoxy-5-fluorocytidine and the implication that these compounds will be activated by hydrolysis under normal physiological conditions to provide 5'-deoxy-5-fluorocytidine.

[000204] A series of cytarabine N4-carbamates has been by reported by Fadl et al (Pharmazie. 1995, 50, 382-7, herein incorporated by reference) in which compounds were designed to convert into cytarabine in the liver and plasma. WO
2004/041203, herein incorporated by reference, discloses prodrugs of gemcitabine, where some of the prodrugs are N4-carbamates. These compounds were designed to overcome the gastrointestinal toxicity of gemcitabine and were intended to provide gemcitabine by hydrolytic release in the liver and plasma after absorption of the intact prodrug from the gastrointestinal tract. Nomura et al (Bioorg Med. Chem. 2003, 11, 2453-61, herein incorporated by reference) have described acetal derivatives of 1-(3-C-ethyny1-13-D-ribo-pentofaranosyl) cytosine which, on bioreduction, produced an intermediate that required further hydrolysis under acidic conditions to produce a cytotoxic nucleoside compound.

[000205] Cytotoxic nucleotides which may be chemotherapeutic also include, but are not limited to, pyrazolo [3,4-D]-pyrimidines, allopurinol, azathioprine, capecitabine, cytosine arabinoside, fluorouracil, mercaptopurine, 6-thioguanine, acyclovir, ara-adenosine, ribavirin, 7-deaza-adenosine, 7-deaza-guanosine, 6-aza-uracil, 6-aza-cytidine, thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloro-purine, and inosine, or combinations thereof Flanking Regions: Untranslated Regions (UTRs) [000206] Untranslated regions (UTRs) of a gene are transcribed but not translated. The 5'UTR starts at the transcription start site and continues to the start codon but does not include the start codon; whereas, the 3'UTR starts immediately following the stop codon and continues until the transcriptional termination signal. There is growing body of evidence about the regulatory roles played by the UTRs in terms of stability of the nucleic acid molecule and translation. The regulatory features of a UTR can be incorporated into the polynucleotides, primary constructs and/or mmRNA of the present invention to enhance the stability of the molecule. The specific features can also be incorporated to ensure controlled down-regulation of the transcript in case they are misdirected to undesired organs sites.
5' UTR and Translation Initiation [000207] Natural 5'UTRs bear features which play roles in for translation initiation.
They harbor signatures like Kozak sequences which are commonly known to be involved in the process by which the ribosome initiates translation of many genes.
Kozak sequences have the consensus CCR(A/G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), which is followed by another 'G'. 5'UTR also have been known to form secondary structures which are involved in elongation factor binding.

[000208] By engineering the features typically found in abundantly expressed genes of specific target organs, one can enhance the stability and protein production of the polynucleotides, primary constructs or mmRNA of the invention. For example, introduction of 5' UTR of liver-expressed mRNA, such as albumin, serum amyloid A, Apolipoprotein A/B/E, transferrin, alpha fetoprotein, erythropoietin, or Factor VIII, could be used to enhance expression of a nucleic acid molecule, such as a mmRNA, in hepatic cell lines or liver. Likewise, use of 5' UTR from other tissue-specific mRNA
to improve expression in that tissue is possible for muscle (MyoD, Myosin, Myoglobin, Myogenin, Herculin), for endothelial cells (Tie-1, CD36), for myeloid cells (C/EBP, AML1, G-CSF, GM-CSF, CD1 lb, MSR, Fr-1, i-NOS), for leukocytes (CD45, CD18), for adipose tissue (CD36, GLUT4, ACRP30, adiponectin) and for lung epithelial cells (SP-A/B/C/D).

[000209] Other non-UTR sequences may be incorporated into the 5' (or 3' UTR) UTRs.
For example, introns or portions of introns sequences may be incorporated into the flanking regions of the polynucleotides, primary constructs or mmRNA of the invention.
Incorporation of intronic sequences may increase protein production as well as mRNA
levels.
3' UTR and the AU Rich Elements [000210] 3' UTRs are known to have stretches of Adenosines and Uridines embedded in them. These AU rich signatures are particularly prevalent in genes with high rates of turnover. Based on their sequence features and functional properties, the AU
rich elements (AREs) can be separated into three classes (Chen et al, 1995): Class I AREs contain several dispersed copies of an AUUUA motif within U-rich regions. C-Myc and MyoD contain class I AREs. Class II AREs possess two or more overlapping UUAUUUA(U/A)(U/A) nonamers. Molecules containing this type of AREs include GM-CSF and TNF-a. Class III ARES are less well defined. These U rich regions do not contain an AUUUA motif. c-Jun and Myogenin are two well-studied examples of this class. Most proteins binding to the AREs are known to destabilize the messenger, whereas members of the ELAV family, most notably HuR, have been documented to increase the stability of mRNA. HuR binds to AREs of all the three classes.
Engineering the HuR specific binding sites into the 3' UTR of nucleic acid molecules will lead to HuR
binding and thus, stabilization of the message in vivo.

[000211] Introduction, removal or modification of 3' UTR AU rich elements (AREs) can be used to modulate the stability of polynucleotides, primary constructs or mmRNA
of the invention. When engineering specific polynucleotides, primary constructs or mmRNA, one or more copies of an ARE can be introduced to make polynucleotides, primary constructs or mmRNA of the invention less stable and thereby curtail translation and decrease production of the resultant protein. Likewise, AREs can be identified and removed or mutated to increase the intracellular stability and thus increase translation and production of the resultant protein. Transfection experiments can be conducted in relevant cell lines, using polynucleotides, primary constructs or mmRNA of the invention and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different ARE-engineering molecules and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, and 7 days post-transfection.
Incorporating microRNA Binding Sites [000212] microRNAs (or miRNA) are 19-25 nucleotide long noncoding RNAs that bind to the 3'UTR of nucleic acid molecules and down-regulate gene expression either by reducing nucleic acid molecule stability or by inhibiting translation. The polynucleotides, primary constructs or mmRNA of the invention may comprise one or more microRNA target sequences, microRNA seqences, or microRNA seeds. Such sequences may correspond to any known microRNA such as those taught in US
Publication U52005/0261218 and US Publication U52005/0059005, the contents of which are incorporated herein by reference in their entirety.

[000213] A microRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which sequence has perfect Watson-Crick complementarity to the miRNA target sequence. A microRNA seed may comprise positions 2-8 or 2-7 of the mature microRNA. In some embodiments, a microRNA
seed may comprise 7 nucleotides (e.g., nucleotides 2-8 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked by an adenine (A) opposed to microRNA position 1. In some embodiments, a microRNA seed may comprise 6 nucleotides (e.g., nucleotides 2-7 of the mature microRNA), wherein the seed-complementary site in the corresponding miRNA target is flanked byan adenine (A) opposed to microRNA position 1. See for example, Grimson A, Farh KK, Johnston WK, Garrett-Engele P, Lim LP, Bartel DP; Mol Cell. 2007 Jul 6;27(1):91-105; each of which is herein incorporated by reference in their entirety. The bases of the microRNA seed have complete complementarity with the target sequence. By engineering microRNA
target sequences into the 3'UTR of polynucleotides, primary constructs or mmRNA of the invention one can target the molecule for degradation or reduced translation, provided the microRNA in question is available. This process will reduce the hazard of off target effects upon nucleic acid molecule delivery. Identification of microRNA, microRNA
target regions, and their expression patterns and role in biology have been reported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 201118:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20.
doi: 10.1038/1eu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al, Cell, 2007 129:1401-1414; each of which is herein incorporated by reference in its entirety).

[000214] For example, if the nucleic acid molecule is an mRNA and is not intended to be delivered to the liver but ends up there, then miR-122, a microRNA abundant in liver, can inhibit the expression of the gene of interest if one or multiple target sites of miR-122 are engineered into the 3' UTR of the polynucleotides, primary constructs or mmRNA.
Introduction of one or multiple binding sites for different microRNA can be engineered to further decrease the longevity, stability, and protein translation of a polynucleotides, primary constructs or mmRNA.

[000215] As used herein, the term "microRNA site" refers to a microRNA target site or a microRNA recognition site, or any nucleotide sequence to which a microRNA
binds or associates. It should be understood that "binding" may follow traditional Watson-Crick hybridization rules or may reflect any stable association of the microRNA with the target sequence at or adjacent to the microRNA site.

[000216] Conversely, for the purposes of the polynucleotides, primary constructs or mmRNA of the present invention, microRNA binding sites can be engineered out of (i.e.
removed from) sequences in which they naturally occur in order to increase protein expression in specific tissues. For example, miR-122 binding sites may be removed to improve protein expression in the liver. Regulation of expression in multiple tissues can be accomplished through introduction or removal or one or several microRNA
binding sites.

[000217] Examples of tissues where microRNA are known to regulate mRNA, and thereby protein expression, include, but are not limited to, liver (miR-122), muscle (miR-133, miR-206, miR-208), endothelial cells (miR-17-92, miR-126), myeloid cells (miR-142-3p, miR-142-5p, miR-16, miR-21, miR-223, miR-24, miR-27), adipose tissue (let-7, miR-30c), heart (miR-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). MicroRNA can also regulate complex biological processes such as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 201118:171-176; herein incorporated by reference in its entirety). In the polynucleotides, primary constructs or mmRNA of the present invention, binding sites for microRNAs that are involved in such processes may be removed or introduced, in order to tailor the expression of the polynucleotides, primary constructs or mmRNA
expression to biologically relevant cell types or to the context of relevant biological processes. A listing of MicroRNA, miR sequences and miR binding sites is listed in Table 9 of U.S. Provisional Application No. 61/753,661 filed January 17, 2013, in Table 9 of U.S. Provisional Application No. 61/754,159 filed January 18, 2013, and in Table 7 of U.S. Provisional Application No. 61/758,921 filed January 31, 2013, each of which are herein incorporated by reference in their entireties.

[000218] Lastly, through an understanding of the expression patterns of microRNA in different cell types, polynucleotides, primary constructs or mmRNA can be engineered for more targeted expression in specific cell types or only under specific biological conditions. Through introduction of tissue-specific microRNA binding sites, polynucleotides, primary constructs or mmRNA could be designed that would be optimal for protein expression in a tissue or in the context of a biological condition. Examples of use of microRNA to drive tissue or disease-specific gene expression are listed (Getner and Naldini, Tissue Antigens. 2012, 80:393-403; herein incoroporated by reference in its entirety). In addition, microRNA seed sites can be incorporated into mRNA to decrease expression in certain cells which results in a biological improvement. An example of this is incorporation of miR-142 sites into a UGT1A1-expressing lentiviral vector.
The presence of miR-142 seed sites reduced expression in hematopoietic cells, and as a consequence reduced expression in antigen-presentating cells, leading to the absence of an immune response against the virally expressed UGT1A1 (Schmitt et al., Gastroenterology 2010; 139:999-1007; Gonzalez-Asequinolaza et al.
Gastroenterology 2010, 139:726-729; both herein incorporated by reference in its entirety) .
Incorporation of miR-142 sites into modified mRNA could not only reduce expression of the encoded protein in hematopoietic cells, but could also reduce or abolish immune responses to the mRNA-encoded protein. Incorporation of miR-142 seed sites (one or multiple) into mRNA would be important in the case of treatment of patients with complete protein deficiencies (UGT1A1 type I, LDLR-deficient patients, CRIM-negative Pompe patients, etc.) .

[000219] Transfection experiments can be conducted in relevant cell lines, using engineered polynucleotides, primary constructs or mmRNA and protein production can be assayed at various time points post-transfection. For example, cells can be transfected with different microRNA binding site-engineering polynucleotides, primary constructs or mmRNA and by using an ELISA kit to the relevant protein and assaying protein produced at 6 hour, 12 hour, 24 hour, 48 hour, 72 hour and 7 days post-transfection. In vivo experiments can also be conducted using microRNA-binding site-engineered molecules to examine changes in tissue-specific expression of formulated polynucleotides, primary constructs or mmRNA.
5' Capping [000220] The 5' cap structure of an mRNA is involved in nuclear export, increasing mRNA stability and binds the mRNA Cap Binding Protein (CBP), which is responsibile for mRNA stability in the cell and translation competency through the association of CBP
with poly(A) binding protein to form the mature cyclic mRNA species. The cap further assists the removal of 5' proximal introns removal during mRNA splicing.

[000221] Endogenous mRNA molecules may be 5'-end capped generating a 5'-ppp-5'-triphosphate linkage between a terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap may then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and/or anteterminal transcribed nucleotides of the 5' end of the mRNA
may optionally also be 2'-0-methylated. 5'-decapping through hydrolysis and cleavage of the guanylate cap structure may target a nucleic acid molecule, such as an mRNA
molecule, for degradation.

[000222] Modifications to the polynucleotides, primary constructs, and mmRNA
of the present invention may generate a non-hydrolyzable cap structure preventing decapping and thus increasing mRNA half-life. Because cap structure hydrolysis requires cleavage of 5'-ppp-5' phosphorodiester linkages, modified nucleotides may be used during the capping reaction. For example, a Vaccinia Capping Enzyme from New England Biolabs (Ipswich, MA) may be used with a-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate linkage in the 5'-ppp-5' cap.

Additional modified guanosine nucleotides may be used such as a-methyl-phosphonate and seleno-phosphate nucleotides.

[000223] Additional modifications include, but are not limited to, 2'-0-methylation of the ribose sugars of 5'-terminal and/or 5'-anteterminal nucleotides of the mRNA (as mentioned above) on the 2'-hydroxyl group of the sugar ring. Multiple distinct 5'-cap structures can be used to generate the 5'-cap of a nucleic acid molecule, such as an mRNA molecule.

[000224] Cap analogs, which herein are also referred to as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from natural (i.e. endogenous, wild-type or physiological) 5'-caps in their chemical structure, while retaining cap function. Cap analogs may be chemically (i.e. non-enzymatically) or enzymatically synthesized and/or linked to a nucleic acid molecule.

[000225] For example, the Anti-Reverse Cap Analog (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, wherein one guanine contains an methyl group as well as a 3'-0-methyl group (i.e., N7,3'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m7G-3'mppp-G; which may equivaliently be designated 3' 0-Me-m7G(5')ppp(5')G). The 3'-0 atom of the other, unmodified, guanine becomes linked to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g. an mRNA or mmRNA). The N7- and 3'-0-methlyated guanine provides the terminal moiety of the capped nucleic acid molecule (e.g. mRNA or mmRNA).

[000226] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-methyl group on guanosine (i.e., N7,2'-0-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m7Gm-ppp-G).

[000227] While cap analogs allow for the concomitant capping of a nucleic acid molecule in an in vitro transcription reaction, up to 20% of transcripts can remain uncapped. This, as well as the structural differences of a cap analog from an endogenous 5'-cap structures of nucleic acids produced by the endogenous, cellular transcription machinery, may lead to reduced translational competency and reduced cellular stability.

[000228] Polynucleotides, primary constructs and mmRNA of the invention may also be capped post-transcriptionally, using enzymes, in order to generate more authentic 5'-cap structures. As used herein, the phrase "more authentic" refers to a feature that closely mirrors or mimics, either structurally or functionally, an endogenous or wild type feature. That is, a "more authentic" feature is better representative of an endogenous, wild-type, natural or physiological cellular function and/or structure as compared to synthetic features or analogs, etc., of the prior art, or which outperforms the corresponding endogenous, wild-type, natural or physiological feature in one or more respects. Non-limiting examples of more authentic 5'cap structures of the present invention are those which, among other things, have enhanced binding of cap binding proteins, increased half life, reduced susceptibility to 5' endonucleases and/or reduced 5'decapping, as compared to synthetic 5'cap structures known in the art (or to a wild-type, natural or physiological 5'cap structure). For example, recombinant Vaccinia Virus Capping Enzyme and recombinant 2'-0-methyltransferase enzyme can create a canonical 5'-5'-triphosphate linkage between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide wherein the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-0-methyl. Such a structure is termed the Capl structure. This cap results in a higher translational-competency and cellular stability and a reduced activation of cellular pro-inflammatory cytokines, as compared, e.g., to other 5'cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (cap 0), 7mG(5')ppp(5')NlmpNp (cap 1), and 7mG(5')-ppp(5')NlmpN2mp (cap 2).

[000229] Because the polynucleotides, primary constructs or mmRNA may be capped post-transcriptionally, and because this process is more efficient, nearly 100% of the polynucleotides, primary constructs or mmRNA may be capped. This is in contrast to ¨80% when a cap analog is linked to an mRNA in the course of an in vitro transcription reaction.

[000230] According to the present invention, 5' terminal caps may include endogenous caps or cap analogs. According to the present invention, a 5' terminal cap may comprise a guanine analog. Useful guanine analogs include, but are not limited to, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.
Viral Sequences [000231] Additional viral sequences such as, but not limited to, the translation enhancer sequence of the barley yellow dwarf virus (BYDV-PAV), the Jaagsiekte sheep retrovirus (JSRV) and/or the Enzootic nasal tumor virus (See e.g., International Pub. No.

W02012129648; herein incorporated by reference in its entirety) can be engineered and inserted in the 3' UTR of the polynucleotides, primary constructs or mmRNA of the invention and can stimulate the translation of the construct in vitro and in vivo.
Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.
IRES Sequences [000232] Further, provided are polynucleotides, primary constructs or mmRNA
which may contain an internal ribosome entry site (IRES). First identified as a feature Picorna virus RNA, IRES plays an important role in initiating protein synthesis in absence of the 5' cap structure. An IRES may act as the sole ribosome binding site, or may serve as one of multiple ribosome binding sites of an mRNA. Polynucleotides, primary constructs or mmRNA containing more than one functional ribosome binding site may encode several peptides or polypeptides that are translated independently by the ribosomes ("multicistronic nucleic acid molecules"). When polynucleotides, primary constructs or mmRNA are provided with an IRES, further optionally provided is a second translatable region. Examples of IRES sequences that can be used according to the invention include without limitation, those from picornaviruses (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).
Poly-A tails [000233] During RNA processing, a long chain of adenine nucleotides (poly-A
tail) may be added to a polynucleotide such as an mRNA molecules in order to increase stability. Immediately after transcription, the 3' end of the transcript may be cleaved to free a 3' hydroxyl. Then poly-A polymerase adds a chain of adenine nucleotides to the RNA. The process, called polyadenylation, adds a poly-A tail that can be between, for example, approximately 100 and 250 residues long.

[000234] It has been discovered that unique poly-A tail lengths provide certain advantages to the polynucleotides, primary constructs or mmRNA of the present invention.

[000235] Generally, the length of a poly-A tail of the present invention is greater than 30 nucleotides in length. In another embodiment, the poly-A tail is greater than 35 nucleotides in length (e.g., at least or greater than about 35, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides). In some embodiments, the polynucleotide, primary construct, or mmRNA
includes from about 30 to about 3,000 nucleotides (e.g., from 30 to 50, from 30 to 100, from 30 to 250, from 30 to 500, from 30 to 750, from 30 to 1,000, from 30 to 1,500, from 30 to 2,000, from 30 to 2,500, from 50 to 100, from 50 to 250, from 50 to 500, from 50 to 750, from 50 to 1,000, from 50 to 1,500, from 50 to 2,000, from 50 to 2,500, from 50 to 3,000, from 100 to 500, from 100 to 750, from 100 to 1,000, from 100 to 1,500, from 100 to 2,000, from 100 to 2,500, from 100 to 3,000, from 500 to 750, from 500 to 1,000, from 500 to 1,500, from 500 to 2,000, from 500 to 2,500, from 500 to 3,000, from 1,000 to 1,500, from 1,000 to 2,000, from 1,000 to 2,500, from 1,000 to 3,000, from 1,500 to 2,000, from 1,500 to 2,500, from 1,500 to 3,000, from 2,000 to 3,000, from 2,000 to 2,500, and from 2,500 to 3,000).

[000236] In one embodiment, the poly-A tail is designed relative to the length of the overall polynucleotides, primary constructs or mmRNA. This design may be based on the length of the coding region, the length of a particular feature or region (such as the first or flanking regions), or based on the length of the ultimate product expressed from the polynucleotides, primary constructs or mmRNA.

[000237] In this context the poly-A tail may be 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100% greater in length than the polynucleotides, primary constructs or mmRNA
or feature thereof The poly-A tail may also be designed as a fraction of polynucleotides, primary constructs or mmRNA to which it belongs. In this context, the poly-A
tail may be 10, 20, 30, 40, 50, 60, 70, 80, or 90% or more of the total length of the construct or the total length of the construct minus the poly-A tail. Further, engineered binding sites and conjugation of polynucleotides, primary constructs or mmRNA for Poly-A binding protein may enhance expression.

[000238] Additionally, multiple distinct polynucleotides, primary constructs or mmRNA may be linked together to the PABP (Poly-A binding protein) through the 3'-end using modified nucleotides at the 3'-terminus of the poly-A tail.
Transfection experiments can be conducted in relevant cell lines at and protein production can be assayed by ELISA at 12hr, 24hr, 48hr, 72 hr and day 7 post-transfection.

[000239] In one embodiment, the polynucleotide primary constructs of the present invention are designed to include a polyA-G Quartet. The G-quartet is a cyclic hydrogen bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G-quartet is incorporated at the end of the poly-A tail. The resultant mmRNA construct is assayed for stability, protein production and other parameters including half-life at various time points. It has been discovered that the polyA-G quartet results in protein production equivalent to at least 75% of that seen using a poly-A tail of 120 nucleotides alone.
Quantification [000240] In one embodiment, the polynucleotides, primary constructs or mmRNA
of the present invention may be quantified in exosomes derived from one or more bodily fluid. As used herein "bodily fluids" include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood. Alternatively, exosomes may be retrieved from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testis, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

[000241] In the quantification method, a sample of not more than 2mL is obtained from the subject and the exosomes isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof In the analysis, the level or concentration of a polynucleotide, primary construct or mmRNA may be an expression level, presence, absence, truncation or alteration of the administered construct. It is advantageous to correlate the level with one or more clinical phenotypes or with an assay for a human disease biomarker. The assay may be performed using construct specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or combinations thereof while the exosomes may be isolated using immunohistochemical methods such as enzyme linked immunosorbent assay (ELISA) methods. Exosomes may also be isolated by size exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunoabsorbent capture, affinity purification, microfluidic separation, or combinations thereof [000242] These methods afford the investigator the ability to monitor, in real time, the level of polynucleotides, primary constructs or mmRNA remaining or delivered.
This is possible because the polynucleotides, primary constructs or mmRNA of the present invention differ from the endogenous forms due to the structural or chemical modifications.
II. Design and synthesis of mmRNA

[000243] Polynucleotides, primary constructs or mmRNA for use in accordance with the invention may be prepared according to any available technique including, but not limited to chemical synthesis, enzymatic synthesis, which is generally termed in vitro transcription (IVT) or 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).

[000244] The process of design and synthesis of the primary constructs of the invention generally includes the steps of gene construction, mRNA production (either with or without modifications) and purification. In the enzymatic synthesis method, a target polynucleotide sequence encoding the polypeptide of interest is first selected for incorporation into a vector which will be amplified to produce a cDNA
template.
Optionally, the target polynucleotide sequence and/or any flanking sequences may be codon optimized. The cDNA template is then used to produce mRNA through in vitro transcription (IVT). After production, the mRNA may undergo purification and clean-up processes. The steps of which are provided in more detail below.
Gene Construction [000245] The step of gene construction may include, but is not limited to gene synthesis, vector amplification, plasmid purification, plasmid linearization and clean-up, and cDNA template synthesis and clean-up.
Gene Synthesis [000246] Once a polypeptide of interest, or target, is selected for production, a primary construct is designed. Within the primary construct, a first region of linked nucleosides encoding the polypeptide of interest may be constructed using an open reading frame (ORF) of a selected nucleic acid (DNA or RNA) transcript. The ORF may comprise the wild type ORF, an isoform, variant or a fragment thereof As used herein, an "open reading frame" or "ORF" is meant to refer to a nucleic acid sequence (DNA or RNA) which is capable of encoding a polypeptide of interest. ORFs often begin with the start codon, ATG and end with a nonsense or termination codon or signal.

[000247] Further, the nucleotide sequence of the first region may be codon optimized.
Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include to match codon frequencies in target and host organisms to ensure proper folding, bias GC content to increase mRNA
stability or reduce secondary structures, minimize tandem repeat codons or base runs that may impair gene construction or expression, customize transcriptional and translational control regions, insert or remove protein trafficking sequences, remove/add post translation modification sites in encoded protein (e.g. glycosylation sites), add, remove or shuffle protein domains, insert or delete restriction sites, modify ribosome binding sites and mRNA degradation sites, to adjust translational rates to allow the various domains of the protein to fold properly, or to reduce or eliminate problem secondary structures within the mRNA. Codon optimization tools, algorithms and services are known in the art, non-limiting examples include services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and/or proprietary methods. In one embodiment, the ORF sequence is optimized using optimization algorithms. Codon options for each amino acid are given in Table 1.
Table 1. Codon Options Amino Acid Single Letter Code Codon Options Isoleucine I ATT, ATC, ATA
Leucine L CTT, CTC, CTA, CTG, TTA, TTG
Valine V GTT, GTC, GTA, GTG
Phenylalanine F TTT, TTC
Methionine M ATG
Cysteine C TGT, TGC
Alanine A GCT, GCC, GCA, GCG
Glycine G GGT, GGC, GGA, GGG
Proline P CCT, CCC, CCA, CCG
Threonine T ACT, ACC, ACA, ACG
Serine S TCT, TCC, TCA, TCG, AGT, AGC
Tyrosine Y TAT, TAC
Tryptophan W TGG
Glutamine Q CAA, CAG
Asparagine N AAT, AAC
Histidine H CAT, CAC
Glutamic acid E GAA, GAG
Aspartic acid D GAT, GAC
Lysine K AAA, AAG
Arginine R CGT, CGC, CGA, CGG, AGA, AGG
Selenocysteine Sec UGA in mRNA in presence of Selenocystein insertion element (SECIS) Stop codons Stop TAA, TAG, TGA

[000248] Features, which may be considered beneficial in some embodiments of the present invention, may be encoded by the primary construct and may flank the ORF as a first or second flanking region. The flanking regions may be incorporated into the primary construct before and/or after optimization of the ORF. It is not required that a primary construct contain both a 5' and 3' flanking region. Examples of such features include, but are not limited to, untranslated regions (UTRs), Kozak sequences, an oligo(dT) sequence, and detectable tags and may include multiple cloning sites which may have XbaI recognition.

[000249] In some embodiments, a 5' UTR and/or a 3' UTR may be provided as flanking regions. Multiple 5' or 3' UTRs may be included in the flanking regions and may be the same or of different sequences. Any portion of the flanking regions, including none, may be codon optimized and any may independently contain one or more different structural or chemical modifications, before and/or after codon optimization.
Combinations of features may be included in the first and second flanking regions and may be contained within other features. For example, the ORF may be flanked by a 5' UTR which may contain a strong Kozak translational initiation signal and/or a 3' UTR which may include an oligo(dT) sequence for templated addition of a poly-A tail. 5'UTR may comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and/or different genes such as the 5'UTRs described in US Patent Application Publication No.
20100293625, herein incorporated by reference in its entirety.

[000250] Tables 2 and 3 provide a listing of exemplary UTRs which may be utilized in the primary construct of the present invention as flanking regions. Shown in Table 2 is a listing of a 5'-untranslated region of the invention. Variants of 5' UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C
or G.
Table 2. 5'-Untranslated Regions 5' UTR Name/Desc SEQ ID
Sequence Identifier ription NO.
Upstream GGGAAATAAGAGAGAAAAGAAGAGTA

UTR AGAAGAAATATAAGAGCCACC
Upstream GGGAGATCAGAGAGAAAAGAAGAGTA

UTR AGAAGAAATATAAGAGCCACC
GGAATAAAAGTCTCAACACAACATATA
CAAAACAAACGAATCTCAAGCAATCAA
Upstream GCATTCTACTTCTATTGCAGCAATTTAA

UTR ATCATTTCTTTTAAAGCAAAAGCAATTT
TCTGAAAATTTTCACCATTTACGAACGA
TAGCAAC
5UTR 004 Upstream GGGAGACAAGCUUGGCAUUCCGGUAC 4 -UTR UGUUGGUAAAGCCACC

[000251] Shown in Table 3 is a representative listing of 3'-untranslated regions of the invention. Variants of 3' UTRs may be utilized wherein one or more nucleotides are added or removed to the termini, including A, T, C or G.
Table 3. 3'-Untranslated Regions 3' UTR Name/Desc SEQ
Sequence ID
Identifier ription NO.
GCGCCTGCCCACCTGCCACCGACTGCTGGA
ACCCAGCCAGTGGGAGGGCCTGGCCCACC
AGAGTCCTGCTCCCTCACTCCTCGCCCCGC
CCCCTGTCCCAGAGTCCCACCTGGGGGCTC
TCTCCACCCTTCTCAGAGTTCCAGTTTCAAC
C reatine CAGAGTTCCAACCAATGGGCTCCATCCTCT

Kinase AGGGTCCTCTTCTTTTCCCAGAGCTCCACCC
CAACCAGGAGCTCTAGTTAATGGAGAGCTC
CCAGCACACTCGGAGCTTGTGCTTTGTCTC
CACGCAAAGCGATAAATAAAAGCATTGGT
GGCCTTTGGTCTTTGAATAAAGCCTGAGTA
GGAAGTCTAGA
GCCCCTGCCGCTCCCACCCCCACCCATCTG
GGCCCCGGGTTCAAGAGAGAGCGGGGTCT
GATCTCGTGTAGCCATATAGAGTTTGCTTC
TGAGTGTCTGCTTTGTTTAGTAGAGGTGGG
CAGGAGGAGCTGAGGGGCTGGGGCTGGGG
TGTTGAAGTTGGCTTTGCATGCCCAGCGAT
GCGCCTCCCTGTGGGATGTCATCACCCTGG
GAACCGGGAGTGGCCCTTGGCTCACTGTGT
TCTGCATGGTTTGGATCTGAATTAATTGTCC
3UTR-002 Myoglobin TTTCTTCTAAATCCCAACCGAACTTCTTCCA 6 ACCTCCAAACTGGCTGTAACCCCAAATCCA
AGCCATTAACTACACCTGACAGTAGCAATT
GTCTGATTAATCACTGGCCCCTTGAAGACA
GCAGAATGTCCCTTTGCAATGAGGAGGAG
ATCTGGGCTGGGCGGGCCAGCTGGGGAAG
CATTTGACTATCTGGAACTTGTGTGTGCCTC
CTCAGGTATGGCAGTGACTCACCTGGTTTT
AATAAAACAACCTGCAACATCTCATGGTCT
TTGAATAAAGCCTGAGTAGGAAGTCTAGA
ACACACTCCACCTCCAGCACGCGACTTCTC
AGGACGACGAATCTTCTCAATGGGGGGGC
GGCTGAGCTCCAGCCACCCCGCAGTCACTT
C. T TTTGTAACAACTTCCGTTGCTGCCATCGT
3UTR-003 ct-actmAAACTGACACAGTGTTTATAACGTGTACAT 7 ACATTAACTTATTACCTCATTTTGTTATTTT
TCGAAACAAAGCCCTGTGGAAGAAAATGG
AAAACTTGAAGAAGCATTAAAGTCATTCTG
TTAAGCTGCGTAAATGGTCTTTGAATAAAG

CCTGAGTAGGAAGTCTAGA
CATCACATTTAAAAGCATCTCAGCCTACCA
TGAGAATAAGAGAAAGAAAATGAAGATCA
AAAGCTTATTCATCTGTTTTTCTTTTTCGTT
GGTGTAAAGCCAACACCCTGTCTAAAAAAC
ATAAATTTCTTTAATCATTTTGCCTCTTTTC
Alb umm C. T TGTGCTTCAATTAATAAAAAATGGAAAG

TTTCAAAGATGTGTTGCTATCCTGAAAATT
CTGTAGGTTCTGTGGAAGTTCCAGTGTTCT
CTCTTATTCCACTTCGGTAGAGGATTTCTAG
TTTCTTGTGGGCTAATTAAATAAATCATTA
ATACTCTTCTAATGGTCTTTGAATAAAGCC
TGAGTAGGAAGTCTAGA
GCTGCCTTCTGCGGGGCTTGCCTTCTGGCC
a-globin ATGCCCTTCTTCTCTCCCTTGCACCTGTACC

TCTTGGTCTTTGAATAAAGCCTGAGTAGGA
AGGCGGCCGCTCGAGCATGCATCTAGA
GCCAAGCCCTCCCCATCCCATGTATTTATCT
CTATTTAATATTTATGTCTATTTAAGCCTCA
TATTTAAAGACAGGGAAGAGCAGAACGGA
GCCCCAGGCCTCTGTGTCCTTCCCTGCATTT
CTGAGTTTCATTCTCCTGCCTGTAGCAGTG
AGAAAAAGCTCCTGTCCTCCCATCCCCTGG
ACTGGGAGGTAGATAGGTAAATACCAAGT
ATTTATTACTATGACTGCTCCCCAGCCCTG
GCTCTGCAATGGGCACTGGGATGAGCCGCT
GTGAGCCCCTGGTCCTGAGGGTCCCCACCT
GGGACCCTTGAGAGTATCAGGTCTCCCACG
TGGGAGACAAGAAATCCCTGTTTAATATTT
AAACAGCAGTGTTCCCCATCTGGGTCCTTG
CACCCCTCACTCTGGCCTCAGCCGACTGCA
CAGCGGCCCCTGCATCCCCTTGGCTGTGAG

TGGGAGGCATGGCCCTGGGGTCCCACGAAT
TTGCTGGGGAATCTCGTTTTTCTTCTTAAGA
CTTTTGGGACATGGTTTGACTCCCGAACAT
CACCGACGCGTCTCCTGTTTTTCTGGGTGG
CCTCGGGACACCTGCCCTGCCCCCACGAGG
GTCAGGACTGTGACTCTTTTTAGGGCCAGG
CAGGTGCCTGGACATTTGCCTTGCTGGACG
GGGACTGGGGATGTGGGAGGGAGCAGACA
GGAGGAATCATGTCAGGCCTGTGTGTGAAA
GGAAGCTCCACTGTCACCCTCCACCTCTTC
ACCCCCCACTCACCAGTGTCCCCTCCACTG
TCACATTGTAACTGAACTTCAGGATAATAA
AGTGTTTGCCTCCATGGTCTTTGAATAAAG
CCTGAGTAGGAAGGCGGCCGCTCGAGCAT
GCATCTAGA
3UTR-007 Col 1 a2; ACTCAATCTAAATTAAAAAAGAAAGAAAT 11 collagen, TTGAAAAAACTTTCTCTTTGCCATTTCTTCT
type I, alpha TCTTCTTTTTTAACTGAAAGCTGAATCCTTC

GTGGGCAAAAGAGAAAAAGAAGGATTGAT
CAGAGCATTGTGCAATACAGTTTCATTAAC
TCCTTCCCCCGCTCCCCCAAAAATTTGAATT
TTTTTTTCAACACTCTTACACCTGTTATGGA
AAATGTCAACCTTTGTAAGAAAACCAAAAT
AAAAATTGAAAAATAAAAACCATAAACAT
TTGCACCACTTGTGGCTTTTGAATATCTTCC
ACAGAGGGAAGTTTAAAACCCAAACTTCC
AAAGGTTTAAACTACCTCAAAACACTTTCC
CATGAGTGTGATCCACATTGTTAGGTGCTG
ACCTAGACAGAGATGAACTGAGGTCCTTGT
TTTGTTTTGTTCATAATACAAAGGTGCTAAT
TAATAGTATTTCAGATACTTGAAGAATGTT
GATGGTGCTAGAAGAATTTGAGAAGAAAT
ACTCCTGTATTGAGTTGTATCGTGTGGTGT
ATTTTTTAAAAAATTTGATTTAGCATTCATA
TTTTCCATCTTATTCCCAATTAAAAGTATGC
AGATTATTTGCCCAAATCTTCTTCAGATTCA
GCATTTGTTCTTTGCCAGTCTCATTTTCATC
TTCTTCCATGGTTCCACAGAAGCTTTGTTTC
TTGGGCAAGCAGAAAAATTAAATTGTACCT
ATTTTGTATATGTGAGATGTTTAAATAAAT
TGTGAAAAAAATGAAATAAAGCATGTTTG
GTTTTCCAAAAGAACATAT
CGCCGCCGCCCGGGCCCCGCAGTCGAGGGT
CGTGAGCCCACCCCGTCCATGGTGCTAAGC
GGGCCCGGGTCCCACACGGCCAGCACCGCT
Co16a2; GCTCACTCGGACGACGCCCTGGGCCTGCAC
collagen, CTCTCCAGCTCCTCCCACGGGGTCCCCGTA

type VI, GCCCCGGCCCCCGCCCAGCCCCAGGTCTCC
alpha 2 CCAGGCCCTCCGCAGGCTGCCCGGCCTCCC
TCCCCCTGCAGCCATCCCAAGGCTCCTGAC
CTACCTGGCCCCTGAGCTCTGGAGCAAGCC
CTGACCCAATAAAGGCTTTGAACCCAT
GGGGCTAGAGCCCTCTCCGCACAGCGTGGA
GACGGGGCAAGGAGGGGGGTTATTAGGAT
TGGTGGTTTTGTTTTGCTTTGTTTAAAGCCG
TGGGAAAATGGCACAACTTTACCTCTGTGG
GAGATGCAACACTGAGAGCCAAGGGGTGG
GAGTTGGGATAATTTTTATATAAAAGAAGT
RPN1; TTTTCCACTTTGAATTGCTAAAAGTGGCATT

rthophorin I TTTCCTATGTGCAGTCACTCCTCTCATTTCT
AAAATAGGGACGTGGCCAGGCACGGTGGC
TCATGCCTGTAATCCCAGCACTTTGGGAGG
CCGAGGCAGGCGGCTCACGAGGTCAGGAG
ATCGAGACTATCCTGGCTAACACGGTAAAA
CCCTGTCTCTACTAAAAGTACAAAAAATTA
GCTGGGCGTGGTGGTGGGCACCTGTAGTCC

CAGCTACTCGGGAGGCTGAGGCAGGAGAA
AGGCATGAATCCAAGAGGCAGAGCTTGCA
GTGAGCTGAGATCACGCCATTGCACTCCAG
CCTGGGCAACAGTGTTAAGACTCTGTCTCA
AATATAAATAAATAAATAAATAAATAAAT
AAATAAATAAAAATAAAGCGAGATGTTGC
CCTCAAA
GGCCCTGCCCCGTCGGACTGCCCCCAGAAA
GCCTCCTGCCCCCTGCCAGTGAAGTCCTTC
AGTGAGCCCCTCCCCAGCCAGCCCTTCCCT
GGCCCCGCCGGATGTATAAATGTAAAAATG
AAGGAATTACATTTTATATGTGAGCGAGCA
AGCCGGCAAGCGAGCACAGTATTATTTCTC
CATCCCCTCCCTGCCTGCTCCTTGGCACCCC
CATGCTGCCTTCAGGGAGACAGGCAGGGA
GGGCTTGGGGCTGCACCTCCTACCCTCCCA
CCAGAACGCACCCCACTGGGAGAGCTGGT
LRP1; l ow GGTGCAGCCTTCCCCTCCCTGTATAAGACA
CTTTGCCAAGGCTCTCCCCTCTCGCCCCATC
density. CCTGCTTGCCCGCTCCCACAGCTTCCTGAG
lm 3UTR-010 ipoproteGGCTAATTCTGGGAAGGGAGAGTTCTTTGC 14 receptor-TGCCCCTGTCTGGAAGACGTGGCTCTGGGT
related GAGGTAGGCGGGAAAGGATGGAGTGTTTT
protein 1 AGTTCTTGGGGGAGGCCACCCCAAACCCCA
GCCCCAACTCCAGGGGCACCTATGAGATGG
CCATGCTCAACCCCCCTCCCAGACAGGCCC
TCCCTGTCTCCAGGGCCCCCACCGAGGTTC
CCAGGGCTGGAGACTTCCTCTGGTAAACAT
TCCTCCAGCCTCCCCTCCCCTGGGGACGCC
AAGGAGGTGGGCCACACCCAGGAAGGGAA
AGCGGGCAGCCCCGTTTTGGGGACGTGAAC
GTTTTAATAATTTTTGCTGAATTCCTTTACA
ACTAAATAACACAGATATTGTTATAAATAA
AATTGT
ATATTAAGGATCAAGCTGTTAGCTAATAAT
GCCACCTCTGCAGTTTTGGGAACAGGCAAA
TAAAGTATCAGTATACATGGTGATGTACAT
CTGTAGCAAAGCTCTTGGAGAAAATGAAG
ACTGAAGAAAGCAAAGCAAAAACTGTATA
GAGAGATTTTTCAAAAGCAGTAATCCCTCA
Nntl; ATTTTAAAAAAGGATTGAAAATTCTAAATG
cardiotrophi TCTTTCTGTGCATATTTTTTGTGTTAGGAAT
3UTR-011 n-like CAAAAGTATTTTATAAAAGGAGAAAGAAC 15 cytokine AGCCTCATTTTAGATGTAGTCCTGTTGGATT
factor 1 TTTTATGCCTCCTCAGTAACCAGAAATGTTT
TAAAAAACTAAGTGTTTAGGATTTCAAGAC
AACATTATACATGGCTCTGAAATATCTGAC
ACAATGTAAACATTGCAGGCACCTGCATTT
TATGTTTTTTTTTTCAACAAATGTGACTAAT
TTGAAACTTTTATGAACTTCTGAGCTGTCCC
CTTGCAATTCAACCGCAGTTTGAATTAATC

ATATCAAATCAGTTTTAATTTTTTAAATTGT
ACTTCAGAGTCTATATTTCAAGGGCACATT
TTCTCACTACTATTTTAATACATTAAAGGA
CTAAATAATCTTTCAGAGATGCTGGAAACA
AATCATTTGCTTTATATGTTTCATTAGAATA
CCAATGAAACATACAACTTGAAAATTAGTA
ATAGTATTTTTGAAGATCCCATTTCTAATTG
GAGATCTCTTTAATTTCGATCAACTTATAAT
GTGTAGTACTATATTAAGTGCACTTGAGTG
GAATTCAACATTTGACTAATAAAATGAGTT
CATCATGTTGGCAAGTGATGTGGCAATTAT
CTCTGGTGACAAAAGAGTAAAATCAAATAT
TTCTGCCTGTTACAAATATCAAGGAAGACC
TGCTACTATGAAATAGATGACATTAATCTG
TCTTCACTGTTTATAATACGGATGGATTTTT
TTTCAAATCAGTGTGTGTTTTGAGGTCTTAT
GTAATTGATGACATTTGAGAGAAATGGTGG
CTTTTTTTAGCTACCTCTTTGTTCATTTAAG
CACCAGTAAAGATCATGTCTTTTTATAGAA
GTGTAGATTTTCTTTGTGACTTTGCTATCGT
GCCTAAAGCTCTAAATATAGGTGAATGTGT
GATGAATACTCAGATTATTTGTCTCTCTATA
TAATTAGTTTGGTACTAAGTTTCTCAAAAA
ATTATTAACACATGAAAGACAATCTCTAAA
CCAGAAAAAGAAGTAGTACAAATTTTGTTA
CTGTAATGCTCGCGTTTAGTGAGTTTAAAA
CACACAGTATCTTTTGGTTTTATAATCAGTT
TCTATTTTGCTGTGCCTGAGATTAAGATCTG
TGTATGTGTGTGTGTGTGTGTGTGCGTTTGT
GTGTTAAAGCAGAAAAGACTTTTTTAAAAG
TTTTAAGTGATAAATGCAATTTGTTAATTG
ATCTTAGATCACTAGTAAACTCAGGGCTGA
ATTATACCATGTATATTCTATTAGAAGAAA
GTAAACACCATCTTTATTCCTGCCCTTTTTC
TTCTCTCAAAGTAGTTGTAGTTATATCTAG
AAAGAAGCAATTTTGATTTCTTGAAAAGGT
AGTTCCTGCACTCAGTTTAAACTAAAAATA
ATCATACTTGGATTTTATTTATTTTTGTCAT
AGTAAAAATTTTAATTTATATATATTTTTAT
TTAGTATTATCTTATTCTTTGCTATTTGCCA
ATCCTTTGTCATCAATTGTGTTAAATGAATT
GAAAATTCATGCCCTGTTCATTTTATTTTAC
TTTATTGGTTAGGATATTTAAAGGATTTTTG
TATATATAATTTCTTAAATTAATATTCCAAA
AGGTTAGTGGACTTAGATTATAAATTATGG
CAAAAATCTAAAAACAACAAAAATGATTTT
TATACATTCTATTTCATTATTCCTCTTTTTCC
AATAAGTCATACAATTGGTAGATATGACTT
ATTTTATTTTTGTATTATTCACTATATCTTT
ATGATATTTAAGTATAAATAATTAAAAAAA
TTTATTGTACCTTATAGTCTGTCACCAAAA

AAAAAAAATTATCTGTAGGTAGTGAAATGC
TAATGTTGATTTGTCTTTAAGGGCTTGTTAA
CTATCCTTTATTTTCTCATTTGTCTTAAATT
AGGAGTTTGTGTTTAAATTACTCATCTAAG
CAAAAAATGTATATAAATCCCATTACTGGG
TATATACCCAAAGGATTATAAATCATGCTG
CTATAAAGACACATGCACACGTATGTTTAT
TGCAGCACTATTCACAATAGCAAAGACTTG
GAACCAACCCAAATGTCCATCAATGATAGA
CTTGATTAAGAAAATGTGCACATATACACC
ATGGAATACTATGCAGCCATAAAAAAGGA
TGAGTTCATGTCCTTTGTAGGGACATGGAT
AAAGCTGGAAACCATCATTCTGAGCAAACT
ATTGCAAGGACAGAAAACCAAACACTGCA
TGTTCTCACTCATAGGTGGGAATTGAACAA
TGAGAACACTTGGACACAAGGTGGGGAAC
ACCACACACCAGGGCCTGTCATGGGGTGG
GGGGAGTGGGGAGGGATAGCATTAGGAGA
TATACCTAATGTAAATGATGAGTTAATGGG
TGCAGCACACCAACATGGCACATGTATACA
TATGTAGCAAACCTGCACGTTGTGCACATG
TACCCTAGAACTTAAAGTATAATTAAAAAA
AAAAAGAAAACAGAAGCTATTTATAAAGA
AGTTATTTGCTGAAATAAATGTGATCTTTC
CCATTAAAAAAATAAAGAAATTTTGGGGTA
AAAAAACACAATATATTGTATTCTTGAAAA
ATTCTAAGAGAGTGGATGTGAAGTGTTCTC
ACCACAAAAGTGATAACTAATTGAGGTAAT
GCACATATTAATTAGAAAGATTTTGTCATT
CCACAATGTATATATACTTAAAAATATGTT
ATACACAATAAATACATACATTAAAAAATA
AGTAAATGTA
CCCACCCTGCACGCCGGCACCAAACCCTGT
CCTCCCACCCCTCCCCACTCATCACTAAAC
AGAGTAAAATGTGATGCGAATTTTCCCGAC
CAACCTGATTCGCTAGATTTTTTTTAAGGA
AAAGCTTGGAAAGCCAGGACACAACGCTG
CTGCCTGCTTTGTGCAGGGTCCTCCGGGGC
TCAGCCCTGAGTTGGCATCACCTGCGCAGG
C o16 a 1. GCCCTCTGGGGCTCAGCCCTGAGCTAGTGT
, CACCTGCACAGGGCCCTCTGAGGCTCAGCC
3UTR-012 collagen, type VI, TGGGGCTCAGCCCTGAGCTGGCCTCACCTG
alpha 1 GGTTCCCCACCCCGGGCTCTCCTGCCCTGC
CCTCCTGCCCGCCCTCCCTCCTGCCTGCGCA
GCTCCTTCCCTAGGCACCTCTGTGCTGCATC
CCACCAGCCTGAGCAAGACGCCCTCTCGGG
GCCTGTGCCGCACTAGCCTCCCTCTCCTCTG
TCCCCATAGCTGGTTTTTCCCACCAATCCTC
ACCTAACAGTTACTTTACAATTAAACTCAA
AGCAAGCTCTTCTCCTCAGCTTGGGGCAGC

CATTGGCCTCTGTCTCGTTTTGGGAAACCA
AGGTCAGGAGGCCGTTGCAGACATAAATCT
CGGCGACTCGGCCCCGTCTCCTGAGGGTCC
TGCTGGTGACCGGCCTGGACCTTGGCCCTA
CAGCCCTGGAGGCCGCTGCTGACCAGCACT
GACCCCGACCTCAGAGAGTACTCGCAGGG
GCGCTGGCTGCACTCAAGACCCTCGAGATT
AACGGTGCTAACCCCGTCTGCTCCTCCCTC
CCGCAGAGACTGGGGCCTGGACTGGACAT
GAGAGCCCCTTGGTGCCACAGAGGGCTGTG
TCTTACTAGAAACAACGCAAACCTCTCCTT
CCTCAGAATAGTGATGTGTTCGACGTTTTA
TCAAAGGCCCCCTTTCTATGTTCATGTTAGT
TTTGCTCCTTCTGTGTTTTTTTCTGAACCAT
ATCCATGTTGCTGACTTTTCCAAATAAAGG
TTTTCACTCCTCTC
AGAGGCCTGCCTCCAGGGCTGGACTGAGG
CCTGAGCGCTCCTGCCGCAGAGCTGGCCGC
GCCAAATAATGTCTCTGTGAGACTCGAGAA
CTTTCATTTTTTTCCAGGCTGGTTCGGATTT
GGGGTGGATTTTGGTTTTGTTCCCCTCCTCC
ACTCTCCCCCACCCCCTCCCCGCCCTTTTTT
TTTTTTTTTTTTAAACTGGTATTTTATCTTTG
ATTCTCCTTCAGCCCTCACCCCTGGTTCTCA
TCTTTCTTGATCAACATCTTTTCTTGCCTCT
Calr; GTCCCCTTCTCTCATCTCTTAGCTCCCCTCC

calreticulin AACCTGGGGGGCAGTGGTGTGGAGAAGCC
ACAGGCCTGAGATTTCATCTGCTCTCCTTCC
TGGAGCCCAGAGGAGGGCAGCAGAAGGGG
GTGGTGTCTCCAACCCCCCAGCACTGAGGA
AGAACGGGGCTCTTCTCATTTCACCCCTCC
CTTTCTCCCCTGCCCCCAGGACTGGGCCAC
TTCTGGGTGGGGCAGTGGGTCCCAGATTGG
CTCACACTGAGAATGTAAGAACTACAAAC
AAAATTTCTATTAAATTAAATTTTGTGTCTC
C
CTCCCTCCATCCCAACCTGGCTCCCTCCCAC
CCAACCAACTTTCCCCCCAACCCGGAAACA
GACAAGCAACCCAAACTGAACCCCCTCAA
AAGCCAAAAAATGGGAGACAATTTCACAT
GGACTTTGGAAAATATTTTTTTCCTTTGCAT
C oll a 1 = TCATCTCTCAAACTTAGTTTTTATCTTTGAC
' CAACCGAACATGACCAAAAACCAAAAGTG
3UTR-014 collagen, type I, alpha AAAAGAATAAATAAATAACTTTTTAAAAA

AGGAAGCTTGGTCCACTTGCTTGAAGACCC
ATGCGGGGGTAAGTCCCTTTCTGCCCGTTG
GGCTTATGAAACCCCAATGCTGCCCTTTCT
GCTCCTTTCTCCACACCCCCCTTGGGGCCTC
CCCTCCACTCCTTCCCAAATCTGTCTCCCCA
GAAGACACAGGAAACAATGTATTGTCTGCC

CAGCAATCAAAGGCAATGCTCAAACACCC
AAGTGGCCCCCACCCTCAGCCCGCTCCTGC
CCGCCCAGCACCCCCAGGCCCTGGGGGACC
TGGGGTTCTCAGACTGCCAAAGAAGCCTTG
CCATCTGGCGCTCCCATGGCTCTTGCAACA
TCTCCCCTTCGTTTTTGAGGGGGTCATGCCG
GGGGAGCCACCAGCCCCTCACTGGGTTCGG
AGGAGAGTCAGGAAGGGCCACGACAAAGC
AGAAACATCGGATTTGGGGAACGCGTGTC
AATCCCTTGTGCCGCAGGGCTGGGCGGGAG
AGACTGTTCTGTTCCTTGTGTAACTGTGTTG
CTGAAAGACTACCTCGTTCTTGTCTTGATGT
GTCACCGGGGCAACTGCCTGGGGGCGGGG
ATGGGGGCAGGGTGGAAGCGGCTCCCCAT
TTTATACCAAAGGTGCTACATCTATGTGAT
GGGTGGGGTGGGGAGGGAATCACTGGTGC
TATAGAAATTGAGATGCCCCCCCAGGCCAG
CAAATGTTCCTTTTTGTTCAAAGTCTATTTT
TATTCCTTGATATTTTTCTTTTTTTTTTTTTT
TTTTTGTGGATGGGGACTTGTGAATTTTTCT
AAAGGTGCTATTTAACATGGGAGGAGAGC
GTGTGCGGCTCCAGCCCAGCCCGCTGCTCA
CTTTCCACCCTCTCTCCACCTGCCTCTGGCT
TCTCAGGCCTCTGCTCTCCGACCTCTCTCCT
CTGAAACCCTCCTCCACAGCTGCAGCCCAT
CCTCCCGGCTCCCTCCTAGTCTGTCCTGCGT
CCTCTGTCCCCGGGTTTCAGAGACAACTTC
CCAAAGCACAAAGCAGTTTTTCCCCCTAGG
GGTGGGAGGAAGCAAAAGACTCTGTACCT
ATTTTGTATGTGTATAATAATTTGAGATGTT
TTTAATTATTTTGATTGCTGGAATAAAGCA
TGTGGAAATGACCCAAACATAATCCGCAGT
GGCCTCCTAATTTCCTTCTTTGGAGTTGGGG
GAGGGGTAGACATGGGGAAGGGGCTTTGG
GGTGATGGGCTTGCCTTCCATTCCTGCCCTT
TCCCTCCCCACTATTCTCTTCTAGATCCCTC
CATAACCCCACTCCCCTTTCTCTCACCCTTC
TTATACCGCAAACCTTTCTACTTCCTCTTTC
ATTTTCTATTCTTGCAATTTCCTTGCACCTT
TTCCAAATCCTCTTCTCCCCTGCAATACCAT
ACAGGCAATCCACGTGCACAACACACACA
CACACTCTTCACATCTGGGGTTGTCCAAAC
CTCATACCCACTCCCCTTCAAGCCCATCCA
CTCTCCACCCCCTGGATGCCCTGCACTTGG
TGGCGGTGGGATGCTCATGGATACTGGGAG
GGTGAGGGGAGTGGAACCCGTGAGGAGGA
CCTGGGGGCCTCTCCTTGAACTGACATGAA
GGGTCATCTGGCCTCTGCTCCCTTCTCACCC
ACGCTGACCTCCTGCCGAAGGAGCAACGC
AACAGGAGAGGGGTCTGCTGAGCCTGGCG
AGGGTCTGGGAGGGACCAGGAGGAAGGCG

TGCTCCCTGCTCGCTGTCCTGGCCCTGGGG
GAGTGAGGGAGACAGACACCTGGGAGAGC
TGTGGGGAAGGCACTCGCACCGTGCTCTTG
GGAAGGAAGGAGACCTGGCCCTGCTCACC
ACGGACTGGGTGCCTCGACCTCCTGAATCC
CCAGAACACAACCCCCCTGGGCTGGGGTG
GTCTGGGGAACCATCGTGCCCCCGCCTCCC
GCCTACTCCTTTTTAAGCTT
TTGGCCAGGCCTGACCCTCTTGGACCTTTCT
TCTTTGCCGACAACCACTGCCCAGCAGCCT
CTGGGACCTCGGGGTCCCAGGGAACCCAGT
CCAGCCTCCTGGCTGTTGACTTCCCATTGCT
CTTGGAGCCACCAATCAAAGAGATTCAAA
GAGATTCCTGCAGGCCAGAGGCGGAACAC
ACCTTTATGGCTGGGGCTCTCCGTGGTGTT
CTGGACCCAGCCCCTGGAGACACCATTCAC
TTTTACTGCTTTGTAGTGACTCGTGCTCTCC
Plod 1; AACCTGTCTTCCTGAAAAACCAAGGCCCCC
procollagen- TTCCCCCACCTCTTCCATGGGGTGAGACTT
lysine, 2- GAGCAGAACAGGGGCTTCCCCAAGTTGCCC
3UTR-015 oxoglutarate AGAAAGACTGTCTGGGTGAGAAGCCATGG 19 CCAGAGCTTCTCCCAGGCACAGGTGTTGCA
dioxygenase CCAGGGACTTCTGCTTCAAGTTTTGGGGTA

CCCTGAGTCTGGGACTTCTGCCTCCATGGC
TGGTCATGAGAGCAAACCGTAGTCCCCTGG
AGACAGCGACTCCAGAGAACCTCTTGGGA
GACAGAAGAGGCATCTGTGCACAGCTCGA
TCTTCTACTTGCCTGTGGGGAGGGGAGTGA
CAGGTCCACACACCACACTGGGTCACCCTG
TCCTGGATGCCTCTGAAGAGAGGGACAGA
CCGTCAGAAACTGGAGAGTTTCTATTAAAG
GTCATTTAAACCA
TCCTCCGGGACCCCAGCCCTCAGGATTCCT
GATGCTCCAAGGCGACTGATGGGCGCTGG
ATGAAGTGGCACAGTCAGCTTCCCTGGGGG
CTGGTGTCATGTTGGGCTCCTGGGGCGGGG
GCACGGCCTGGCATTTCACGCATTGCTGCC
ACCCCAGGTCCACCTGTCTCCACTTTCACA
GCCTCCAAGTCTGTGGCTCTTCCCTTCTGTC
CTCCGAGGGGCTTGCCTTCTCTCGTGTCCA
Nucbl.
. ' . GTGAGGTGCTCAGTGATCGGCTTAACTTAG
3UTR-016 nucleobmdt 20 AGAAGCCCGCCCCCTCCCCTTCTCCGTCTG
n 1 TCCCAAGAGGGTCTGCTCTGAGCCTGCGTT
CCTAGGTGGCTCGGCCTCAGCTGCCTGGGT
TGTGGCCGCCCTAGCATCCTGTATGCCCAC
AGCTACTGGAATCCCCGCTGCTGCTCCGGG
CCAAGCTTCTGGTTGATTAATGAGGGCATG
GGGTGGTCCCTCAAGACCTTCCCCTACCTT
TTGTGGAACCAGTGATGCCTCAAAGACAGT
GTCCCCTCCACAGCTGGGTGCCAGGGGCAG

GGGATCCTCAGTATAGCCGGTGAACCCTGA
TACCAGGAGCCTGGGCCTCCCTGAACCCCT
GGCTTCCAGCCATCTCATCGCCAGCCTCCT
CCTGGACCTCTTGGCCCCCAGCCCCTTCCC
CACACAGCCCCAGAAGGGTCCCAGAGCTG
ACCCCACTCCAGGACCTAGGCCCAGCCCCT
CAGCCTCATCTGGAGCCCCTGAAGACCAGT
CCCACCCACCTTTCTGGCCTCATCTGACACT
GCTCCGCATCCTGCTGTGTGTCCTGTTCCAT
GTTCCGGTTCCATCCAAATACACTTTCTGG
AACAAA
GCTGGAGCCTCGGTGGCCATGCTTCTTGCC
3 UTR- 01 a-globin CCTTGGGCCTCCCCCCAGCCCCTCCTCCCCT 21 TCCTGCACCCGTACCCCCGTGGTCTTTGAA
TAAAGTCTGAGTGGGCGGC

[000252] It should be understood that those listed in the previous tables are examples and that any UTR from any gene may be incorporated into the respective first or second flanking region of the primary construct. Furthermore, multiple wild-type UTRs of any known gene may be utilized. It is also within the scope of the present invention to provide artificial UTRs which are not variants of wild type genes. These UTRs or portions thereof may be placed in the same orientation as in the transcript from which they were selected or may be altered in orientation or location. Hence a 5' or 3' UTR may be inverted, shortened, lengthened, made chimeric with one or more other 5' UTRs or 3' UTRs. As used herein, the term "altered" as it relates to a UTR sequence, means that the UTR has been changed in some way in relation to a reference sequence. For example, a 3' or 5' UTR may be altered relative to a wild type or native UTR by the change in orientation or location as taught above or may be altered by the inclusion of additional nucleotides, deletion of nucleotides, swapping or transposition of nucleotides. Any of these changes producing an "altered" UTR (whether 3' or 5') comprise a variant UTR.

[000253] In one embodiment, a double, triple or quadruple UTR such as a 5' or 3' UTR
may be used. As used herein, a "double" UTR is one in which two copies of the same UTR are encoded either in series or substantially in series. For example, a double beta-globin 3' UTR may be used as described in US Patent publication 20100129877, the contents of which are incorporated herein by reference in its entirety.

[000254] It is also within the scope of the present invention to have patterned UTRs. As used herein "patterned UTRs" are those UTRs which reflect a repeating or alternating pattern, such as ABABAB or AABBAABBAABB or ABCABCABC or variants thereof repeated once, twice, or more than 3 times. In these patterns, each letter, A, B, or C
represent a different UTR at the nucleotide level.

[000255] In one embodiment, flanking regions are selected from a family of transcripts whose proteins share a common function, structure, feature of property. For example, polypeptides of interest may belong to a family of proteins which are expressed in a particular cell, tissue or at some time during development. The UTRs from any of these genes may be swapped for any other UTR of the same or different family of proteins to create a new chimeric primary transcript. As used herein, a "family of proteins" is used in the broadest sense to refer to a group of two or more polypeptides of interest which share at least one function, structure, feature, localization, origin, or expression pattern.

[000256] After optimization (if desired), the primary construct components are reconstituted and transformed into a vector such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes. For example, the optimized construct may be reconstituted and transformed into chemically competent E. coli, yeast, neurospora, maize, drosophila, etc. where high copy plasmid-like or chromosome structures occur by methods described herein.

[000257] The untranslated region may also include translation enhancer elements (TEE). As a non-limiting example, the TEE may include those described in US
Application No. 20090226470, herein incorporated by reference in its entirety, and those known in the art.
Stop Codons [000258] In one embodiment, the primary constructs of the present invention may include at least two stop codons before the 3' untranslated region (UTR). The stop codon may be selected from TGA, TAA and TAG. In one embodiment, the primary constructs of the present invention include the stop codon TGA and one additional stop codon. In a further embodiment the addition stop codon may be TAA. In another embodiment, the primary constructs of the present invention include three stop codons.
Vector Amplification [000259] The vector containing the primary construct is then amplified and the plasmid isolated and purified using methods known in the art such as, but not limited to, a maxi prep using the Invitrogen PURELNKTM HiPure Maxiprep Kit (Carlsbad, CA).
Plasmid Linearization [000260] The plasmid may then be linearized using methods known in the art such as, but not limited to, the use of restriction enzymes and buffers. The linearization reaction may be purified using methods including, for example Invitrogen's PURELINKTM
PCR
Micro Kit (Carlsbad, CA), and HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC
(RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) and Invitrogen's standard PURELINKTM PCR Kit (Carlsbad, CA). The purification method may be modified depending on the size of the linearization reaction which was conducted. The linearized plasmid is then used to generate cDNA for in vitro transcription (IVT) reactions.
cDNA Template Synthesis [000261] A cDNA template may be synthesized by having a linearized plasmid undergo polymerase chain reaction (PCR). Table 4 is a listing of primers and probes that may be usefully in the PCR reactions of the present invention. It should be understood that the listing is not exhaustive and that primer-probe design for any amplification is within the skill of those in the art. Probes may also contain chemically modified bases to increase base-pairing fidelity to the target molecule and base-pairing strength. Such modifications may include 5-methyl-Cytidine, 2, 6-di-amino-purine, 2'-fluoro, phosphoro-thioate, or locked nucleic acids.
Table 4. Primers and Probes Primer/SEQ
Hybridization Probe Sequence (5'-3') ID
Identifier target NO.
TTGGACCCTCGTACAGAAGCTAA
UFP TACG cDNA Template 22 URP TxmoCTTCCTACTCAGGCTTTATTC
C Templ 23 ate AAAGACCA
CCTTGACCTTCTGGAACTTC Acid glucocerebrosidase CCAAGCACTGAAACGGATAT Acid glucocerebrosidase GATGAAAAGTGCTCCAAGGA
LUC1 Luciferase 26 AACCGTGATGAAAAGGTACC
LUC2 Luciferase 27 TCATGCAGATTGGAAAGGTC
LUC3 Luciferase 28 CTTCTTGGACTGTCCAGAGG

GCAGTCCCTGATACAAGAAC

GATTGAAGGTGGCTCGCTAC

*UFP is universal forward primer; URP is universal reverse primer.

[000262] In one embodiment, the cDNA may be submitted for sequencing analysis before undergoing transcription.
mRNA Production [000263] The process of mRNA or mmRNA production may include, but is not limited to, in vitro transcription, cDNA template removal and RNA clean-up, and mRNA
capping and/or tailing reactions.
In Vitro Transcription [000264] The cDNA produced in the previous step may be transcribed using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be manufactured in house, may be selected from a supplier, or may be synthesized as described herein. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids.
RNA Polymerases [000265] Any number of RNA polymerases or variants may be used in the design of the primary constructs of the present invention.

[000266] RNA polymerases may be modified by inserting or deleting amino acids of the RNA polymerase sequence. As a non-limiting example, the RNA polymerase may be modified to exhibit an increased ability to incorporate a 2'-modified nucleotide triphosphate compared to an unmodified RNA polymerase (see International Publication W02008078180 and U.S. Patent 8,101,385; herein incorporated by reference in their entireties).

[000267] Variants may be obtained by evolving an RNA polymerase, optimizing the RNA polymerase amino acid and/or nucleic acid sequence and/or by using other methods known in the art. As a non-limiting example, T7 RNA polymerase variants may be evolved using the continuous directed evolution system set out by Esvelt et at. (Nature (2011) 472(7344):499-503; herein incorporated by reference in its entirety) where clones of T7 RNA polymerase may encode at least one mutation such as, but not limited to, lysine at position 93 substituted for threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, 5128R, A136T, N1655, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, 5228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A3545, E356D, L360P, A383V, Y385C, D388Y, 5397R, M401T, N4105, K450R, P45 1T, G452V, E484A, H523L, H524N, G542V, E565K, K577E, K577M, N6015, 5684Y, L699I, K713E, N748D, Q754R, E775K, A827V, D851N or L864F. As another non-limiting example, T7 RNA polymerase variants may encode at least mutation as described in U.S. Pub. Nos. 20100120024 and 20070117112; herein incorporated by reference in their entireties. Variants of RNA polymerase may also include, but are not limited to, substitutional variants, conservative amino acid substitution, insertional variants, deletional variants and/or covalent derivatives.

[000268] In one embodiment, the primary construct may be designed to be recognized by the wild type or variant RNA polymerases. In doing so, the primary construct may be modified to contain sites or regions of sequence changes from the wild type or parent primary construct.

[000269] In one embodiment, the primary construct may be designed to include at least one substitution and/or insertion upstream of an RNA polymerase binding or recognition site, downstream of the RNA polymerase binding or recognition site, upstream of the TATA box sequence, downstream of the TATA box sequence of the primary construct but upstream of the coding region of the primary construct, within the 5'UTR, before the 5'UTR and/or after the 5'UTR.

[000270] In one embodiment, the 5'UTR of the primary construct may be replaced by the insertion of at least one region and/or string of nucleotides of the same base. The region and/or string of nucleotides may include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 nucleotides and the nucleotides may be natural and/or unnatural. As a non-limiting example, the group of nucleotides may include 5-8 adenine, cytosine, thymine, a string of any of the other nucleotides disclosed herein and/or combinations thereof.

[000271] In one embodiment, the 5'UTR of the primary construct may be replaced by the insertion of at least two regions and/or strings of nucleotides of two different bases such as, but not limited to, adenine, cytosine, thymine, any of the other nucleotides disclosed herein and/or combinations thereof For example, the 5'UTR may be replaced by inserting 5-8 adenine bases followed by the insertion of 5-8 cytosine bases. In another example, the 5'UTR may be replaced by inserting 5-8 cytosine bases followed by the insertion of 5-8 adenine bases.

[000272] In one embodiment, the primary construct may include at least one substitution and/or insertion downstream of the transcription start site which may be recognized by an RNA polymerase. As a non-limiting example, at least one substitution and/or insertion may occur downstream the transcription start site by substituting at least one nucleic acid in the region just downstream of the transcription start site (such as, but not limited to, +1 to +6). Changes to region of nucleotides just downstream of the transcription start site may affect initiation rates, increase apparent nucleotide triphosphate (NTP) reaction constant values, and increase the dissociation of short transcripts from the transcription complex curing initial transcription (Brieba et al, Biochemistry (2002) 41: 5144-5149; herein incorporated by reference in its entirety).
The modification, substitution and/or insertion of at least one nucleic acid may cause a silent mutation of the nucleic acid sequence or may cause a mutation in the amino acid sequence.

[000273] In one embodiment, the primary construct may include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12 or at least 13 guanine bases downstream of the transcription start site.

[000274] In one embodiment, the primary construct may include the substitution of at least 1, at least 2, at least 3, at least 4, at least 5 or at least 6 guanine bases in the region just downstream of the transcription start site. As a non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 adenine nucleotides. In another non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 cytosine bases. In another non-limiting example, if the nucleotides in the region are GGGAGA the guanine bases may be substituted by at least 1, at least 2, at least 3 or at least 4 thymine, and/or any of the nucleotides described herein.

[000275] In one embodiment, the primary construct may include at least one substitution and/or insertion upstream of the start codon. For the purpose of clarity, one of skill in the art would appreciate that the start codon is the first codon of the protein coding region whereas the transcription start site is the site where transcription begins.
The primary construct may include, but is not limited to, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7 or at least 8 substitutions and/or insertions of nucleotide bases. The nucleotide bases may be inserted or substituted at 1, at least 1, at least 2, at least 3, at least 4 or at least 5 locations upstream of the start codon. The nucleotides inserted and/or substituted may be the same base (e.g., all A or all C or all T
or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C and T or A, C and T) or at least four different bases. As a non-limiting example, the guanine base upstream of the coding region in the primary construct may be substituted with adenine, cytosine, thymine, or any of the nucleotides described herein.
In another non-limiting example the substitution of guanine bases in the primary construct may be designed so as to leave one guanine base in the region downstream of the transcription start site and before the start codon (see Esvelt et at.
Nature (2011) 472(7344):499-503; herein incorporated by reference in its entirety). As a non-limiting example, at least 5 nucleotides may be inserted at 1 location downstream of the transcription start site but upstream of the start codon and the at least 5 nucleotides may be the same base type.
cDNA Template Removal and Clean-Up [000276] The cDNA template may be removed using methods known in the art such as, but not limited to, treatment with Deoxyribonuclease I (DNase I). RNA clean-up may also include a purification method such as, but not limited to, AGENCOURTO
CLEANSEQO system from Beckman Coulter (Danvers, MA), HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC) .
Capping and/or Tailing Reactions [000277] The primary construct or mmRNA may also undergo capping and/or tailing reactions. A capping reaction may be performed by methods known in the art to add a 5' cap to the 5' end of the primary construct. Methods for capping include, but are not limited to, using a Vaccinia Capping enzyme (New England Biolabs, Ipswich, MA).

[000278] A poly-A tailing reaction may be performed by methods known in the art, such as, but not limited to, 2' 0-methyltransferase and by methods as described herein. If the primary construct generated from cDNA does not include a poly-T, it may be beneficial to perform the poly-A-tailing reaction before the primary construct is cleaned.
mRNA Purification [000279] Primary construct or mmRNA purification may include, but is not limited to, mRNA or mmRNA clean-up, quality assurance and quality control. mRNA or mmRNA
clean-up may be performed by methods known in the arts such as, but not limited to, AGENCOURTO beads (Beckman Coulter Genomics, Danvers, MA), poly-T beads, LNATM oligo-T capture probes (EXIQONO Inc, Vedbaek, Denmark) or HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term "purified" when used in relation to a polynucleotide such as a "purified mRNA or mmRNA" refers to one that is separated from at least one contaminant. As used herein, a "contaminant" is any substance which makes another unfit, impure or inferior. Thus, a purified polynucleotide (e.g., DNA and RNA) is present in a form or setting different from that in which it is found in nature, or a form or setting different from that which existed prior to subjecting it to a treatment or purification method.

[000280] A quality assurance and/or quality control check may be conducted using methods such as, but not limited to, gel electrophoresis, UV absorbance, or analytical HPLC.

[000281] In another embodiment, the mRNA or mmRNA may be sequenced by methods including, but not limited to reverse-transcriptase-PCR.

[000282] In one embodiment, the mRNA or mmRNA may be quantified using methods such as, but not limited to, ultraviolet visible spectroscopy (UVNis). A non-limiting example of a UVNis spectrometer is a NANODROPO spectrometer (ThermoFisher, Waltham, MA). The quantified mRNA or mmRNA may be analyzed in order to determine if the mRNA or mmRNA may be of proper size, check that no degradation of the mRNA or mmRNA has occurred. Degradation of the mRNA and/or mmRNA may be checked by methods such as, but not limited to, agarose gel electrophoresis, HPLC based purification methods such as, but not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), liquid chromatography-mass spectrometry (LCMS), capillary electrophoresis (CE) and capillary gel electrophoresis (CGE).
Signal Sequences [000283] The primary constructs or mmRNA may also encode additional features which facilitate trafficking of the polypeptides to therapeutically relevant sites. One such feature which aids in protein trafficking is the signal sequence. As used herein, a "signal sequence" or "signal peptide" is a polynucleotide or polypeptide, respectively, which is from about 9 to 200 nucleotides (3-60 amino acids) in length which is incorporated at the 5' (or N-terminus) of the coding region or polypeptide encoded, respectively.
Addition of these sequences result in trafficking of the encoded polypeptide to the endoplasmic reticulum through one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidase after the proteins are transported.

[000284] Table 5 is a representative listing of protein signal sequences which may be incorporated for encoding by the polynucleotides, primary constructs or mmRNA
of the invention.

Table 5. Signal Sequences ID Description NUCLEOTIDE SEQUENCE SEQ ENCODED SEQ ID
(5'-3') ID PEPTIDE NO.
NO.
SS-001 a-1- ATGATGCCATCCTCAGTCTCA 32 MMPSSVSWGI 94 antitrypsin TGGGGTATTTTGCTCTTGGCG LLAGLCCLVP
GGTCTGTGCTGTCTCGTGCCG VSLA
GTGTCGCTCGCA

TCGCCCATGAAACTCATGGCC KLMALQLLLW
CTGCAGTTGTTGCTTTGGCAC HSALWTVQEA
TCAGCCCTCTGGACCGTCCAA
GAGGCG
SS-003 Factor IX ATGCAGAGAGTGAACATGATT 34 MQRVNMIMAE 96 ATGGCCGAGTCCCCATCGCTC SP SLITICLLGY
ATCACAATCTGCCTGCTTGGT LLSAECTVFLD
ACCTGCTTTCCGCCGAATGCA HENANKILNRP
CTGTCTTTCTGGATCACGAGA KR
ATGCGAATAAGATCTTGAACC
GACCCAAACGG
SS-004 Prolactin ATGAAAGGATCATTGCTGTTG 35 MKGSLLLLLV 97 CTCCTCGTGTCGAACCTTCTG SNLLLCQSVAP
CTTTGCCAGTCCGTAGCCCCC
SS-005 Albumin ATGAAATGGGTGACGTTCATC 36 MKWVTFISLLF 98 TCACTGTTGTTTTTGTTCTCGT LFSSAYSRG
CCGCCTACTCCAGGGGAGTAT VFRR
TCCGCCGA

CTGCTCCTGTTGCTCCTCTTGC LLLLLLPMWA
TGTGGCCCATGGTGTGGGCA
MLS- ornithine TGCTCTTTAACCTCCGCATCCT 38 MLFNLRILLNN 100 001 carbamoyltr GTTGAATAACGCTGCGTTCCG AAFRNGHNFM
ansferase AAATGGGCATAACTTCATGGT VRNFRCGQPL
ACGCAACTTCAGATGCGGCCA Q
GCCACTCCAG
MLS- Cytochrome ATGTCCGTCTTGACACCCCTG 39 MSVLTPLLLRG 101 002 C Oxidase CTCTTGAGAGGGCTGACGGGG LTGSARRLPVP
subunit 8A TCCGCTAGACGCCTGCCGGTA RAKIHSL
CCGCGAGCGAAGATCCACTCC
CTG
MLS- Cytochrome ATGAGCGTGCTCACTCCGTTG 40 MSVLTPLLLRG 102 003 C Oxidase CTTCTTCGAGGGCTTACGGGA LTGSARRLPVP
subunit 8A TCGGCTCGGAGGTTGCCCGTC RAKIHSL
CCGAGAGCGAAGATCCATTCG
TTG
SS-007 Type III, TGACAAAAATAACTTTATCTC 41 MVTKITLSPQN 103 bacterial CCCAGAATTTTAGAATCCAAA FRIQKQETTLL
AACAGGAAACCACACTACTA KEKSTEKNSLA
AAAGAAAAATCAACCGAGAA KSILAVKNHFI
AAATTCTTTAGCAAAAAGTAT ELRSKLSERFIS

TCTCGCAGTAAAAATCACTTC HKNT
ATCGAATTAAGGTCAAAATTA
TCGGAACGTTTTATTTCGCAT
AAGAACACT
SS-008 Viral ATGCTGAGCTTTGTGGATACC 42 MLSFVDTRTLL 104 CGCACCCTGCTGCTGCTGGCG LLAVTSCLATC
GTGACCAGCTGCCTGGCGACC Q
TGCCAG
SS-009 viral ATGGGCAGCAGCCAGGCGCC 43 MGS SQAPRMG 105 GCGCATGGGCAGCGTGGGCG SVGGHGLMAL
GCCATGGCCTGATGGCGCTGC LMAGLILPGIL
TGATGGCGGGCCTGATTCTGC A
CGGGCATTCTGGCG
SS-010 Viral ATGGCGGGCATTTTTTATTTTC 44 MAGIFYFLF SF 106 TGTTTAGCTTTCTGTTTGGCAT LFGICD
TTGCGAT
SS-011 Viral ATGGAAAACCGCCTGCTGCGC 45 MENRLLRVFL 107 GTGTTTCTGGTGTGGGCGGCG VWAALTMDG
CTGACCATGGATGGCGCGAGC ASA
GCG
SS-012 Viral ATGGCGCGCCAGGGCTGCTTT 46 MARQGCFGSY 108 GGCAGCTATCAGGTGATTAGC QVISLFTFAIGV
CTGTTTACCTTTGCGATTGGC NLCLG
GTGAACCTGTGCCTGGGC
SS-013 Bacillus ATGAGCCGCCTGCCGGTGCTG 47 MSRLPVLLLLQ 109 CTGCTGCTGCAGCTGCTGGTG LLVRPGLQ
CGCCCGGGCCTGCAG
SS-014 Bacillus ATGAAACAGCAGAAACGCCT 48 MKQQKRLYAR 110 GTATGCGCGCCTGCTGACCCT LLTLLFALIFLL
GCTGTTTGCGCTGATTTTTCTG PHS SASA
CTGCCGCATAGCAGCGCGAGC
GCG
SS-015 Secretion ATGGCGACGCCGCTGCCTCCG 49 MATPLPPPSPR 111 signal CCCTCCCCGCGGCACCTGCGG HLRLLRLLL SG
CTGCTGCGGCTGCTGCTCTCC
GCCCTCGTCCTCGGC
SS-016 Secretion ATGAAGGCTCCGGGTCGGCTC 50 MKAPGRLVLII 112 signal GTGCTCATCATCCTGTGCTCC LCSVVFS
GTGGTCTTCTCT
SS-017 Secretion ATGCTTCAGCTTTGGAAACTT 51 MLQLWKLLCG 113 signal GTTCTCCTGTGCGGCGTGCTC VLT
ACT
SS-018 Secretion ATGCTTTATCTCCAGGGTTGG 52 MLYLQGWSM 114 signal AGCATGCCTGCTGTGGCA PAVA
SS-019 Secretion ATGGATAACGTGCAGCCGAA 53 MDNVQPKIKH 115 signal AATAAAACATCGCCCCTTCTG RPFCFSVKGHV
CTTCAGTGTGAAAGGCCACGT KMLRLDIINSL
GAAGATGCTGCGGCTGGATAT VTTVFMLIVSV
TATCAACTCACTGGTAACAAC LALIP
AGTATTCATGCTCATCGTATC

TGTGTTGGCACTGATACCA
SS-020 Secretion ATGCCCTGCCTAGACCAACAG 54 MPCLDQQLTV 116 signal CTCACTGTTCATGCCCTACCCT HALPCPAQPS S
GCCCTGCCCAGCCCTCCTCTC LAFCQVGFLT
TGGCCTTCTGCCAAGTGGGGT A
TCTTAACAGCA
SS-021 Secretion ATGAAAACCTTGTTCAATCCA 55 MKTLFNPAPAI 117 signal GCCCCTGCCATTGCTGACCTG ADLDPQFYTLS
GATCCCCAGTTCTACACCCTC DVFCCNESEAE
TCAGATGTGTTCTGCTGCAAT ILTGLTVGSAA
GAAAGTGAGGCTGAGATTTTA DA
ACTGGCCTCACGGTGGGCAGC
GCTGCAGATGCT
SS-022 Secretion ATGAAGCCTCTCCTTGTTGTG 56 MKPLLVVFVF 118 signal TTTGTCTTTCTTTTCCTTTGGG LFLWDPVLA
ATCCAGTGCTGGCA
SS-023 Secretion ATGTCCTGTTCCCTAAAGTTT 57 MSCSLKFTLIVI 119 signal ACTTTGATTGTAATTTTTTTTT FFTCTLS SS
ACTGTTGGCTTTCATCCAGC
SS-024 Secretion ATGGTTCTTACTAAACCTCTTC 58 MVLTKPLQRN 120 signal AAAGAAATGGCAGCATGATG GSMMSFENVK
AGCTTTGAAAATGTGAAAGAA EKSREGGPHA
AAGAGCAGAGAAGGAGGGCC HTPEEELCFVV
CCATGCACACACACCCGAAGA THTPQVQTTL
AGAATTGTGTTTCGTGGTAAC NLFFHIFKVLT
ACACTACCCTCAGGTTCAGAC QPLSLLWG
CACACTCAACCTGTTTTTCCAT
ATATTCAAGGTTCTTACTCAA
CCACTTTCCCTTCTGTGGGGT
SS-025 Secretion ATGGCCACCCCGCCATTCCGG 59 MATPPFRLIRK 121 signal CTGATAAGGAAGATGTTTTCC MFSFKVSRWM
TTCAAGGTGAGCAGATGGATG GLACFRSLAAS
GGGCTTGCCTGCTTCCGGTCC
CTGGCGGCATCC
SS-026 Secretion ATGAGCTTTTTCCAACTCCTG 60 MSFFQLLMKR 122 signal ATGAAAAGGAAGGAACTCAT KELIPLVVFMT
TCCCTTGGTGGTGTTCATGAC VAAGGASS
TGTGGCGGCGGGTGGAGCCTC
ATCT
SS-027 Secretion ATGGTCTCAGCTCTGCGGGGA 61 MVSALRGAPLI 123 signal GCACCCCTGATCAGGGTGCAC RVHS SPVS SPS
TCAAGCCCTGTTTCTTCTCCTT VSGPAALVSCL
CTGTGAGTGGACCACGGAGGC SSQSSALS
TGGTGAGCTGCCTGTCATCCC
AAAGCTCAGCTCTGAGC
SS-028 Secretion ATGATGGGGTCCCCAGTGAGT 62 MMGSPVSHLL 124 signal CATCTGCTGGCCGGCTTCTGT AGFCVWVVLG
GTGTGGGTCGTCTTGGGC
SS-029 Secretion ATGGCAAGCATGGCTGCCGTG 63 MASMAAVLT 125 signal CTCACCTGGGCTCTGGCTCTT WALALLSAFS

CTTTCAGCGTTTTCGGCCACC ATQA
CAGGCA
SS-030 Secretion ATGGTGCTCATGTGGACCAGT 64 MVLMWTSGD 126 signal GGTGACGCCTTCAAGACGGCC AFKTAYFLLK
TACTTCCTGCTGAAGGGTGCC GAPLQFSVCGL
CCTCTGCAGTTCTCCGTGTGC LQVLVDLAILG
GGCCTGCTGCAGGTGCTGGTG QATA
GACCTGGCCATCCTGGGGCAG
GCCTACGCC
SS-031 Secretion ATGGATTTTGTCGCTGGAGCC 65 MDFVAGAIGG 127 signal ATCGGAGGCGTCTGCGGTGTT VCGVAVGYPL
GCTGTGGGCTACCCCCTGGAC DTVKVRIQTEP
ACGGTGAAGGTCAGGATCCA LYTGIWHCVR
GACGGAGCCAAAGTACACAG DTYHRERVWG
GCATCTGGCACTGCGTCCGGG FYRGLSLPVCT
ATACGTATCACCGAGAGCGCG VSLVS S
TGTGGG
GCTTCTACCGGGGCCTCTCGC
TGCCCGTGTGCACGGTGTCCC
TGGTATCTTCC
SS-032 Secretion ATGGAGAAGCCCCTCTTCCCA 66 MEKPLFPLVPL 128 signal TTAGTGCCTTTGCATTGGTTTG HWFGFGYTAL
GCTTTGGCTACACAGCACTGG VVSGGIVGYV
TTGTTTCTGGTGGGATCGTTG KTGSVPSLAA
GCTATGTAAAAACAGGCAGC GLLFGSLA
GTGCCGTCCCTGGCTGCAGGG
CTGCTCTTCGGCAGTCTAGCC
SS-033 Secretion ATGGGTCTGCTCCTTCCCCTG 67 MGLLLPLALCI 129 signal GCACTCTGCATCCTAGTCCTG LVLC
TGC
SS-034 Secretion ATGGGGATCCAGACGAGCCCC 68 MGIQTSPVLLA 130 signal GTCCTGCTGGCCTCCCTGGGG SLGVGLVTLL
GTGGGGCTGGTCACTCTGCTC GLAVG
GGCCTGGCTGTGGGC
SS-035 Secretion ATGTCGGACCTGCTACTACTG 69 MSDLLLLGLIG 131 signal GGCCTGATTGGGGGCCTGACT GLTLLLLLTLL
CTCTTACTGCTGCTGACGCTG AFA
CTAGCCTTTGCC
SS-036 Secretion ATGGAGACTGTGGTGATTGTT 70 METVVIVAIGV 132 signal GCCATAGGTGTGCTGGCCACC LATIFLAS FAA
ATGTTTCTGGCTTCGTTTGCAG LVLVCRQ
CCTTGGTGCTGGTTTGCAGGC
AG
SS-037 Secretion ATGCGCGGCTCTGTGGAGTGC 71 MAGSVECTWG 133 signal ACCTGGGGTTGGGGGCACTGT WGHCAPSPLL
GCCCCCAGCCCCCTGCTCCTT LWTLLLFAAPF
TGGACTCTACTTCTGTTTGCA GLLG
GCCCCATTTGGCCTGCTGGGG
SS-038 Secretion ATGATGCCGTCCCGTACCAAC 72 MMPSRTNLAT 134 signal CTGGCTACTGGAATCCCCAGT GIPS SKVKYSR

AGTAAAGTGAAATATTCAAGG LSSTDDGYIDL
CTCTCCAGCACAGACGATGGC QFKKTPPKIPY
TACATTGACCTTCAGTTTAAG KAIALATVLFL
AAAACCCCTCCTAAGATCCCT IGA
TATAAGGCCATCGCACTTGCC
ACTGTGCTGTTTTTGATTGGC
GCC
SS-039 Secretion ATGGCCCTGCCCCAGATGTGT 73 MALPQMCDGS 135 signal GACGGGAGCCACTTGGCCTCC HLASTLRYCM
ACCCTCCGCTATTGCATGACA TVSGTVVLVA
GTCAGCGGCACAGTGGTTCTG GTLCFA
GTGGCCGGGACGCTCTGCTTC
GCT
SS-041 Vrg-6 TGAAAAAGTGGTTCGTTGCTG 74 CCGGCATCGGCGCTGCCGGAC GAGLLML S SA
TCATGCTCTCCAGCGCCGCCA A
SS-042 PhoA ATGAAACAGAGCACCATTGCG 75 CTGGCGCTGCTGCCGCTGCTG PLLFTPVTKA
TTTACCCCGGTGACCAAAGCG
SS-043 OmpA ATGAAAAAAACCGCGATTGC 76 MKKTAIAIAV 138 GATTGCGGTGGCGCTGGCGGG ALAGFATVAQ
CTTTGCGACCGTGGCGCAGGC A
G

ATTTTTTTTAGCGTGCTGAGCT VLSFPSFSQS
TTCCGAGCTTTAGCCAGAGC

CTGCTGGCGAGCATGTTTGTG MFVFSIATNAY
TTTAGCATTGCGACCAACGCG A
TATGCG
SS-046 Amylase ATGTTTGCGAAACGCTTTAAA 79 MFAKRFKTSL 141 ACCAGCCTGCTGCCGCTGTTT LPLFAGFLLLF
GCGGGCTTTCTGCTGCTGTTTC HLVLAGPAAA
ATCTGGTGCTGGCGGGCCCGG S
CGGCGGCGAGC
SS-047 Alpha ATGCGCTTTCCGAGCATTTTT 80 Factor ACCGCGGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-048 Alpha ATGCGCTTTCCGAGCATTTTT 81 Factor ACCACCGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-049 Alpha ATGCGCTTTCCGAGCATTTTT 82 Factor ACCAGCGTGCTGTTTGCGGCG AAS SALA
AGCAGCGCGCTGGCG
SS-050 Alpha ATGCGCTTTCCGAGCATTTTT 83 Factor ACCCATGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-051 Alpha ATGCGCTTTCCGAGCATTTTT 84 Factor ACCATTGTGCTGTTTGCGGCG AAS SALA
AGCAGCGCGCTGGCG

SS-052 Alpha ATGCGCTTTCCGAGCATTTTT 85 MRFPSIFTFVLF 147 Factor ACCTTTGTGCTGTTTGCGGCG AASSALA
AGCAGCGCGCTGGCG
SS-053 Alpha ATGCGCTTTCCGAGCATTTTT 86 MRFPSIFTEVL 148 Factor ACCGAAGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-054 Alpha ATGCGCTTTCCGAGCATTTTT 87 MRFPSIFTGVL 149 Factor ACCGGCGTGCTGTTTGCGGCG FAAS SALA
AGCAGCGCGCTGGCG
SS-055 Endoglucan ATGCGTTCCTCCCCCCTCCTCC 88 MRSSPLLRSAV 150 ase V GCTCCGCCGTTGTGGCCGCCC VAALPVLALA
TGCCGGTGTTGGCCCTTGCC
SS-056 Secretion ATGGGCGCGGCGGCCGTGCGC 89 MGAAAVRWH 151 signal TGGCACTTGTGCGTGCTGCTG LCVLLALGTR
GCCCTGGGCACACGCGGGCG GRL
GCTG
SS-057 Fungal ATGAGGAGCTCCCTTGTGCTG 90 MRSSLVLFFVS 152 TTCTTTGTCTCTGCGTGGACG AWTALA
GCCTTGGCCAG
SS-058 Fibronectin ATGCTCAGGGGTCCGGGACCC 91 MLRGPGPGRL 153 GGGCGGCTGCTGCTGCTAGCA LLLAVLCLGTS
GTCCTGTGCCTGGGGACATCG VRCTETGKSK
GTGCGCTGCACCGAAACCGGG R
AAGAGCAAGAGG
SS-059 Fibronectin ATGCTTAGGGGTCCGGGGCCC 92 MLRGPGPGLL 154 GGGCTGCTGCTGCTGGCCGTC LLAVQCLGTA
CAGCTGGGGACAGCGGTGCCC VPSTGA
TCCACG
SS-060 Fibronectin ATGCGCCGGGGGGCCCTGACC 93 MRRGALTGLL 155 GGGCTGCTCCTGGTCCTGTGC LVLCLSVVLR
CTGAGTGTTGTGCTACGTGCA AAPSATSKKR
GCCCCCTCTGCAACAAGCAAG R
AAGCGCAGG

[000285] In the table, SS is secretion signal and MLS is mitochondrial leader signal.
The primary constructs or mmRNA of the present invention may be designed to encode any of the signal sequences of SEQ ID NOs 94-155, or fragments or variants thereof These sequences may be included at the beginning of the polypeptide coding region, in the middle or at the terminus or alternatively into a flanking region.
Further, any of the polynucleotide primary constructs of the present invention may also comprise one or more of the sequences defined by SEQ ID NOs 32-93. These may be in the first region or either flanking region.

[000286] Additional signal sequences which may be utilized in the present invention include those taught in, for example, databases such as those found at http://www.signalpeptide.de/ or http://proline.bic.nus.edu.sg/spdb/. Those described in US Patents 8,124,379; 7,413,875 and 7,385,034 are also within the scope of the invention and the contents of each are incorporated herein by reference in their entirety.
Target Selection [000287] According to the present invention, the primary constructs comprise at least a first region of linked nucleosides encoding at least one polypeptide of interest. The polypeptides of interest or "Targets" of the present invention are listed in Table 6. Shown in Table 6, in addition to the name and description of the gene encoding the polypeptide of interest are the ENSEMBL Transcript ID (ENST), the ENSEMBL Protein ID
(ENSP) and when available the optimized transcript sequence ID (Optim Trans SEQ ID) or optimized open reading frame sequence ID (Optim ORF SEQ ID). For any particular gene there may exist one or more variants or isoforms. Where these exist, they are shown in the table as well. It will be appreciated by those of skill in the art that disclosed in the Table are potential flanking regions. These are encoded in each ENST
transcript either to the 5' (upstream) or 3' (downstream) of the ORF or coding region. The coding region is definitively and specifically disclosed by teaching the ENSP sequence.
Consequently, the sequences taught flanking that encoding the protein are considered flanking regions.
It is also possible to further characterize the 5' and 3' flanking regions by utilizing one or more available databases or algorithms. Databases have annotated the features contained in the flanking regions of the ENST transcripts and these are available in the art.
Table 6. Targets Target Target ENST Trans ENSP Peptide Optim ORF Optim No. Description SEQ SEQ ID SEQ ID Trans ID NO NO SEQ ID
NO
1 4- 289004 156 289004 769 1673, 2293, hydroxyphenylpyr 2913, 3533, uvate dioxygenase 4153,5433-2 4- 543163 157 441677 770 1674,2294, hydroxyphenylpyr 2914, 3534, uvate dioxygenase 4154 3 4- 545969 158 437419 771 1675,2295, hydroxyphenylpyr 2915, 3535, uvate dioxygenase 4155 4 6- 280362 159 280362 772 1676,2296, pyruvoyltetrahydr 2916, 3536, opterin synthase 4156, 4773, 5103,5520-A disintegrin and 563979 160 456186 773 1677, 2297, metalloproteinase 2917, 3537, with 4157, 4774, thrombospondin 5104, 5607-motifs 13 isoform 5693 1 preproprotein 6 activity-dependent 349014 161 342905 774 1678, 2298, neuroprotector 2918, 3538, homeobox 4158, 4775, 5105, 5694-7 activity-dependent 371602 162 360662 775 1679, 2299, neuroprotector 2919, 3539, homeobox 4159, 4776, 8 activity-dependent 396029 163 379346 776 1680, 2300, neuroprotector 2920, 3540, homeobox 4160, 4777, 9 activity-dependent 396032 164 379349 777 1681, 2301, neuroprotector 2921, 3541, homeobox 4161, 4778, activity-dependent 534467 165 436181 778 1682, 2302, neuroprotector 2922, 3542, homeobox 4162, 4779, 11 ADAM 338351 166 345120 779 1683,2303, metallopeptidase 2923, 3543, with 4163, 4780, thrombospondin 5110 type 1 motif, 13 12 ADAM 355699 167 347927 780 1393, 1684, metallopeptidase 2304, 2924, with 3544, 4164, thrombospondin 4781, 5111 type 1 motif, 13 13 ADAM 356589 168 348997 781 1394, 1685, metallopeptidase 2305, 2925, with 3545, 4165, thrombospondin 4782, 5112 type 1 motif, 13 14 ADAM 371910 169 360978 782 1395, 1686, metallopeptidase 2306, 2926, with 3546, 4166, thrombospondin 4783, 5113 type 1 motif, 13 ADAM 371911 170 360979 783 1396, 1687, metallopeptidase 2307, 2927, with 3547, 4167, thrombospondin 4784, 5114 type 1 motif, 13 16 ADAM 371916 171 360984 784 1397, 1688, metallopeptidase 2308, 2928, with 3548, 4168, thrombospondin 4785, 5115 type 1 motif, 13 17 ADAM 371929 172 360997 785 1398, 1689, 1610 metallopeptidase 2309, 2929, with 3549, 4169, thrombospondin 4786, 5116 type 1 motif, 13 18 ADAM 536611 173 444504 786 1399, 1690, metallopeptidase 2310, 2930, with 3550, 4170, thrombospondin 4787, 5117 type 1 motif, 13 19 AIF sh 20 Albiglutide;
albiglutide 21 aldolase A 563060 174 455800 787 1691,2311, fructose- 2931, 3551, bisphosphate 4171 22 aldolase A 564546 175 455917 788 1692,2312, fructose- 2932, 3552, bisphosphate 4172 23 aldolase A 564595 176 457468 789 1693, 2313, fructose- 2933, 3553, bisphosphate 4173 24 aldolase A, 338110 177 336927 790 1694, 2314, fructose- 2934, 3554, bisphosphate 4174 25 aldolase A, 395240 178 378661 791 1695,2315, fructose- 2935, 3555, bisphosphate 4175 26 aldolase A, 395248 179 378669 792 1696,2316, fructose- 2936, 3556, bisphosphate 4176, 5781-27 aldolase A, 412304 180 400452 793 1697, 2317, fructose- 2937, 3557, bisphosphate 4177 28 ameloblastin 322937 181 313809 794 1698,2318, (enamel matrix 2938, 3558, protein) 4178, 4788, 5118, 5868-29 ameloblastin 449493 182 391234 795 1699,2319, (enamel matrix 2939, 3559, protein) 4179, 4789, 30 ameloblastin 538728 183 445605 796 1700, 2320, (enamel matrix 2940, 3560, protein) 4180, 4790, 31 amelogenin, X- 348912 184 335312 797 1701,2321, linked 2941, 3561, 4181, 4791, 32 amelogenin, X- 380712 185 370088 798 1702,2322, linked 2942, 3562, 4182, 4792, 5122, 5955-33 amelogenin, X- 380714 186 370090 799 1703, 2323, linked 2943, 3563, 4183, 4793, 34 amelogenin, Y- 215479 187 215479 800 1704, 2324, linked 2944, 3564, 35 amelogenin, Y- 383036 188 372505 801 1705,2325, linked 2945, 3565, 36 amelogenin, Y- 383037 189 372506 802 1706, 2326, linked 2946, 3566, 37 amylo-alpha-1, 6- 294724 190 294724 803 1707, 2327, glucosidase, 4- 2947, 3567, alpha- 4187 glucanotransferase 38 amylo-alpha-1, 6- 361302 191 354971 804 1708,2328, glucosidase, 4- 2948, 3568, alpha- 4188 glucanotransferase 39 amylo-alpha-1, 6- 361522 192 354635 805 1709,2329, glucosidase, 4- 2949, 3569, alpha- 4189 glucanotransferase 40 amylo-alpha-1, 6- 361915 193 355106 806 1710,2330, glucosidase, 4- 2950, 3570, alpha- 4190, 6042-glucanotransferase 6128 41 amylo-alpha-1, 6- 370161 194 359180 807 1711,2331, glucosidase, 4- 2951, 3571, alpha- 4191 glucanotransferase 42 amylo-alpha-1, 6- 370163 195 359182 808 1712,2332, glucosidase, 4- 2952, 3572, alpha- 4192 glucanotransferase 43 amylo-alpha-1, 6- 370165 196 359184 809 1713,2333, glucosidase, 4- 2953, 3573, alpha- 4193 glucanotransferase 44 amyloid P 255040 197 255040 810 1714,2334, component, serum 2954, 3574, 4194, 6129-45 angiotensin I 252519 198 252519 811 1715,2335, converting enzyme 2955, 3575, (peptidyl- 4195, 6216-dipeptidase A) 2 6302 46 angiotensin I 427411 199 389326 812 1716, 2336, converting enzyme 2956, 3576, (peptidyl- 4196 dipeptidase A) 2 47 Anti-TNFalpha (TNFRFusion;
Enbrel) 48 AP0A1 Milano; 813 1400, 1717, 1613 apo A- 2337, 2957, I(R173C)Milano , 3577, 4197, ETC-216 4794, 5124 49 AP0A1 Paris; apo 814 1401, 1718, A-I(R151C)Paris 2338, 2958, 3578, 4198, 4795, 5125 50 ApoA-a optimized 1614 mRNA
51 apolipoprotein A-I 236850 200 236850 815 1402, 1719, 1615 2339, 2959, 3579, 4199, 4796, 5126, 52 apolipoprotein A-I 359492 201 352471 816 1720, 2340, 1615 2960, 3580, 4200, 4797, 53 apolipoprotein A-I 375320 202 364469 817 1721, 2341, 1615 2961, 3581, 4201, 4798, 54 apolipoprotein A-I 375323 203 364472 818 1722, 2342, 1615 2962, 3582, 4202, 4799, 55 apolipoprotein B 233242 204 233242 819 1723, 2343, (including Ag(x) 2963, 3583, antigen) 4203 56 apolipoprotein B 535079 205 439731 820 1724,2344, (including Ag(x) 2964, 3584, antigen) 4204 57 aquaporin 5 293599 206 293599 821 1725,2345, 1616 2965,3585, 4205,4800, 5130,6564-58 arginase, liver 356962 207 349446 822 1726,2346, 2966,3586, 4206, 59 arginase, liver 368087 208 357066 823 1727,2347, 2967,3587, 4207,6651-60 arginase, liver 476845 209 417694 824 1728, 2348, 2968, 3588, 61 argininosuccinate 304874 210 307188 825 1729, 2349, lyase 2969, 3589, 4209, 6738-62 argininosuccinate 380839 211 370219 826 1730, 2350, lyase 2970, 3590, 63 argininosuccinate 395331 212 378740 827 1731, 2351, lyase 2971, 3591, 64 argininosuccinate 395332 213 378741 828 1732, 2352, lyase 2972, 3592, 65 argininosuccinate 502022 214 441778 829 1733, 2353, lyase 2973, 3593, 66 argininosuccinate 334909 215 361470 830 1403, 1734, synthase 1 2354, 2974, 3594, 4214 67 argininosuccinate 352480 216 253004 831 1735, 2355, synthase 1 2975, 3595, 4215, 6877-68 argininosuccinate 372386 217 361461 832 1404, 1736, synthase 1 2356, 2976, 3596, 4216 69 argininosuccinate 372393 218 361469 833 1737, 2357, synthase 1 2977, 3597, 70 argininosuccinate 372394 219 361471 834 1738, 2358, synthase 1 2978, 3598, 71 argininosuccinate 422569 220 394212 835 1405, 1739, synthase 1 2359, 2979, 3599, 4219 72 argininosuccinate 443588 221 397785 836 1406, 1740, synthase 1 2360, 2980, 3600, 4220 73 artemin 372354 222 361429 837 1741, 2361, 2981, 3601, 4221, 4801, 5131, 6964-74 artemin 372359 223 361434 838 1742, 2362, 2982, 3602, 4222, 4802, 75 artemin 414809 224 387435 839 1743, 2363, 2983, 3603, 4223, 4803, 76 artemin 471394 225 435804 840 1744, 2364, 2984, 3604, 4224, 4804, 77 artemin 474592 226 434856 841 1745, 2365, 2985, 3605, 4225, 4805, 78 artemin 477048 227 434784 842 1746, 2366, 2986, 3606, 4226, 4806, 79 artemin 491846 228 436149 843 1747, 2367, 2987, 3607, 4227, 4807, 80 artemin 498139 229 436727 844 1748, 2368, 2988, 3608, 4228, 4808, 81 arylsulfatase B 264914 230 264914 845 1749,2369, 2989,3609, 4229,4809, 5139,7051-82 arylsulfatase B 264914 231 264914 846 1750, 2370, 2990, 3610, 4230, 4810, 83 arylsulfatase B 396151 232 379455 847 1751,2371, 2991,3611, 4231,4811, 84 arylsulfatase B 521117 233 428611 848 1752,2372, 2992,3612, 4232,4812, 85 asparaginase 299234 234 299234 849 1753, 2373, homolog (S. 2993, 3613, cerevisiae) 4233, 4813, 86 asparaginase 455920 235 389003 850 1754, 2374, homolog (S. 2994, 3614, cerevisiae) 4234, 4814, 87 asparaginase 550583 236 446856 851 1755, 2375, homolog (S. 2995, 3615, cerevisiae) 4235, 4815, 88 asparaginase 551177 237 450040 852 1756, 2376, homolog (S. 2996, 3616, cerevisiae) 4236, 4816, 89 aspartoacylase 263080 238 263080 853 1757, 2377, 2997, 3617, 90 aspartoacylase 456349 239 409976 854 1758, 2378, 2998, 3618, 4238, 7138-91 ATPase, 283684 240 283684 855 1407, 1759, aminophospholipi 2379, 2999, d transporter, class 3619, 4239, I, type 8B, 7225-7398 member 1 92 ATPase, 536015 241 445359 856 1760, 2380, aminophospholipi 3000, 3620, d transporter, class 4240 I, type 8B, member 1 93 ATPase, Cu++ 242839 242 242839 857 1761,2381, transporting, beta 3001, 3621, polypeptide 4241, 4817, 5147, 7399-94 ATPase, Cu++ 344297 243 342559 858 1762,2382, transporting, beta 3002, 3622, polypeptide 4242, 4818, 95 ATPase, Cu++ 400366 244 383217 859 1763,2383, transporting, beta 3003, 3623, polypeptide 4243, 4819, 96 ATPase, Cu++ 400370 245 383221 860 1764,2384, transporting, beta 3004, 3624, polypeptide 4244, 4820, 97 ATPase, Cu++ 417240 246 390360 861 1765,2385, transporting, beta 3005, 3625, polypeptide 4245, 4821, 98 ATPase, Cu++ 418097 247 393343 862 1766,2386, transporting, beta 3006, 3626, polypeptide 4246, 4822, 99 ATPase, Cu++ 448424 248 416738 863 1767,2387, transporting, beta 3007, 3627, polypeptide 4247, 4823, 100 ATPase, Cu++ 542656 249 443128 864 1768, 2388, transporting, beta 3008, 3628, polypeptide 4248, 4824, 101 ATP-binding 263817 250 263817 865 1408, 1769, cassette, sub- 2389, 3009, family B 3629, 4249 (MDR/TAP), member 11 102 ATP-binding 265723 251 265723 866 1409, 1770, cassette, sub- 2390, 3010, family B 3630, 4250, (MDR/TAP), 7486-7659 member 4 103 ATP-binding 358400 252 351172 867 1410, 1771, cassette, sub- 2391, 3011, family B 3631, 4251 (MDR/TAP), member 4 104 ATP-binding 359206 253 352135 868 1411, 1772, cassette, sub- 2392, 3012, family B 3632, 4252 (MDR/TAP), member 4 105 ATP-binding 417608 254 394511 869 1412, 1773, cassette, sub- 2393, 3013, family B 3633, 4253 (MDR/TAP), member 4 106 ATP-binding 453593 255 392983 870 1774, 2394, cassette, sub- 3014, 3634, family B 4254 (MDR/TAP), member 4 107 ATP-binding 545634 256 437465 871 1775, 2395, cassette, sub- 3015, 3635, family B 4255 (MDR/TAP), member 4 108 bactericidal/perme 262865 257 262865 872 1776, 2396, ability-increasing 3016, 3636, protein 4256, 7660-109 basic helix-loop- 242728 258 242728 873 1413, 1777, helix family, 2397, 3017, member e41 3637, 4257, 110 basic helix-loop- 540731 259 437369 874 1778, 2398, helix family, 3018, 3638, member e41 4258 111 bone 378827 260 368104 875 1414, 1779, morphogenetic 2399, 3019, protein 2 3639, 4259, 112 bone 395863 261 379204 876 1415, 1780, morphogenetic 2400, 3020, protein 7 3640, 4260, 4825, 5155, 113 bone 395864 262 379205 877 1416, 1781, morphogenetic 2401, 3021, protein 7 3641, 4261, 4826, 5156 114 Cap18 115 carbamoyl- 233072 263 233072 878 1417,1782, phosphate 2402,3022, synthase 1, 3642,4262 mitochondrial 116 carbamoyl- 417946 264 388496 879 1418, 1783, phosphate 2403, 3023, synthase 1, 3643, 4263 mitochondrial 117 carbamoyl- 430249 265 402608 880 1419, 1784, phosphate 2404, 3024, synthase 1, 3644, 4264, mitochondrial 8008-8146 118 carbamoyl- 451903 266 406136 881 1420, 1785, phosphate 2405, 3025, synthase 1, 3645, 4265 mitochondrial 119 carbamoyl- 518043 267 430697 882 1786, 2406, phosphate 3026, 3646, synthase 1, 4266, mitochondrial 120 carbamoyl- 536125 268 445539 883 1787,2407, phosphate 3027,3647, synthase 1, 4267 mitochondrial 121 carbamoyl- 539150 269 444139 884 1788, 2408, phosphate 3028, 3648, synthase 1, 4268 mitochondrial 122 carbamoyl- 544169 270 442790 885 1789,2409, phosphate 3029,3649, synthase 1, 4269 mitochondrial 123 cardiotrophin 1 279804 271 279804 886 1790, 2410, 3030, 3650, 4270, 8147-124 cardiotrophin 1 395019 272 378465 887 1791,2411, 3031,3651, 125 cathelicidin 296435 273 296435 888 1792,2412, antimicrobial 3032, 3652, peptide 4272, 4827, 5157, 8234-126 cathelicidin 576243 274 458149 889 1793,2413, antimicrobial 3033, 3653, peptide 4273, 4828, 127 Cbp/p300- 372638 275 361721 890 1421, 1794, interacting 2414, 3034, transactivator, 3654, 4274, with Glu/Asp-rich 8408-8494 carboxy-terminal domain, 4 128 ceruloplasmin 264613 276 264613 891 1422, 1795, (ferroxidase) 2415, 3035, 3655, 4275, 4829, 5159, 129 Cheetah Program 2; hyaluronidase (human); insulin lispro 130 ciliary 361987 277 355370 892 1796, 2416, neurotrophic 3036, 3656, factor 4276 131 coagulation factor 311907 278 308541 893 1423, 1797, II (thrombin) 2417, 3037, 3657, 4277, 4830, 5160, 132 coagulation factor 446804 279 406403 894 1798, 2418, II (thrombin) 3038, 3658, 4278, 4831, 133 coagulation factor 530231 280 433907 895 1424, 1799, II (thrombin) 2419, 3039, 3659, 4279, 4832, 5162 134 coagulation factor 334047 281 334145 896 1425, 1800, III 2420, 3040, (thromboplastin, 3660, 4280, tissue factor) 8669-8755 135 coagulation factor 370207 282 359226 897 1426, 1801, III 2421, 3041, (thromboplastin, 3661, 4281, tissue factor) 8756-8842 136 coagulation factor 218099 283 218099 898 1427, 1802, 1621 IX 2422, 3042, 3662, 4282, 137 coagulation factor 394090 284 377650 899 1428, 1803, 1621 IX 2423, 3043, 3663, 4283, 4833, 5163 138 coagulation factor 367796 285 356770 900 1429, 1804, V (proaccelerin, 2424, 3044, labile factor) 3664, 4284 139 coagulation factor 367797 286 356771 901 1430, 1805, V (proaccelerin, 2425, 3045, labile factor) 3665, 4285, 140 coagulation factor 546081 287 439664 902 1431, 1806, V (proaccelerin, 2426, 3046, labile factor) 3666, 4286 141 coagulation factor 346342 288 329546 903 1432, 1807, 1622 VII (serum 2427, 3047, prothrombin 3667, 4287, conversion 4834, 5164 accelerator) 142 coagulation factor 375581 289 364731 904 1433, 1808, 1622 VII (serum 2428, 3048, prothrombin 3668, 4288, conversion 4835, 5165, accelerator) 9156-9242 143 coagulation factor 541084 290 442051 905 1434, 1809, 1622 VII (serum 2429, 3049, prothrombin 3669, 4289, conversion 4836, 5166 accelerator) 144 coagulation factor 330287 291 327895 906 1810, 2430, VIII procoagulant 3050, 3670, component 4290, 4837, 145 coagulation factor 360256 292 353393 907 1435, 1811, 1623 VIII, procoagulant 2431, 3051, component 3671, 4291, 4838, 5168 146 coagulation factor 375551 293 364701 908 1436, 1812, X 2432, 3052, 3672, 4292, 4839, 5169 147 coagulation factor 375559 294 364709 909 1437, 1813, X 2433, 3053, 3673, 4293, 4840, 5170, 148 coagulation factor 409306 295 387092 910 1438, 1814, X 2434, 3054, 3674, 4294, 4841, 5171 149 coagulation factor 264692 296 264692 911 1439, 1815, 1624 XI 2435, 3055, 3675, 4295, 4842, 5172 150 coagulation factor 403665 297 384957 912 1440, 1816, 1624 XI 2436, 3056, 3676, 4296, 4843, 5173, 151 coagulation factor 452239 298 397401 913 1441, 1817, XI 2437, 3057, 3677, 4297, 4844, 5174, 152 coagulation factor 264870 299 264870 914 1442, 1818, XIII, Al 2438, 3058, polypeptide 3678, 4298, 153 coagulation factor 414279 300 413334 915 1443, 1819, XIII, Al 2439, 3059, polypeptide 3679, 4299 154 coagulation factor 441301 301 416127 916 1820, 2440, XIII, Al 3060, 3680, polypeptide 4300 155 collagen, type VII, 328333 302 332371 917 1821,2441, 1625 alpha 1 3061,3681, 4301, 4845, 5175, 9556-156 collagen, type VII, 454817 303 412569 918 1822, 2442, alpha 1 3062, 3682, 4302, 4846, 5176, 157 colony stimulating 296871 304 296871 919 1823, 2443, 1626 factor 2 3063, 3683, (granulocyte- 4303, 4847, macrophage) 5177, 9643-158 colony stimulating 225474 305 225474 920 1824, 2444, factor 3 3064, 3684, (granulocyte) 4304, 9782-159 colony stimulating 331769 306 327766 921 1825, 2445, factor 3 3065, 3685, (granulocyte) 4305, 4848, 5178, 9869-160 colony stimulating 394148 307 377704 922 1826, 2446, 1627 factor 3 3066, 3686, (granulocyte) 4306, 4849, 5179, 161 colony stimulating 394149 308 377705 923 1827, 2447, 1627 factor 3 3067, 3687, (granulocyte) 4307, 4850, 5180, 9956-162 complement factor 367429 309 356399 924 1444, 1828, H 2448, 3068, 3688, 4308, 4851, 5181 163 complement factor 391986 310 375846 925 1829, 2449, H 3069, 3689, 4309, 4852, 164 complement factor 439155 311 402656 926 1445, 1830, H 2450, 3070, 3690, 4310, 4853, 5183 165 copper metabolism 311832 312 308236 927 1446, 1831, (Murrl) domain 2451, 3071, containing 1 3691, 4311, 166 copper metabolism 427417 313 413207 928 1447, 1832, (Murrl) domain 2452, 3072, containing 1 3692, 4312 167 copper metabolism 444166 314 410050 929 1448, 1833, (Murrl) domain 2453, 3073, containing 1 3693, 4313 168 copper metabolism 458337 315 401236 930 1834, 2454, (Murrl) domain 3074, 3694, containing 1 4314 169 copper metabolism 538736 316 438961 931 1449, 1835, (Murrl) domain 2455, 3075, containing 1 3695, 4315 170 corticotropin 276571 317 276571 932 1836, 2456, releasing hormone 3076, 3696, 4316, 4854, 171 CTLA4-Ig (Orencia) 172 cystic fibrosis 3084 318 3084 933 1450, 1837, 1628 transmembrane 2457, 3077, conductance 3697, 4317, regulator (ATP- 4855, 5185, binding cassette 10130-10216 sub-family C, member 7) 173 cystic fibrosis 426809 319 389119 934 1451, 1838, transmembrane 2458, 3078, conductance 3698, 4318, regulator (ATP- 4856, 5186 binding cassette sub-family C, member 7) 174 cystic fibrosis 454343 320 403677 935 1452, 1839, transmembrane 2459, 3079, conductance 3699, 4319, regulator (ATP- 4857, 5187 binding cassette sub-family C, member 7) 175 cystic fibrosis 468795 321 419254 936 1453, 1840, transmembrane 2460, 3080, conductance 3700, 4320, regulator (ATP- 4858, 5188 binding cassette sub-family C, member 7) 176 cystinosin 381870 322 371294 937 1841,2461, lysosomal cystine 3081, 3701, transporter 4321 177 cystinosin, 46640 323 46640 938 1454, 1842, lysosomal cystine 2462, 3082, transporter 3702, 4322, 4859, 5189, 178 cystinosin, 414524 324 395471 939 1455, 1843, lysosomal cystine 2463, 3083, transporter 3703, 4323 179 cystinosin, 441220 325 411465 940 1456, 1844, lysosomal cystine 2464, 3084, transporter 3704, 4324 180 cystinosin, 452111 326 408652 941 1457, 1845, lysosomal cystine 2465, 3085, transporter 3705, 4325 181 defensin, beta 314357 327 320951 942 1846,2466, 103A 3086,3706, 4326,4860, 182 defensin, beta 318124 328 324633 943 1847,2467, 103B 3087,3707, 4327,4861, 5191,10356-183 defensin, beta 4A 302247 329 303532 944 1848, 2468, 1631 3088, 3708, 4328, 4862, 5192, 10443-184 defensin, beta 4B 318157 330 424598 945 1849, 2469, 1632 3089, 3709, 4329, 4863, 185 DegludecPlus; insulin aspart; insulin degludec 186 deoxyribonuclease 246949 331 246949 946 1850, 2470, 3090, 3710, 4330, 4864, 5194, 10530-187 deoxyribonuclease 407479 332 385905 947 1851, 2471, 1633 3091, 3711, 4331, 4865, 188 deoxyribonuclease 414110 333 416699 948 1852, 2472, 3092, 3712, 4332, 4866, 189 dysferlin, limb 258104 334 258104 949 1458, 1853, girdle muscular 2473, 3093, dystrophy 2B 3713, 4333, (autosomal 4867, 5197 recessive) 190 dysferlin, limb 394120 335 377678 950 1459, 1854, girdle muscular 2474, 3094, dystrophy 2B 3714, 4334, (autosomal 4868, 5198, recessive) 10617-10703 191 dysferlin, limb 409366 336 386512 951 1460, 1855, girdle muscular 2475, 3095, dystrophy 2B 3715,4335, (autosomal 4869, 5199 recessive) 192 dysferlin, limb 409582 337 386547 952 1461, 1856, girdle muscular 2476, 3096, dystrophy 2B 3716, 4336, (autosomal 4870, 5200 recessive) 193 dysferlin, limb 409651 338 386683 953 1462, 1857, girdle muscular 2477, 3097, dystrophy 2B 3717, 4337, (autosomal 4871, 5201 recessive) 194 dysferlin, limb 409744 339 386285 954 1463, 1858, girdle muscular 2478, 3098, dystrophy 2B 3718, 4338, (autosomal 4872, 5202 recessive) 195 dysferlin, limb 409762 340 387137 955 1464, 1859, girdle muscular 2479, 3099, dystrophy 2B 3719, 4339, (autosomal 4873, 5203 recessive) 196 dysferlin, limb 410020 341 386881 956 1465,1860, girdle muscular 2480, 3100, dystrophy 2B 3720, 4340, (autosomal 4874, 5204 recessive) 197 dysferlin, limb 410041 342 386617 957 1466,1861, girdle muscular 2481, 3101, dystrophy 2B 3721, 4341, (autosomal 4875, 5205 recessive) 198 dysferlin, limb 413539 343 407046 958 1467, 1862, girdle muscular 2482, 3102, dystrophy 2B 3722, 4342, (autosomal 4876, 5206 recessive) 199 dysferlin, limb 429174 344 398305 959 1468, 1863, girdle muscular 2483, 3103, dystrophy 2B 3723, 4343, (autosomal 4877, 5207 recessive) 200 ectodysplasin A 338901 345 340611 960 1864, 2484, 3104, 3724, 4344, 4878, 201 ectodysplasin A 374552 346 363680 961 1865, 2485, 3105, 3725, 4345, 4879, 202 ectodysplasin A 374553 347 363681 962 1866, 2486, 3106, 3726, 4346, 4880, 5210, 10704-203 ectodysplasin A 525810 348 434195 963 1867, 2487, 3107, 3727, 4347, 4881, 204 ectodysplasin A 524573 349 432585 964 1868, 2488, 3108, 3728, 4348, 4882, 205 ectodysplasin A 527388 350 434861 965 1869, 2489, 3109, 3729, 4349, 4883, 206 enamelin 396073 351 379383 966 1870, 2490, 3110, 3730, 4350, 4884, 207 erythropoietin 252723 352 252723 967 1871,2491, 3111,3731, 4351,4885, 5215,10791-208 Exenafide 968 1872,2492, 3112,3732, 4352,4886, 209 family with 252530 353 252530 969 1873,2493, sequence 3113,3733, similarity 98, 4353 member C
210 family with 343358 354 340348 970 1874,2494, sequence 3114,3734, similarity 98, 4354 member C
211 fibrinogen alpha 302053 355 306361 971 1469, 1875, chain 2495,3115, 3735, 4355, 212 fibrinogen alpha 403106 356 385981 972 1470, 1876, chain 2496,3116, 3736, 4356 213 fibrinogen alpha 457487 357 407891 973 1877, 2497, chain 3117, 3737, 214 fibrinogen beta 302068 358 306099 974 1471,1878, chain 2498,3118, 3738,4358, 215 fibrinogen beta 537843 359 437727 975 1879, 2499, chain 3119, 3739, 216 fibrinogen gamma 336098 360 336829 976 1472, 1880, chain 2500,3120, 3740, 4360, 217 fibrinogen gamma 404648 361 384860 977 1473, 1881, chain 2501,3121, 3741, 4361 218 fibroblast growth 222157 362 222157 978 1474, 1882, factor 21 2502, 3122, 3742, 4362 219 fibroblast growth 593756 363 471477 979 1883,2503, factor 21 3123,3743, 4363, 11469-220 fibronectin type III 373470 364 362569 980 1884, 2504, domain containing 3124, 3744, 4364, 221 fibronectin type III 373471 365 362570 981 1885,2505, domain containing 3125, 3745, 5 4365, 11556-222 follicle stimulating 254122 366 254122 982 1886, 2506, hormone, beta 3126, 3746, polypeptide 4366, 4887, 5217, 11643-223 follicle stimulating 417547 367 416606 983 1887, 2507, hormone, beta 3127, 3747, polypeptide 4367, 4888, 224 follicle stimulating 533718 368 433424 984 1888,2508, hormone, beta 3128, 3748, polypeptide 4368, 4889, 225 fumarylacetoacetat 261755 369 261755 985 1475, 1889, e hydrolase 2509, 3129, (fumarylacetoaceta 3749, 4369 se) 226 fumarylacetoacetat 407106 370 385080 986 1890, 2510, e hydrolase 3130, 3750, (fumarylacetoaceta 4370, 11730-se) 11868 227 fumarylacetoacetat 561421 371 453347 987 1891, 2511, e hydrolase 3131,3751, (fumarylacetoaceta 4371 se) 228 galactokinase 1 225614 372 225614 988 1892,2512, 3132,3752, 229 galactokinase 1 375188 373 364334 989 1893,2513, 3133,3753, 230 galactokinase 1 437911 374 406305 990 1894,2514, 3134,3754, 231 galactokinase 1 588479 375 465930 991 1895, 2515, 3135, 3755, 4375, 11869-232 galactosamine (N- 268695 376 268695 992 1476, 1896, 1638 acetyl)-6-sulfate 2516, 3136, sulfatase 3756, 4376, 4890, 5220, 233 galactosamine (N- 439266 377 402127 993 1897, 2517, acetyl)-6-sulfate 3137, 3757, sulfatase 4377, 4891, 234 galactosamine (N- 542788 378 438197 994 1477, 1898, acetyl)-6-sulfate 2518, 3138, sulfatase 3758, 4378, 4892, 5222 235 galactose-1- 378842 379 368119 995 1899,2519, phosphate 3139,3759, uridylyltransferase 4379, 12043-236 galactose-1- 450095 380 401956 996 1900, 2520, phosphate 3140, 3760, uridylyltransferase 4380 237 galactose-1- 554550 381 451435 997 1901,2521, phosphate 3141,3761, uridylyltransferase 4381 238 galactosidase, 218516 382 218516 998 1902, 2522, 1639 alpha 3142, 3762, 4382, 12182-239 Galectin 3 383 999 1640 inhibitor (A9) 240 Galectin 3 384 1000 1641 inhibitor (C12) 241 glial cell derived 326524 385 317145 1001 1903, 2523, neurotrophic 3143, 3763, factor 4383, 4893, 242 glial cell derived 344622 386 339703 1002 1904, 2524, neurotrophic 3144, 3764, factor 4384, 4894, 243 glial cell derived 427982 387 409007 1003 1905, 2525, 1642 neurotrophic 3145, 3765, factor 4385, 4895, 5225, 12269-244 glial cell derived 502572 388 423557 1004 1906,2526, neurotrophic 3146, 3766, factor 4386, 4896, 245 glial cell derived 510177 389 424592 1005 1907, 2527, neurotrophic 3147, 3767, factor 4387, 4897, 246 glial cell derived 515058 390 425928 1006 1908, 2528, neurotrophic 3148, 3768, factor 4388, 4898, 247 glial cell derived 381826 391 371248 1007 1909, 2529, neurotrophic 3149, 3769, factor 4389, 4899, 248 GLP-1 Fc;
dulaglutide 249 glucagon 375497 392 364647 1008 1910, 2530, 3150, 3770, 4390, 4900, 250 glucagon 418842 393 387662 1009 1911, 2531, 3151, 3771, 4391, 4901, 5231, 12356-251 glucan (1,4-alpha- 264326 394 264326 1010 1912,2532, ), branching 3152, 3772, enzyme 1 4392 252 glucan (1,4-alpha- 429644 395 410833 1011 1478, 1913, ), branching 2533, 3153, enzyme 1 3773, 4393, 253 glucan (1,4-alpha- 536832 396 445365 1012 1914,2534, ), branching 3154, 3774, enzyme 1 4394 254 glucose-6- 253801 397 253801 1013 1915, 2535, phosphatase, 3155, 3775, catalytic subunit 4395, 12843-255 glucosidase, alpha; 302262 398 305692 1014 1916, 2536, 1644 acid 3156, 3776, 4396, 4902, 5232, 12982-256 glucosidase, alpha; 390015 399 374665 1015 1917, 2537, 1644 acid 3157, 3777, 4397, 4903, 257 glucosidase, beta, 327247 400 314508 1016 1918,2538, 1645 acid 3158, 3778, 4398, 4904, 258 glucosidase, beta, 368373 401 357357 1017 1919,2539, acid 3159, 3779, 4399, 4905, 259 glucosidase, beta, 402928 402 385813 1018 1920, 2540, 1645 acid 3160, 3780, 4400, 4906, 260 glucosidase, beta, 427500 403 402577 1019 1921,2541, acid 3161, 3781, 4401, 4907, 261 glucosidase, beta, 428024 404 397986 1020 1922, 2542, 1645 acid 3162, 3782, 4402, 4908, 262 glucosidase, beta, 536555 405 446457 1021 1923,2543, acid 3163, 3783, 4403, 4909, 263 glucosidase, beta, 536770 406 445560 1022 1924, 2544, 1645 acid 3164, 3784, 4404, 4910, 264 glycogen synthase 261195 407 261195 1023 1925, 2545, 2 (liver) 3165, 3785, 4405, 13156-265 glycoprotein 369582 408 358595 1024 1926,2546, hormones, alpha 3166, 3786, polypeptide 4406, 4911, 5241, 13243-266 GP2013;
rituximab 267 Granulysin 9 kDa 1479 (non-secreted) 268 Granulysin 9 kDa 1480 (secreted) 269 growth hormone 1 323322 409 312673 1025 1927,2547, 3167, 3787, 4407, 4912, 5242, 13330-270 growth hormone 1 342364 410 339278 1026 1928,2548, 3168, 3788, 4408, 4913, 271 growth hormone 1 351388 411 343791 1027 1929,2549, 3169, 3789, 4409, 4914, 272 growth hormone 1 458650 412 408486 1028 1930,2550, 3170, 3790, 4410, 4915, 273 growthhormone 2 332800 413 333157 1029 1931,2551, 3171,3791, 4411,4916, 274 growth hormone 2 423893 414 409294 1030 1932,2552, 3172,3792, 4412,4917, 275 growthhormone 2 449787 415 410618 1031 1933,2553, 3173,3793, 4413,4918, 276 growthhormone 2 456543 416 394122 1032 1934,2554, 3174,3794, 4414,4919, 277 (111) 395514 417 378890 1033 1935,2555, 1648 cyclohydrolase 1 3175,3795, 4415,4920, 5250,13417-278 (111) 395524 418 378895 1034 1936,2556, cyclohydrolase 1 3176,3796, 4416,4921, 279 (111) 491895 419 419045 1035 1937,2557, 1649 cyclohydrolase 1 3177,3797, 4417,4922, 280 (111) 536224 420 445246 1036 1938,2558, cyclohydrolase 1 3178,3798, 4418,4923, 281 (111) 543643 421 444011 1037 1939,2559, cyclohydrolase 1 3179,3799, 4419,4924, 282 hemochromatosis 309234 422 311698 1038 1940,2560, 3180,3800, 283 hemochromatosis 317896 423 313776 1039 1941,2561, 3181,3801, 284 hemochromatosis 336625 424 337819 1040 1942,2562, 3182,3802, 285 hemochromatosis 349999 425 259699 1041 1943,2563, 3183,3803, 286 hemochromatosis 352392 426 315936 1042 1944,2564, 3184,3804, 287 hemochromatosis 353147 427 312342 1043 1945, 2565, 3185, 3805, 288 hemochromatosis 357618 428 417404 1044 1946, 2566, 3186, 3806, 289 hemochromatosis 397022 429 380217 1045 1947, 2567, 3187, 3807, 290 hemochromatosis 461397 430 420802 1046 1948, 2568, 3188, 3808, 291 hemochromatosis 470149 431 419725 1047 1949, 2569, 3189, 3809, 292 hemochromatosis 488199 432 420559 1048 1950, 2570, 3190, 3810, 293 hemochromatosis 535098 433 445872 1049 1951, 2571, 3191, 3811, 294 hemochromatosis 539147 434 445098 1050 1952, 2572, 3192, 3812, 295 hemochromatosis 336751 435 337014 1051 1481, 1953, type 2 (juvenile) 2573, 3193, 3813, 4433, 4925, 5255, 296 hemochromatosis 357836 436 350495 1052 1482, 1954, type 2 (juvenile) 2574, 3194, 3814, 4434, 4926, 5256 297 hemochromatosis 421822 437 411863 1053 1483, 1955, type 2 (juvenile) 2575, 3195, 3815, 4435, 4927, 5257 298 hemochromatosis 475797 438 425716 1054 1484, 1956, type 2 (juvenile) 2576, 3196, 3816, 4436, 4928, 5258 299 hemochromatosis 497365 439 421820 1055 1957, 2577, type 2 (juvenile) 3197, 3817, 4437, 4929, 300 hemochromatosis 577520 440 463276 1056 1958, 2578, type 2 (juvenile) 3198, 3818, 301 hemochromatosis 580693 441 464413 1057 1959, 2579, type 2 (juvenile) 3199, 3819, 302 hepatocyte growth 222390 442 222390 1058 1960, 2580, factor (hepapoietin 3200, 3820, A; scatter factor) 4440, 13591-303 hepatocyte growth 354224 443 346164 1059 1961, 2581, factor (hepapoietin 3201, 3821, A; scatter factor) 4441 304 hepatocyte growth 394769 444 378250 1060 1962, 2582, factor (hepapoietin 3202, 3822, A; scatter factor) 4442 305 hepatocyte growth 412881 445 396307 1061 1963, 2583, factor (hepapoietin 3203, 3823, A; scatter factor) 4443 306 hepatocyte growth 421558 446 388592 1062 1964, 2584, factor (hepapoietin 3204, 3824, A; scatter factor) 4444 307 hepatocyte growth 423064 447 413829 1063 1965, 2585, factor (hepapoietin 3205, 3825, A; scatter factor) 4445 308 hepatocyte growth 444829 448 389854 1064 1966, 2586, factor (hepapoietin 3206, 3826, A; scatter factor) 4446 309 hepatocyte growth 453411 449 408270 1065 1967, 2587, factor (hepapoietin 3207, 3827, A; scatter factor) 4447 310 hepatocyte growth 457544 450 391238 1066 1968, 2588, factor (hepapoietin 3208, 3828, A; scatter factor) 4448 311 hepcidin 222304 451 222304 1067 14851969, antimicrobial 2589, 3209, peptide 3829, 4449 312 hepcidin 598398 452 471894 1068 1970, 2590, antimicrobial 3210, 3830, peptide 4450, 13678-313 Herceptin Heavy 1069 1971, 2591, Chain 3211, 3831, 4451, 4930, 314 Herceptin Light 1070 1972, 2592, Chain 3212, 3832, 4452, 4931, 315 hypoxanthine 298556 453 298556 1071 1486, 1973, 1650 phosphoribosyltra 2593, 3213, nsferase 1 3833, 4453, 4932, 5262, 316 hypoxanthine 370796 454 359832 1072 1974,2594, phosphoribosyltra 3214, 3834, nsferase 1 4454, 4933, 317 IDegLira; insulin degludec;
liraglutide 318 iduronate 2- 340855 455 339801 1073 1975, 2595, 1651 sulfatase 3215, 3835, 4455, 4934, 319 kluronate2- 370441 456 359470 1074 1976,2596, 1651 sulfalase 3216,3836, 4456,4935, 320 kluronate2- 370443 457 359472 1075 1977,2597, 1651 sulfalase 3217,3837, 4457,4936, 321 kluronate2- 428056 458 390241 1076 1978,2598, 1651 sulfalase 3218,3838, 4458,4937, 322 kluronate2- 521702 459 429745 1077 1979,2599, 1651 sulfalase 3219,3839, 4459,4938, 323 kluronate2- 537071 460 440324 1078 1980,2600, 1651 sulfalase 3220,3840, 4460,4939, 324 kluronate2- 541269 461 441261 1079 1981,2601, 1651 sulfalase 3221,3841, 4461,4940, 325 iduronidase, alpha- 247933 462 247933 1080 1982,2602, L, 3222,3842, 4462,4941, 5271,13852-326 iduronidase, alpha- 453894 463 396458 1081 1983,2603, L, 3223,3843, 4463,4942, 327 IgIVI Heavy 328 IgNnight 329 1NS-IGF2 397270 464 380440 1082 1984,2604, readthrough 3224,3844, 4464,4943, 330 insulin 250971 465 250971 1083 1985,2605, 1653 3225,3845, 4465,4944, 5274,13939-331 insulin 381330 466 370731 1084 1986,2606, 1653 3226,3846, 4466,4945, 332 insulin 397262 467 380432 1085 1987,2607, 1653 3227,3847, 4467,4946, 5276,14287-333 insulin 512523 468 424008 1086 1988, 2608, 3228, 3848, 4468, 4947, 334 Insulin Aspart 1087 1989, 2609, 3229, 3849, 4469, 4948, 335 Insulin Glargine 1088 1990,2610, 3230, 3850, 4470, 4949, 336 Insulin Glulisine 1089 1991,2611, 3231, 3851, 4471, 4950, 337 Insulin Lispro 1090 1992, 2612, 3232, 3852, 4472, 4951, 338 interferon, alpha 357374 469 369566 1091 1993,2613, 3233, 3853, 4473, 4952, 339 interferon, alpha 449498 470 394494 1092 1994, 2614, 13 3234, 3854, 4474, 4953, 340 interferon, alpha 380222 471 369571 1093 1995, 2615, 14 3235, 3855, 4475, 4954, 341 interferon, alpha 380216 472 369564 1094 1996, 2616, 16 3236, 3856, 4476, 4955, 342 interferon, alpha 413767 473 411940 1095 1997, 2617, 17 3237, 3857, 4477, 4956, 343 interferon, alpha 2 380206 474 369554 1096 1998, 2618, 1654 3238, 3858, 4478, 4957, 5287, 14374-344 interferon, alpha 380225 475 369574 1097 1999, 2619, 21 3239, 3859, 4479, 4958, 345 interferon, alpha 4 421715 476 412897 1098 2000, 2620, 3240, 3860, 4480, 4959, 346 interferon, alpha 5 259555 477 259555 1099 2001, 2621, 3241, 3861, 4481, 4960, 347 interferon, alpha 6 380210 478 369558 1100 2002, 2622, 3242, 3862, 4482, 4961, 348 interferon, alpha 7 239347 479 239347 1101 2003, 2623, 3243, 3863, 4483, 4962, 349 interferon, alpha 8 380205 480 369553 1102 2004, 2624, 3244, 3864, 4484, 4963, 350 interferon, beta 1, 380232 481 369581 1103 2005,2625, fibroblast 3245, 3865, 4485, 4964, 5294, 14461-351 interleukin 10 423557 482 412237 1104 1487,2006, 2626,3246, 3866,4486, 352 interleukin 21 264497 483 264497 1105 2007,2627, 3247,3867, 4487,14635-353 interleukin 7 263851 484 263851 1106 1488,2008, 2628,3248, 3868,4488, 4965,5295, 354 interleukin 7 379113 485 368408 1107 1489,2009, 2629,3249, 3869,4489, 4966,5296 355 interleukin 7 379114 486 368409 1108 2010,2630, 3250, 3870, 4490, 4967, 356 interleukin 7 518982 487 430272 1109 2011,2631, 3251,3871, 4491,4968, 357 interleukin 7 520215 488 428364 1110 1490,2012, 2632,3252, 3872,4492, 4969,5299 358 interleukin 7 520269 489 427750 1111 1491,2013, 2633,3253, 3873,4493, 4970,5300 359 interleukin 7 520317 490 427800 1112 2014,2634, 3254,3874, 4494,4971, 360 interleukin 7 541183 491 438922 1113 1492, 2015, 2635, 3255, 3875, 4495, 4972, 5302 361 KIT ligand 228280 492 228280 1114 2016, 2636, 3256, 3876, 4496, 14861-362 KIT ligand 347404 493 54216 1115 2017, 2637, 3257, 3877, 363 KIT ligand 537835 494 438889 1116 2018,2638, 3258, 3878, 364 klotho 380099 495 369442 1117 1493, 2019, 2639, 3259, 3879, 4499, 365 klotho 426690 496 399513 1118 14942020, 2640, 3260, 3880, 4500 366 Kruppel-like 374672 497 363804 1119 2021, 2641, factor 4 (gut) 3261,3881, 367 Kruppel-like 411706 498 399921 1120 2022, 2642, factor 4 (gut) 3262, 3882, 368 Kruppel-like 420475 499 404922 1121 2023, 2643, factor 4 (gut) 3263, 3883, 369 Kruppel-like 439281 500 396294 1122 2024, 2644, factor 4 (gut) 3264, 3884, 370 lecithin- 264005 501 264005 1123 1495, 2025, cholesterol 2645, 3265, acyltransferase 3885, 4505, 371 lectin, galactoside- 254301 502 254301 1124 2026, 2646, binding, soluble, 3 3266, 3886, 4506, 15435-372 lin-28 homolog A 254231 503 254231 1125 2027, 2647, (C. elegans) 3267, 3887, 373 lin-28 homolog A 326279 504 363314 1126 2028,2648, (C. elegans) 3268, 3888, 4508, 15522-374 lipase A, 282673 505 282673 1127 1496 , 2029, lysosomal acid, 2649, 3269, cholesterol 3889, 4509, esterase 4973, 5303 375 lipase A, 336233 506 337354 1128 1497,2030, 1657 lysosomal acid, 2650, 3270, cholesterol 3890, 4510, esterase 4974, 5304, 376 lipase A, 354621 507 360895 1129 2031,2651, lysosomal acid, 3271, 3891, cholesterol 4511, 4975, esterase 5305 377 lipase A, 371829 508 360894 1130 2032,2652, 1657 lysosomal acid, 3272, 3892, cholesterol 4512, 4976, esterase 5306 378 lipase A, 371837 509 360903 1131 1498,2033, lysosomal acid, 2653, 3273, cholesterol 3893, 4513, esterase 4977, 5307 379 lipase A, 425287 510 392037 1132 1499,2034, lysosomal acid, 2654, 3274, cholesterol 3894, 4514, esterase 4978, 5308 380 lipase A, 428800 511 388415 1133 1500,2035, 1657 lysosomal acid, 2655, 3275, cholesterol 3895, 4515, esterase 4979, 5309 381 lipase A, 456827 512 413019 1134 2036,2656, 1657 lysosomal acid, 3276, 3896, cholesterol 4516, 4980, esterase 5310 382 lipase A, 540050 513 439727 1135 2037,2657, lysosomal acid, 3277, 3897, cholesterol 4517, 4981, esterase 5311 383 lipase A, 541980 514 438127 1136 2038,2658, 1657 lysosomal acid, 3278, 3898, cholesterol 4518, 4982, esterase 5312 384 lipase A, 542307 515 437564 1137 1501,2039, 1657 lysosomal acid, 2659, 3279, cholesterol 3899, 4519, esterase 4983, 5313 385 lipoprotein lipase 311322 516 309757 1138 1502,2040, 2660, 3280, 3900, 4520, 4984, 5314, 386 lipoprotein lipase 520959 517 428496 1139 1503, 2041, 2661, 3281, 3901, 4521, 4985, 5315 387 lipoprotein lipase 522701 518 428557 1140 1504, 2042, 2662, 3282, 3902, 4522, 4986, 5316 388 lipoprotein lipase 524029 519 428237 1141 1505,2043, 2663, 3283, 3903, 4523, 4987, 5317 389 lipoprotein lipase 535763 520 438606 1142 2044, 2664, 3284, 3904, 4524, 4988, 390 lipoprotein lipase 538071 521 439407 1143 2045, 2665, 3285, 3905, 4525, 4989, 391 liraglutide native 1506 392 liraglutide native 1507 393 liraglutide native 1508 394 liraglutide; GLP1 ; 1509 Victoza 395 LL-37; 1144 1620 Cathelicidin anti-microbial peptide 396 low density 455727 522 397829 1145 1510, 2046, lipoprotein 2666, 3286, receptor 3906, 4526 397 low density 535915 523 440520 1146 1511, 2047, lipoprotein 2667, 3287, receptor 3907, 4527 398 low density 545707 524 437639 1147 1512, 2048, lipoprotein 2668, 3288, receptor 3908, 4528 399 low density 558013 525 453346 1148 1513,2049, lipoprotein 2669, 3289, receptor 3909, 4529 400 low density 558518 526 454071 1149 1514 , 2050, lipoprotein 2670, 3290, receptor 3910, 4530, 401 low density 561343 527 454147 1150 2051,2671, lipoprotein 3291, 3911, receptor 4531 402 low density 252444 528 252444 1151 2052, 2672, lipoprotein 3292, 3912, receptor 4532 403 luteinizing 221421 529 221421 1152 2053, 2673, hormone beta 3293, 3913, polypeptide 4533 404 luteinizing 391870 530 375743 1153 2054, 2674, hormone beta 3294, 3914, polypeptide 4534, 4990, 5320, 405 LY2963016;
insulin glargine 406 Ly6/neurotoxin 1 317543 531 319846 1154 2055,2675, 3295, 3915, 407 MabThera SC;
rituximab 408 mannosidase alpha 221363 532 221363 1155 2056,2676, class 2B member 1 3296, 3916, 409 mannosidase, 433513 533 390583 1156 1515, 2057, alpha, class 2B, 2677, 3297, member 1 3917, 4537 410 mannosidase, 456935 534 395473 1157 1516, 2058, alpha, class 2B, 2678, 3298, member 1 3918, 4538, 411 mannosidase, 536796 535 438266 1158 2059, 2679, alpha, class 2B, 3299, 3919, member 1 4539 412 meteorin glial cell 568223 536 455068 1159 2060, 2680, differentiation 3300, 3920, regulator 4540, 16566-413 meteorin, glial cell 219542 537 219542 1160 2061,2681, differentiation 3301, 3921, regulator 4541 414 methylmalonyl 274813 538 274813 1161 1517,2062, CoAnnAase 2682,3302, 3922,4542, 4991,5321, 415 methylmalonyl 540138 539 445535 1162 2063,2683, CoAnnAase 3303,3923, 4543,4992, 5322, 416 microsomal 265517 540 265517 1163 1518,2064, higlycelide 2684,3304, transfer protein 3924, 4544, 417 microsomal 457717 541 400821 1164 2065,2685, h*glyceride 3305,3925, transfer protein 4545 418 microsomal 506883 542 426755 1165 1519,2066, higlycelide 2686,3306, transfer protein 3926, 4546 419 microsomal 538053 543 437358 1166 2067,2687, triglyceride 3307, 3927, transfer protein 4547 420 N- 299314 544 299314 1167 1520, 2068, acetylglucos amine 2688, 3308, -1-phosphate 3928, 4548, transferase, alpha 16879-17017 and beta subunits 421 N- 549940 545 449150 1168 1521, 2069, acetylglucos amine 2689, 3309, -1-phosphate 3929, 4549 transferase, alpha and beta subunits 422 N-acetylglutamate 293404 546 293404 1169 2070, 2690, synthase 3310, 3930, 4550, 17018-423 N-acetylglutamate 541745 547 441262 1170 2071, 2691, synthase 3311, 3931, 424 N-acylsphingosine 262097 548 262097 1171 1522, 2072, amidohydrolase 2692, 3312, (acid ceramidase) 3932, 425 N-acylsphingosine 314146 549 326970 1172 1523, 2073, amidohydrolase 2693, 3313, (acid ceramidase) 3933, 4553 426 N-acylsphingosine 381733 550 371152 1173 1524 , 2074, amidohydrolase 2694, 3314, (acid ceramidase) 3934, 4554 427 N-acylsphingosine 417108 551 394125 1174 1525 , 2075, amidohydrolase 2695, 3315, (acid ceramidase) 3935, 4555 428 N-acylsphingosine 520781 552 427751 1175 1526, 2076, amidohydrolase 2696, 3316, (acid ceramidase) 3936, 4556 429 natriuretic peptide 376468 553 365651 1176 2077, 2697, B 3317, 3937, 4557, 4993, 5323, 430 nerve growth 369512 554 358525 1177 2078, 2698, factor (beta 3318, 3938, polypeptide) 4558, 4994, 5324, 17296-431 neuregulin 1 287840 555 287840 1178 2079,2699, 3319, 3939, 432 neuregulin 1 287842 556 287842 1179 2080, 2700, 3320, 3940, 4560, 17383-433 neuregulin 1 287845 557 287845 1180 2081,2701, 3321, 3941, 434 neuregulin 1 338921 558 343395 1181 2082,2702, 3322, 3942, 435 neuregulin 1 341377 559 340497 1182 2083,2703, 3323, 3943, 436 neuregulin 1 356819 560 349275 1183 2084,2704, 3324, 3944, 437 neuregulin 1 405005 561 384620 1184 2085, 2705, 3325, 3945, 438 neuregulin 1 519301 562 429582 1185 2086, 2706, 3326, 3946, 439 neuregulin 1 520407 563 434640 1186 2087, 2707, 3327, 3947, 4567, 17470-440 neuregulin 1 520502 564 433289 1187 2088, 2708, 3328, 3948, 441 neuregulin 1 521670 565 428828 1188 2089, 2709, 3329, 3949, 442 neuregulin 1 539990 566 439276 1189 2090, 2710, 3330, 3950, 443 neuregulin 1 523079 567 430120 1190 2091, 2711, 3331, 3951, 444 OMOMYC 1191 1527, 17557-445 OMOMYC90 1528, 17644-(90AA truncated 17784 domain) 446 Orencia S.C.;
abatacept 447 ornithine 39007 568 39007 1192 1529,2092, 1659 carbamoyltransfer 2712, 3332, ase 3952, 4572, 4995, 5325, 448 parathyroid 282091 569 282091 1193 2093,2713, hormone 3333,3953, 4573,4996, 449 parathyroid 529816 570 433208 1194 2094, 2714, hormone 3334, 3954, 4574, 4997, 450 PBO-326;
rituximab 451 phenylalanine 546844 571 446658 1195 1530, 2095, hydroxylase 2715, 3335, 3955, 4575, 4998, 5328 452 phenylalanine 551337 572 447620 1196 1531, 2096, hydroxylase 2716, 3336, 3956, 4576, 4999, 5329 453 phenylalanine 553106 573 448059 1197 1532, 2097, 1660 hydroxylase 2717, 3337, 3957, 4577, 5000, 5330, 454 phosphorylase 566044 574 456729 1198 2098, 2718, kinase beta 3338, 3958, 455 phosphorylase 563588 575 455607 1199 2099,2719, kinase gamma 2 3339, 3959, (testis) 4579, 17959-456 phosphorylase 379942 576 369274 1200 2100, 2720, kinase, alpha 2 3340, 3960, (liver) 4580, 18046-457 phosphorylase 299167 577 299167 1201 2101, 2721, kinase, beta 3341, 3961, 458 phosphorylase 323584 578 313504 1202 2102, 2722, kinase, beta 3342, 3962, 4582, 18133-459 phosphorylase 455779 579 414345 1203 2103, 2723, kinase, beta 3343, 3963, 460 phosphorylase 328273 580 329968 1204 2104, 2724, kinase, gamma 2 3344, 3964, (testis) 4584 461 phosphorylase 424889 581 388571 1205 2105, 2725, kinase, gamma 2 3345, 3965, (testis) 4585 462 phosphorylase, 216392 582 216392 1206 2106, 2726, glycogen, liver 3346, 3966, 463 phosphorylase, 544180 583 443787 1207 2107,2727, glycogen, liver 3347, 3967, 464 plasminogen 308192 584 308938 1208 1533,2108, 2728,3348, 3968,4588, 465 plasminogen 316325 585 321466 1209 2109,2729, 3349,3969, 466 plasminogen 366924 586 355891 1210 1534, 2110, 2730, 3350, 3970, 4590 467 plasminogen 220809 587 220809 1211 1535,2111, activator, tissue 2731, 3351, 3971, 4591, 468 plasminogen 270189 588 270189 1212 1536,2112, 1661 activator, tissue 2732, 3352, 3972, 4592, 5001, 5331 469 plasminogen 352041 589 270188 1213 1537,2113, 1661 activator, tissue 2733, 3353, 3973, 4593, 5002, 5332 470 plasminogen 429089 590 392045 1214 2114,2734, 1661 activator, tissue 3354, 3974, 4594, 5003, 471 plasminogen 429710 591 407861 1215 1538,2115, activator, tissue 2735, 3355, 3975, 4595, 5004, 5334 472 plasminogen 519510 592 428886 1216 1539,2116, activator, tissue 2736, 3356, 3976, 4596, 5005, 5335 473 plasminogen 520523 593 428797 1217 1540, 2117, activator, tissue 2737, 3357, 3977, 4597, 5006, 5336 474 plasminogen 521694 594 429801 1218 1541,2118, 1661 activator, tissue 2738, 3358, 3978, 4598, 5007, 5337 475 plasminogen 524009 595 429401 1219 1542,2119, activator, tissue 2739, 3359, 3979, 4599, 5008, 5338 476 POU class 5 259915 596 259915 1220 2120, 2740, homeobox 1 3360, 3980, 477 POU class 5 376243 597 365419 1221 2121, 2741, homeobox 1 3361, 3981, 478 POU class 5 383524 598 373016 1222 2122, 2742, homeobox 1 3362, 3982, 479 POU class 5 412166 599 387646 1223 2123, 2743, homeobox 1 3363, 3983, 480 POU class 5 419095 600 413622 1224 2124, 2744, homeobox 1 3364, 3984, 481 POU class 5 429603 601 392877 1225 2125, 2745, homeobox 1 3365, 3985, 482 POU class 5 433063 602 405041 1226 2126, 2746, homeobox 1 3366, 3986, 483 POU class 5 433348 603 412665 1227 2127, 2747, homeobox 1 3367, 3987, 484 POU class 5 434616 604 388842 1228 2128, 2748, homeobox 1 3368, 3988, 4608, 18359-485 POU class 5 448657 605 416165 1229 2129, 2749, homeobox 1 3369, 3989, 486 POU class 5 451077 606 391507 1230 2130, 2750, homeobox 1 3370, 3990, 487 POU class 5 454714 607 400047 1231 2131, 2751, homeobox 1 3371, 3991, 488 POU class 5 546505 608 448154 1232 2132, 2752, homeobox 1 3372, 3992, 489 POU class 5 547234 609 449442 1233 2133, 2753, homeobox 1 3373, 3993, 490 POU class 5 547658 610 446962 1234 2134, 2754, homeobox 1 3374, 3994, 491 POU class 5 548682 611 446815 1235 2135, 2755, homeobox 1 3375, 3995, 492 POU class 5 550059 612 447874 1236 2136, 2756, homeobox 1 3376, 3996, 493 POU class 5 550521 613 447969 1237 2137, 2757, homeobox 1 3377, 3997, 494 POU class 5 550572 614 448254 1238 2138, 2758, homeobox 1 3378, 3998, 495 POU class 5 553069 615 448231 1239 2139, 2759, homeobox 1 3379, 3999, 496 POU class 5 553206 616 446757 1240 2140, 2760, homeobox 1 3380, 4000, 497 proprotein 302118 617 303208 1241 2141,2761, convertase 3381,4001, subtilisin/kexin 4621 type 9 498 proprotein 452118 618 401598 1242 2142,2762, convertase 3382,4002, subtilisin/kexin 4622 type 9 499 proprotein 543384 619 441859 1243 2143,2763, convertase 3383,4003, subtilisin/kexin 4623 type 9 500 protein C 234071 620 234071 1244 2144,2764, (inactivator of 3384, 4004, coagulation factors 4624 Va and Villa) 501 protein C 409048 621 386679 1245 2145,2765, (inactivator of 3385, 4005, coagulation factors 4625, 5009, Va and Villa) 5339 502 protein C 422777 622 409543 1246 2146,2766, (inactivator of 3386, 4006, coagulation factors 4626, 5010, Va and VIIIa) 5340 503 protein C 427769 623 406295 1247 2147,2767, (inactivator of 3387, 4007, coagulation factors 4627, 5011, Va and VIIIa) 5341 504 protein C 429925 624 412697 1248 2148,2768, (inactivator of 3388, 4008, coagulation factors 4628, 5012, Va and VIIIa) 5342 505 protein C 453608 625 404030 1249 2149,2769, (inactivator of 3389, 4009, coagulation factors 4629, 5013, Va and VIIIa) 5343 506 protein C 537436 626 442106 1250 2150,2770, (inactivator of 3390, 4010, coagulation factors 4630, 5014, Va and VIIIa) 5344 507 relaxin 2 308420 627 308018 1251 2151,2771, 3391,4011, 508 Raxh12 381627 628 371040 1252 2152,2772, 3392,4012, 4632,18446-509 Reverse Caspase 3 1543 (cleavable) 510 Reverse Caspase 3 1544 (non-cleavable) 511 Reverse Caspase 6 1545 512 rhodopsin 296271 629 296271 1253 2153,2773, 3393,4013, 4633,5015, 5345,18533-513 Rituximab;
rituximab 514 serpin peptidase 355814 630 348068 1254 2154, 2774, 1662 inhibitor, clade A 3394, 4014, (alpha-1 4634, 5016, antiproteinase, 5346, 18620-antitrypsin), 18706 member 1 515 serpin peptidase 393087 631 376802 1255 2155, 2775, 1662 inhibitor, clade A 3395, 4015, (alpha-1 4635, 5017, antiproteinase, 5347 antitrypsin), member 1 516 serpin peptidase 393088 632 376803 1256 2156, 2776, 1662 inhibitor, clade A 3396, 4016, (alpha-1 4636, 5018, antiproteinase, 5348 antitrypsin), member 1 517 serpin peptidase 402629 633 386094 1257 2157, 2777, inhibitor, clade A 3397, 4017, (alpha-1 4637, 5019, antiproteinase, 5349 antitrypsin), member 1 518 serpin peptidase 404814 634 385960 1258 2158, 2778, 1662 inhibitor, clade A 3398, 4018, (alpha-1 4638, 5020, antiproteinase, 5350 antitrypsin), member 1 519 serpin peptidase 437397 635 408474 1259 2159, 2779, 1662 inhibitor, clade A 3399, 4019, (alpha-1 4639, 5021, antiproteinase, 5351 antitrypsin), member 1 520 serpin peptidase 440909 636 390299 1260 2160, 2780, 1662 inhibitor, clade A 3400, 4020, (alpha-1 4640, 5022, antiproteinase, 5352 antitrypsin), member 1 521 serpin peptidase 448921 637 416066 1261 2161,2781, inhibitor, clade A 3401, 4021, (alpha-1 4641, 5023, antiproteinase, 5353 antitrypsin), member 1 522 serpin peptidase 449399 638 416354 1262 2162, 2782, 1662 inhibitor, clade A 3402, 4022, (alpha-1 4642, 5024, antiproteinase, 5354 antitrypsin), member 1 523 serpin peptidase 553327 639 452480 1263 2163, 2783, inhibitor, clade A 3403, 4023, (alpha-1 4643, 5025, antiproteinase, 5355, 18707-antitrypsin), 18793 member 1 524 serpin peptidase 556091 640 452169 1264 2164, 2784, inhibitor, clade A 3404, 4024, (alpha-1 4644, 5026, antiproteinase, 5356 antitrypsin), member 1 525 serpin peptidase 556955 641 451098 1265 2165, 2785, inhibitor, clade A 3405, 4025, (alpha-1 4645, 5027, antiproteinase, 5357 antitrypsin), member 1 526 serpin peptidase 557118 642 451826 1266 2166, 2786, inhibitor, clade A 3406, 4026, (alpha-1 4646, 5028, antiproteinase, 5358 antitrypsin), member 1 527 serpin peptidase 557492 643 452452 1267 2167, 2787, inhibitor, clade A 3407, 4027, (alpha-1 4647, 5029, antiproteinase, 5359 antitrypsin), member 1 528 serpin peptidase 351522 644 307953 1268 2168, 2788, inhibitor, clade C 3408, 4028, (antithrombin), 4648, 5030, member 1 5360 529 serpin peptidase 367698 645 356671 1269 1546, 2169, inhibitor, clade C 2789, 3409, (antithrombin), 4029, 4649, member 1 5031, 5361, 530 serpin peptidase 324015 646 321853 1270 1547, 2170, inhibitor, clade F 2790, 3410, (alpha-2 4030, 4650 antiplasmin, pigment epithelium derived factor), member 2 531 serpin peptidase 382061 647 371493 1271 2171, 2791, inhibitor, clade F 3411, 4031, (alpha-2 4651, 18933-antiplasmin, 19071 pigment epithelium derived factor), member 2 532 serpin peptidase 450523 648 403877 1272 1548, 2172, inhibitor, clade F 2792, 3412, (alpha-2 4032, 4652 antiplasmin, pigment epithelium derived factor), member 2 533 serpin peptidase 453066 649 402286 1273 1549 , 2173, inhibitor, clade F 2793, 3413, (alpha-2 4033, 4653 antiplasmin, pigment epithelium derived factor), member 2 534 serpin peptidase 278407 650 278407 1274 1550, 2174, inhibitor, clade G 2794, 3414, (Cl inhibitor), 4034, 4654, member 1 5032, 5362, 535 serpin peptidase 378323 651 367574 1275 1551 ,2175, inhibitor, clade G 2795, 3415, (Cl inhibitor), 4035, 4655, member 1 5033, 5363 536 serpin peptidase 378324 652 367575 1276 1552, 2176, inhibitor, clade G 2796, 3416, (Cl inhibitor), 4036, 4656, member 1 5034, 5364 537 serpin peptidase 405496 653 384561 1277 1553, 2177, inhibitor, clade G 2797, 3417, (Cl inhibitor), 4037, 4657, member 1 5035, 5365 538 serpin peptidase 433668 654 399800 1278 2178, 2798, inhibitor, clade G 3418, 4038, (Cl inhibitor), 4658, 5036, member 1 5366 539 sirtuin 1 212015 655 212015 1279 1554, 2179, 1663 2799, 3419, 4039, 4659, 540 sirtuin 1 403579 656 384063 1280 1555,2180, 2800, 3420, 4040, 4660, 5037, 5367 541 sirtuin 1 406900 657 384508 1281 2181,2801, 3421, 4041, 4661, 5038, 542 sirtuin 1 432464 658 409208 1282 1556,2182, 1664 2802, 3422, 4042, 4662, 5039, 5369 543 sirtuin 6 305232 659 305310 1283 1557, 2183, 2803, 3423, 4043, 4663, 5040, 5370 544 sirtuin 6 337491 660 337332 1284 1558,2184, 1665 2804,3424, 4044,4664, 5041,5371, 545 sh-tuin6 381935 661 371360 1285 1559,2185, 2805,3425, 4045,4665, 5042,5372 546 solute carrier 314251 662 323568 1286 1560,2186, family 2 2806, 3426, (facilitated glucose 4046, 4666, transporter), 19385-19523 member 2 547 solute carrier 382808 663 372258 1287 1561,2187, family 2 2807, 3427, (facilitated glucose 4047, 4667 transporter), member 2 548 solute carrier 577093 664 461344 1288 2188,2808, family 37 3428, 4048, (glucose-6- 4668õ 19524-phosphate 19610 transporter) member 4 549 solute carrier 590663 665 464769 1289 2189, 2809, family 37 3429, 4049, (glucose-6- 4669 phosphate transporter) member 4 550 solute carrier 261024 666 261024 1290 2190,2810, family 40 (iron- 3430, 4050, regulated 4670 transporter), member 1 551 solute carrier 427241 667 390005 1291 2191,2811, family 40 (iron- 3431, 4051, regulated 4671 transporter), member 1 552 solute carrier 427419 668 392730 1292 2192,2812, family 40 (iron- 3432, 4052, regulated 4672 transporter), member 1 553 solute carrier 440626 669 396134 1293 2193,2813, family 40 (iron- 3433, 4053, regulated 4673 transporter), member 1 554 solute carrier 455320 670 413549 1294 2194,2814, family 40 (iron- 3434, 4054, regulated 4674 transporter), member 1 555 solute carrier 481497 671 446069 1295 2195,2815, family 40 (iron- 3435, 4055, regulated 4675 transporter), member 1 556 solute carrier 544056 672 444582 1296 2196,2816, family 40 (iron- 3436, 4056, regulated 4676 transporter), member 1 557 sortilin 1 256637 673 256637 1297 1562,2197, 2817,3437, 4057,4677, 5043,5373 558 sortilhll 538502 674 438597 1298 1563,2198, 2818,3438, 4058,4678, 5044,5374, 559 sperm adhesion 223028 675 223028 1299 2199,2819, molecule 1 (PH-20 3439, 4059, hyaluronidase, 4679 zona pellucida binding) 560 sperm adhesion 340011 676 345849 1300 2200,2820, molecule 1 (PH-20 3440, 4060, hyaluronidase, 4680, 5045, zona pellucida 5375 binding) 561 sperm adhesion 402183 677 386028 1301 2201, 2821, molecule 1 (PH-20 3441, 4061, hyaluronidase, 4681, 5046, zona pellucida 5376 binding) 562 sperm adhesion 413927 678 391491 1302 2202,2822, molecule 1 (PH-20 3442, 4062, hyaluronidase, 4682, 5047, zona pellucida 5377 binding) 563 sperm adhesion 439500 679 402123 1303 2203,2823, molecule 1 (PH-20 3443, 4063, hyaluronidase, 4683, 5048, zona pellucida 5378 binding) 564 sperm adhesion 460182 680 417934 1304 2204, 2824, molecule 1 (PH-20 3444, 4064, hyaluronidase, 4684, 5049, zona pellucida 5379 binding) 565 sphingomyelin 299397 681 299397 1305 1564,2205, phosphodiesterase 2825, 3445, 1, acid lysosomal 4065, 4685, 5050, 5380, 566 sphingomyelin 342245 682 340409 1306 1565, 2206, phosphodiesterase 2826, 3446, 1, acid lysosomal 4066, 4686, 5051, 5381, 567 sphingomyelin 356761 683 349203 1307 1566, 2207, phosphodiesterase 2827, 3447, 1, acid lysosomal 4067, 4687, 5052, 5382 568 sphingomyelin 527275 684 435350 1308 1567, 2208, phosphodiesterase 2828, 3448, 1, acid lysosomal 4068, 4688, 5053, 5383 569 sphingomyelin 533123 685 435950 1309 1568,2209, phosphodiesterase 2829, 3449, 1, acid lysosomal 4069, 4689, 5054, 5384 570 sphingomyelin 534405 686 434353 1310 1569, 2210, phosphodiesterase 2830, 3450, 1, acid lysosomal 4070, 4690, 5055, 5385 571 SRY (sex 325404 687 323588 1311 2211,2831, determining region 3451, 4071, Y)-box 2 4691 572 SRY (sex 431565 688 439111 1312 2212,2832, determining region 3452, 4072, Y)-box 2 4692, 19785-573 Subcutaneous Herceptin;
trastuzumab 574 surfactant protein 393822 689 377409 1313 2213, 2833, B 3453, 4073, 575 surfactant protein 409383 690 386346 1314 2214, 2834, B 3454, 4074, 576 surfactant protein 441838 691 395757 1315 2215,2835, B 3455, 4075, 577 surfactant protein 519937 692 428719 1316 2216, 2836, B 3456, 4076, 578 Synagis 579 thrombomodulin 377103 693 366307 1317 1570, 2217, 2837, 3457, 4077, 4697, 580 thrombomodulin 503590 694 440119 1318 2218, 2838, 3458, 4078, 581 thrombopoietin 204615 695 204615 1319 1571,2219, 2839,3459, 4079,4699, 5056,5386, 582 thrombopoietin 353488 696 341335 1320 2220, 2840, 3460, 4080, 4700, 5057, 583 thrombopoietin 421442 697 411704 1321 1572,2221, 2841,3461, 4081,4701, 5058,5388 584 thrombopoietin 445696 698 410763 1322 1573, 2222, 2842,3462, 4082,4702, 5059,5389 585 TL011; rituximab 586 transfenin 223051 699 223051 1323 2223, 2843, receptor 2 3463, 4083, 587 transfenin 431692 700 413905 1324 2224, 2844, receptor 2 3464, 4084, 588 transfenin 462107 701 420525 1325 2225, 2845, receptor 2 3465, 4085, 589 transfenin 544242 702 443656 1326 2226, 2846, receptor 2 3466, 4086, 590 transforming 221930 703 221930 1327 2227,2847, growth factor, beta 3467, 4087, 1 4707 , 20046-591 transforming 366929 704 355896 1328 2228, 2848, growth factor, beta 3468, 4088, 592 transforming 366930 705 355897 1329 2229,2849, growth factor, beta 3469, 4089, 593 transforming 238682 706 238682 1330 2230, 2850, growth factor, beta 3470, 4090, 3 4710, 5060, 5390, 20133-594 transforming 556285 707 451110 1331 2231, 2851, growth factor, beta 3471, 4091, 3 4711, 5061, 595 transthyretin 237014 708 237014 1332 2232,2852, 3472,4092, 4712,20220-596 transthyretin 432547 709 396762 1333 2233,2853, 3473,4093, 597 transthyretin 541025 710 442134 1334 2234,2854, 3474,4094, 598 tufMinl 353024 711 343781 1335 2235,2855, 1669 3475,4095, 4715,5062, 599 tufMinl 368848 712 357841 1336 2236,2856, 1669 3476,4096, 4716,5063, 600 tufUhll 368849 713 357842 1337 2237,2857, 1669 3477,4097, 4717,5064, 5394,20359-601 tufUhll 507671 714 443643 1338 2238,2858, 1669 3478,4098, 4718,5065, 602 tufUhll 538902 715 437997 1339 2239,2859, 3479,4099, 4719,5066, 603 tufUh11 544350 716 441557 1340 2240,2860, 1669 3480,4100, 4720,5067, 604 tumorproteinp53 269305 717 269305 1341 1574,2241, 1670 2861,3481, 4101,4721, 5068,5398, 605 tumorproteinp53 359597 718 352610 1342 1575,2242, 2862,3482, 4102,4722, 5069,5399 606 tumorproteinp53 396473 719 379735 1343 2243,2863, 3483,4103, 4723,5070, 607 tumorproteinp53 413465 720 410739 1344 1576,2244, 2864,3484, 4104,4724, 5071,5401 608 tumorproteinp53 414315 721 394195 1345 2245,2865, 3485,4105, 4725,5072, 609 tumorproteinp53 419024 722 402130 1346 2246,2866, 3486,4106, 4726,5073, 610 tumor protein p53 420246 723 391127 1347 1577, 2247, 2867, 3487, 4107, 4727, 5074, 5404 611 tumor protein p53 445888 724 391478 1348 2248, 2868, 1670 3488, 4108, 4728, 5075, 612 tumor protein p53 455263 725 398846 1349 1578, 2249, 2869, 3489, 4109, 4729, 5076, 5406 613 tumor protein p53 503591 726 426252 1350 1579 , 2250, 2870, 3490, 4110, 4730, 5077, 5407 614 tumor protein p53 508793 727 424104 1351 1580, 2251, 2871, 3491, 4111, 4731, 5078, 5408 615 tumor protein p53 509690 728 425104 1352 1581õ
2252, 2872, 3492, 4112, 4732, 5079, 5409 616 tumor protein p53 514944 729 423862 1353 1582, 2253, 2873, 3493, 4113, 4733, 5080, 5410 617 tumor protein p53 545858 730 437792 1354 2254, 2874, 3494, 4114, 4734, 5081, 618 tyrosinase 263321 731 263321 1355 2255,2875, (oculocutaneous 3495, 4115, albinism IA) 4735õ , 20533-619 tyrosine 355962 732 348234 1356 1583, 2256, aminotransferase 2876, 3496, 4116, 4736õ , 620 UDP 305208 733 304845 1357 1584, 2257, glucuronosyltransf 2877, 3497, erase 1 family, 4117, 4737õ , polypeptide Al 20759-20845 621 UDP 360418 734 353593 1358 1585, 2258, glucuronosyltransf 2878, 3498, erase 1 family, 4118, 4738 polypeptide Al 1359 2259,2879, glucuronosyltransf 3499, 4119, erase 1 family, 4739 polypeptide A10 623 UDP 482026 736 418532 1360 2260, 2880, glucuronosyltransf 3500, 4120, erase 1 family, 4740 polypeptide A3 624 UDP 513300 737 427404 1361 2261, 2881, glycosyltransferas 3501, 4121, e 3 family 4741, 5082, polypeptide A2 5412 1362 2262,2882, 1671 glycosyltransferas 3502, 4122, e 3 family, 4742, 5083, polypeptide A2 5413 626 UDP 515131 739 420865 1363 2263,2883, glycosyltransferas 3503, 4123, e 3 family, 4743, 5084, polypeptide A2 5414 1364 2264,2884, glycosyltransferas 3504, 4124, e 3 family, 4744, 5085, polypeptide A2 5415 628 vascular 230480 741 230480 1365 1586,2265, endothelial growth 2885, 3505, factor A 4125, 4745 629 vascular 324450 742 317598 1366 1587,2266, endothelial growth 2886, 3506, factor A 4126, 4746, 5086, 5416 630 vascular 372055 743 361125 1367 1588,2267, endothelial growth 2887, 3507, factor A 4127, 4747, 5087, 5417 631 vascular 372064 744 361134 1368 1589, 2268, endothelial growth 2888, 3508, factor A 4128, 4748, 5088, 5418 632 vascular 372067 745 361137 1369 1590, 2269, endothelial growth 2889, 3509, factor A 4129, 4749, 5089, 5419, 633 vascular 372077 746 361148 1370 1591,2270, endothelial growth 2890, 3510, factor A 4130, 4750, 5090, 5420 634 vascular 413642 747 389864 1371 1592, 2271, endothelial growth 2891, 3511, factor A 4131,4751, 5091, 5421 635 vascular 417285 748 388663 1372 1593,2272, endothelial growth 2892, 3512, factor A 4132, 4752, 5092, 5422 636 vascular 425836 749 388465 1373 1594,2273, endothelial growth 2893, 3513, factor A 4133, 4753, 5093, 5423 637 vascular 457104 750 409911 1374 1595,2274, endothelial growth 2894, 3514, factor A 4134, 4754, 5094, 5424 638 vascular 482630 751 421561 1375 1596,2275, endothelial growth 2895, 3515, factor A 4135, 4755, 5095, 5425 639 vascular 518689 752 430829 1376 1597, 2276, endothelial growth 2896, 3516, factor A 4136, 4756, 5096, 5426 640 vascular 518824 753 430002 1377 1598,2277, endothelial growth 2897, 3517, factor A 4137, 4757, 5097, 5427 641 vascular 519767 754 430594 1378 1599, 2278, endothelial growth 2898, 3518, factor A 4138, 4758, 5098, 5428 642 vascular 520948 755 428321 1379 1600, 2279, endothelial growth 2899, 3519, factor A 4139, 4759, 5099, 5429 643 vascular 523125 756 429008 1380 1601,2280, endothelial growth 2900, 3520, factor A 4140, 4760, 5100, 5430 644 vascular 523873 757 430479 1381 1602, 2281, endothelial growth 2901, 3521, factor A 4141,4761, 5101, 5431 645 vascular 523950 758 429643 1382 1603,2282, endothelial growth 2902, 3522, factor A 4142, 4762, 5102, 5432 646 vascular 280193 759 280193 1383 1604,2283, endothelial growth 2903, 3523, factor C 4143, 4763, 647 vasoactive 367243 760 356212 1384 1605,2284, intestinal peptide 2904, 3524, 4144, 4764 648 vasoactive 367244 761 356213 1385 1606,2285, intestinal peptide 2905, 3525, 4145, 4765, 649 vasoactive 431366 762 410356 1386 1607, 2286, intestinal peptide 2906, 3526, 4146, 4766 650 v-myc 259523 763 259523 1387 2287,2907, myelocytomatosis 3527, 4147, viral oncogene 4767 homolog (avian) 651 v-myc 377970 764 367207 1388 2288, 2908, myelocytomatosis 3528, 4148, viral oncogene 4768, 21107-homolog (avian) 21110 652 v-myc 454617 765 405312 1389 2289, 2909, myelocytomatosis 3529, 4149, viral oncogene 4769 homolog (avian) 653 v-myc 524013 766 430235 1390 2290, 2910, myelocytomatosis 3530, 4150, viral oncogene 4770,21111-homolog (avian) 21284 654 von Willebrand 261405 767 261405 1391 1608,2291, factor 2911,3531, 4151, 4771, 655 zinc finger, 403903 768 384434 1392 2292,2912, GATA-like 3532, 4152, protein 1 4772 Protein Cleavage Signals and Sites [000288] In one embodiment, the polypeptides of the present invention may include at least one protein cleavage signal containing at least one protein cleavage site. The protein cleavage site may be located at the N-terminus, the C-terminus, at any space between the N- and the C- termini such as, but not limited to, half-way between the N-and C-termini, between the N-terminus and the half way point, between the half way point and the C-terminus, and combinations thereof.
[000289] The polypeptides of the present invention may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin or Factor Xa protein cleavage signal. Proprotein convertases are a family of nine proteinases, comprising seven basic amino acid-specific subtilisin-like serine proteinases related to yeast kexin, known as prohormone convertase 1/3 (PC1/3), PC2, furin, PC4, PC5/6, paired basic amino-acid cleaving enzyme 4 (PACE4) and PC7, and two other subtilases that cleave at non-basic residues, called subtilisin kexin isozyme 1 (SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9). Non-limiting examples of protein cleavage signal amino acid sequences are listing in Table 7. In Table 7, "X" refers to any amino acid, "n" may be 0, 2, 4 or 6 amino acids and "*" refers to the protein cleavage site. In Table 7, SEQ ID
NO: 21426 refers to when n=4 and SEQ ID NO: 21427 refers to when n=6.

Table 7. Protein Cleavage Site Sequences Protein Cleavage Signal Amino Acid Cleavage Sequence SEQ ID NO
Proprotein convertase R-X-X-R* 21424 R-X-K/R-R* 21425 K/R-Xn-K/R* 21426 or 21427 Thrombin L-V-P-R*-G-S 21428 L-V-P-R* 21429 A/F/G/I/L/TN/M-A/F/G/I/L/TN/W-P-R* 21430 Factor Xa I-E-G-R* 21431 I-D-G-R* 21432 A-E-G-R* 21433 A/F/G/I/L/TN/M-D/E-G-R* 21434 [000290] In one embodiment, the primary constructs and the mmRNA of the present invention may be engineered such that the primary construct or mmRNA contains at least one encoded protein cleavage signal. The encoded protein cleavage signal may be located before the start codon, after the start codon, before the coding region, within the coding region such as, but not limited to, half way in the coding region, between the start codon and the half way point, between the half way point and the stop codon, after the coding region, before the stop codon, between two stop codons, after the stop codon and combinations thereof [000291] In one embodiment, the primary constructs or mmRNA of the present invention may include at least one encoded protein cleavage signal containing at least one protein cleavage site. The encoded protein cleavage signal may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin and/or Factor Xa protein cleavage signal. One of skill in the art may use Table 1 above or other known methods to determine the appropriate encoded protein cleavage signal to include in the primary constructs or mmRNA of the present invention. For example, starting with the signal of Table 7 and considering the codons of Table 1 one can design a signal for the primary construct which can produce a protein signal in the resulting polypeptide.
[000292] In one embodiment, the polypeptides of the present invention include at least one protein cleavage signal and/or site.

[000293] As a non-limiting example, U.S. Pat. No. 7,374,930 and U.S. Pub. No.
20090227660, herein incorporated by reference in their entireties, use a furin cleavage site to cleave the N-terminal methionine of GLP-1 in the expression product from the Golgi apparatus of the cells. In one embodiment, the polypeptides of the present invention include at least one protein cleavage signal and/or site with the proviso that the polypeptide is not GLP-1.
[000294] In one embodiment, the primary constructs or mmRNA of the present invention includes at least one encoded protein cleavage signal and/or site.
[000295] In one embodiment, the primary constructs or mmRNA of the present invention includes at least one encoded protein cleavage signal and/or site with the proviso that the primary construct or mmRNA does not encode GLP-1.
[000296] In one embodiment, the primary constructs or mmRNA of the present invention may include more than one coding region. Where multiple coding regions are present in the primary construct or mmRNA of the present invention, the multiple coding regions may be separated by encoded protein cleavage sites. As a non-limiting example, the primary construct or mmRNA may be signed in an ordered pattern. On such pattern follows AXBY form where A and B are coding regions which may be the same or different coding regions and/or may encode the same or different polypeptides, and X and Y are encoded protein cleavage signals which may encode the same or different protein cleavage signals. A second such pattern follows the form AXYBZ where A and B
are coding regions which may be the same or different coding regions and/or may encode the same or different polypeptides, and X, Y and Z are encoded protein cleavage signals which may encode the same or different protein cleavage signals. A third pattern follows the form ABXCY where A, B and C are coding regions which may be the same or different coding regions and/or may encode the same or different polypeptides, and X and Y are encoded protein cleavage signals which may encode the same or different protein cleavage signals.
[000297] In one embodiment, the polypeptides, primary constructs and mmRNA can also contain sequences that encode protein cleavage sites so that the polypeptides, primary constructs and mmRNA can be released from a carrier region or a fusion partner by treatment with a specific protease for said protein cleavage site.

[000298] In one embodiment, the polypeptides, primary constructs and mmRNA of the present invention may include a sequence encoding the 2A peptide. In one embodiment, this sequence may be used to separate the coding region of two or more polypeptides of interest. As a non-limiting example, the sequence encoding the 2A peptide may be between coding region A and coding region B (A-2Apep-B). The presence of the peptide would result in the cleavage of one long protein into protein A, protein B and the 2A peptide. Protein A and protein B may be the same or different polypeptides of interest. In another embodiment, the 2A peptide may be used in the polynucleotides, primary constructs and/or mmRNA of the present invention to produce two, three, four, five, six, seven, eight, nine, ten or more proteins.
Incorporating Post Transcriptional Control Modulators [000299] In one embodiment, the polynucleotides, primary constructs and/or mmRNA
of the present invention may include at least one post transcriptional control modulator.
These post transcriptional control modulators may be, but are not limited to, small molecules, compounds and regulatory sequences. As a non-limiting example, post transcriptional control may be achieved using small molecules identified by PTC
Therapeutics Inc. (South Plainfield, NJ) using their GEMSTm (Gene Expression Modulation by Small-Moleclues) screening technology.
[000300] The post transcriptional control modulator may be a gene expression modulator which is screened by the method detailed in or a gene expression modulator described in International Publication No. W02006022712, herein incorporated by reference in its entirety. Methods identifying RNA regulatory sequences involved in translational control are described in International Publication No.
W02004067728, herein incorporated by reference in its entirety; methods identifying compounds that modulate untranslated region dependent expression of a gene are described in International Publication No. W02004065561, herein incorporated by reference in its entirety.
[000301] In one embodiment, the polynucleotides, primary constructs and/or mmRNA
of the present invention may include at least one post transcriptional control modulator is located in the 5' and/or the 3' untranslated region of the polynucleotides, primary constructs and/or mmRNA of the present invention [000302] In another embodiment, the polynucleotides, primary constructs and/or mmRNA of the present invention may include at least one post transcription control modulator to modulate premature translation termination. The post transcription control modulators may be compounds described in or a compound found by methods outlined in International Publication Nso. W02004010106, W02006044456, W02006044682, W02006044503 and W02006044505, each of which is herein incorporated by reference in its entirety. As a non-limiting example, the compound may bind to a region of the 28S
ribosomal RNA in order to modulate premature translation termination (See e.g., W02004010106, herein incorporated by reference in its entirety).
[000303] In one embodiment, polynucleotides, primary constructs and/or mmRNA
of the present invention may include at least one post transcription control modulator to alter protein expression. As a non-limiting example, the expression of VEGF
may be regulated using the compounds described in or a compound found by the methods described in International Publication Nos. W02005118857, W02006065480, W02006065479 and W02006058088, each of which is herein incorporated by reference in its entirety.
[000304] The polynucleotides, primary constructs and/or mmRNA of the present invention may include at least one post transcription control modulator to control translation. In one embodiment, the post transcription control modulator may be a RNA
regulatory sequence. As a non-limiting example, the RNA regulatory sequence may be identified by the methods described in International Publication No.
W02006071903, herein incorporated by reference in its entirety.
III. Modifications [000305] Herein, in a polynucleotide (such as a primary construct or an mRNA
molecule), the terms "modification" or, as appropriate, "modified" refer to modification with respect to A, G, U or C ribonucleotides. Generally, herein, these terms are not intended to refer to the ribonucleotide modifications in naturally occurring 5'-terminal mRNA cap moieties. In a polypeptide, the term "modification" refers to a modification as compared to the canonical set of 20 amino acids, moiety) [000306] The modifications may be various distinct modifications. In some embodiments, the coding region, the flanking regions and/or the terminal regions may contain one, two, or more (optionally different) nucleoside or nucleotide modifications.
In some embodiments, a modified polynucleotide, primary construct, or mmRNA
introduced to a cell may exhibit reduced degradation in the cell, as compared to an unmodified polynucleotide, primary construct, or mmRNA.
[000307] The polynucleotides, primary constructs, and mmRNA can include any useful modification, such as to the sugar, the nucleobase, or the internucleoside linkage (e.g. to a linking phosphate / to a phosphodiester linkage / to the phosphodiester backbone). One or more atoms of a pyrimidine nucleobase may be replaced or substituted with optionally substituted amino, optionally substituted thiol, optionally substituted alkyl (e.g., methyl or ethyl), or halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and the internucleoside linkage. Modifications according to the present invention may be modifications of ribonucleic acids (RNAs) to deoxyribonucleic acids (DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs) or hybrids thereof). Additional modifications are described herein.
[000308] As described herein, the polynucleotides, primary constructs, and mmRNA of the invention do not substantially induce an innate immune response of a cell into which the mRNA is introduced. Featues of an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc, and/or 3) termination or reduction in protein translation.
[000309] In certain embodiments, it may desirable to intracellularly degrade a modified nucleic acid molecule introduced into the cell. For example, degradation of a modified nucleic acid molecule may be preferable if precise timing of protein production is desired. Thus, in some embodiments, the invention provides a modified nucleic acid molecule containing a degradation domain, which is capable of being acted on in a directed manner within a cell. In another aspect, the present disclosure provides polynucleotides comprising a nucleoside or nucleotide that can disrupt the binding of a major groove interacting, e.g. binding, partner with the polynucleotide (e.g., where the modified nucleotide has decreased binding affinity to major groove interacting partner, as compared to an unmodified nucleotide).
[000310] The polynucleotides, primary constructs, and mmRNA can optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNA, RNAs that induce triple helix formation, aptamers, vectors, etc.). In some embodiments, the polynucleotides, primary constructs, or mmRNA may include one or more messenger RNAs (mRNAs) and one or more modified nucleoside or nucleotides (e.g., mmRNA molecules). Details for these polynucleotides, primary constructs, and mmRNA follow.
Polynucleotides and Primary Constructs [000311] The polynucleotides, primary constructs, and mmRNA of the invention includes a first region of linked nucleosides encoding a polypeptide of interest, a first flanking region located at the 5' terminus of the first region, and a second flanking region located at the 3' terminus of the first region.
[000312] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (Ia) or Formula (Ia-1):
__ yl y5 I B ________________ yl _y5 R3 L.L4,R4 /
% R4 R1),rt R5 ; (1, j R54-1 \4r mil mil y2 y2 \ m, Y3=131 __________________________ Y3=131 ______________ _ (Ia) (Ia-1) or a pharmaceutically acceptable salt or stereoisomer thereof, [000313] wherein [000314] U is 0, S, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl;
[000315] - -- is a single bond or absent;
[000316] each of R1', R2', R1", R2", Rl, R2, R3, R4, and R5 is, independently, if present, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl,or absent; wherein the combination of R3 with one or more of R1', R1", R2', R2", or R5 (e.g., the combination of R1' and R3, the combination of Rl"
and R3, the combination of R2' and R3, the combination of R2" and R3, or the combination of R5 and R3) can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); wherein the combination of R5 with one or more of R1', R1", R2', or R2" (e.g., the combination of R1' and R5, the combination of Rl" and R5, the combination of R2' and R5, or the combination of R2" and R5) can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); and wherein the combination of R4 and one or more of R1' , R3, or R5 can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocycly1);each of m' and m" is, independently, an integer from 0 to 3 (e.g., from 0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);
[000317] each of Yl, Y2, and Y3, is, independently, 0, S, Se, -NRN1-, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent;
[000318] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;

[000319] each Y5 is, independently, 0, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;
[000320] n is an integer from 1 to 100,000; and [000321] B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof), wherein the combination of B and Ry, the combination of B and R2', the combination of B and or the combination of B and R2" can, taken together with the carbons to which they are attached, optionally form a bicyclic group (e.g., a bicyclic heterocycly1) or wherein the combination of B, Ri", and R3 or the combination of B, R2", and R3 can optionally form a tricyclic or tetracyclic group (e.g., a tricyclic or tetracyclic heterocyclyl, such as in Formula (IIo)-(IIp) herein),In some embodiments, the polynucleotide, primary construct, or mmRNA includes a modified ribose. In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (Ia-2)-(Ia-5) or a pharmaceutically acceptable salt or stereoisomer thereof __ yl v5 __________________ y1 y5 ' U v-U
IV' 1R4 y2 R3 -C2/R4 R R\
y2 \R2 ' y3=I? ______________________ y3I m =I? ___________________________________________ y4 ¨ (ia-2) ¨ ¨ (Ia-3) _____ 15u0 y1 y5 4 R5/ iroR)' Y2 rnI u y-2 k m \ nn' Y3=I? ______________________ Y3=1? ___________ y4 ¨ ¨ ¨ (Ia-5).
[000322] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (Ib) or Formula (lb-1):

R3" u B U B
1iyR3.¨<1 y . R
RI3N1--- R4 \I--- R4 Y3=I? _________________ y=1? __ I 4 yI4 Y
_ ¨ (Ib), _ _ (lb-1) or a pharmaceutically acceptable salt or stereoisomer thereof, [000323] wherein [000324] U is 0, S, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2 and each Ru is, independently, H, halo, or optionally substituted alkyl;
[000325] - - - is a single bond or absent;
[000326] each of Rl, R3', R3", and R4 is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent; and wherein the combination of Rl and R3' or the combination of Rl and R3" can be taken together to form optionally substituted alkylene or optionally substituted heteroalkylene (e.g., to produce a locked nucleic acid);
[000327] each R5 is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, or absent;
[000328] each of Yl, Y2, and Y3 is, independently, 0, S, Se, -NRN1-, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;
[000329] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
[000330] n is an integer from 1 to 100,000; and [000331] B is a nucleobase.
[000332] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (Ic):
__ 1 5 Y ¨Y B3 q _.---- U Rb3 R5.1' ' 4, - "K
Y2 I b1 Rb2 i R
y3=p _______________ I

¨ ¨ (Ic), or a pharmaceutically acceptable salt or stereoisomer thereof, [000333] wherein [000334] U is 0, S, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl;
[000335] - - - is a single bond or absent;
[000336] each of B', B2, and B3 is, independently, a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof, as described herein), H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl, wherein one and only one of Bl, B2, and B3 is a nucleobase;
[000337] each of Rbl, Rb2, Rb3, R3, and R5 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl or optionally substituted aminoalkynyl;
[000338] each of Yl, Y2, and Y3, is, independently, 0, S, Se, -NRN1-, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;
[000339] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
[000340] each Y5 is, independently, 0, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;
[000341] n is an integer from 1 to 100,000; and [000342] wherein the ring including U can include one or more double bonds.
[000343] In particular embodiments, the ring including U does not have a double bond between U-CB3Rb3 or between CB3Rb3_cB2Rb2.
[000344] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (Id):
, B
__ yl y., 1 -J

y2 y3I=R _______ I

¨ (Id), or a pharmaceutically acceptable salt or stereoisomer thereof, [000345] wherein [000346] U is 0, S, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2 and each Ru is, independently, H, halo, or optionally substituted alkyl;
[000347] each R3 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl;
[000348] each of Yl, Y2, and Y3, is, independently, 0, S, Se, -NRN1-, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;
[000349] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
[000350] each Y5 is, independently, 0, S, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;
[000351] n is an integer from 1 to 100,000; and [000352] B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof).
[000353] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (le):
N
yu"
R6 N __________ ¨ (le), or a pharmaceutically acceptable salt or stereoisomer thereof, [000354] wherein [000355] each of U' and U" is, independently, 0, S, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2 and each Ru is, independently, H, halo, or optionally substituted alkyl;
[000356] each R6 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl;
[000357] each Y5' is, independently, 0, S, optionally substituted alkylene (e.g., methylene or ethylene), or optionally substituted heteroalkylene;
[000358] n is an integer from 1 to 100,000; and [000359] B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof).
[000360] In some embodiments, the polynucleotide, primary construct, or mmRNA
(e.g., the first region, first flanking region, or second flanking region) includes n number of linked nucleosides having Formula (If) or (If-1):
__ y1 y5 g ¨ ¨
R3LrUt yl R1y5 g -R4 )õ_,:-.....Ut õ -R2/ 11j R2" R2' y R2"
y2 y2 Y3=P ___________________________ Y3=P ______ I (zi y4 _ _ (If), ¨ ¨ (If-1), or a pharmaceutically acceptable salt or stereoisomer thereof, [000361] wherein [000362] each of U' and U" is, independently, 0, S, N, N(Ru)õu, or C(Ru)õu, wherein nu is an integer from 0 to 2 and each RU is, independently, H, halo, or optionally substituted alkyl (e.g., U' is 0 and U" is N);
[000363] - - - is a single bond or absent;
[000364] each of R1', R2', Ri", R2", R3, and R4 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent; and wherein the combination of R1' and R3, the combination of Ri" and R3, the combination of R2' and R3, or the combination of R2" and R3 can be taken together to form optionally substituted alkylene or optionally substituted heteroalkylene (e.g., to produce a locked nucleic acid);each of m' and m" is, independently, an integer from 0 to 3 (e.g., from 0 to 2, from 0 to 1, from 1 to 3, or from 1 to 2);
[000365] each of Yl, Y2, and Y3, is, independently, 0, S, Se, -NRN1-, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent;
[000366] each Y4 is, independently, H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
[000367] each Y5 is, independently, 0, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;
[000368] n is an integer from 1 to 100,000; and [000369] B is a nucleobase (e.g., a purine, a pyrimidine, or derivatives thereof).
[000370] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If), and (IIa)-(IIp)), the ring including U has one or two double bonds.
[000371] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each of Rl, R1', and Ri", if present, is H. In further embodiments, each of R2, R2', and R2", if present, is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is -(CH2)s2(0CH2CH2)s1(CH2)s30R', wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or C1_20 alkyl). In some embodiments, s2 is 0, sl is 1 or 2, s3 is 0 or 1, and R' is Ci_6 alkyl.
[000372] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each of R2, R2', and R2", if present, is H. In further embodiments, each of Rl, Ry, and Ri", if present, is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In particular embodiments, alkoxyalkoxy is -(CH2)s2(OCH2CH2)si(CH2)s3OR', wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or Ci_20 alkyl). In some embodiments, s2 is 0, sl is 1 or 2, s3 is 0 or 1, and R' is Ci_6 alkyl.
[000373] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each of R3, R4, and R5 is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In particular embodiments, R3 is H, R4 is H, R5 is H, or R3, R4, and R5 are all H. In particular embodiments, R3 is C1_ 6 alkyl, R4 is Ci_6 alkyl, R5 is Ci_6 alkyl, or R3, R4, and R5 are all Ci_6 alkyl. In particular embodiments, R3 and R4 are both H, and R5 is Ci_6 alkyl.
[000374] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R3 and R5 join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl, such as trans-3',4' analogs, wherein R3 and R5 join together to form heteroalkylene (e.g., -(CH2)biO(CH2)b20(CH2)b3-, wherein each of bl, b2, and b3 are, independently, an integer from 0 to 3).
[000375] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R3 and one or more of R1' , or R5 join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl, R3 and one or more of R1' , or R5 join together to form heteroalkylene (e.g., -(CH2)1,10(CH2)b20(CH2)b3-, wherein each of bl, b2, and b3 are, independently, an integer from 0 to 3).
[000376] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), R5 and one or more of Ry, Ri", R2', or R2" join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl, R5 and one or more of Ry, Ri", R2', or R2" join together to form heteroalkylene (e.g., -(CH2)biO(CH2)b20(CH2)b3-, wherein each of bl, b2, and b3 are, independently, an integer from 0 to 3).
[000377] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each Y2 is, independently, 0, S, or -NRN1-, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl. In particular embodiments, Y2 is NR -, wherein RN1 is H or optionally substituted alkyl (e.g., C1-6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl).
[000378] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each Y3 is, independently, 0 or S.
[000379] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), Rl is H; each R2 is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g., -(CH2)s2(0CH2CH2),1(CH2),30R', wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or C1_20 alkyl, such as wherein s2 is 0, sl is 1 or 2, s3 is 0 or 1, and R' is C1_6 alkyl); each Y2 is, independently, 0 or -NRN1-, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted alkyl (e.g., C1-6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y3 is, independently, 0 or S (e.g., S). In further embodiments, R3 is H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In yet further embodiments, each Y1 is , independently, 0 or -NRN1-, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted alkyl (e.g., Ci_6 alkyl, such as methyl, ethyl, isopropyl, or n-propy1)); and each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.
[000380] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), each R1 is, independently, H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g., -(CH2)s2(0CH2CH2),1(CH2),30R', wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or C1_20 alkyl, such as wherein s2 is 0, sl is 1 or 2, s3 is 0 or 1, and R' is C1_6 alkyl); R2 is H; each Y2 is, independently, 0 or -NRN1-, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted alkyl (e.g., Cl -6 alkyl, such as methyl, ethyl, isopropyl, or n-propyl)); and each Y3 is, independently, 0 or S (e.g., S). In further embodiments, R3 is H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In yet further embodiments, each Y1 is , independently, 0 or -NRN1-, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., wherein RN1 is H or optionally substituted alkyl (e.g., Ci_6 alkyl, such as methyl, ethyl, isopropyl, or n-propy1)); and each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.
[000381] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), the ring including U is in the I3-D
(e.g., I3-D-ribo) configuration.
[000382] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), the ring including U is in the a-L
(e.g., a-L-ribo) configuration.
[000383] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), one or more B is not pseudouridine (y) or 5-methyl-cytidine (m5C). In some embodiments, about 10% to about 100% of n number of B nucleobases is not y or m5C (e.g., from 10% to 20%, from 10% to 35%, from 10% to 50%, from 10% to 60%, from 10% to 75%, from 10% to 90%, from 10% to 95%, from 10% to 98%, from 10% to 99%, from 20% to 35%, from 20% to 50%, from 20% to 60%, from 20% to 75%, from 20% to 90%, from 20% to 95%, from 20% to 98%, from 20%
to 99%, from 20% to 100%, from 50% to 60%, from 50% to 75%, from 50% to 90%, from 50% to 95%, from 50% to 98%, from 50% to 99%, from 50% to 100%, from 75% to 90%, from 75% to 95%, from 75% to 98%, from 75% to 99%, and from 75% to 100%
of n number of B is not y or m5C). In some embodiments, B is not y or m5C.
[000384] In some embodiments of the polynucleotides, primary constructs, or mmRNA
(e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (llb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr)), when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Yl, Y2, or Y3 is not 0.
[000385] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a modified ribose. In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (IIa)-(IIc):

________ y1 y5 B ____yl_y5 B
\1U,RI
U

R3 __ 02 R4 R4 y2 Y2 Fµ
y3=pI 1 Y3=1? ______________________________________ I I
Y4 y4 ¨ ¨ (Ha), _ _ (IIb), or _______________ yi y5 u g R3 ____________ R4 y2 I
Y3=Pi ____________ - - (lie), or a pharmaceutically acceptable salt or stereoisomer thereof In particular embodiments, U is 0 or C(Ru)nu, wherein nu is an integer from 0 to 2 and each Ru is, independently, H, halo, or optionally substituted alkyl (e.g., U is ¨CH2¨
or ¨CH¨). In other embodiments, each of Rl, R2, R3, R4, and R5 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent (e.g., each Rl and R2 is, independently, H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy; each R3 and R4 is, independently, H
or optionally substituted alkyl; and R5 is H or hydroxy), and --- is a single bond or double bond.
[000386] In particular embodiments, the polynucleotidesor mmRNA includes n number of linked nucleosides having Formula (IIb- 1 )-(IIb -2) :

_ _y1_y5 u g _y 1 _y5 B

y2 R2' y2 y3=1? _________________________ Y3=I? _____ yi4 yi4 - - (IIb-1) or ¨ ¨ (IIb-2) or a pharmaceutically acceptable salt or stereoisomer thereof In some embodiments, U is 0 or C(Ru)õõ, wherein nu is an integer from 0 to 2 and each Ru is, independently, H, halo, or optionally substituted alkyl (e.g., U is ¨CH2¨ or ¨CH¨). In other embodiments, each of Rl and R2 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent (e.g., each Rl and R2 is, independently, H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, e.g., H, halo, hydroxy, alkyl, or alkoxy). In particular embodiments, R2 is hydroxy or optionally substituted alkoxy (e.g., methoxy, ethoxy, or any described herein).
[000387] In particular embodiments, the polynucleotide, primary construct, or mmRNA
includes n number of linked nucleosides having Formula (IIc-1)-(IIc-4):

_______ Y1 Y5 n2 n2 Y2 rµ Y2 rµ
y3=pI 3_ I
y _p __ ¨ (lie-1), ¨ (IIc-2), _______ Y1 Y5 R3 __ n2 Y2 rµ Y2 rµ
y3=pI 3_ I
y _p __________________________________________ ¨ (IIc-3), or ¨ (IIc-4), or a pharmaceutically acceptable salt or stereoisomer thereof In some embodiments, U is 0 or C(Ru)nu, wherein nu is an integer from 0 to 2 and each Ru is, independently, H, halo, or optionally substituted alkyl (e.g., U is ¨CH2¨ or ¨CH¨). In some embodiments, each of Rl, R2, and R3 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent (e.g., each Rl and R2 is, independently, H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, e.g., H, halo, hydroxy, alkyl, or alkoxy; and each R3 is, independently, H or optionally substituted alkyl)). In particular embodiments, R2 is optionally substituted alkoxy (e.g., methoxy or ethoxy, or any described herein). In particular embodiments, Rl is optionally substituted alkyl, and R2 is hydroxy. In other embodiments, Rl is hydroxy, and R2 is optionally substituted alkyl. In further embodiments, R3 is optionally substituted alkyl.
[000388] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes an acyclic modified ribose. In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (IId)-(IIf):

___ Y1 Y5 B 4 _______ Y1 Y5 B 4 __________________________________________________ Y1 Y5 C r R31Lifp U R R1 lUV

R5 R5 R5 m.2 n2 Cm.2 Y2 rµ Y2 rµ Y2 rµ

¨ ¨ (lid), ¨ ¨ (He), or ¨ ¨
(Ili), or a pharmaceutically acceptable salt or stereoisomer thereof [000389] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes an acyclic modified hexitol. In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides Formula (IIg)-(IID:
__ y1 y5 B _____________ y1 y5 B
R3'ij "'WI R3:riji "R4 R5 i .., .IR'' R1" R5 R'i ' R1"
y2 12 y2 12' y31=1? _______________________ y3=P1 ____________ Y
_ _ (hg), _ _ (IIh), _ __ y1 y5 B3 ______________ y1 y5 B3 R3'lj '''Rb3 R32rUliiRb3 "Rb2 - iRb2 y2 ib1 y2 : b1 R
I I
y3=1? _________________________ y3=1? ___________ _ _ (Iii), or _ _ (IIj), or a pharmaceutically acceptable salt or stereoisomer thereof [000390] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a sugar moiety having a contracted or an expanded ribose ring. In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (IIk)-(IIm):

_______________ y1_y5 u g __ yl_y5 U D
R5 '1 y2 m y2 y3=1? __________________________ y3=P1 _____ Y4 y4 - (Ilk), ¨ (Ill), or __ y1 y5 R3% '" R4 R5> R1"
= R2"
y2 Y3=1? _______________ (IIm),or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each of R1', Ri", R2', and R2" is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, or absent; and wherein the combination of R2' and R3 or the combination of R2" and R3 can be taken together to form optionally substituted alkylene or optionally substituted heteroalkylene.
[000391] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a locked modified ribose. In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (In):
__ y 1 y5 R3\ ____________ R4 y3=pI
¨ (IIn), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R3' is 0, S, or -NRN1-, wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl and R3" is optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-) or optionally substituted heteroalkylene (e.g., -CH2NH-, -CH2CH2NH-, -CH2OCH2-, or -CH2CH2OCH2-)(e.g., R3' is 0 and R3" is optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-)).
[000392] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes n number of linked nucleosides having Formula (IIn-1)-(II-n2):
__ yi y5 u B1 5 _______________________________ Y -Y
U B
R __________________________________ 3\
R ----2-R-' y3=pI 3_ I
y _p __ I I

¨ ¨ (IIn-1) or ¨ ¨ (IIn-2), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R3' is 0, S, or wherein RN1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl and R3" is optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-) or optionally substituted heteroalkylene (e.g., -CH2NH-, -CH2CH2NH-, -CH2OCH2-, or -CH2CH2OCH2-) (e.g., R3' is 0 and R3" is optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -)).
[000393] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a locked modified ribose that forms a tetracyclic heterocyclyl. In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) includes n number of linked nucleosides having Formula (Ho):

___ Y1 Y5 ______________________________ Y1 Y5 UR4 -r2T' 2õ U R4 72' "

R3" ______ ( \N--N ¨R12a R3 .,,. N---"\N_R12a v1+1-1"
v 12c R
T1' y2 y2 y3=pI 3_ I
y _p ______________________________________________________ I I

¨ ¨ (Ho) or ¨ ¨ (Hp), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R12a5 R12c5 T1', Tr5T2', T2", V1, and V3 are as described herein.
[000394] Any of the formulas for the polynucleotides, primary constructs, or mmRNA
can include one or more nucleobases described herein (e.g., Formulas (b1)-(b43)).
[000395] In one embodiment, the present invention provides methods of preparing a polynucleotide, primary construct, or mmRNA, wherein the polynucleotide comprises n number of nucleosides having Formula (Ia), as defined herein:
_______________________ y1 y5 B
R3.V-U
R5,,,( R) RI

..n,, y2 152' 1 ' x MI
Y3=P1 _________ Y
¨ ¨ (Ia), the method comprising reacting a compound of Formula (Ma), as defined herein:
( y3 \
y6 __________________________ II:i y1 y5 B
IiLl / RR5 >--"U ,õR4 ...
.. .7,4 71i 1,,, R) M
/ y \
R 2) R m.
I
y3=1? ________________________________ y7 \ /
/ a (Ma), with an RNA polymerase, and a cDNA template.

[000396] In a further embodiment, the present invention provides methods of amplifying a polynucleotide, primary construct, or mmRNA comprising at least one nucleotide (e.g., mmRNA molecule), the method comprising: reacting a compound of Formula (Ma), as defined herein, with a primer, a cDNA template, and an RNA
polymerase.
[000397] In one embodiment, the present invention provides methods of preparing a polynucleotide, primary construct, or mmRNA comprising at least one nucleotide (e.g., mmRNA molecule), wherein the polynucleotide comprises n number of nucleosides having Formula (Ia), as defined herein:
_______________________ yl y5 B
U,,,,R4 -----;= . 1 .
(Ri, R5 s 1,K 2..
: \R õ
y2 ikq m, M
I
y3=1? ______________________________________ Y
_ _ (Ia-1), the method comprising reacting a compound of Formula (IIIa-1), as defined herein:
I I
y6 ____________________ p y 1 __ y5 B
\I /
y4 r R5/ R =) R)' .....-- .1.,õ
7 mõ
Y3=1? _____________________________ Y7 \ ir,4/ /
a (IIIa-1), with an RNA polymerase, and a cDNA template.
[000398] In a further embodiment, the present invention provides methods of amplifying a polynucleotide, primary construct, or mmRNA comprising at least one nucleotide (e.g., mmRNA molecule), the method comprising:
[000399] reacting a compound of Formula (IIIa-1), as defined herein, with a primer, a cDNA template, and an RNA polymerase.

[000400] In one embodiment, the present invention provides methods of preparing a modified mRNA comprising at least one nucleotide (e.g., mmRNA molecule), wherein the polynucleotide comprises n number of nucleosides having Formula (Ia-2), as defined herein:
_________________________ yl_y5 B
U
5c.44 y2 mi I
Y3=131 ____________________________________ Niii4 - - (Ia-2), the method comprising reacting a compound of Formula (IIIa-2), as defined herein:
u y6 _____________________ p yl y5 B
I U
\ y4 / 4 7 y2 \ m I µ
y3=Pi __ y7 \ itzt /
/ a (IIIa-2), with an RNA polymerase, and a cDNA template.
[000401] In a further embodiment, the present invention provides methods of amplifying a modified mRNA comprising at least one nucleotide (e.g., mmRNA
molecule), the method comprising:
[000402] reacting a compound of Formula (IIIa-2), as defined herein, with a primer, a cDNA template, and an RNA polymerase.
[000403] In some embodiments, the reaction may be repeated from 1 to about 7,000 times. In any of the embodiments herein, B may be a nucleobase of Formula (b1)-(b43).
[000404] The polynucleotides, primary constructs, and mmRNA can optionally include 5' and/or 3' flanking regions, which are described herein.

Modified RNA (mmRNA) Molecules [000405] The present invention also includes building blocks, e.g., modified ribonucleosides, modified ribonucleotides, of modified RNA (mmRNA) molecules.
For example, these building blocks can be useful for preparing the polynucleotides, primary constructs, or mmRNA of the invention.
In some embodiments, the building block molecule has Formula (Ma) or (IIIa-1):
/ y3 \
y6 __ p yl __ y5 y6 __ p yl __ y5 H B
yl4 /
"R4 \!4. 1 Pk_ R1, " R5( Ri) R2,, ====\ R /mi, y2 \ rn y2 62, rµ rµ
Y3=I? Y7 ____ Y3=I? Y7 (Ma), \ y4 /
/ q (IIIa-1) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the substituents are as described herein (e.g., for Formula (Ia) and (Ia-1)), and wherein when B is an unmodified nucleobase selected from cytosine, guanine, uracil and adenine, then at least one of Yl, Y2, or Y3 is not 0.
[000406] In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IVa)-(IVb):
\i(3 y6 __ Fi y5 B
7y3 xi 1y 5 \ Y
0,0 r p (IVa) or HO OH (IVb), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, Formula (IVa) or (IVb) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula ()1), (b8), (b28), (b29), or (b30)). In particular embodiments, Formula (IVa) or (IVb) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, Formula (IVa) or (IVb) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, Formula (IVa) or (IVb) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).
[000407] In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IVc)-(IVk):
II I I
y6 __ p y _____ 5 y6 __ p vl .
1 ' t-----Y`' n \ 4 r R3µµ ' Y'vU R1 \ y4 /r *
HO I-2 (IVO, HO 12(ivd.), / y3 \ (3\
ii y6 __ A y1,_ y6 __ p y1 5,....._ 5 \''4 ir ,\y4 / r HO R2(IVe), HO R2(ivf), 7 y3 \
(y3\
I I
y6 __ p yl,...... 5 y6 __ A y1 5 \y4 1 ir Y,,u13 y4 / 3µµ('v r U /3 R ____ R1 m(IVg), Ho bCH3(IVh), 7 y3 \ (3\
i I
y6 __ p yip__ 5 y6 __ A yl 5 \4 / r 3YvU /3RI y4 iR3µµ r Yvuy Rµµ R1 Ho F (Ivo, Ha bCH3(Ivi), 7 y3 \ 7 y3 \
y6 __ p NI/I5 y6 __ p y 1 5 \ yi 4 / V ' r Y.,u13 i I U
\ y4 i R R ' __________________________________ / rR3õ - Ri Ho "CI (IVk), or Ha i (IV1), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IVc)-(IVk) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (bl), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IVc)-(IVk) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, one of Formulas (IVc)-(IVk) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IVc)-(IVk) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).
[000408] In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (Va) or (Vb):

/3 \/ ____---1\1, 27 I I y3 \ V7 \ ----1:Z
y6 __ Fr yl ________ D N
y6 ________________________________ ig yl I N
\ y4 4 R3y,1,, , ili 1 4 U
.7.- -(-:. r =

rn (Va) or y-7 -1:z2 (Vb), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)).
[000409] In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IXa)-(IXd):

l y3 \ 7 Y3 II
y6 ___________ p ylr_____ 5 __ y6 ig yl 5 \(4 Jr VI \i4Jr V/3 HO F (IXa), Ho Br (IXb), y6 __ ig yl 5 V/
yoa ____________________________ II 4 .... __ HO al (IXc), or H6 1 (IXd), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IXa)-(IXd) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula ()1), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IXa)-(IXd) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)).
In particular embodiments, one of Formulas (IXa)-(IXd) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IXa)-(IXd) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).
[000410] In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IXe)-(IXg):

y 3 \ y 3 \
I I I I
y6 __ p k 5 y6 __ p y1, 5 k I I
BH2 ir V/31 \H2 /r V/3 - _ Ha R.- 2 (IXe), HO 2(IXf), or S
lie y6 __ p yl, 5 NI(4 r V/3 Ha R2 (IXg), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IXe)-(IXg) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula ()1), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IXe)-(IXg) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, one of Formulas (IXe)-(IXg) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IXe)-(IXg) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).
[000411] In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IXh)-(Da):

II II
___ p yl 5 y6 y6 R1 \
1 (L1 jr 0/3 \ y4 /Y

(.0 OH
.. __________________________________________ ..
HO 0 (IXh), Ho t H 3 (IM), / y3 7 y3 y6 __ IlLy1 5 y6 __ ig yl 5 \43, Y r 'Y,,__, \y4 r %.,n3 H3C ... ..
Ho OH (IXj), or Ho OH (Da), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IXh)-(IXk) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (bl), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IXh)-(IXk) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, one of Formulas (IXh)-(IXk) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IXh)-(IXk) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).
[000412] In other embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, has Formula (IX1)-(IXr):

/0 \ /079 \/
HO __ P 0 ___ P ¨o .-.L B HO PO ________ PO B
/r2 I I I I
\OH \OH / r2 \ \ CH3 rl09 / \ /
õ
Ha bH(IX1), HO bH(IXm), /0 \ /Se \ /0 HO __ 1¨O ___ l¨o B HO-- IJ-0 B
I
\OH j / r2\ 0H A09 / r \OH Jr 0) HO OH(IXn), HO -.F (Ixo), /so II
/0 \ /0 p1 B I
r r .. _ - ____________________________________ _ ¨
Ha al (IXp), HO br(IXq), or Ha 0CH3 (IXr) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each rl and r2 is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5) and B is as described herein (e.g., any one of (b1)-(b43)). In particular embodiments, one of Formulas (IX1)-(IXr) is combined with a modified uracil (e.g., any one of formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), such as formula (bl), (b8), (b28), (b29), or (b30)). In particular embodiments, one of Formulas (IX1)-(IXr) is combined with a modified cytosine (e.g., any one of formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), such as formula (b10) or (b32)). In particular embodiments, one of Formulas (IX1)-(IXr) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In particular embodiments, one of Formulas (IX1)-(IXr) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).
[000413] In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be selected from the group consisting of:

NH N
(:
N,...)--...N /0 \ N 3 I 1 N ----N-Ho H0L0 0 P-0-yi N NH2 I I r /
H ir \OH
Ha OH (BB- 1), Ha -OH (BB- 2), NH CI
NN/L N--.../L.N
/9 \ I 0 NN- NN
HO-P-0 HO-(11L0 I O/
\OH iA I r i OH A
Ho OH (BB- 3), Ho OH (BB- 4), N) .CH3 1\1)*L
)1 /0 \ I NH
H0+0 N.'"i\r NN

\OH /A i I r /
\OH A
Ho OH (BB- 5), Ho b1-1 (BB- 6), N,.../N
(--)1N /0 \ I
/9 \ N'. II N ----". N":;-1-- N
HO-T-O-LA .0i N NO-H3-0A04 H2 I
\OH ir QH ir _ _____________________________________________ /
Ha OH (BB- 7), Ho OH (BB- 8), N)LN=CH2 FI).L
/0 \ I
,1/4,----..., 4:2) \ oN 1 r HO-P-0 " N NH2 õ,,,..., .....;-.A., I Ao/ HO-ILO " N NH2 \OH /r I AO' \OH ir Ha OH (BB- 9), Ho OH

CI
N--......./L.N
II
HO'-FrO-O+ NH2 \OH ir (BB- 10), Ha OH (BB- 11), and II
HO-1?-0 N----NNH2 \OH /-r r.-\ ' Ha OH (BB- 12), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).
[000414] In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be selected from the group consisting of:

c, rN-...N, _NI
N N
/0 \ N
/0 \ I 1 N----õ,-, p 1-1044-0-Noy - " HO-ILO
, I oq \OH /1. H /r ... ___________________________________________ (.
HO OH (BB- 13), HO -OH (BB- 14), e 9 o s \ I\(),) N N N N
/0 \ /0 NNH04-11=1)-0-yi N
\OH /r H01-P-0-y/
I
Ho OH (BB- 15), Ho OH (BB- 16), NH2 e 1\1-__N-0 N-----r\j' H01-0-yi \OH ir Ho OH (BB- 17), H'NOhNNH2 /0 il(NH
si N-..../LN HOi-\0Pi -HO r "0 NN
HO-T-0 r 0"
',)F1 Ha bil (BB- 18), (BB- 19), /0 (NH
---µ
HO¨ILO N 0 I
\OH
r _______________ õ
and Ha OH (BB- 20), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5) and sl is as described herein.
[000415] In some embodiments, the building block molecule, which may be incorporated into a nucleic acid (e.g., RNA, mRNA, polynucleotide, primary construct, or mmRNA), is a modified uridine (e.g., selected from the group consisting of:

H3CA HOJ.L
Y3 1 li / y3 1 11H
( y6 __ ilLy1 N 0 y6 __ p yl N 0 I I
y4r 0) \ y4 0) r Fib OH (BB- 21), HO OH (BB- 22), I ).( / y3 \ I X
II
y6 __ p_y1 N 0 I
\ y4 r ___________ ) Ha OH (BB- 23), H2N.L NH
7Y3 \ \ ANN

II NLO
y6 __ p yl y6 __ p yl N 0 I I
\y4 -iA0) \Y4 ---"'s."0 r _____________________________________ r __ ) HO OH (BB- 24), Ho OH (BB- 25), A
/y3\ )-NH / y3 I X
y6 __ ig_y1 NLID y6 _ilLy1 N 0 I
\(L1 ----'sV) \ y4 0) r ______________________________ \ r __ Ha OH (BB- 26), Ho OH (BB- 27), / y3 H)Arti /y3\ ?L NH
N,N=L0 y6 _p_yl y6_yl (L1- 1 0 \ y4 -----":\õ0õ) \ r __ Ha OH (BB- 28), Ho OH (BB- 29), H3C,NANH Y3 \
/y3 \ y6 __ ig yl ii ro y6 __ p _y1 ( \ yl 4 ---4-:\,0 zi Ho OH (BB- 30), Ho OH (BB- 31), F3CA )c70LL

/y3 \ t XI HN 1 y6 __ pli_y1 N 0 i\i Y ¨Pi -Y)1 s-' n -\i4 i 0) (Lt 0) r r Ho OH (BB- 32), HO OH (BB-)-7.y0CH3 HNNH2 \ HN
( 3 \ j y3 \ j +4 ii7r0) Y
Y 171 Y/I-A1 CI Oi 33), Ha OH (BB- 34), Ho OH (BB-HN)-1-.....7-,..NCF3 Y3 \ Y3 \ HN 1 NH2 ii 0 N H 7 j ii SN
y6_p_y1 y6_p_y1 \ y4 /7::\r0) 35), Ho OH (BB- 36), H6 OH

)=

y3 ),,1 H
y ¨ig¨yi S- -1\1 6( )!/4 0) r (BB- 37), Ha OH (BB- 38), H)\1!Vri 0 y6(ig_yi 0 N y Y3 \ i /1 0) ilLyi 0 N 0 CF3 1-7 \,0) - --__ HO OH (BB- 39), Ho OH (BB-1-11\1N--Thr 6(I(I 3N OCF3C) Y Pi ¨Y1 N;f4 0) r ),_ 40), Ha OH (BB- 41), HN)-N.r0H
HN)-N.r0H
/ y3 (:) ),,\ N j H
0 / v3 ) S
H

ii " - III "-N
µ,61_0¨µ,1 µ,61_0¨µ,1 ' 'I ' ' 'I ' \ y4 o \ y4 0 r r _ ___________ ..
Ha OH (BB- 42), Ha OH

N ...r0H
HN)CV
Y3 \ 0 N 0 y _ilLyi j )1(4 ) / r ______ H
6( (BB- 43), Ha 0CH3 (BB- 44), HN)c,Nr0Fmoc _1_13 \ 1 H 0 y yl Nir4 /AO) 6( i Ha OH (BB- 45), )czN .r0Fmoc , HN - - N
Y6( 14-3 Y/17.4\r\i S j H

- -HO OH (BB-46), HN )czN.r0Fmoc _ir \ H
i y _Nit 6( 0 Nj NI(4 'Ai 0) / r y6 il t_L
r Ho 6cNH3 (BB- 47), o coN2F:Fmoc 3 oc y4 0) - ____________ _ HO OH (BB- 48), ).L
/e N NH2 \ t y6 _y1 \ -I
\ y4 irT-V)) Ha ol-I (BB- 49), ),,O0Fmoc HN)70-LOH
/ y3 ),s\rHI j y3 \ j y6_ p_yl Y
r Ho OH (BB- 50), Ho OH
0 OFmoc OFmoc \ HNri _YA3 \i ON" 0 y _y 6( NI(4 -AO) / r z s_ (BB- 51), HO OH (BB- 52), OH HN)yy0Me (\ HN)YY y3 i 0 r 3 \ i y _Nit 6( 0 N

NI(4 ;TAO) y _ilLy1 N
(LI 0) HO OH (BB- 53), Ho OH (BB-OMe ),y0Me , HN).VY , HN 1 / Y3 \ ONi 0 Y3 \ ON 0 ii Y6 _ p _y1 y _IlLy1 \ 2r7V)) ilLi ;AO) 54), 1-16 OH (BB- 55), Ha OH

).r0Me , HN 1 7 Y3 \
ONi 0 y6 _ilLy1 \ y4 / 1,0) (BB-56), H6 0- CH3 (BB-57), )cv.r0Me 7 Y3 ), \ Y3 H
\
ii S N 0 7 II 0 N j p_y 1 y6 _ p _y Y6 _Y) \ y4 0) y4 /1,C)) r Ha OH (BB- 58), Ha OH

H
y6_1Ly1 S N
\ !r4 0) (BB- 59), HO OH (BB- 60), HN)CN -CH

y _Ig_yi Se N I H
6( (L1 0) r Ha OH (BB-61), HN).r 7 y3 I 0 yo ,, I I 1 0 N

\ y4 0) r Ha OH (BB- 62), HN
7 y3 \ ACcN H2 y6_p_yl \ y4 /1-7-\/0) Ho oCH3 (BB- 63), 0 CO2Fmoc H3CNANNHFmoc Y3 \

v ( ig yl 0 Ho OH (BB- 64), H3C,NANNH2 Y3 \

v ( ig yl 0 Ho OH (BB- 65), )czy0H

y6_p_yl NI(4 0) r Ho OH (BB- 66), yF3 ))yr0 MOC

y ig_yi 0 N
6( (41 0) r Ho OH (BB- 67), H N )C7 N
7 y3 y6F, j H

) ryi 0 N
\ y4 0 r Ho OH (BB-68), H N
7 y3 j 11 y6 yi S N
\ yl 4 0) r Ho OH (BB- 69), H N
7 y3 ! il y6 il:Lyi - (-) N
\ (zi 0) r Ho oCH3 (BB- 70), HNAN
H3C,,,AN-CH3 /y3\ IN
, ,, y6 __ ilLy1 o yo_Fry 1 (LI -(:) \ y4 0 \ r r ____________ Ha OH (BB- 71), Ho OH (BB- 72), 6( \ HNAN/ \ HNAN
y3 \
y ¨_yl / r Y3 \
ii Y _ry1 ( y4 --'---4\r ir Ha OH (BB- 73), Ha OH (BB-HNAN HNAN'' /y3\ /y3\
y6HLy1 0 y6_,g_y1 0 r r 74), Ho OH (BB- 75), Ho OH

µ HNAN
ye(y3 \
ig_y1 I
(BB- 76), Ha OH (BB- 77), HN A NrOH HN)C71 Y3 / y3 ), i , 1 1 y6(_y).._.C:lri yo_p_y1 0 N

y4 0 \ y4 r 0) r Ha OH (BB- 78), Ho OH (BB-HNI o).1 ), i HN)I
Y3 \
7 y3 0 N i y6_ig_yl 1 y _ig 0 N
\ y4 r 0)Ty) 79), Ho OH (BB- 80), Ha OH

).c7\
7 y3 HN j y6_p_y l \ ( 0) r ),, (BB- 81), Ho OH (BB- 82), HN HNOH
) / y3 \ j 0 N / y3 \ j µ,6_ig_µ,1 µ,6_0_µ,1 0 1 I 1 1 'T ' HO OH (BB- 83), Ha OH (BB-)*LI ).NH
/ y3 NH / \I(13 t I
v6 __________ ig yi N .L0 y6 __ p yi N 0 \4 ONJ \f4 r H
r iCH3 84), Ho OH (BB- 85), HO tH3 (BB- 86), O o INH A, NH
7 Y 3 / Y3 \ &
v6 _______ ig yi N .LO v6 __ ig y1 N 0 r ,-H3d ____________ Ho OH (BB- 87), H(57:6 (BB- 88), INH tAyH
Y y3 y6 _______ ig yi N y6( A yi NO
I, \
(zi 0) r r Ha -I (BB- 89), Ho CI (BB- 90), o o AI NH AI NH
/ y3 /y3\
y6 _______ ig yi NO y6 __ ig yi N
I I
\(4 0) \(4 0) r r H6 Br (BB- 91), Ho -I (BB- 92), A ANH
/y3\ t 11F1 / y3 \ t I I
y6 __ ig yl 'N 'O y6 __ p y1 N 0 I
\ y4r 0) \y4 Ha tH3 (BB- 93), HO Ocl--13 (BB- 94), A ,cH3 1-13c0 y3 t y6 __ ig y I NO v6 __ i¨yl N 0 I I
\ y-rA I r 0) \ y4 r 0) Ho OH (BB- 95), HO OH (BB- 96), H3C 0 s ANH
/ y3 t yH HN
y6 __ ig yi N 0 y6 __ ir yl r0 \ y-r, r 0) (z1 r 0 Ho OH (BB- 97), HO OH (BB- 98), HNANH H3C,NANH
Y3 \

v6 \ )rS v6 __ ig yl S
D yl "1 1 1 y4 0 y4 0 Ho OH (BB- 99), Ho OH (BB- 100), S

7Y3 \
y6 __ ig y1 \)r0 y4 0 Ho OH (BB-1O1), HN
AN N V\ SO3H
-7 i(13 H
) y6_p_y1 \ y4 r 0 Ho OH (BB-102), HNAN/\ N SO3Fmoc / y3 H
y6 ii:Ly1 \ y4 0 r Ho OH (BB-103), ...11-...\ ,...------........õ.....S03H

O N H
y6_,g_y1 \ (41 0) r Ha OH (BB-104), )-7\ SO3FMOC
HN N
/ y3 ), N j H
y6 __y1 0 \ y4 0 r Ho OH (BB-105), )'cZ\
HN N

y3 H
y6(11:Ly1 S N
(zi 0) r Ho OH (BB-106), HN)=cz N SO3Fmoc / y3 ) S
j H
II N
y6 _p_yl \r Ha OH (BB-107), \I(3 \ Th\10 y6 p y1 ( 1 yl4 )T' ) HO OH (BB- 108), )..L

y3 j Y6(11'1-Y1 0 N
Izt 0) r Ho -OH (BB-109), ).c HN 1z NH2 y3 y6(11:Lyi 0 N
r Ho OH (BB-11O), H3Cj-L
1 Ir HN
),_ )C)Cj OC H 3 y6 ( A yl NS y3 yi 4 y _I
0 ilLy1 O N
) \A 0) r r Ha OH (BB-111), Ha OH

HN ( )c2OI ICH 3 7 y3 ),_ y6 ON O N 0 \ (zt 0) r (BB- 112), Ha OH (BB- 113), / y3 \ AI NH

/ y3 \
y6 __ py1 y6 __ A _y N'O
A
\ \!/4 \ \I /17-4\,0) H
HO OCH3 (BB- 114), a OCH3 (BB- 115), HN
A
NH A
HN NH
y3 \ y3 \

6 __ ( " 1 LL

L0 y6 ( ig y1 r) y P 1(,4,, yl yl 0 r Ho F (BB-116), Ho ol (BB-117), HNA NH HNA NH
7 __ NI(13) 0 6 1(3 rLO
N/ ( igy ,),..
i4 1 1 0 y 4 \y y6 p yL'I- r __ Ha OCH3 (BB-118), HO 1 (BB-119), HNANH HNANH
/
y6 __ p-yl 0 y6 I

\
\ y4 Ni(3 6 ( " 1 )rL0 P y,i_.
yl4 ...I.-4NC) Ha tH3 (BB- 120), Ha OCH3 (BB- 121), A
HN NH HNANH
/Y3 \ Y3 \
II 0 y6 __ 1g y 1 cr 0 y6 __ p yl \
______________________ CH3 Ho OH (BB- 122), HO OcH3(BB- 123), 7 y3 )1NANH / y3 HNA NH
ii )r=L II r y6 __ p yl 0 y6 __ p yl 0 \ y4 r 0 \ y4 r 0 : =---_ Ho OH (BB- 124), and HO 0 (BB-125), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Yl, Y3, Y4, Y6, and r are as described herein (e.g., each r is, independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3, or from 1 to 5)).
[000416] In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, is a modified cytidine (e.g., selected from the group consisting of:

I Id3CN
7 Y3 \ Y3 \ 1 y6 __ A yi NO y6 __ A y......õ\1 1\1LQ
yi4 \ yi 4 AO
HO OH (BB-126), HO OH (BB-127), ), rN
(3\ HN - N

\ _____________________________________ y 4 Ao N 0 v6 D v I
"1 1 r ________________________________________________ ) .. ______________ _ Ha old (BB- 128), HO OH (BB- 129), y3 \ 1 1 A yl N - S
( f.4 -VONJ

y6 1 y6 ____________________________________ A 1 NO
I Y ()NI
y4 r __________________________________________________ /
.. ______________ :.
Ha old (BB-130), Ho OH (BB-131), ,CH3 NH HN
)-L ,CH3 y6 7 A yi NO y6 ( yl N 0 N!izt 0) NI(4 () \ r ________________________________ r __ Ho OH (BB-132), Ha OH (BB-133), ,CH3 HN H3C,N,CH3 /y3 t 11 /y3 t 11 y6 __ A y 1 N 0 y6 __ A y 1 N 0 \y4 0)0) r __________________________________ \ r __ HO oCH3 (BB-134), Ho OH (BB-135), Fi3C,N,CH3 7y3 HO
y3 \ 1 N
NO
\ 11 y6 __ p _y1 N 0 y6 __ :i_y1 \Y4 0) y4 A. 0) HO oCH3 (BB- 136), Ho OH ( BB-137), NHAc NH2 Ac0 N TBDMS,oN

\
/y3 \ 1\1LC) y3 \ N 0 y6 __ p y1 y6 __ ig _y 1 \
y1 A.
\ 4 yi 4 A
r 0) 0) Ha OH (BB-138), Ho OH (BB-139), .....,...õ--'L
/ X3 t / y3 t 11 y6 ____________________________________ A y1 N 0 y6 __ 15 y1 NO 1 y14y4 0 0) \ r __ HO OH (BB-140), HO bl--13 (BB-141), )1 N
/y3 \ 1 I I
Y6( \IIDIC3 yl r\i'Lci y6 __ ig_y1 1\1 0 I
y14 0) \ Y4 A-o/CH3 H30' _______________________________________________ HO OH (BB- 142), Ha OH (BB- 143), ((),,\) Y ¨11"¨Y1 N-10 y/¨_yi N-10 I
y14 0) y4 0 HO 0 (BB- 144), HO 0 (BB- 145), /y3 1 y3 1 N
y6 __ A yl NOy6 ________________ A y1 \ NO
)\(L1 y14 0) r r*
HO Br (BB- 146), HO OH (BB- 147), NHAc )N
NO
\i3 1 I / (13 y6 __ p y1 N 0 v6 __ D v:)s.1, ) /c IA "I I
\y-r 0) \Y4 0 r _________________________________________ r ____ HO tH3 (BB- 148), Ho OH (BB- 149), NHAc NH
OHCN

/y3 N 0 y3 \ N 0 y6 __ II:i yl ) y6 \ y4 0 y4 0 r ______________ Ha OCH3 (BB-150), HO OH
(BB-151), OHCJ
I 11 H3C,NAN
7Y3 \ N 0 / y3 \

y6 __ ilLy1 y6py1 \Y4 ---A, 0) \(4 A5, r Ha OCH3 (BB- 152), HO OH (BB- 153), Br y Br 7 y3 \ t y6 __ A yl N 0 y6 __ A yl 1\10 I
\ y14 AO) \ r _____________________ \ y4 r 0) Ho -OH (BB- 154), Ho OH (BB- 155), HO N 1\1 t tN/0 /y3 \ k N 0 43 \ 1 y6_p_e_yi...,...\/ ) y6,_p_y_1..,..\/ ) \ +4 0 \ y4 r 0 r __________ Ho 01-1 (BB- 156), Ho OH (BB- 157), NH
/ y3 \ ). N CO2Fmoc II I JL
y6y1 N N NHFmoc I H
\Y4 AO
r ) Ho OH (BB- 158), and NH

).N CO2H
Y3 I *
y6 __ ilLy1 N N NH2 itzl- 0) H
\ r __ Ha OH (BB- 159), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Yl, Y3, Y4, Y6, and r are as described herein (e.g., each r is, independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3, or from 1 to 5)). For example, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be:

H3CI 1111-i) H
ii )......
HO-H=1-0 N 0 H0,¨P-0 N 0 \O r H 0) \OH 0) r \:
- ______________________________________________ _ HO OH (BB- 160) or Ho OH (BB- 161), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).
[000417] In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, is a modified adenosine (e.g., selected from the group consisting of:

/y3 \ e p N N
/y3 \ I II
, 1 1 yo __ p _y1 1\1---- N y6Ly1 N ----- N

\Y4 7\O \Y4 AO
r _________________ /CH3 r __ /OH
HO OH (BB- 162), Ha al-13 (BB- 163), N,)m N,)N
r 1 1 1/3 H3d _____________________________________________________ 1 y6 __ p y 1 N-----N- y6 ( A y 1 N-----N
/y3 \

y 4 0) r ,, r ,, _ .).
) ____________________________________________________ :----Hici OH (BB- 164), HO b (BB-7 NN N,)N

y6 ______ 11:i yl N-----N- y6 __ A y 1 N----N-\Y4 fLI
0) 0) r ________________________________________________________ r __________________ 165), Ha F (BB-166), Ha bl NN
/y3 1 y6 __________ 11:i yl N-----N
I
\Y4 0) r _______________________ (BB- 167), Hici Br (BB- 168), N

p yl N N
y6 __ p yl N--"N y6 __ y4 10) 0) \Y4 r _____ r ______________ Ha 1 (BB- 169), Ho tH3 (BB-/y3 \ N-....)N

I I
y6 ______ p y1 N---N
VI Ao r ) 170), Ha OCH3 (BB-171), N

/y3 \ ,)L
N......)m /y3 N..... m :.Q
.1 µ,6 _\/1 n ii Ni, 4 wo_4 N N
N N I ' T 'T ' y4 0) \Y4 7o \ r Ha OH (BB- 172), HO OH (BB-N......) /y3 \
N1-...,N
1 /y3 \ </ ---I N
ii ii y-Ni6_o_yl N---N 1 v "i 6_1 N N OCH3 \Y4 .A0) " 1 \ y4 AO
r _______________ ) 173), Ha OH (BB- 174), Ho OH
(BB- 175), OH

HN
/y3 \ /1\1-,./IN
N....,) < I / y3 \ i N
< I
y6Ly1 r )N---"kr SCH 3 II
y6_,_p_yl 1,.4 A0)1\1----N SCH3 I
\Y4 AO
\ r Ha OH (BB- 176), Ha OH
OH
HN
N....,) /y3 I N
y6 N
yl ----\ %L
1,4 0)I N OCH3 \ r __ (BB- 177), HO OH (BB- 178), N,) HN /y3 \ H3C- I N

II
/y3 \ N...1 N y6 __ p yl NN
y6 ____ p_y1 N."---Nr \\ y4 I
\ y4 Arj) -O) 1.-Ha OH (BB- 179), Ho OH (BB-------L
/y3 \ e 1 )\1 11 y3 ( _____ eN
y6 ______ p yl N----N y6 NN
___________________________________________________ yl N----N
\ fzi.fzi.
0) 0) r _______________________________ r __ 180), Ha OH (BB- 181), Ha OH

(y3 e,N
y6 __ p yl NN
y4 r 0) (BB- 182), Ho OH (BB- 183), (Y p113 1 _11\1 /Y113 Br¨e_INI
Y6( yl N---"N y6 __ p yl N----N
fz1 0) \ y14 0) r ____________________________________________ r __ Ha OH (BB- 184), Ha OH (BB-N,) 6'13 \ Cl¨e 1 _11\1 Yll3 1¨ 1 _11\1 v6 _______ wl ni y6 __ ' 'T ' y14 \Y4 r ________________________________________________________ 185), Ha OH (BB-186), Ha OH

y6 ___________ p yl N.---\Y4 0) r ________________________ (BB- 187), Ha OH (BB- 188), 7 Yi 1 3 \S¨e NI N,/L
( Y 04 113 )s_ 1 1 y6 ______ p y 1 N ---1 N y6 ________ yi N---N
\ y4 0) 0) r ____________________________________________ r ___ Ha old (BB- 189), Ha OH (BB-7 Y3 S¨e,INI /Y113 S¨e 1 NI
II , yo ___________ p y 1 N----N y6 \Y4 !r ) \Y4 0) r ________________________________________________________ 190), Ho OH (BB-191), Ha OH
\

I I
yoa p yl N"--N!
\Y4 AO
r ________________________ ) (BB- 192), Ho OH (BB- 193), 4113 H N-NI\II /y3 \ NN
y i': yl 2 N---- \ I
6 __ I N y6 _______ ilj) y1 N N CH3 ---..., ---\Y4 0) \Y4 TA )0, 1 r _______________________________________ i r __ 7 Ho ohi (BB- 194), Ho OH (BB-HN
7y3 I I
NN
y6 _____ A yl N-..--tN
I
\Y4 0) r __________________ 195), HO OH (BB-196), HN
N,/L
' N
( Y6( __ E.) yl N----N, yI4 0) r ______________ Ha old (BB-197), HN
N,/L
' N
IY13 ), 1 Y6( F., yl N--\j ( Th, yI4 0) r ______________ Ha old (BB-198), HN
N,) i N
Y3 \ I
y6( ig yl N-----N
yI4 A- ) Ha OH (BB- 199), and N,) N
( ,(23 ), 1 y6 __ !Jr yl N----\N, yi4 0) 5 r ______________ Ho OH (BB- 200) or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Yl, Y3, Y4, Y6, and r are as described herein (e.g., each r is, independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3, or from 1 to 5)).
[000418] In some embodiments, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, is a modified guanosine (e.g., selected from the group consisting of:

( y6 p_yl _ N __ õA NH
jic3 \ ( I
(.4 Ao N---"N NH2 7 y3 N.....,A
1 r y6_y1 N-----NNH2 \ Y
/ r/OH CH3 r ___________ Ha -OH (BB- 201), Ha tH3 /y3 ),, NNH
y6 _p_yl \y4 0) r .-H3d :... .:
(BB- 202), HO OH (BB- 203), /y3 ): NNH /y3 ii I 1 y6 _p_yl N---- kr N H2 y6Ly1 \y4 0) \(.4 r 0) r _ HO 0 (BB- 204), HO

/y3 ________ \ ,N -......,--11-, NH
II _________ I
y6 _________ p _y 1 I
\ y4 0) \ A-(BB- 205), Ha CI (BB- 206), /y3 \
N NH /y3 N 1 NH
/I
1 y6 N N H2 --- L
y614-yA1 oil ---- Nr NH2 0) 1\1 \ r ________________________ \ r __ .7 s ¨
HO Br (BB- 207), Ha CI (BB-208), N,ANH
43 \ J,N NH /y3 \ < I
ii K I
y6 p)_y1 1\1-NN H2 v6_1_ o _v1 N----Nr NH2 I
' 'T ' \y4 Ao) \Y4 AO) r r Ha tH3 (BB- 209), Ho oCH3 N,ANH
/y3\ I
Ni_µ,1 NN N
1 I 1 ,0) H
r ____________________ (BB-210), Ho OH (BB-211), 43 \i N "")Li NH
H < I /y3 \

N/6 __ n_v1 N N N y6 __ p y1 NNN
\Y4 ' if ' I
.AO) H \Y4 -;:\,o) H
r _________ Ho OH (BB- 212), Ha OH (BB-(:) N

43 \

NN 3 \
II \ I
y6_yl N N NH2 y6_Hp_yl 1\1---N NH I
\Nk ....-'N\1 2 \Y4 A. 0) r 213), Ha old (BB- 214), Ha el o\/
N) /y3 \ // I N
\ I
II
y6_1.1,-_: -y1 N---- NNH2 \Y4 i0) r V
(BB-215), Ha OH (BB-216), N )LNH
/y3 \
N) /y3 \
y6_y1 ,,, N NH 1 H3C- I .L
I NI II
ii ....- 1 N-----N NH2 'A
.1(4 cl,1 2 v6_1_o_v \4 Ha OH (BB- 217), Ha ol-1 (BB-/y3 \\ ______________________ e-).(1 NH

y6ziTyl a IN -N NH2 \ r .. 7 218), Ho OH (BB-219), I
NjLNH
/y3 \( __ < N).cH /y3 I
y6_yl N----NL NH2 \,6_4_\,1 N----1\1 NH2 I
" I 1 ) \Y4 AO
\y4 "r\' r __________ ) Ha OH (BB- 220), Ha OH (BB-N,A
7 y3 \ F_ 1 NH
il y6 _____ p_y1 N--- NNH 2 14 Acd \ Y r __ 1 -___ 221), HO OH (BB- 222), N,A NH N,)(NH
43 Cl- I / 3 \ //
X Br-\ I
.---y6 __ ig _y1 N - N NH2 y6_p_p_yl N---"N NH

\y4 0) r \ / r \ /
. _ _ Ho OH (BB- 223), HO OH (BB-N,A NH
/Y3 \
N,A NH 43 \ HS I
1¨ Iii - -6 _i_11:i _s =1 Y ifzi Y,,\ ,cd1\1---NNH2 -6-_-1 Y 1--1:4 1r,,\ .0, iii--NLNEI2 \ ir v __ 7 \Y
v _ __ 1 224), Ho OH (BB- 225), HO OH

/ _ \ \ N......}..
NH
i ri N s¨ I
,6_,_,g_õ1 N N NH2 \Y4 -N\,0 r __ ) (BB- 226), Ho OH (BB- 227), 0 (1:311 r ____________ N,A
/y s_ I I] Fl y3 \ s_N NH
II
y6 __ ilLy1 N N NH2 v/6 ig_wi N-----1 (zi. M A N NH2 \
I
yl 0) / r _________________________________________________ _ 7 Ha OH (BB- 228), Ha OH

V
/y3 s_e NH
ii ---1 y6_y1 N -N NH2 I
\y4 0) r (BB- 229), Ha OH (BB- 230), \

N NH /y3 \
N.....)LNH
/y3 \s¨
\ I
II
L
y6 y1 N------ N N H2 y6_Nit',1A m, ......i, ......
¨ N N
y4 AO) \Y4 0) H
\ r r .. ________________________________________________ ._ Ho OH (BB- 231), Ho ol-I (BB-N 43 \ -.....A NH N.,...)L NH
I <
ii I
y6_y1 N----,..N%L.N..--' y6_y1 1\1----N NH
I
\Y4 0) I (.4 VICI) 2 \
r ___________________________________________________ / r \
232), Ha OH (BB- 233), Ho OH
S
/y3 \
I
ii y6_y1 N N NH2 \Y4 AO) - ____________________ ..
(BB- 234), Ha OH (BB- 235), N-õA ,CH3 0 /ii3 I 11 /Y3 \ H N-N.----)1NH
y6 __ p_y1 ii 0)N----N NH2 2 _____,....
1.1,1,....
I y6_Hp_yl \ yzi \ NI(4 -AO) r r _ Ho OH (BB- 236), and Ha ol-I
(BB- 237), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Yl, Y3, Y4, Y6, and r are as described herein (e.g., each r is, independently, an integer from 0 to 5, such as from 0 to 3, from 1 to 3, or from 1 to 5)).
[000419] In some embodiments, the chemical modification can include replacement of C group at C-5 of the ring (e.g., for a pyrimidine nucleoside, such as cytosine or uracil) with N (e.g., replacement of the >CH group at C-5 with >NRN1 group, wherein RN1 is H
or optionally substituted alkyl). For example, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be:

HNANH H3C,NANH
/0 /0 \
II
HO¨A-0 cr0 HO¨P-0 c)r.L0 I I
\OH r \OH /A
r Ha OH (BB- 238) or Ha OH (BB-239) 0 H3Ci A CH3 HO _________ 0 CH3 \IN-õ ro HO
PI \\A
\ OH 0 OH O
\
/ r ______________________________________________ r Or Ho OH (BB- 240) or Ho OH
(BB- 241), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).
[000420] In another embodiment, the chemical modification can include replacement of the hydrogen at C-5 of cytosine with halo (e.g., Br, Cl, F, or I) or optionally substituted alkyl (e.g., methyl). For example, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be:

/0 \tNc) /9 t ii HO¨P-0 HO-nP-0 N 0 I , I
\OH -70 \OH 0) r ) r Ha OH (BB- 242) or HO OH (BB- 243) NH2 NHAc TBDMS,oN AcON

( NO i 0 HO II:' 0) ) I
OH 0 \
HO
I o OH At / r Or Ha OH (BB- 244) or Ha OH (BB-245), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).
[000421] In yet a further embodiment, the chemical modification can include a fused ring that is formed by the NH2 at the C-4 position and the carbon atom at the position. For example, the building block molecule, which may be incorporated into a polynucleotide, primary construct, or mmRNA, can be:

/ NH

I
\OH () r : __ s Ha OH (BB- 246), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is, independently, an integer from 0 to 5 (e.g., from 0 to 3, from 1 to 3, or from 1 to 5).
Modifications on the Sugar [000422] The modified nucleosides and nucleotides (e.g., building block molecules), which may be incorporated into a polynucleotide, primary construct, or mmRNA
(e.g., RNA or mRNA, as described herein), can be modified on the sugar of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with a number of different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halo, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6_10 aryloxy; optionally substituted C3-8 cycloalkyl;
optionally substituted C3-8 cycloalkoxy; optionally substituted C6_10 aryloxy;
optionally substituted C6_10 aryl-C1_6 alkoxy, optionally substituted C1_12 (heterocyclyl)oxy; a sugar (e.g., ribose, pentose, or any described herein); a polyethyleneglycol (PEG), -0(CH2CH20)õCH2CH2OR, where R is H or optionally substituted alkyl, and n is an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20); "locked" nucleic acids (LNA) in which the 2'-hydroxyl is connected by a C1_6 alkylene or C1_6 heteroalkylene bridge to the 4'-carbon of the same ribose sugar, where exemplary bridges included methylene, propylene, ether, or amino bridges;
aminoalkyl, as defined herein; aminoalkoxy, as defined herein; amino as defined herein;
and amino acid, as defined herein Generally, RNA includes the sugar group ribose, which is a 5-membered ring having an oxygen. Exemplary, non-limiting modified nucleotides include replacement of the oxygen in ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition of a double bond (e.g., to replace ribose with cyclopentenyl or cyclohexenyl);
ring contraction of ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of ribose (e.g., to form a 6- or 7-membered ring having an additional carbon or heteroatom, such as for anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino that also has a phosphoramidate backbone); multicyclic forms (e.g., tricyclo; and "unlocked" forms, such as glycol nucleic acid (GNA) (e.g., R-GNA
or S-GNA, where ribose is replaced by glycol units attached to phosphodiester bonds), threose nucleic acid (TNA, where ribose is replace with a-L-threofuranosyl-(3'¨>2)) , and peptide nucleic acid (PNA, where 2-amino-ethyl-glycine linkages replace the ribose and phosphodiester backbone). The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a polynucleotide, primary construct, or mmRNA molecule can include nucleotides containing, e.g., arabinose, as the sugar.
Modifications on the Nucleobase [000423] The present disclosure provides for modified nucleosides and nucleotides. As described herein "nucleoside" is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof in combination with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as "nucleobase"). As described herein, "nucleotide" is defined as a nucleoside including a phosphate group. The modified nucleotides may by synthesized by any useful method, as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides).
[000424] The modified nucleotide base pairing encompasses not only the standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and/or modified nucleotides comprising non-standard or modified bases, wherein the arrangement of hydrogen bond donors and hydrogen bond acceptors permits hydrogen bonding between a non-standard base and a standard base or between two complementary non-standard base structures. One example of such non-standard base pairing is the base pairing between the modified nucleotide inosine and adenine, cytosine or uracil.
[000425] The modified nucleosides and nucleotides can include a modified nucleobase.
Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobase found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases can be modified or wholly replaced to provide polynucleotides, primary constructs, or mmRNA
molecules having enhanced properties, e.g., resistance to nucleases through disruption of the binding of a major groove binding partner. Table 8 below identifies the chemical faces of each canonical nucleotide. Circles identify the atoms comprising the respective chemical regions.

Table 8 Watson-Crick Major Groove Minor Groove Base-pairing Face Face Face I-Cyt Wine; 0P01 )l .4 .)0 . 0470 &E(SE4 H
Pyrimidines OHO OHO
I./
r,----mi 0 1 f,:
0 o...., _ 0- :.I
=5 ,I <
Uridinel 0-P-I -4 " (3.4!õ..,..:-. -,-, <õ0 c''' 3 s'y ---1.
oi-ioii OtiOil (MON
$012 N
.-,> .4 Adenosine: 0-P7P-W:- `N- 031:0 Purines tict-i. 01-04 01-c=H
0 0 n 110.1:
/ 1 .
GUanasine: 0--P-.0, '.:Thi*iz ol-t6H 01-0N 01-8)H
[000426] In some embodiments, B is a modified uracil. Exemplary modified uracils include those having Formula (b1)-(b5):
Tv\ /Tv' R12c R12c R12c )c R12a N NIA
Rio N Rio m 12a , N
I
I :2 I
/\ 2"
V...., ....... T2" N
N Rii c) Rii N T N '0 ' T2' 1 T2' 1 ' (bl), n't (b2),rvvvv, ' (b3),,vs.A.,-, ' (b4), io R.....,õ.õ.õ...,... _Ri2c N
I
N, ,,,,,L,,, Or (b5), or a pharmaceutically acceptable salt or stereoisomer thereof, [000427] wherein [000428] is a single or double bond;

[000429] each of T", Ti", T2', and T2" is, independently, H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or the combination of Tr and Ti" or the combination of T2' and T2" join together (e.g., as in T2) to form 0 (oxo), S (thio), or Se (seleno);
[000430] each of Vl and V2 is, independently, 0, S, N(R), or C(Rvb), wherein nv is an integer from 0 to 2 and each Rvb is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl);
[000431] Rm is H, halo, optionally substituted amino acid, hydroxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aminoalkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl;
[000432] R" is H or optionally substituted alkyl;
[000433] R12a is H, optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl; and [000434] R12c is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl.
[000435] Other exemplary modified uracils include those having Formula (b6)-(b9):
Ri2c R12c T1 T1 R12c R12b X IR12a F.D12b ....., ,......--..., N N N N
1:1 ill W-1m2 \---Tf W` 2T2 T '2' W
NVIAP T 'SNAP
-7-- (b6), ---r" (b7), I (b8), or I
(b9), or a pharmaceutically acceptable salt or stereoisomer thereof, [000436] wherein [000437] is a single or double bond;
[000438] each of Ty, Tr, T2', and T2" is, independently, H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or the combination of T1' and T" join together (e.g., as in T1) or the combination of T2' and T2"
join together (e.g., as in T2) to form 0 (oxo), S (thio), or Se (seleno), or each Tl and T2 is, independently, 0 (oxo), S (thio), or Se (seleno);
[000439] each of Wl and W2 is, independently, N(Rwa)õ, or C(Rwa), wherein nw is an integer from 0 to 2 and each ea is, independently, H, optionally substituted alkyl, or optionally substituted alkoxy;
[000440] each V3 is, independently, 0, S, N(R), or C(Rva), wherein nv is an integer from 0 to 2 and each Rva is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy , optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an 0-protecting group), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl), and wherein Rva and R12c taken together with the carbon atoms to which they are attached can form optionally substituted cycloalkyl, optionally substituted aryl, or optionally substituted heterocyclyl (e.g., a 5- or 6-membered ring);
[000441] R12a is H, optionally substituted alkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an 0-protecting group), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, optionally substituted carbamoylalkyl, or absent;
[000442] Rub is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkaryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted amino acid, optionally substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an 0-protecting group), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl, [000443] wherein the combination of R12b and Ty or the combination of R12b and R12c can join together to form optionally substituted heterocyclyl; and [000444] R12c is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl.
[000445] Further exemplary modified uracils include those having Formula (b28)-(b31):
T1 T1 Ti Vb 12a R',......................õ,õ.......õ N .õ, R m,, Vb' IA ....................-----.., , R12a R12b R12a RVb"/\ k , T2 (b28), (b29), '-n-r`. (b30), or Rvio. ,R 12a N
\ NT2 (b31), or a pharmaceutically acceptable salt or stereoisomer thereof, [000446] wherein [000447] each of T1 and T2 is, independently, 0 (oxo), S (thio), or Se (seleno);
[000448] each Rvb' and Rvb" is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl), optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkyl (e.g., optionally substituted with hydroxy and/or an 0-protecting group), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl) (e.g., Rvb' is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted aminoalkyl, e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl);
[000449] R12a is H, optionally substituted alkyl, optionally substituted carboxyaminoalkyl, optionally substituted aminoalkyl (e.g., e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl;
and [000450] Rub is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl (e.g., e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl), [000451] optionally substituted alkoxycarbonylacyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl.
[000452] In particular embodiments, Tl is 0 (oxo), and T2 is S (thio) or Se (seleno). In other embodiments, Tl is S (thio), and T2 is 0 (oxo) or Se (seleno). In some embodiments, Rvb' is H, optionally substituted alkyl, or optionally substituted alkoxy.
[000453] In other embodiments, each R12a and R12b is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted hydroxyalkyl. In particular embodiments, R12a is H. In other embodiments, both R12a and R12b are H.

[000454] In some embodiments, each Rvb' of Rub =s5 1 independently, optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, such as any described herein, e.g., trifluoroacetyl). In some embodiments, the amino and/or alkyl of the optionally substituted aminoalkyl is substituted with one or more of optionally substituted alkyl, optionally substituted alkenyl, optionally substituted sulfoalkyl, optionally substituted carboxy (e.g., substituted with an 0-protecting group), optionally substituted hydroxy (e.g., substituted with an 0-protecting group), optionally substituted carboxyalkyl (e.g., substituted with an 0-protecting group), optionally substituted alkoxycarbonylalkyl (e.g., substituted with an 0-protecting group), or N-protecting group. In some embodiments, optionally substituted aminoalkyl is substituted with an optionally substituted sulfoalkyl or optionally substituted alkenyl. In particular embodiments, R12a and Rvb"
are both H.
In particular embodiments, Tl is 0 (oxo), and T2 is S (thio) or Se (seleno).
[000455] In some embodiments, Rvb' is optionally substituted alkoxycarbonylalkyl or optionally substituted carbamoylalkyl.
[000456] In particular embodiments, the optional substituent for R12a5 R12b5 R12c5 or RVa is a polyethylene glycol group (e.g., -(CH2)s2(0CH2CH2)s1(CH2)s30R', wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or C1_20 alkyl); or an amino-polyethylene glycol group (e.g., -NRNi(CH2)s2(CH2CH20)si(CH2)s3NRN1, wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted Ci_6 alkyl).
[000457] In some embodiments, B is a modified cytosine. Exemplary modified cytosines include compounds of Formula (b10)-(b14):

R13a ,R13b R13b R1 3aN,R13b R1 3aN,R13b , ,R16 N

i 5 3"

R15 R15 R 3.
Ti T'5 T
(b10), (b11), (b12), T-.
(b13), or (b14), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein [000458] each of T3' and T3" is, independently, H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or the combination of T3' and T3" join together (e.g., as in T3) to form 0 (oxo), S (thio), or Se (seleno);
[000459] each V4 is, independently, 0, S, N(Rvc)õ,,, or C(Rvc)õ,,, wherein nv is an integer from 0 to 2 and each Rvc is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl), wherein the combination of R13b and Rvc can be taken together to form optionally substituted heterocyclyl;
[000460] each V5 is, independently, N(R), or C(Rvd), wherein nv is an integer from 0 to 2 and each Rvd is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl) (e.g., V5 is ¨CH or N);
[000461] each of R13a and R13b is, independently, H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy, wherein the combination of R13b and R14 can be taken together to form optionally substituted heterocyclyl;
[000462] each R14 is, independently, H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl (e.g., substituted with an 0-protecting group), optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted acyloxyalkyl, optionally substituted amino (e.g., -NHR, wherein R is H, alkyl, aryl, or phosphoryl), azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkyl; and [000463] each of R15 and R16 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.
[000464] Further exemplary modified cytosines include those having Formula (b32)-(b35):
R13a ,R13b ,R13b N
N Ti I
R14N R14 ,R16 R14 A

I I
R15 N 3 R15 N T I 3 N-R13a T
I
NW, I
NW, I 3b , (b32), , (b33), ' Ri (b34), or R13aN,R13b R15 \N/
(b35), or a pharmaceutically acceptable salt or stereoisomer thereof, [000465] wherein [000466] each of T1 and T3 is, independently, 0 (oxo), S (thio), or Se (seleno);
[000467] each of R13' and R13b is, independently, H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy, wherein the combination of R13b and R14 can be taken together to form optionally substituted heterocyclyl;
[000468] each R14 is, independently, H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl (e.g., substituted with an 0-protecting group), optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted acyloxyalkyl, optionally substituted amino (e.g., -NHR, wherein R is H, alkyl, aryl, or phosphoryl), azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted aminoalkyl (e.g., hydroxyalkyl, alkyl, alkenyl, or alkynyl), optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl; and [000469] each of R15 and R16 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl (e.g., R15 is H, and R16 is H or optionally substituted alkyl).
[000470] In some embodiments, R15 is H, and R16 is H or optionally substituted alkyl.
In particular embodiments, R14 is H, acyl, or hydroxyalkyl. In some embodiments, R14 is halo. In some embodiments, both R14 and R15 are H. In some embodiments, both and R16 are H. In some embodiments, each of R14 and R15 and R16 is H. In further embodiments, each of R13a and R13b is independently, H or optionally substituted alkyl.
[000471] Further non-limiting examples of modified cytosines include compounds of Formula (b36):
_Rub N
R14a I
N

R14b ?
NIIIIIP
i (b36) or a pharmaceutically acceptable salt or stereoisomer thereof, [000472] wherein [000473] each R13b is, independently, H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy, wherein the combination of Ri3b and Ri4b can be taken together to form optionally substituted heterocyclyl;
[000474] each Rma and R14b is, independently, H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted haloalkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl (e.g., substituted with an 0-protecting group), optionally substituted hydroxyalkenyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted acyloxyalkyl, optionally substituted amino (e.g., -NHR, wherein R is H, alkyl, aryl, phosphoryl, optionally substituted aminoalkyl, or optionally substituted carboxyaminoalkyl), azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl; and [000475] each of le is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.
[000476] In particular embodiments, R14b is an optionally substituted amino acid (e.g., optionally substituted lysine). In some embodiments, Rma is H.
[000477] In some embodiments, B is a modified guanine. Exemplary modified guanines include compounds of Formula (b15)-(b17):
"

m R21 " R24 \ N ,R19a N N

R (b15), , R22 (b16), or T5 T5"

R17flN
6"
N T.
N

(b17), or a pharmaceutically acceptable salt or stereoisomer thereof, [000478] wherein [000479] each of T4', T4", T5', T5", T6', and T6" is, independently, H, optionally substituted alkyl, or optionally substituted alkoxy, and wherein the combination of T4' and T4" (e.g., as in T4) or the combination of T5' and T5" (e.g., as in T5) or the combination of T6' and T6" (e.g., as in T6) join together form 0 (oxo), S (thio), or Se (seleno);
[000480] each of V5 and V6 is, independently, 0, S, N(R), or C(Rvd)õv, wherein nv is an integer from 0 to 2 and each Rvd is, independently, H, halo, thiol, optionally substituted amino acid, cyano, amidine, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl) , optionally substituted thioalkoxy, or optionally substituted amino;
and [000481] each of R17, R18, R19a, R191), R21, R22, R235 and R24 is, independently, H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted amino acid.
[000482] Exemplary modified guanosines include compounds of Formula (b37)-(b40):
T4 T4' T4 N-_,./IcN.R18 //N N
-----i N ,R18 1 \ 1 &C
N N.R19a I 19b 1 N N
19b , R (b37), ' (b38), ' R19b (b39), Nõ....>c N .R18 R21 __ N---\ %\ N .R19a I N
=I.n.P.Nl I
I
or R19b (b40), or a pharmaceutically acceptable salt or stereoisomer thereof, [000483] wherein [000484] each of T4' is, independently, H, optionally substituted alkyl, or optionally substituted alkoxy, and each T4 is, independently, 0 (oxo), S (thio), or Se (seleno);

[000485] each of R185 R19a, R191)5 and R21 is, independently, H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted amino acid.
[000486] In some embodiments, R18 is H or optionally substituted alkyl. In further embodiments, T4 is oxo. In some embodiments, each of R19a and R19b is, independently, H or optionally substituted alkyl.
[000487] In some embodiments, B is a modified adenine. Exemplary modified adenines include compounds of Formula (b18)-(b20):
R26a R2613 R2613 _R28 R25 __________________________ R25 'NAM
(b18), (b19), or VN

(b20), or a pharmaceutically acceptable salt or stereoisomer thereof, [000488] wherein [000489] each V7 is, independently, 0, S, N(R), or C(Rve), wherein nv is an integer from 0 to 2 and each RVe is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, such as those selected from (1)-(21) for alkyl);
[000490] each R25 is, independently, H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino;
[000491] each of R26a and R26b is, independently, H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoylalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, or polyethylene glycol group (e.g., -(CH2)s2(OCH2CH2)s1(CH2)s3OR', wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or C1_20 alkyl); or an amino-polyethylene glycol group (e.g., -NRNi(CH2)s2(CH2CH20)si(CH2)s3NRN1, wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl);
[000492] each R27 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy or optionally substituted amino;
[000493] each R28 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and [000494] each R29 is, independently, H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoylalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted alkoxy, or optionally substituted amino.
[000495] Exemplary modified adenines include compounds of Formula (b41)-(b43):
R26a R2 6b R26a R26b R26a R26b N N N
N....,....../N N-.........N N-..,...,./N
1 R25 ____ 1 i 1 j N -NR2 7 N---e N"---N
[000496] , (b41), , (b42), or , (b43), or a pharmaceutically acceptable salt or stereoisomer thereof, [000497] wherein [000498] each R25 is, independently, H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino;

[000499] each of R26a and R26b is, independently, H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoylalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted alkoxy, or polyethylene glycol group (e.g., -(CH2)s2(OCH2CH2)s1(CH2)s3OR', wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or C1_20 alkyl); or an amino-polyethylene glycol group (e.g., -NRNi(CH2)s2(CH2CH20)si(CH2)s3NRN1, wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted C1-6 alkyl); and [000500] each R27 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy, or optionally substituted amino.
[000501] In some embodiments, R26a is H, and R26b is optionally substituted alkyl. In some embodiments, each of R26a and R26b is, independently, optionally substituted alkyl.
In particular embodiments, R27 is optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy. In other embodiments, R25 is optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy.
[000502] In particular embodiments, the optional substituent for R26a, R26b, or R29 is a polyethylene glycol group (e.g., -(CH2)s2(OCH2CH2)s1(CH2)s3OR', wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R' is H or C1_20 alkyl); or an amino-polyethylene glycol group (e.g., -NRNi(CH2)s2(CH2CH20)si(CH2)s3NRN1, wherein sl is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each RN1 is, independently, hydrogen or optionally substituted Ci_6 alkyl).
[000503] In some embodiments, B may have Formula (b21):

ex12 R12a (b21), wherein X'2 is, independently, 0, S, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene, xa is an integer from 0 to 3, and R12a and T2 are as described herein.
[000504] In some embodiments, B may have Formula (b22):

R10 it ,R12a N
H I

'NNW
(b22), wherein R1 ' is, independently, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl, and R11, R12a5 T15 and T2 are as described herein.
[000505] In some embodiments, B may have Formula (b23):

IR10N R1 2a N T
fvvvv, (b23), wherein Rlo is optionally substituted heterocyclyl (e.g., optionally substituted furyl, optionally substitued thienyl, or optionally substitued pyrrolyl), optionally substituted aryl (e.g., optionally substituted phenyl or optionally substituted naphthyl), or any substituent described herein (e.g., for R1 ) ;and wherein R"
(e.g., H or any substituent described herein), R12a (e.g., H or any substituent described herein), Tl (e.g., oxo or any substituent described herein), and T2 (e.g., oxo or any substituent described herein) are as described herein.
In some embodiments, B may have Formula (b24):
Ri 3aN, R13b R14' )1 N

N T
(b24), wherein R14' is, independently, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkaryl, optionally substituted alkheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl, and R13a, R13b, R15, and T3 are as described herein.
[000506] In some embodiments, B may have Formula (b25):
R13a R13b R14' N N
R15 N-r3 (b25), wherein R14' is optionally substituted heterocyclyl (e.g., optionally substituted furyl, optionally substitued thienyl, or optionally substitued pyrrolyl), optionally substituted aryl (e.g., optionally substituted phenyl or optionally substituted naphthyl), or any substituent described herein (e.g., for R14 or R14'); and wherein R13a (e.g., H or any substituent described herein), R13b (e.g., H or any substituent described herein), R15 (e.g., H or any substituent described herein), and T3 (e.g., oxo or any substituent described herein) are as described herein.
[000507] In some embodiments, B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil. In some embodiments, B may be:

N
'N NH2 e N N
N N N
(b26) or + (b27).
[000508] In some embodiments, the modified nucleobase is a modified uracil.
Exemplary nucleobases and nucleosides having a modified uracil include pseudouridine (y), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethy1-2-thio-uridine (mcm5s2U), 5-aminomethy1-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 5-methylaminomethy1-2-thio-uridine (mnm5s2U), 5-methylaminomethy1-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), carboxymethylaminomethy1-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (Tm5U), 1-taurinomethyl-pseudouridine, taurinomethy1-2-thio-uridine('rm5s2U), 1-taurinomethy1-4-thio-pseudouridine, 5-methyl-uridine (m5U, i.e., having the nucleobase deoxythymine), 1-methylpseudouridine (mly), 5-methy1-2-thio-uridine (m5s2U), 1-methy1-4-thio-pseudouridine (m1s4lif), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m3y), 2-thio-1-methyl-pseudouridine, 1-methyl-1 -deaza-pseudouridine, 2-thio- 1 -methyl- 1 -de aza-ps eudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine (also known as 1-methylpseudouridine (mly)), 3-(3-amino-3-carboxypropyl)uridine (acp3U), 1-methy1-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 kv), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-0-methyl-uridine (Um), 5,2'-0-dimethyl-uridine (m5Um), 2'-0-methyl-pseudouridine (wm), 2-thio-2'-0-methyl-uridine (s2Um), 5-methoxycarbonylmethy1-2'-0-methyl-uridine (mcm5Um), 5-carbamoylmethy1-2'-0-methyl-uridine (ncm5Um), 5-carboxymethylaminomethy1-2'-0-methyl-uridine (cmnm5Um), 3,2'-0-dimethyl-uridine (m3Um), 5-(isopentenylaminomethyl)-2'-0-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-0H-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)uridine.
[000509] In some embodiments, the modified nucleobase is a modified cytosine.
Exemplary nucleobases and nucleosides haying a modified cytosine include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m3C), N4-acetyl-cytidine (ac4C), 5-formyl-cytidine (f5C), N4-methyl-cytidine (m4C), 5-methyl-cytidine (m5C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s2C), 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, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), a-thio-cytidine, 2'-0-methyl-cytidine (Cm), 5,2'-0-dimethyl-cytidine (m5Cm), N4-acety1-2'-0-methyl-cytidine (ac4Cm), N4,2'-0-dimethyl-cytidine (m4Cm), 5-formy1-2'-0-methyl-cytidine (f5Cm), N4,N4,2'-0-trimethyl-cytidine (m42Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-0H-ara-cytidine.
[000510] In some embodiments, the modified nucleobase is a modified adenine.
Exemplary nucleobases and nucleosides having a modified adenine include 2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine (m6A), 2-methylthio-N6-methyl-adenosine (ms2m6A), N6-isopentenyl-adenosine (i6A), 2-methylthio-N6-isopentenyl-adenosine (ms2i6A), N6-(cis-hydroxyisopentenyl)adenosine (io6A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms2io6A), N6-glycinylcarbamoyl-adenosine (g6A), N6-threonylcarbamoyl-adenosine (t6A), N6-methyl-N6-threonylcarbamoyl-adenosine (m6t6A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms2g6A), N6,N6-dimethyl-adenosine (m62A), N6-hydroxynorvalylcarbamoyl-adenosine (hn6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms2hn6A), N6-acetyl-adenosine (ac6A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, a-thio-adenosine, 2'-0-methyl-adenosine (Am), N6,2'-0-dimethyl-adenosine (m6Am), N6,N6,2'-0-trimethyl-adenosine (m62Am), 1,2'-0-dimethyl-adenosine (mlAm), 2'-0-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecy1)-adenosine.
[000511] In some embodiments, the modified nucleobase is a modified guanine.
Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (mil), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW), peroxywybutosine (o2yW), hydroxywybutosine (OHyW), undermodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queuosine (Q), epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethy1-7-deaza-guanosine (preQi), archaeosine (G d), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m7G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m' G), N2-methyl-guanosine (m2G), N2,N2-dimethyl-guanosine (m22G), N2,7-dimethyl-guanosine (m2'7G), N2, N2,7-dimethyl-guanosine (m2'2'7G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methy1-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethy1-6-thio-guanosine, a-thio-guanosine, 2'-0-methyl-guanosine (Gm), N2-methy1-2'-0-methyl-guanosine (m2Gm), N2,N2-dimethy1-2'-0-methyl-guanosine (m22Gm), 1-methy1-2'-0-methyl-guanosine (m' Gm), N2,7-dimethy1-2'-0-methyl-guanosine (m2'7Gm), 2'-0-methyl-inosine (Im), 1,2'-0-dimethyl-inosine (m1Im), and 2'-0-ribosylguanosine (phosphate) (Gr(p)).
[000512] The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine or pyrimidine analog. For example, the nucleobase can each be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase can also include, for example, naturally-occurring and synthetic derivatives of a base, including pyrazolo[3,4-d]pyrimidines, 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazine, pyridazine; and 1,3,5 triazine.
When the nucleotides are depicted using the shorthand A, G, C, T or U, each letter refers to the representative base and/or derivatives thereof, e.g., A includes adenine or adenine analogs, e.g., 7-deaza adenine).
Modifications on the Internucleoside Linkage [000513] The modified nucleotides, which may be incorporated into a polynucleotide, primary construct, or mmRNA molecule, can be modified on the internucleoside linkage (e.g., phosphate backbone). Herein, in the context of the polynucleotide backbone, the phrases "phosphate" and "phosphodiester" are used interchangeably. Backbone phosphate groups can be modified by replacing one or more of the oxygen atoms with a different substituent. Further, the modified nucleosides and nucleotides can include the wholesale replacement of an unmodified phosphate moiety with another internucleoside linkage as described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioate, phosphoroselenates, boranophosphates,boranophosphate esters, hydrogen phosphonates, phosphoramidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. The phosphate linker can also be modified by the replacement of a linking oxygen with nitrogen (bridged phosphoramidates), sulfur (bridged phosphorothioates), and carbon (bridged methylene-phosphonates).
[000514] 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 polynucleotides, primary constructs, or mmRNA molecules are expected to also reduce the innate immune response through weaker binding/activation of cellular innate immune molecules.
[000515] In specific embodiments, a modified nucleoside includes an alpha-thio-nucleoside (e.g., 5'-0-(1-thiophosphate)-adenosine, 5'-0-(1-thiophosphate)-cytidine (a-thio-cytidine), 5'-0-(1-thiophosphate)-guanosine, 5'-0-(1-thiophosphate)-uridine, or 5'-0-(1-thiophosphate)-pseudouridine).
[000516] Other internucleoside linkages that may be employed according to the present invention, including internucleoside linkages which do not contain a phosphorous atom, are described herein below.
Combinations of Modified Sugars, Nucleobases, and Internucleoside Linkages [000517] The polynucleotides, primary constructs, and mmRNA of the invention can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein. For examples, any of the nucleotides described herein in Formulas (Ia), (Ia-1)-(Ia-3), (Ib)-(If), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IV1), and (IXa)-(IXr) can be combined with any of the nucleobases described herein (e.g., in Formulas (b1)-(b43) or any other described herein).
Synthesis of Polypeptides, Primary Constructs, and mmRNA Molecules [000518] The polypeptides, primary constructs, and mmRNA molecules for use in accordance with the invention may be prepared according to any useful technique, as described herein. The modified nucleosides and nucleotides used in the synthesis of polynucleotides, primary constructs, and mmRNA molecules disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. Where typical or preferred process conditions (e.g., reaction temperatures, times, mole ratios of reactants, solvents, pressures, etc.) are provided, a skilled artisan would be able to optimize and develop additional process conditions.
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.
[000519] 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.
[000520] Preparation of polypeptides, primary constructs, and mmRNA molecules of the present invention 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.
[000521] 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.
[000522] 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 nucleosides and nucleotides (e.g., building block molecules) can be prepared according to the synthetic methods described in Ogata et al., J. Org. Chem.
74:2585-2588 (2009); Purmal et al., Nucl. Acids Res. 22(1): 72-78, (1994); Fukuhara et al., Biochemistry, 1(4): 563-568 (1962); and Xu et al., Tetrahedron, 48(9): 1729-(1992), each of which are incorporated by reference in their entirety.
[000523] The polypeptides, primary constructs, and mmRNA of the invention may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotide (e.g., purine or pyrimidine, or any one or more or all of A, G, U, C) may or may not be uniformly modified in a polynucleotide of the invention, or in a given predetermined sequence region thereof (e.g. one or more of the sequence regions represented in Figure 1). In some embodiments, all nucleotides X in a polynucleotide of the invention (or in a given sequence region thereof) are modified, wherein X may any one of nucleotides A, G, U, C, or any one of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C, G+U+C or A+G+C.
[000524] Different sugar modifications, nucleotide modifications, and/or internucleoside linkages (e.g., backbone structures) may exist at various positions in the polynucleotide, primary construct, or mmRNA. 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 polynucleotide, primary construct, or mmRNA such that the function of the polynucleotide, primary construct, or mmRNA is not substantially decreased. A
modification may also be a 5' or 3' terminal modification. The polynucleotide, primary construct, or mmRNA may contain from about 1% to about 100% modified nucleotides (either in relation to overall nucleotide content, or in relation to one or more types of nucleotide, i.e. any one or more of A, G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100%).
[000525] In some embodiments, the polynucleotide, primary construct, or mmRNA
includes a modified pyrimidine (e.g., a modified uracil/uridine/U or modified cytosine/cytidine/C). In some embodiments, the uracil or uridine (generally:
U) in the polynucleotide, primary construct, or mmRNA molecule may be replaced with from about 1% to about 100% of a modified uracil or modified uridine (e.g., from 1%
to 20%, from 1% to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100% of a modified uracil or modified uridine). The modified uracil or uridine can be replaced by a compound having a single unique structure or by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures, as described herein). In some embodiments, the cytosine or cytidine (generally: C) in the polynucleotide, primary construct, or mmRNA molecule may be replaced with from about 1% to about 100%
of a modified cytosine or modified cytidine (e.g., from 1% to 20%, from 1% to 25%, from 1%
to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%, from 1% to 90%, from 1%
to 95%, from 10% to 20%, from 10% to 25%, from 10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10% to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to 50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%, from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%, from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%, from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%, from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%, from 90% to 100%, and from 95% to 100% of a modified cytosine or modified cytidine). The modified cytosine or cytidine can be replaced by a compound having a single unique structure or by a plurality of compounds having different structures (e.g., 2, 3, 4 or more unique structures, as described herein).
In some embodiments, the present disclosure provides methods of synthesizing a polynucleotide, primary construct, or mmRNA (e.g., the first region, first flanking region, or second flanking region) including n number of linked nucleosides having Formula (Ia-1):
___ Y1 Y5 R-R1-(RR2 Y"m Y3=P _____________ - (Ia-1), comprising:
a) reacting a nucleotide of Formula (IV-1):
B
3 =
RR4 \
(tk;r2 P2 m (IV-1), with a phosphoramidite compound of Formula (V-1):
P -Y-Y5 Ui R31_ R5 9 2) 3 Y m P\ 1 O-P\ _( (V-1), wherein Y9 is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino, thiol, optionally substituted amino acid, or a peptide (e.g., including from 2 to 12 amino acids); and each Pl, P2, and P3 is, independently, a suitable protecting group; and 0 denotes a solid support;
to provide a polynucleotide, primary construct, or mmRNA of Formula (VI-1):

R5v2 Y9-P2)m P\3 I
O-P
vi B
R3 1-Lj cr2Y9-4m (VI-1), and b) oxidizing or sulfurizing the polynucleotide, primary construct, or mmRNA of Formula (V) to yield a polynucleotide, primary construct, or mmRNA of Formula (V11-1):
R3 1-Lji R5 2 (Y9-P2im p3 Y
' \ I 3 y5 Cr2 \Y9-P)m (VII-1), and c) removing the protecting groups to yield the polynucleotide, primary construct, or mmRNA of Formula (Ia).
[000526] In some embodiments, steps a) and b) are repeated from 1 to about 10,000 times. In some embodiments, the methods further comprise a nucleotide (e.g., mmRNA
molecule) selected from the group consisting of A, C, G and U adenosine, cytosine, guanosine, and uracil. In some embodiments, the nucleobase may be a pyrimidine or derivative thereof In some embodiments, the polynucleotide, primary construct, or mmRNA is translatable.

[000527] Other components of polynucleotides, primary constructs, and mmRNA
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 nucleotide modifications. In such embodiments, nucleotide modifications may also be present in the translatable region. Also provided are polynucleotides, primary constructs, and mmRNA containing a Kozak sequence.
[000528] Exemplary syntheses of modified nucleotides, which are incorporated into a modified nucleic acid or mmRNA, e.g., RNA or mRNA, are provided below in Scheme 1 through Scheme 11. Scheme 1 provides a general method for phosphorylation of nucleosides, including modified nucleosides.
Scheme 1 /1.1¨/ /1.1¨/
------(cL-- --------N ------(Ct--------N
Z \ N)(N \ ) ii N 1) POCI3 0 0 0 HO _________________________ IP e0¨P¨O¨P¨O¨P-0 )0 2) Pyrophosphate 1 1 1 0 o e oe ..----o----?
OH OH OH OH
[000529] Various protecting groups may be used to control the reaction. For example, Scheme 2 provides the use of multiple protecting and deprotecting steps to promote phosphorylation at the 5' position of the sugar, rather than the 2' and 3' hydroxyl groups.
Scheme 2 _"-------N _----------N
\ ) Acetone/H+ HO /k1 HO N \ N) N
0-..,..\j Ac20 H

_---"---------N
_---"-------N N \ N) N \ N) Ac0 Ac0 Dowex Ii+ N
______________________________________________ ( ( 0 ph3c.

1, H2N
I) OH-_--- ----N
_------------N rij \ ) N \
Ac0 N) 0 0 0 2) POCI3 N
%1:,p//
N 3) Pyrophosphate 8 0 0/ 0 ....õ...-0.-____ 4) H+
e e e ____ OH OH

Ph3C/ CPh3 [000530] Modified nucleotides can be synthesized in any useful manner. Schemes 3, 4, and 7 provide exemplary methods for synthesizing modified nucleotides having a modified purine nucleobase; and Schemes 5 and 6 provide exemplary methods for synthesizing modified nucleotides having a modified pseudouridine or pseudoisocytidine, respectively.

Scheme 3 o o N----N........,NCH3 NH
< 1 < 1 N"-N"----NNH2 N NH2 CH3I/heat HO
HO
___________________________________ 0/1 c0 <L1 OH OH
OH OH
1) POCI3 2) Pyrophosphate V

Ne0H3 0 0 0 ( 1 N.......-........ ,.."-.., 0 0--o--o¨i-0 I , 01 e 0 e 01e <LI
OH OH
Scheme 4 e o e l o e \
< 1 0 ll 0 ll 0 11 Nr-----.'"eL'NH2 o 0¨P¨O¨P¨O¨P-0.
N"----'-N-----.'"NF12 I) POCI3 I
HO ol e O e o e c _ c0 2) Pyrophosphate OH OH
OH OH
Scheme 5 HN/\NH N NH
RBr/Heat R = alkyl, alkenyl, HO _________________ ally!, and benzyl __ HO c0 CcL>
OH OH OH OH
1) POCI3 2) Pyrophosphate N NH

8 0¨P ¨0¨P ¨0¨P-0 oe oe oe OH OH

Scheme 6 R...N..k.õ.N
HNN
0 RBr/Heat R = alkyl, alkenyl, HO _____________________________ allyl, and benzyl HO
c0 (cLI
OH OH OH OH
1) POCI3 2) Pyrophosphate V

R
N ' N
===.1 %. ..,....õ....-..õ

II II II
eo¨P¨O¨P¨O¨P-0-Oe oe oe OH OH

Scheme 7 CI

N-................,N
< 1 N.......,..õ..-"'\\...N
N-------NI.---NH2 < 1 , N,...----,,, HO CH3N H2/ Heat N NH2 ---õ,..
HO
00. ====,....
c0 OH OH
OH OH
1) POCI3 ,1 2) Pyrophosphate N.................N
< 1 8 e e OH OH
[000531] Schemes 8 and 9 provide exemplary syntheses of modified nucleotides.
Scheme 10 provides a non-limiting biocatalytic method for producing nucleotides.

Scheme 8 AcOOH cc Ph Pd(0) 3 Ac0Cr OAc Ac20 Enzymatic Hydrolysis (/ INN Ph3Pd(0) H0.0õ,...N--\<
Hahn4.0Ac Uracil (1) 0504 (2) Acetone, Ts0H/......, ID
(/ INF! 01 ) (/ NH
H016.24=N--\( (1) (Et0)2POCH2C7Ts 0...2.00AN-i _õ,_ 0,0 00 (2)TMSil (1) DCC, Morpholine (2) Pyrophosphate - , 0 *
(/ NH
- 0...20N--c( Scheme 9 HO

zzlo rstz-NH2 Fin \.......
Ph3P(Pd) _N ......
).. _ \

HO OH

1 1) H-2) -OH, heat 0 Frs1 H
H __________ 1 ¨N
\Z 1) POCI3 HO/----sON 2) Pyrophosphate HO OH
pO¨OH
H

(:)=1:(-0H
b P:¨OH

HO,p' 0' %
OH
Scheme 10 HO -0,1,0 B enzyme, ATP P B
_0_?1 yeast enzymes, B
, -4 OH OH
[000532] Scheme 11 provides an exemplary synthesis of a modified uracil, where the Ni position is modified with Rim, as provided elsewhere, and the 5'-position of ribose is phosphorylated. Tl, T2, R12a, R12b, and r are as provided herein. This synthesis, as well as optimized versions thereof, can be used to modify other pyrimidine nucleobases and purine nucleobases (see e.g., Formulas (b1)-(b43)) and/or to install one or more phosphate groups (e.g., at the 5' position of the sugar). This alkylating reaction can also be used to include one or more optionally substituted alkyl group at any reactive group (e.g., amino group) in any nucleobase described herein (e.g., the amino groups in the Watson-Crick base-pairing face for cytosine, uracil, adenine, and guanine).
Scheme 11 Ti Ti Ti ,R12a R12b/R12a R12b,, ,R12a HN N N N

R12bX/ileat 7 0 \
1) POC13 (X is halo) HO-2) Pyi ()phosphate HO
c0 0 \ 01H /r OH OH OH OH OH OH
Combinations of Nucleotides in mmRNA
[000533] Further examples of modified nucleotides and modified nucleotide combinations are provided below in Table 9. These combinations of modified nucleotides can be used to form the polypeptides, primary constructs, or mmRNA
of the invention. Unless otherwise noted, the modified nucleotides may be completely substituted for the natural nucleotides of the modified nucleic acids or mmRNA
of the invention. As a non-limiting example, the natural nucleotide uridine may be substituted with a modified nucleoside described herein. In another non-limiting example, the natural nucleotide uridine may be partially substituted (e.g., about 0.1%, 1%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99.9%) with at least one of the modified nucleoside disclosed herein.
Table 9 Modified Nucleotide Modified Nucleotide Combination a-thio-cytidine a-thio-cytidine/5-iodo-uridine a-thio-cytidine/Nl-methyl-pseudouridine a-thio-cytidine/a-thio-uridine a-thio-cytidine/5-methy1-uridine a-thio-cytidine/pseudo-uridine about 50% of the cytosines are a-thio-cytidine pseudoisocytidine pseudoisocytidine/5-iodo-uridine pseudoisocytidine/Nl-methyl-pseudouridine pseudoisocytidine/a-thio-uridine pseudoisocytidine/5-methyl-uridine pseudoisocytidine/pseudouridine about 25% of cytosines are pseudoisocytidine pseudoisocytidine/about 50% of uridines are N1-methyl-pseudouridine and about 50% of uridines are pseudouridine pseudoisocytidine/about 25% of uridines are N1-methyl-pseudouridine and about 25% of uridines are pseudouridine pyrrolo-cytidine pyrrolo-cytidine/5-iodo-uridine pyrrolo-cytidine/Nl-methyl-pseudouridine pyrrolo-cytidine/a-thio-uridine pyrrolo-cytidine/5-methyl-uridine pyrrolo-cytidine/pseudouridine about 50% of the cytosines are pyrrolo-cytidine 5-methyl-cytidine 5-methyl-cytidine/5-iodo-uridine 5-methyl-cytidine/N1-methyl-pseudouridine 5-methyl-cytidine/a-thio-uridine 5-methyl-cytidine/5-methyl-uridine 5-methyl-cytidine/pseudouridine about 25% of cytosines are 5-methyl-cytidine about 50% of cytosines are 5-methyl-cytidine 5-methyl-cytidine/5-methoxy-uridine 5-methyl-cytidine/5-bromo-uridine 5-methyl-cytidine/2-thio-uridine 5-methyl-cytidine/about 50% of uridines are 2-thio-uridine about 50% of uridines are 5-methyl-cytidine/ about 50%
of uridines are 2-thio-uridine N4-acetyl-cytidine N4-acetyl-cytidine /5-iodo-uridine N4-acetyl-cytidine /Nl-methyl-pseudouridine N4-acetyl-cytidine /a-thio-uridine N4-acetyl-cytidine /5-methyl-uridine N4-acetyl-cytidine /pseudouridine about 50% of cytosines are N4-acetyl-cytidine about 25% of cytosines are N4-acetyl-cytidine N4-acetyl-cytidine /5-methoxy-uridine N4-acetyl-cytidine /5-bromo-uridine N4-acetyl-cytidine /2-thio-uridine about 50% of cytosines are N4-acetyl-cytidine/ about 50% of uridines are 2-thio-uridine [000534] Further examples of modified nucleotide combinations are provided below in Table 10. These combinations of modified nucleotides can be used to form the polypeptides, primary constructs, or mmRNA of the invention.
Table 10 Modified Nucleotide __ Modified Nucleotide Combination _ modified cytidine modified cytidine with (b10)/pseudouridine having one or more modified cytidine with (b10)/N1-methyl-pseudouridine nucleobases of Formula modified cytidine with (b10)/5-methoxy-uridine (b10) modified cytidine with (b10)/5-methyl-uridine modified cytidine with (b10)/5-bromo-uridine modified cytidine with (b10)/2-thio-uridine about 50% of cytidine substituted with modified cytidine (b10)/
about 50% of uridines are 2-thio-uridine modified cytidine modified cytidine with (b32)/pseudouridine having one or more modified cytidine with (b32)/N1-methyl-pseudouridine nucleobases of Formula modified cytidine with (b32)/5-methoxy-uridine (b32) modified cytidine with (b32)/5-methyl-uridine modified cytidine with (b32)/5-bromo-uridine modified cytidine with (b32)/2-thio-uridine about 50% of cytidine substituted with modified cytidine (b32)/
about 50% of uridines are 2-thio-uridine modified uridine modified uridine with (b 1)! N4-acetyl-cytidine having one or more modified uridine with (b 1)! 5-methyl-cytidine nucleobases of Formula (b 1) modified uridine modified uridine with (b8)/ N4-acetyl-cytidine having one or more modified uridine with (b8)/ 5-methyl-cytidine nucleobases of Formula (b8) modified uridine modified uridine with (b28)/ N4-acetyl-cytidine having one or more modified uridine with (b28)/ 5-methyl-cytidine nucleobases of Formula (b28) modified uridine modified uridine with (b29)/ N4-acetyl-cytidine having one or more modified uridine with (b29)/ 5-methyl-cytidine nucleobases of Formula (b29) modified uridine modified uridine with (b30)/ N4-acetyl-cytidine having one or more modified uridine with (b30)/ 5-methyl-cytidine nucleobases of Formula (b30) [000535] In some embodiments, at least 25% of the cytosines are replaced by a compound of Formula (b10)-(b14) (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%).
[000536] In some embodiments, at least 25% of the uracils are replaced by a compound of Formula (b1)-(b9) (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%).

[000537] In some embodiments, at least 25% of the cytosines are replaced by a compound of Formula (b10)-(b14), and at least 25% of the uracils are replaced by a compound of Formula (b1)-(b9) (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%).
IV. Pharmaceutical Compositions Formulation, Administration, Delivery and Dosing [000538] The present invention provides polynucleotides, primary constructs and mmRNA compositions and complexes in combination with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions may optionally comprise one or more additional active substances, e.g. therapeutically and/or prophylactically active substances. 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).
[000539] In some embodiments, compositions are administered to humans, human patients or subjects. For the purposes of the present disclosure, the phrase "active ingredient" generally refers to polynucleotides, primary constructs and mmRNA
to be delivered as described herein.
[000540] 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 any other animal, e.g., to non-human animals, e.g.
non-human mammals. 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 poultry, chickens, ducks, geese, and/or turkeys.
[000541] 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, dividing, shaping and/or packaging the product into a desired single- or multi-dose unit.
[000542] A pharmaceutical composition in accordance with the invention 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.
[000543] Relative amounts of the active ingredient, the pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention 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%, e.g., between .5 and 50%, between 1-30%, between 5-80%, at least 80% (w/w) active ingredient.
Formulations [000544] The polynucleotide, primary construct, and mmRNA of the invention can be formulated using one or more excipients to: (1) increase stability; (2) increase cell transfection; (3) permit the sustained or delayed release (e.g., from a depot formulation of the polynucleotide, primary construct, or mmRNA); (4) alter the biodistribution (e.g., target the polynucleotide, primary construct, or mmRNA to specific tissues or cell types);
(5) increase the translation of encoded protein in vivo; and/or (6) alter the release profile of encoded protein in vivo. In addition to traditional excipients such as 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, excipients of the present invention can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, peptides, proteins, cells transfected with polynucleotide, primary construct, or mmRNA
(e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics and combinations thereof. Accordingly, the formulations of the invention can include one or more excipients, each in an amount that together increases the stability of the polynucleotide, primary construct, or mmRNA, increases cell transfection by the polynucleotide, primary construct, or mmRNA, increases the expression of polynucleotide, primary construct, or mmRNA encoded protein, and/or alters the release profile of polynucleotide, primary construct, or mmRNA encoded proteins.
Further, the primary construct and mmRNA of the present invention may be formulated using self-assembled nucleic acid nanoparticles.
[000545] 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 associating the active ingredient with an excipient and/or one or more other accessory ingredients.
[000546] 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" refers to a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient.
The amount of the active ingredient may generally be equal to the dosage of the active ingredient which would be administered to a subject and/or a convenient fraction of such a dosage including, but not limited to, one-half or one-third of such a dosage.
[000547] 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 may vary, depending upon the identity, size, and/or condition of the subject being treated and further depending upon the route by which the composition is to be administered. For example, the composition may comprise between 0.1% and 99% (w/w) of the active ingredient.

[000548] In some embodiments, the formulations described herein may contain at least one mmRNA. As a non-limiting example, the formulations may contain 1, 2, 3, 4 or 5 mmRNA. In one embodiment the formulation may contain modified mRNA encoding proteins selected from categories such as, but not limited to, human proteins, veterinary proteins, bacterial proteins, biological proteins, antibodies, immunogenic proteins, therapeutic peptides and proteins, secreted proteins, plasma membrane proteins, cytoplasmic and cytoskeletal proteins, intrancellular membrane bound proteins, nuclear proteins, proteins associated with human disease and/or proteins associated with non-human diseases. In one embodiment, the formulation contains at least three modified mRNA encoding proteins. In one embodiment, the formulation contains at least five modified mRNA encoding proteins.
[000549] Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes, but is not limited to, 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, and the like, as suited to the particular dosage form desired.
Various excipients for formulating pharmaceutical compositions and techniques for preparing the composition are known in the art (see Remington: The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006;
incorporated herein by reference in its entirety). The use of a conventional excipient medium may be contemplated within the scope of the present disclosure, except insofar as any conventional excipient medium may be 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.
[000550] In some embodiments, the particle size of the lipid nanoparticle may be increased and/or decreased. The change in particle size may be able to help counter biological reaction such as, but not limited to, inflammation or may increase the biological effect of the modified mRNA delivered to mammals.
[000551] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, surface active agents and/or emulsifiers, preservatives, buffering agents, lubricating agents, and/or oils.
Such excipients may optionally be included in the pharmaceutical formulations of the invention.
Lipidoids [000552] The synthesis of lipidoids has been extensively described and formulations containing these compounds are particularly suited for delivery of polynucleotides, primary constructs or mmRNA (see Mahon et al., Bioconjug Chem. 2010 21:1448-1454;
Schroeder et al., J Intern Med. 2010 267:9-21; Akinc et al., Nat Biotechnol.
2008 26:561-569; Love et al., Proc Natl Acad Sci U S A. 2010 107:1864-1869; Siegwart et al., Proc Natl Acad Sci U S A. 2011108:12996-3001; all of which are incorporated herein in their entireties).
[000553] While these lipidoids have been used to effectively deliver double stranded small interfering RNA molecules in rodents and non-human primates (see Akinc et al., Nat Biotechnol. 2008 26:561-569; Frank-Kamenetsky et al., Proc Natl Acad Sci U
S A.
2008 105:11915-11920; Akinc et al., Mol Ther. 2009 17:872-879; Love et al., Proc Natl Acad Sci USA. 2010 107:1864-1869; Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; all of which is incorporated herein in their entirety), the present disclosure describes their formulation and use in delivering single stranded polynucleotides, primary constructs, or mmRNA. Complexes, micelles, liposomes or particles can be prepared containing these lipidoids and therefore, can result in an effective delivery of the polynucleotide, primary construct, or mmRNA, as judged by the production of an encoded protein, following the injection of a lipidoid formulation via localized and/or systemic routes of administration. Lipidoid complexes of polynucleotides, primary constructs, or mmRNA can be administered by various means including, but not limited to, intravenous, intramuscular, or subcutaneous routes.
[000554] In vivo delivery of nucleic acids may be affected by many parameters, including, but not limited to, the formulation composition, nature of particle PEGylation, degree of loading, oligonucleotide to lipid ratio, and biophysical parameters such as, but not limited to, particle size (Akinc et al., Mol Ther. 2009 17:872-879; herein incorporated by reference in its entirety). As an example, small changes in the anchor chain length of poly(ethylene glycol) (PEG) lipids may result in significant effects on in vivo efficacy.

Formulations with the different lipidoids, including, but not limited to penta[3-(1-laurylaminopropionyl)]-triethylenetetramine hydrochloride (TETA-5LAP; aka 98N12-5, see Murugaiah et al., Analytical Biochemistry, 401:61(2010); herein incorporated by reference in its entirety), C12-200 (including derivatives and variants), and MD1, can be tested for in vivo activity.
[000555] The lipidoid referred to herein as "98N12-5" is disclosed by Akinc et al., Mol Ther. 2009 17:872-879 and is incorporated by reference in its entirety. (See Figure 2) [000556] The lipidoid referred to herein as "C12-200" is disclosed by Love et al., Proc Natl Acad Sci U S A. 2010 107:1864-1869 (see Figure 2) and Liu and Huang, Molecular Therapy. 2010 669-670 (see Figure 2); both of which are herein incorporated by reference in their entirety. The lipidoid formulations can include particles comprising either 3 or 4 or more components in addition to polynucleotide, primary construct, or mmRNA. As an example, formulations with certain lipidoids, include, but are not limited to, 98N12-5 and may contain 42% lipidoid, 48% cholesterol and 10% PEG (C14 alkyl chain length). As another example, formulations with certain lipidoids, include, but are not limited to, C12-200 and may contain 50% lipidoid, 10%
disteroylphosphatidyl choline, 38.5% cholesterol, and 1.5% PEG-DMG.
[000557] In one embodiment, a polynucleotide, primary construct, or mmRNA
formulated with a lipidoid for systemic intravenous administration can target the liver.
For example, a final optimized intravenous formulation using polynucleotide, primary construct, or mmRNA, and comprising a lipid molar composition of 42% 98N12-5, 48%
cholesterol, and 10% PEG-lipid with a final weight ratio of about 7.5 to 1 total lipid to polynucleotide, primary construct, or mmRNA, and a C14 alkyl chain length on the PEG
lipid, with a mean particle size of roughly 50-60 nm, can result in the distribution of the formulation to be greater than 90% to the liver.(see, Akinc et al., Mol Ther.
2009 17:872-879; herein incorporated by reference in its entirety). In another example, an intravenous formulation using a C12-200 (see US provisional application 61/175,770 and published international application W02010129709, each of which is herein incorporated by reference in their entirety) lipidoid may have a molar ratio of 50/10/38.5/1.5 of C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG, with a weight ratio of 7 to 1 total lipid to polynucleotide, primary construct, or mmRNA, and a mean particle size of 80 nm may be effective to deliver polynucleotide, primary construct, or mmRNA
to hepatocytes (see, Love et al., Proc Natl Acad Sci U S A. 2010 107:1864-1869 herein incorporated by reference in its entirety). In another embodiment, an MD1 lipidoid-containing formulation may be used to effectively deliver polynucleotide, primary construct, or mmRNA to hepatocytes in vivo. The characteristics of optimized lipidoid formulations for intramuscular or subcutaneous routes may vary significantly depending on the target cell type and the ability of formulations to diffuse through the extracellular matrix into the blood stream. While a particle size of less than 150 nm may be desired for effective hepatocyte delivery due to the size of the endothelial fenestrae (see, Akinc et al., Mol Ther. 2009 17:872-879 herein incorporated by reference in its entirety), use of a lipidoid-formulated polynucleotide, primary construct, or mmRNA to deliver the formulation to other cells types including, but not limited to, endothelial cells, myeloid cells, and muscle cells may not be similarly size-limited. Use of lipidoid formulations to deliver siRNA in vivo to other non-hepatocyte cells such as myeloid cells and endothelium has been reported (see Akinc et al., Nat Biotechnol. 2008 26:561-569;
Leuschner et al., Nat Biotechnol. 2011 29:1005-1010; Cho et al. Adv. Funct.
Mater. 2009 19:3112-3118; 8th International Judah Folkman Conference, Cambridge, MA
October 8-9, 2010; each of which is herein incorporated by reference in its entirety).
Effective delivery to myeloid cells, such as monocytes, lipidoid formulations may have a similar component molar ratio. Different ratios of lipidoids and other components including, but not limited to, disteroylphosphatidyl choline, cholesterol and PEG-DMG, may be used to optimize the formulation of the polynucleotide, primary construct, or mmRNA
for delivery to different cell types including, but not limited to, hepatocytes, myeloid cells, muscle cells, etc. For example, the component molar ratio may include, but is not limited to, 50% C12-200, 10% disteroylphosphatidyl choline, 38.5% cholesterol, and %1.5 PEG-DMG (see Leuschner et al., Nat Biotechnol 2011 29:1005-1010; herein incorporated by reference in its entirety). The use of lipidoid formulations for the localized delivery of nucleic acids to cells (such as, but not limited to, adipose cells and muscle cells) via either subcutaneous or intramuscular delivery, may not require all of the formulation components desired for systemic delivery, and as such may comprise only the lipidoid and the polynucleotide, primary construct, or mmRNA.

[000558] Combinations of different lipidoids may be used to improve the efficacy of polynucleotide, primary construct, or mmRNA directed protein production as the lipidoids may be able to increase cell transfection by the polynucleotide, primary construct, or mmRNA; and/or increase the translation of encoded protein (see Whitehead et al., Mol. Ther. 2011, 19:1688-1694, herein incorporated by reference in its entirety).
Liposomes, Lipoplexes, and Lipid Nanoparticles [000559] The polynucleotide, primary construct, and mmRNA of the invention can be formulated using one or more liposomes, lipoplexes, or lipid nanoparticles. In one embodiment, pharmaceutical compositions of polynucleotide, primary construct, or mmRNA include liposomes. Liposomes are artificially-prepared vesicles which may primarily be composed of a lipid bilayer and may be used as a delivery vehicle for the administration of nutrients and pharmaceutical formulations. Liposomes can be of different sizes such as, but not limited to, a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments, a small unicellular vesicle (SUV) which may be smaller than 50 nm in diameter, and a large unilamellar vesicle (LUV) which may be between 50 and 500 nm in diameter. Liposome design may include, but is not limited to, opsonins or ligands in order to improve the attachment of liposomes to unhealthy tissue or to activate events such as, but not limited to, endocytosis.
Liposomes may contain a low or a high pH in order to improve the delivery of the pharmaceutical formulations.
[000560] The formation of liposomes may depend on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients , the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
[000561] In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, WA), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoley1-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety) and liposomes which may deliver small molecule drugs such as, but not limited to, DOXILO from Janssen Biotech, Inc. (Horsham, PA).
[000562] In one embodiment, pharmaceutical compositions described herein may include, without limitation, liposomes such as those formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo (see Wheeler et al. Gene Therapy. 1999 6:271-281; Zhang et al. Gene Therapy. 1999 6:1438-1447; Jeffs et al. Pharm Res. 2005 22:362-372; Morrissey et al., Nat Biotechnol. 2005 2:1002-1007; Zimmermann et al., Nature. 2006 441:111-114;

Heyes et al. J Contr Rel. 2005 107:276-287; Semple et al. Nature Biotech. 2010 28:172-176; Judge et al. J Clin Invest. 2009 119:661-673; deFougerolles Hum Gene Ther. 2008 19:125-132; all of which are incorporated herein in their entireties). The original manufacture method by Wheeler et al. was a detergent dialysis method, which was later improved by Jeffs et al. and is referred to as the spontaneous vesicle formation method.
The liposome formulations are composed of 3 to 4 lipid components in addition to the polynucleotide, primary construct, or mmRNA. As an example a liposome can contain, but is not limited to, 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10%
PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. As another example, certain liposome formulations may contain, but are not limited to, 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30%
cationic lipid, where the cationic lipid can be 1,2-distearloxy-/V,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et al.
[000563] In one embodiment, pharmaceutical compositions may include liposomes which may be formed to deliver mmRNA which may encode at least one immunogen.
The mmRNA may be encapsulated by the liposome and/or it may be contained in an aqueous core which may then be encapsulated by the liposome (see International Pub.

Nos. W02012031046, W02012031043, W02012030901 and W02012006378; each of which is herein incorporated by reference in their entirety). In another embodiment, the mmRNA which may encode an immunogen may be formulated in a cationic oil-in-water emulsion where the emulsion particle comprises an oil core and a cationic lipid which can interact with the mmRNA anchoring the molecule to the emulsion particle (see International Pub. No. W02012006380; herein incorporated by reference in its entirety).
In yet another embodiment, the lipid formulation may include at least cationic lipid, a lipid which may enhance transfection and a least one lipid which contains a hydrophilic head group linked to a lipid moiety (International Pub. No. W02011076807 and U.S.
Pub. No. 20110200582; each of which is herein incorporated by reference in their entirety). In another embodiment, the polynucleotides, primary constructs and/or mmRNA encoding an immunogen may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers (see U.S. Pub. No.
20120177724, herein incorporated by reference in its entirety).
[000564] In one embodiment, the polynucleotides, primary constructs and/or mmRNA
may be formulated in a lipid vesicle which may have crosslinks between functionalized lipid bilayers.
[000565] In one embodiment, the polynucleotides, primary constructs and/or mmRNA
may be formulated in a liposome comprising a cationic lipid. The liposome may have a molar ratio of nitrogen atoms in the cationic lipid to the phophates in the RNA (N:P ratio) of between 1:1 and 20:1 as described in International Publication No.
W02013006825, herein incorporated by reference in its entirety. In another embodiment, the liposome may have a N:P ratio of greater than 20:1 or less than 1:1.
[000566] In one embodiment, the polynucleotides, primary constructs and/or mmRNA
may be formulated in a lipid-polycation complex. The formation of the lipid-polycation complex may be accomplished by methods known in the art and/or as described in U.S.
Pub. No. 20120178702, herein incorporated by reference in its entirety. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide such as, but not limited to, polylysine, polyornithine and/or polyarginine and the cationic peptides described in International Pub. No. W02012013326; herein incorporated by reference in its entirety. In another embodiment, the polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid-polycation complex which may further include a neutral lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).
[000567] The liposome formulation may be influenced by, but not limited to, the selection of the cationic lipid component, the degree of cationic lipid saturation, the nature of the PEGylation, ratio of all components and biophysical parameters such as size. In one example by Semple et al. (Semple et al. Nature Biotech. 2010 28:172-176;
herein incorporated by reference in its entirety), the liposome formulation was composed of 57.1 % cationic lipid, 7.1% dipalmitoylphosphatidylcholine, 34.3 %
cholesterol, and 1.4% PEG-c-DMA. As another example, changing the composition of the cationic lipid could more effectively deliver siRNA to various antigen presenting cells (Basha et al.
Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety).
[000568] In some embodiments, the ratio of PEG in the lipid nanoparticle (LNP) formulations may be increased or decreased and/or the carbon chain length of the PEG
lipid may be modified from C14 to C18 to alter the pharmacokinetics and/or biodistribution of the LNP formulations. As a non-limiting example, LNP
formulations may contain 1-5% of the lipid molar ratio of PEG-c-DOMG as compared to the cationic lipid, DSPC and cholesterol. In another embodiment the PEG-c-DOMG may be replaced with a PEG lipid such as, but not limited to, PEG- DSG (1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol) or PEG-DPG (1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationic lipid may be selected from any lipid known in the art such as, but not limited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.
[000569] In one embodiment, the polynucleotides, primary constructs or mmRNA
may be formulated in a lipid nanoparticle such as those described in International Publication No. W02012170930, herein incorporated by reference in its entirety.
[000570] In one embodiment, the cationic lipid may be selected from, but not limited to, a cationic lipid described in International Publication Nos. W02012040184, W02011153120, W02011149733, W02011090965, W02011043913, W02011022460, W02012061259, W02012054365, W02012044638, W02010080724, W0201021865 and W02008103276, US Patent Nos. 7,893,302, 7,404,969 and 8,283,333 and US
Patent Publication No. US20100036115 and US20120202871; each of which is herein incorporated by reference in their entirety. In another embodiment, the cationic lipid may be selected from, but not limited to, formula A described in International Publication Nos. W02012040184, W02011153120, W02011149733, W02011090965, W02011043913, W02011022460, W02012061259, W02012054365 and W02012044638; each of which is herein incorporated by reference in their entirety. In yet another embodiment, the cationic lipid may be selected from, but not limited to, formula CLI-CL)(XIX of International Publication No. W02008103276, formula CLI-CL)(XIX of US Patent No. 7,893,302, formula CLI-CLXXXXII of US Patent No.
7,404,969 and formula 1-VI of US Patent Publication No. US20100036115; each of which is herein incorporated by reference in their entirety. As a non-limiting example, the cationic lipid may be selected from (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N5N-dimethylpentacosa-16, 19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-amine, (19Z,22Z)-N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21 Z)-N,N-dimethylheptacosa- 18 ,21 -dien-8 ¨amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21 Z ,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimetylheptacos-18-en-10-amine, (17Z)-N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-l-yl] pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethyl eptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-amine, (22Z)-N,N-dimethylhentriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethy1-2-nonylhenicosa-12,15-dien-1¨amine, (13Z,16Z)-N,N-dimethy1-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethy1-1-[(1S,2R)-2-octylcyclopropyl] eptadecan-8-amine, 1- [(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethy1-1 - [(1S ,2R)-2-o ctylcyclopropyl]nonadecan-10-amine, N,N-dimethy1-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine,N,N-dimethyl-1-[(1S,25)-2- { [(1R,2R)-2-pentylcycIopropyl]methyl}
cyclopropyl]nonadecan-10-amine,N,N-dimethy1-1-[(1S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2 S)-2-undecyIcyclopropyl]tetradecan-5 -amine, N,N-dimethy1-3- {

[(1 S ,2R)-2-octylcyclopropyl]heptyl} dodecan-l-amine, 1- [(1R,2S)-2-hepty lcyclopropy1]-N,N-dimethyloctadecan-9-amine, 1- [(1S,2R)-2-decylcyclopropy1]-N,N-dimethylpentadecan-6-amine, N,N-dimethy1-1- [(1S ,2R)-2-octylcyclopropyl]pentadecan-8 -amine, R-N,N-dimethy1-1 - [(9Z,12Z)-octadeca-9,12-dien-1-yloxy] -3 -(octyloxy)propan-2-amine, S-N,N-dimethy1-1 - [(9Z,12Z)-o ctadeca-9,12-dien-1 -yloxy]-3 -(octyloxy)propan-2-amine, 1- {2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-Roctyloxy)methyllethyl}pyrrolidine, (2 S)-N,N-dimethy1-1 -[(9Z,12Z)-octadeca-9,12-dien-1 -yloxy] -3- [(5Z)-oct-5 -en-1 -yloxy]propan-2-amine, 1- {2-[(9Z,12Z)-octadeca-9,12-dien-1 -yloxy]-1 -[(octyloxy)methyl] ethyl} azetidine, (25)-i -(hexyloxy)-N,N-dimethy1-3 -[(9Z,12Z)-octadeca-9,12-dien-1 -yloxy]propan-2-amine, (2 S)-1 -(heptyloxy)-N,N-dimethy1-3 - [(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethy1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-2-amine, N,N-dimethy1-[(9Z)-octadec-9-en-l-yloxy]-3-(octyloxy)propan-2-amine; (2 S)-N,N-dimethy1-1 -[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1 -ylo xy]-3 -(octyloxy)propan-2-amine, (2 S)-1 -[(11Z,14Z)-icosa-11,14-dien-1 -yloxy]-N,N-dimethy1-3 -(pentyloxy)propan-2-amine, (2S)-1 -(hexylo xy)-3 - [(11Z,14Z)-icosa-11,14-dien-l-yloxy]-N,N-dimethylpropan-2 -amine, 1 -[(11Z,14Z)-icosa-11,14-dien-1 -yloxy]-N,N-dimethyl -3 -(octyloxy)propan-2-amine, 1 -[(13Z,16Z)-docosa-13 ,16-dien-l-yloxy]-N,N-dimethy1-3-(octyloxy)propan-2-amine, (2 S)-1 - [(13Z,16Z)-docosa-13 ,16-dien-1 -yloxy] -3 -(hexyloxy)-N,N-dimethylpropan-2-amine, (2 S)-1 -[(13Z)-docos-13-en-1 -yloxy]-3 -(hexyloxy)-N,N-dimethylpropan-2-amine, 1 -[(13Z)-docos-13 -en-1 -yloxy]-N,N-dimethy1-3 -(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethy1-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1 -metoylo ctyl)oxy]-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3 ,7-dimethyloctyl)oxy]-N,N-dimethy1-3- [(9Z,12Z)-octadeca-9,12-dien-1 -yloxy]propan-2-amine, N,N-dimethy1-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl} cyc lopropyl] o ctyl} oxy)propan-2-amine, N,N-dimethy1-1- { [8-(2-oc1ylcyclopropyl)octyl]oxy} -3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.
[000571] In one embodiment, the lipid may be a cleavable lipid such as those described in International Publication No. W02012170889, herein incorporated by reference in its entirety.
[000572] In one embodiment, the cationic lipid may be synthesized by methods known in the art and/or as described in International Publication Nos. W02012040184, W02011153120, W02011149733, W02011090965, W02011043913, W02011022460, W02012061259, W02012054365, W02012044638, W02010080724 and W0201021865; each of which is herein incorporated by reference in their entirety.
[000573] In one embodiment, the LNP formulations of the polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG at 3% lipid molar ratio. In another embodiment, the LNP formulations polynucleotides, primary constructs and/or mmRNA may contain PEG-c-DOMG at 1.5% lipid molar ratio.
[000574] In one embodiment, the pharmaceutical compositions of the polynucleotides, primary constructs and/or mmRNA may include at least one of the PEGylated lipids described in International Publication No. 2012099755, herein incorporated by reference.
[000575] In one embodiment, the LNP formulation may contain PEG-DMG 2000 (1,2-dimyristoyl-sn-glycero-3-phophoethanolamine-N-[methoxy(polyethylene glycol)-2000).
In one embodiment, the LNP formulation may contain PEG-DMG 2000, a cationic lipid known in the art and at least one other component. In another embodiment, the LNP
formulation may contain PEG-DMG 2000, a cationic lipid known in the art, DSPC
and cholesterol. As a non-limiting example, the LNP formulation may contain PEG-DMG
2000, DLin-DMA, DSPC and cholesterol. As another non-limiting example the LNP
formulation may contain PEG-DMG 2000, DLin-DMA, DSPC and cholesterol in a molar ratio of 2:40:10:48 (see e.g., Geall et al., Nonviral delivery of self-amplifying RNA
vaccines, PNAS 2012; PMID: 22908294; herein incorporated by reference in its entirety).
. As another non-limiting example, modified RNA described herein may be formulated in a nanoparticle to be delivered by a parenteral route as described in U.S.
Pub. No.
20120207845; herein incorporated by reference in its entirety.
In one embodiment, the LNP formulation may be formulated by the methods described in International Publication Nos. W02011127255 or W02008103276, each of which is herein incorporated by reference in their entirety. As a non-limiting example, modified RNA described herein may be encapsulated in LNP formulations as described in W02011127255 and/or W02008103276; each of which is herein incorporated by reference in their entirety.
In one embodiment, LNP formulations described herein may comprise a polycationic composition. As a non-limiting example, the polycationic composition may be selected from formula 1-60 of US Patent Publication No. U520050222064; herein incorporated by reference in its entirety. In another embodiment, the LNP formulations comprising a polycationic composition may be used for the delivery of the modified RNA
described herein in vivo and/or in vitro.
[000576] In one embodiment, the LNP formulations described herein may additionally comprise a permeability enhancer molecule. Non-limiting permeability enhancer molecules are described in US Patent Publication No. U52005 0222064; herein incorporated by reference in its entirety.
[000577] In one embodiment, the pharmaceutical compositions may be formulated in liposomes such as, but not limited to, DiLa2 liposomes (Marina Biotech, Bothell, WA), SMARTICLESO (Marina Biotech, Bothell, WA), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
[000578] The nanoparticle formulations may be a carbohydrate nanoparticle comprising a carbohydrate carrier and a modified nucleic acid molecule (e.g., mmRNA). As a non-limiting example, the carbohydrate carrier may include, but is not limited to, an anhydride-modified phytoglycogen or glycogen-type material, phtoglycogen octenyl succinate, phytoglycogen beta-dextrin, anhydride-modified phytoglycogen beta-dextrin.
(See e.g., International Publication No. W02012109121; herein incorporated by reference in its entirety).

[000579] Lipid nanoparticle formulations may be improved by replacing the cationic lipid with a biodegradable cationic lipid which is known as a rapidly eliminated lipid nanoparticle (reLNP). Ionizable cationic lipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, and DLin-MC3-DMA, have been shown to accumulate in plasma and tissues over time and may be a potential source of toxicity. The rapid metabolism of the rapidly eliminated lipids can improve the tolerability and therapeutic index of the lipid nanoparticles by an order of magnitude from a 1 mg/kg dose to a 10 mg/kg dose in rat.
Inclusion of an enzymatically degraded ester linkage can improve the degradation and metabolism profile of the cationic component, while still maintaining the activity of the reLNP formulation. The ester linkage can be internally located within the lipid chain or it may be terminally located at the terminal end of the lipid chain. The internal ester linkage may replace any carbon in the lipid chain.
[000580] In one embodiment, the internal ester linkage may be located on either side of the saturated carbon. Non-limiting examples of reLNPs include, and [000581] In one embodiment, an immune response may be elicited by delivering a lipid nanoparticle which may include a nanospecies, a polymer and an immunogen.
(U.S.
Publication No. 20120189700 and International Publication No. W02012099805;
each of which is herein incorporated by reference in their entirety). The polymer may encapsulate the nanospecies or partially encapsulate the nanospecies. The immunogen may be a recombinant protein, a modified RNA and/or a primary construct described herein. In one embodiment, the lipid nanoparticle may be formulated for use in a vaccine such as, but not limited to, against a pathogen.
[000582] Lipid nanoparticles may be engineered to alter the surface properties of particles so the lipid nanoparticles may penetrate the mucosal barrier. Mucus is located on mucosal tissue such as, but not limted to, oral (e.g., the buccal and esophageal membranes and tonsil tissue), ophthalmic, gastrointestinal (e.g., stomach, small intestine, large intestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal, tracheal and bronchial membranes), genital (e.g., vaginal, cervical and urethral membranes).
Nanoparticles larger than 10-200 nm which are preferred for higher drug encapsulation efficiency and the ability to provide the sustained delivery of a wide array of drugs have been thought to be too large to rapidly diffuse through mucosal barriers.
Mucus is continuously secreted, shed, discarded or digested and recycled so most of the trapped particles may be removed from the mucosla tissue within seconds or within a few hours.
Large polymeric nanoparticles (200nm -500nm in diameter) which have been coated densely with a low molecular weight polyethylene glycol (PEG) diffused through mucus only 4 to 6-fold lower than the same particles diffusing in water (Lai et al.

104(5):1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2): 158-171; each of which is herein incorporated by reference in their entirety). The transport of nanoparticles may be determined using rates of permeation and/or fluorescent microscopy techniques including, but not limited to, fluorescence recovery after photobleaching (FRAP) and high resolution multiple particle tracking (MPT). As a non-limiting example, compositions which can penetrate a mucosal barrier may be made as described in U.S.
Pat. No. 8,241,670, herein incorporated by reference in its entirety.
[000583] The lipid nanoparticle engineered to penetrate mucus may comprise a polymeric material (i.e. a polymeric core) and/or a polymer-vitamin conjugate and/or a tri-block co-polymer. The polymeric material may include, but is not limited to, polyamines, polyethers, polyamides, polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes), polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes, polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates, polyacrylonitriles, and polyarylates. The polymeric material may be biodegradable and/or biocompatible. The polymeric material may additionally be irradiated. As a non-limiting example, the polymeric material may be gamma irradiated (See e.g., International App.
No. W0201282165, herein incorporated by reference in its entirety). Non-limiting examples of specific polymers include poly(caprolactone) (PCL), ethylene vinyl acetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid) (PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide) (PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone), poly(D,L-lactide-co-caprolactone-co-glycolide), poly(D,L-lactide-co-PEO-co-D,L-lactide), poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate, polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA), polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids), polyanhydrides, polyorthoesters, poly(ester amides), polyamides, poly(ester ethers), polycarbonates, polyalkylenes such as polyethylene and polypropylene, polyalkylene glycols such as poly(ethylene glycol) (PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such as poly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinyl ethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halides such as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes, polystyrene (PS), polyurethanes, derivatized celluloses such as alkyl celluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters, nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose, polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA), poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate), poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate), poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate), poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) and copolymers and mixtures thereof, polydioxanone and its copolymers, polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene, poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid), poly(lactide-co-caprolactone), and trimethylene carbonate, polyvinylpyrrolidone .The lipid nanoparticle may be coated or associated with a co-polymer such as, but not limited to, a block co-polymer (such as a branched polyether-polyamide block copolymer described in International Publication No. W02013012476, herein incorporated by reference in its entirety), and (poly(ethylene glycol))-(poly(propylene oxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., US Publication 20120121718 and US Publication 20100003337 and U.S. Pat. No. 8,263,665; each of which is herein incorporated by reference in their entirety). The co-polymer may be a polymer that is generally regarded as safe (GRAS) and the formation of the lipid nanoparticle may be in such a way that no new chemical entities are created. For example, the lipid nanoparticle may comprise poloxamers coating PLGA nanoparticles without forming new chemical entities which are still able to rapidly penetrate human mucus (Yang et al.
Angew. Chem.
Int. Ed. 2011 50:2597-2600; herein incorporated by reference in its entirety).
[000584] The vitamin of the polymer-vitamin conjugate may be vitamin E. The vitamin portion of the conjugate may be substituted with other suitable components such as, but not limited to, vitamin A, vitamin E, other vitamins, cholesterol, a hydrophobic moiety, or a hydrophobic component of other surfactants (e.g., sterol chains, fatty acids, hydrocarbon chains and alkylene oxide chains).
[000585] The lipid nanoparticle engineered to penetrate mucus may include surface altering agents such as, but not limited to, mmRNA, anionic proteins (e.g., bovine serum albumin), surfactants (e.g., cationic surfactants such as for example dimethyldioctadecyl-ammonium bromide), sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids, polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolytic agents (e.g., N-acetylcysteine, mugwort, bromelain, papain, clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone, mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin, gelsolin, thymosin P4 dornase alfa, neltenexine, erdosteine) and various DNases including rhDNase.. The surface altering agent may be embedded or enmeshed in the particle's surface or disposed (e.g., by coating, adsorption, covalent linkage, or other process) on the surface of the lipid nanoparticle. (see e.g., US Publication and US Publication 20080166414; each of which is herein incorporated by reference in their entirety).
[000586] The mucus penetrating lipid nanoparticles may comprise at least one mmRNA
described herein. The mmRNA may be encapsulated in the lipid nanoparticle and/or disposed on the surface of the paricle. The mmRNA may be covalently coupled to the lipid nanoparticle. Formulations of mucus penetrating lipid nanoparticles may comprise a plurality of nanoparticles. Further, the formulations may contain particles which may interact with the mucus and alter the structural and/or adhesive properties of the surrounding mucus to decrease mucoadhesion which may increase the delivery of the mucus penetrating lipid nanoparticles to the mucosal tissue.
[000587] In one embodiment, the polynucleotide, primary construct, or mmRNA is formulated as a lipoplex, such as, without limitation, the ATUPLEXTm system, the DACC system, the DBTC system and other siRNA-lipoplex technology from Silence Therapeutics (London, United Kingdom), STEMFECTTm from STEMGENTO
(Cambridge, MA), and polyethylenimine (PEI) or protamine-based targeted and non-targeted delivery of nucleic acids acids (Aleku et al. Cancer Res. 2008 68:9788-9798;
Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al. Microvasc Res 2010 80:286-293Weide et al. J Immunother. 2009 32:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo Expert Opin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J.
Immunother. 34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al., Proc Natl Acad Sci U S A. 2007 6;104:4095-4100; deFougerolles Hum Gene Ther. 2008 19:125-132; all of which are incorporated herein by reference in its entirety).
[000588] In one embodiment such formulations may also be constructed or compositions altered such that they passively or actively are directed to different cell types in vivo, including but not limited to hepatocytes, immune cells, tumor cells, endothelial cells, antigen presenting cells, and leukocytes (Akinc et al. Mol Ther. 2010 18:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge et al., J
Clin Invest.
2009 119:661-673; Kaufmann et al., Microvasc Res 2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Basha et al., Mol. Ther. 201119:2186-2200;
Fenske and Cullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science.

319:627-630; Peer and Lieberman, Gene Ther. 201118:1127-1133; all of which are incorporated herein by reference in its entirety). One example of passive targeting of formulations to liver cells includes the DLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticle formulations which have been shown to bind to apolipoprotein E and promote binding and uptake of these formulations into hepatocytes in vivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein incorporated by reference in its entirety). Formulations can also be selectively targeted through expression of different ligands on their surface as exemplified by, but not limited by, folate, transferrin, N-acetylgalactosamine (GalNAc), and antibody targeted approaches (Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchio and Torchilin, Front Biosci.
2011 16:1388-1412; Yu et al., Mol Membr Biol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst. 2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhao et al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther. 2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012 820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer 2010 J Control Release. 20:63-68;
Peer et al., Proc Natl Acad Sci U S A. 2007 104:4095-4100; Kim et al., Methods Mol Biol.

721:339-353; Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., Nat Biotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630; Peer and Lieberman, Gene Ther. 201118:1127-1133; all of which are incorporated herein by reference in its entirety)..
[000589] In one embodiment, the polynucleotide, primary construct, or mmRNA is formulated as a solid lipid nanoparticle. A solid lipid nanoparticle (SLN) may be spherical with an average diameter between 10 to 1000 nm. SLN possess a solid lipid core matrix that can solubilize lipophilic molecules and may be stabilized with surfactants and/or emulsifiers. In a further embodiment, the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle (see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; herein incorporated by reference in its entirety).
[000590] Liposomes, lipoplexes, or lipid nanoparticles may be used to improve the efficacy of polynucleotide, primary construct, or mmRNA directed protein production as these formulations may be able to increase cell transfection by the polynucleotide, primary construct, or mmRNA; and/or increase the translation of encoded protein. One such example involves the use of lipid encapsulation to enable the effective systemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 2007 15:713-720;
herein incorporated by reference in its entirety). The liposomes, lipoplexes, or lipid nanoparticles may also be used to increase the stability of the polynucleotide, primary construct, or mmRNA.

[000591] In one embodiment, the the polynucleotides, primary constructs, and/or the mmRNA of the present invention can be formulated for controlled release and/or targeted delivery. As used herein, "controlled release" refers to a pharmaceutical composition or compound release profile that conforms to a particular pattern of release to effect a therapeutic outcome. In one embodiment, the polynucleotides, primary constructs or the mmRNA may be encapsulated into a delivery agent described herein and/or known in the art for controlled release and/or targeted delivery. As used herein, the term "encapsulate"
means to enclose, surround or encase. As it relates to the formulation of the compounds of the invention, encapsulation may be substantial, complete or partial. The term "substitantially encapsulated" means that at least greater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 or greater than 99.999% of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent. "Partially encapsulation" means that less than 10, 10, 20, 30, 40 50 or less of the pharmaceutical composition or compound of the invention may be enclosed, surrounded or encased within the delivery agent. Advantageously, encapsulation may be determined by measuring the escape or the activity of the pharmaceutical composition or compound of the invention using fluorescence and/or electron micrograph. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceutical composition or compound of the invention are encapsulated in the delivery agent.
[000592] In one embodiment, the controlled release formulation may include, but is not limited to, tri-block co-polymers. As a non-limiting example, the formulation may include two different types of tri-block co-polymers (International Pub. No.
W02012131104 and W02012131106; each of which is herein incorporated by reference in its entirety).
[000593] In another embodiment, the the polynucleotides, primary constructs, or the mmRNA may be encapsulated into a lipid nanoparticle or a rapidly eliminated lipid nanoparticle and the lipid nanoparticles or a rapidly eliminated lipid nanoparticle may then be encapsulated into a polymer, hydrogel and/or surgical sealant described herein and/or known in the art. As a non-limiting example, the polymer, hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc), poloxamer, GELSITEO

(Nanotherapeutics, Inc. Alachua, FL), HYLENEXO (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELLO (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEALO (Baxter International, Inc Deerfield, IL).
[000594] In another embodiment, the lipid nanoparticle may be encapsulated into any polymer known in the art which may form a gel when injected into a subject. As another non-limiting example, the lipid nanoparticle may be encapsulated into a polymer matrix which may be biodegradable.
[000595] In one embodiment, the the polynucleotide, primary construct, or mmRNA
formulation for controlled release and/or targeted delivery may also include at least one controlled release coating. Controlled release coatings include, but are not limited to, OPADRYO, polyvinylpyrrolidone/vinyl acetate copolymer, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cellulose, hydroxyethyl cellulose, EUDRAGIT RLO, EUDRAGIT RS and cellulose derivatives such as ethylcellulose aqueous dispersions (AQUACOATO and SURELEASEO).
[000596] In one embodiment, the controlled release and/or targeted delivery formulation may comprise at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof. In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
[000597] In one embodiment, the the polynucleotides, primary constructs, and/or the mmRNA of the present invention may be encapsulated in a therapeutic nanoparticle.
Therapeutic nanoparticles may be formulated by methods described herein and known in the art such as, but not limited to, International Pub Nos. W02010005740, W02010030763, W02010005721, W02010005723, W02012054923, US Pub. Nos.
U520110262491, U520100104645, U520100087337, U520100068285, U520110274759, U520100068286 and U520120288541 and US Pat No. 8,206,747, 8,293,276, 8,318,208 and 8,318,211 each of which is herein incorporated by reference in their entirety. In another embodiment, therapeutic polymer nanoparticles may be identified by the methods described in US Pub No. US20120140790, herein incorporated by reference in its entirety.
[000598] In one embodiment, the therapeutic nanoparticle may be formulated for sustained release. As used herein, "sustained release" refers to a pharmaceutical composition or compound that conforms to a release rate over a specific period of time.
The period of time may include, but is not limited to, hours, days, weeks, months and years. As a non-limiting example, the sustained release nanoparticle may comprise a polymer and a therapeutic agent such as, but not limited to, the the polynucleotides, primary constructs, and mmRNA of the present invention (see International Pub No.
2010075072 and US Pub No. U520100216804, US20110217377 and U520120201859, each of which is herein incorporated by reference in their entirety).
[000599] In one embodiment, the therapeutic nanoparticles may be formulated to be target specific. As a non-limiting example, the thereapeutic nanoparticles may include a corticosteroid (see International Pub. No. W02011084518; herein incorporated by reference in its entirety). In one embodiment, the therapeutic nanoparticles may be formulated to be cancer specific. As a non-limiting example, the therapeutic nanoparticles may be formulated in nanoparticles described in International Pub No.
W02008121949, W02010005726, W02010005725, W02011084521 and US Pub No.
U520100069426, U520120004293 and U520100104655, each of which is herein incorporated by reference in their entirety.
[000600] In one embodiment, the nanoparticles of the present invention may comprise a polymeric matrix. As a non-limiting example, the nanoparticle may comprise two or more polymers such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof.
[000601] In one embodiment, the therapeutic nanoparticle comprises a diblock copolymer. In one embodiment, the diblock copolymer may include PEG in combination with a polymer such as, but not limited to, polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) or combinations thereof [000602] As a non-limiting example the therapeutic nanoparticle comprises a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, each of which is herein incorporated by reference in their entirety). In another non-limiting example, the therapeutic nanoparticle is a stealth nanoparticle comprising a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968 and International Publication No. W02012166923, each of which is herein incorporated by reference in its entirety).
[000603] In one embodiment, the therapeutic nanoparticle may comprise a multiblock copolymer (See e.g., U.S. Pat. No. 8,263,665 and 8,287,910; each of which is herein incorporated by reference in its entirety).
[000604] In one embodiment, the block copolymers described herein may be included in a polyion complex comprising a non-polymeric micelle and the block copolymer. (See e.g., U.S. Pub. No. 20120076836; herein incorporated by reference in its entirety).
[000605] In one embodiment, the therapeutic nanoparticle may comprise at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof [000606] In one embodiment, the therapeutic nanoparticles may comprise at least one cationic polymer described herein and/or known in the art.
[000607] In one embodiment, the therapeutic nanoparticles may comprise at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers, poly(beta-amino esters) (See e.g., U.S. Pat. No.
8,287,849; herein incorporated by reference in its entirety) and combinations thereof.
[000608] In one embodiment, the therapeutic nanoparticles may comprise at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
[000609] In another embodiment, the therapeutic nanoparticle may include a conjugation of at least one targeting ligand. The targeting ligand may be any ligand known in the art such as, but not limited to, a monoclonal antibody. (Kirpotin et al, Cancer Res. 2006 66:6732-6740; herein incorporated by reference in its entirety).
[000610] In one embodiment, the therapeutic nanoparticle may be formulated in an aqueous solution which may be used to target cancer (see International Pub No.

W02011084513 and US Pub No. US20110294717, each of which is herein incorporated by reference in their entirety).
[000611] In one embodiment, the polynucleotides, primary constructs, or mmRNA
may be encapsulated in, linked to and/or associated with synthetic nanocarriers. .
Synthetic nanocarriers include, but are not limited to, those described in International Pub. Nos.
W02010005740, W02010030763, W0201213501, W02012149252, W02012149255, W02012149259, W02012149265, W02012149268, W02012149282, W02012149301, W02012149393, W02012149405, W02012149411, W02012149454 and W02013019669, and US Pub. Nos. US20110262491, U520100104645, U520100087337 and US20120244222, each of which is herein incorporated by reference in their entirety.
The synthetic nanocarriers may be formulated using methods known in the art and/or described herein. As a non-limiting example, the synthetic nanocarriers may be formulated by the methods described in International Pub Nos. W02010005740, W02010030763 and W0201213501and US Pub. Nos. US20110262491, U520100104645, U520100087337 and U52012024422, each of which is herein incorporated by reference in their entirety. In another embodiment, the synthetic nanocarrier formulations may be lyophilized by methods described in International Pub.

No. W02011072218 and US Pat No. 8,211,473; each of which is herein incorporated by reference in their entirety.
[000612] In one embodiment, the synthetic nanocarriers may contain reactive groups to release the polynucleotides, primary constructs and/or mmRNA described herein (see International Pub. No. W020120952552 and US Pub No. U520120171229, each of which is herein incorporated by reference in their entirety).
[000613] In one embodiment, the synthetic nanocarriers may contain an immunostimulatory agent to enhance the immune response from delivery of the synthetic nanocarrier. As a non-limiting example, the synthetic nanocarrier may comprise a Thl immunostimulatory agent which may enhance a Thl-based response of the immune system (see International Pub No. W02010123569 and US Pub. No. US20110223201, each of which is herein incorporated by reference in its entirety).
[000614] In one embodiment, the synthetic nanocarriers may be formulated for targeted release. In one embodiment, the synthetic nanocarrier is formulated to release the polynucleotides, primary constructs and/or mmRNA at a specified pH and/or after a desired time interval. As a non-limiting example, the synthetic nanoparticle may be formulated to release the polynucleotides, primary constructs and/or mmRNA
after 24 hours and/or at a pH of 4.5 (see International Pub. Nos. W02010138193 and W02010138194 and US Pub Nos. U520110020388 and U520110027217, each of which is herein incorporated by reference in their entireties).
[000615] In one embodiment, the synthetic nanocarriers may be formulated for controlled and/or sustained release of the polynucleotides, primary constructs and/or mmRNA described herein. As a non-limiting example, the synthetic nanocarriers for sustained release may be formulated by methods known in the art, described herein and/or as described in International Pub No. W02010138192 and US Pub No.
20100303850, each of which is herein incorporated by reference in their entirety.
[000616] In one embodiment, the synthetic nanocarrier may be formulated for use as a vaccine. In one embodiment, the synthetic nanocarrier may encapsulate at least one polynucleotide, primary construct and/or mmRNA which encode at least one antigen. As a non-limiting example, the synthetic nanocarrier may include at least one antigen and an excipient for a vaccine dosage form (see International Pub No. W02011150264 and US

Pub No. US20110293723, each of which is herein incorporated by reference in their entirety). As another non-limiting example, a vaccine dosage form may include at least two synthetic nanocarriers with the same or different antigens and an excipient (see International Pub No. W02011150249 and US Pub No. US20110293701, each of which is herein incorporated by reference in their entirety). The vaccine dosage form may be selected by methods described herein, known in the art and/or described in International Pub No. W02011150258 and US Pub No. US20120027806, each of which is herein incorporated by reference in their entirety).
[000617] In one embodiment, the synthetic nanocarrier may comprise at least one polynucleotide, primary construct and/or mmRNA which encodes at least one adjuvant.
As non-limiting example, the adjuvant may comprise dimethyldioctadecylammonium-bromide, dimethyldioctadecylammonium-chloride, dimethyldioctadecylammonium-phosphate or dimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or part of said apolar fraction of a total lipid extract of a mycobacterium (See e.g, U.S. Pat.
No. 8,241,610; herein incorporated by reference in its entirety). In another embodiment, the synthetic nanocarrier may comprise at least one polynucleotide, primary construct and/or mmRNA and an adjuvant. As a non-limiting example, the synthetic nanocarrier comprising and adjuvant may be formulated by the methods described in International Pub No. W02011150240 and US Pub No. US20110293700, each of which is herein incorporated by reference in its entirety.
[000618] In one embodiment, the synthetic nanocarrier may encapsulate at least one polynucleotide, primary construct and/or mmRNA which encodes a peptide, fragment or region from a virus. As a non-limiting example, the synthetic nanocarrier may include, but is not limited to, the nanocarriers described in International Pub No.
W02012024621, W0201202629, W02012024632 and US Pub No. US20120064110, U520120058153 and U520120058154, each of which is herein incorporated by reference in their entirety.
[000619] In one embodiment, the synthetic nanocarrier may be coupled to a polynucleotide, primary construct or mmRNA which may be able to trigger a humoral and/or cytotoxic T lymphocyte (CTL) response (See e.g., International Publication No.
W02013019669, herein incorporated by reference in its entirety).

[000620] In one embodiment, the nanoparticle may be optimized for oral administration. The nanoparticle may comprise at least one cationic biopolymer such as, but not limited to, chitosan or a derivative thereof As a non-limiting example, the nanoparticle may be formulated by the methods described in U.S. Pub. No.
20120282343; herein incorporated by reference in its entirety.
Polymers, Biodegradable Nanoparticles, and Core-Shell Nanoparticles [000621] The polynucleotide, primary construct, and mmRNA of the invention can be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers which may be used for delivery include, but are not limited to, DYNAMIC
POLYCONJUGATEO (Arrowhead Reasearch Corp., Pasadena, CA) formulations from MIRUSO Bio (Madison, WI) and Roche Madison (Madison, WI), PHASERXTM polymer formulations such as, without limitation, SMARTT POLYMER TECHNOLOGYTm (PHASERXO, Seattle, WA), DMRI/DOPE, poloxamer, VAXFECTINO adjuvant from Vical (San Diego, CA), chitosan, cyclodextrin from Calando Pharmaceuticals (Pasadena, CA), dendrimers and poly(lactic-co-glycolic acid) (PLGA) polymers. RONDELTM
(RNAi/Oligonucleotide Nanoparticle Delivery) polymers (Arrowhead Research Corporation, Pasadena, CA) and pH responsive co-block polymers such as, but not limited to, PHASERXO (Seattle, WA).
[000622] A non-limiting example of chitosan formulation includes a core of positively charged chitosan and an outer portion of negatively charged substrate (U.S.
Pub. No.
20120258176; herein incorporated by reference in its entirety). Chitosan includes, but is not limited to N-trimethyl chitosan, mono-N-carboxymethyl chitosan (MCC), N-palmitoyl chitosan (NPCS), EDTA-chitosan, low molecular weight chitosan, chitosan derivatives, or combinations thereof [000623] In one embodiment, the polymers used in the present invention have undergone processing to reduce and/or inhibit the attachement of unwanted substances such as, but not limited to, bacteria, to the surface of the polymer. The polymer may be processed by methods known and/or described in the art and/or described in International Pub. No. W02012150467, herein incorporated by reference in its entirety.
[000624] A non-limiting example of PLGA formulations include, but are not limited to, PLGA injectable depots (e.g., ELIGARDO which is formed by dissolving PLGA in 66%

N-methyl-2-pyrrolidone (NMP) and the remainder being aqueous solvent and leuprolide.
Once injected, the PLGA and leuprolide peptide precipitates into the subcutaneous space).
[000625] Many of these polymer approaches have demonstrated efficacy in delivering oligonucleotides in vivo into the cell cytoplasm (reviewed in deFougerolles Hum Gene Ther. 2008 19:125-132; herein incorporated by reference in its entirety). Two polymer approaches that have yielded robust in vivo delivery of nucleic acids, in this case with small interfering RNA (siRNA), are dynamic polyconjugates and cyclodextrin-based nanoparticles. The first of these delivery approaches uses dynamic polyconjugates and has been shown in vivo in mice to effectively deliver siRNA and silence endogenous target mRNA in hepatocytes (Rozema et al., Proc Natl Acad Sci U S A. 2007 104:12982-12887; herein incorporated by reference in its entirety). This particular approach is a multicomponent polymer system whose key features include a membrane-active polymer to which nucleic acid, in this case siRNA, is covalently coupled via a disulfide bond and where both PEG (for charge masking) and N-acetylgalactosamine (for hepatocyte targeting) groups are linked via pH-sensitive bonds (Rozema et al., Proc Natl Acad Sci U
S A. 2007 104:12982-12887; herein incorporated by reference in its entirety).
On binding to the hepatocyte and entry into the endosome, the polymer complex disassembles in the low-pH environment, with the polymer exposing its positive charge, leading to endosomal escape and cytoplasmic release of the siRNA from the polymer.
Through replacement of the N-acetylgalactosamine group with a mannose group, it was shown one could alter targeting from asialoglycoprotein receptor-expressing hepatocytes to sinusoidal endothelium and Kupffer cells. Another polymer approach involves using transferrin-targeted cyclodextrin-containing polycation nanoparticles. These nanoparticles have demonstrated targeted silencing of the EWS-FLI1 gene product in transferrin receptor-expressing Ewing's sarcoma tumor cells (Hu-Lieskovan et at., Cancer Res.2005 65: 8984-8982; herein incorporated by reference in its entirety) and siRNA formulated in these nanoparticles was well tolerated in non-human primates (Heidel et at., Proc Natl Acad Sci USA 2007 104:5715-21; herein incorporated by reference in its entirety). Both of these delivery strategies incorporate rational approaches using both targeted delivery and endosomal escape mechanisms.

[000626] The polymer formulation can permit the sustained or delayed release of polynucleotide, primary construct, or mmRNA (e.g., following intramuscular or subcutaneous injection). The altered release profile for the polynucleotide, primary construct, or mmRNA can result in, for example, translation of an encoded protein over an extended period of time. The polymer formulation may also be used to increase the stability of the polynucleotide, primary construct, or mmRNA. Biodegradable polymers have been previously used to protect nucleic acids other than mmRNA from degradation and been shown to result in sustained release of payloads in vivo (Rozema et al., Proc Natl Acad Sci U S A. 2007 104:12982-12887; Sullivan et al., Expert Opin Drug Deliv.
2010 7:1433-1446; Convertine et al., Biomacromolecules. 2010 Oct 1; Chu et al., Acc Chem Res. 2012 Jan 13; Manganiello et al., Biomaterials. 2012 33:2301-2309;
Benoit et al., Biomacromolecules. 201112:2708-2714; Singha et al., Nucleic Acid Ther.

2:133-147; deFougerolles Hum Gene Ther. 2008 19:125-132; Schaffert and Wagner, Gene Ther. 2008 16:1131-1138; Chaturvedi et al., Expert Opin Drug Deliv. 2011 8:1455-1468; Davis, Mol Pharm. 2009 6:659-668; Davis, Nature 2010 464:1067-1070; each of which is herein incorporated by reference in its entirety).
[000627] In one embodiment, the pharmaceutical compositions may be sustained release formulations. In a further embodiment, the sustained release formulations may be for subcutaneous delivery. Sustained release formulations may include, but are not limited to, PLGA microspheres, ethylene vinyl acetate (EVAc), poloxamer, GELSITEO
(Nanotherapeutics, Inc. Alachua, FL), HYLENEXO (Halozyme Therapeutics, San Diego CA), surgical sealants such as fibrinogen polymers (Ethicon Inc. Cornelia, GA), TISSELLO (Baxter International, Inc Deerfield, IL), PEG-based sealants, and COSEALO (Baxter International, Inc Deerfield, IL).
[000628] As a non-limiting example modified mRNA may be formulated in PLGA
microspheres by preparing the PLGA microspheres with tunable release rates (e.g., days and weeks) and encapsulating the modified mRNA in the PLGA microspheres while maintaining the integrity of the modified mRNA during the encapsulation process.
EVAc are non-biodegradeable, biocompatible polymers which are used extensively in pre-clinical sustained release implant applications (e.g., extended release products Ocusert a pilocarpine ophthalmic insert for glaucoma or progestasert a sustained release progesterone intrauterine deivce; transdermal delivery systems Testoderm, Duragesic and Selegiline; catheters). Poloxamer F-407 NF is a hydrophilic, non-ionic surfactant triblock copolymer of polyoxyethylene-polyoxypropylene-polyoxyethylene having a low viscosity at temperatures less than 5 C and forms a solid gel at temperatures greater than 15 C. PEG-based surgical sealants comprise two synthetic PEG components mixed in a delivery device which can be prepared in one minute, seals in 3 minutes and is reabsorbed within 30 days. GELSITEO and natural polymers are capable of in-situ gelation at the site of administration. They have been shown to interact with protein and peptide therapeutic candidates through ionic ineraction to provide a stabilizing effect.
[000629] Polymer formulations can also be selectively targeted through expression of different ligands as exemplified by, but not limited by, folate, transferrin, and N-acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 201112:2708-2714;
Rozema et al., Proc Natl Acad Sci U S A. 2007 104:12982-12887; Davis, Mol Pharm.
2009 6:659-668; Davis, Nature 2010 464:1067-1070; each of which is herein incorporated by reference in its entirety).
[000630] The modified nucleic acid, and mmRNA of the invention may be formulated with or in a polymeric compound. The polymer may include at least one polymer such as, but not limited to, polyethenes, polyethylene glycol (PEG), poly(1-lysine)(PLL), PEG
grafted to PLL, cationic lipopolymer, biodegradable cationic lipopolymer, polyethyleneimine (PEI), cross-linked branched poly(alkylene imines), a polyamine derivative, a modified poloxamer, a biodegradable polymer, elastic biodegradable polymer, biodegradable block copolymer, biodegradable random copolymer, biodegradable polyester copolymer, biodegradable polyester block copolymer, biodegradable polyester block random copolymer, multiblock copolymers, linear biodegradable copolymer, poly[a-(4-aminobuty1)-L-glycolic acid) (PAGA), biodegradable cross-linked cationic multi-block copolymers, polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates, polycaprolactones, polyamides, polyacetals, polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinyl alcohols, polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates, polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine, poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), acrylic polymers, amine-containing polymers, dextran polymers, dextran polymer derivatives or or combinations thereof.
[000631] As a non-limiting example, the modified nucleic acid or mmRNA of the invention may be formulated with the polymeric compound of PEG grafted with PLL as described in U.S. Pat. No. 6,177,274; herein incorporated by reference in its entirety.
The formulation may be used for transfecting cells in vitro or for in vivo delivery of the modified nucleic acid and mmRNA. In another example, the modified nucleic acid and mmRNA may be suspended in a solution or medium with a cationic polymer, in a dry pharmaceutical composition or in a solution that is capable of being dried as described in U.S. Pub. Nos. 20090042829 and 20090042825; each of which are herein incorporated by reference in their entireties.
[000632] As another non-limiting example the polynucleotides, primary constructs or mmRNA of the invention may be formulated with a PLGA-PEG block copolymer (see US Pub. No. US20120004293 and US Pat No. 8,236,330, herein incorporated by reference in their entireties) or PLGA-PEG-PLGA block copolymers (See U.S.
Pat. No.
6,004,573, herein incorporated by reference in its entirety). As a non-limiting example, the polynucleotides, primary constructs or mmRNA of the invention may be formulated with a diblock copolymer of PEG and PLA or PEG and PLGA (see US Pat No 8,246,968, herein incorporated by reference in its entirety).
[000633] A polyamine derivative may be used to deliver nucleic acids or to treat and/or prevent a disease or to be included in an implantable or injectable device (U.S. Pub. No.
20100260817 herein incorporated by reference in its entirety). As a non-limiting example, a pharmaceutical composition may include the modified nucleic acids and mmRNA and the polyamine derivative described in U.S. Pub. No. 20100260817 (the contents of which are incorporated herein by reference in its entirety. As a non-limiting example the polynucleotides, primary constructs and mmRNA of the present invention may be delivered using a polyaminde polymer such as, but not limited to, a polymer comprising a 1,3-dipolar addition polymer prepared by combining a carbohydrate diazide monomer with a dilkyne unite comprising oligoamines (U.S. Pat. No. 8,236,280;
herein incorporated by reference in its entirety).

[000634] In one embodiment, the polynucleotides, primary constructs or mmRNA
of the present invention may be formulated with at least one polymer and/or derivatives thereof described in International Publication Nos. W02011115862, W02012082574 and W02012068187 and U.S. Pub. No. 20120283427, each of which are herein incorporated by reference in their entireties. In another embodiment, the modified nucleic acid or mmRNA of the present invention may be formulated with a polymer of formula Z
as described in W02011115862, herein incorporated by reference in its entirety.
In yet another embodiment, the modified nucleic acid or mmRNA may be formulated with a polymer of formula Z, Z' or Z" as described in International Pub. Nos.

or W02012068187 and U.S. Pub. No. 2012028342, each of which are herein incorporated by reference in their entireties. The polymers formulated with the modified RNA of the present invention may be synthesized by the methods described in International Pub. Nos. W02012082574 or W02012068187, each of which are herein incorporated by reference in their entireties.
[000635] The polynucleotides, primary constructs or mmRNA of the invention may be formulated with at least one acrylic polymer. Acrylic polymers include but are not limited to, acrylic acid, methacrylic acid, acrylic acid and methacrylic acid copolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkyl methacrylate copolymer, poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates and combinations thereof [000636] Formulations of polynucleotides, primary constructs or mmRNA of the invention may include at least one amine-containing polymer such as, but not limited to polylysine, polyethylene imine, poly(amidoamine) dendrimers or combinations thereof [000637] For example, the modified nucleic acid or mmRNA of the invention may be formulated in a pharmaceutical compound including a poly(alkylene imine), a biodegradable cationic lipopolymer, a biodegradable block copolymer, a biodegradable polymer, or a biodegradable random copolymer, a biodegradable polyester block copolymer, a biodegradable polyester polymer, a biodegradable polyester random copolymer, a linear biodegradable copolymer, PAGA, a biodegradable cross-linked cationic multi-block copolymer or combinations thereof The biodegradable cationic lipopolymer may be made by methods known in the art and/or described in U.S.
Pat. No.

6,696,038, U.S. App. Nos. 20030073619 and 20040142474 each of which is herein incorporated by reference in their entireties. The poly(alkylene imine) may be made using methods known in the art and/or as described in U.S. Pub. No. 20100004315, herein incorporated by reference in its entirety. The biodegradabale polymer, biodegradable block copolymer, the biodegradable random copolymer, biodegradable polyester block copolymer, biodegradable polyester polymer, or biodegradable polyester random copolymer may be made using methods known in the art and/or as described in U.S. Pat.
Nos. 6,517,869 and 6,267,987, the contents of which are each incorporated herein by reference in their entirety. The linear biodegradable copolymer may be made using methods known in the art and/or as described in U.S. Pat. No. 6,652,886. The PAGA
polymer may be made using methods known in the art and/or as described in U.S.
Pat.
No. 6,217,912 herein incorporated by reference in its entirety. The PAGA
polymer may be copolymerized to form a copolymer or block copolymer with polymers such as but not limited to, poly-L-lysine, polyargine, polyornithine, histones, avidin, protamines, polylactides and poly(lactide-co-glycolides). The biodegradable cross-linked cationic multi-block copolymers may be made my methods known in the art and/or as described in U.S. Pat. No. 8,057,821 or U.S. Pub. No. 2012009145 each of which are herein incorporated by reference in their entireties. For example, the multi-block copolymers may be synthesized using linear polyethyleneimine (LPEI) blocks which have distinct patterns as compared to branched polyethyleneimines. Further, the composition or pharmaceutical composition may be made by the methods known in the art, described herein, or as described in U.S. Pub. No. 20100004315 or U.S. Pat. Nos.
6,267,987 and 6,217,912 each of which are herein incorporated by reference in their entireties.
[000638] The polynucleotides, primary constructs, and mmRNA of the invention may be formulated with at least one degradable polyester which may contain polycationic side chains. Degradeable polyesters include, but are not limited to, poly(serine ester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester), and combinations thereof In another embodiment, the degradable polyesters may include a PEG conjugation to form a PEGylated polymer.
[000639] The polynucleotides, primary construct, mmRNA of the invention may be formulated with at least one crosslinkable polyester. Crosslinkable polyesters include those known in the art and described in US Pub. No. 20120269761, herein incorporated by reference in its entirety.
[000640] In one embodiment, the polymers described herein may be conjugated to a lipid-terminating PEG. As a non-limiting example, PLGA may be conjugated to a lipid-terminating PEG forming PLGA-DSPE-PEG. As another non-limiting example, PEG
conjugates for use with the present invention are described in International Publication No. W02008103276, herein incorporated by reference in its entirety. The polymers may be conjugated using a ligand conjugate such as, but not limited to, the conjugates described in U.S. Pat. No. 8,273,363, herein incorporated by reference in its entirety.
[000641] In one embodiment, the modified RNA described herein may be conjugated with another compound. Non-limiting examples of conjugates are described in US
Patent Nos. 7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties. In another embodiment, modified RNA of the present invention may be conjugated with conjugates of formula 1-122 as described in US Patent Nos.
7,964,578 and 7,833,992, each of which are herein incorporated by reference in their entireties. The polynucleotides, primary constructs and/or mmRNA described herein may be conjugated with a metal such as, but not limited to, gold. (See e.g., Giljohann et al.
Journ. Amer.
Chem. Soc. 2009 131(6): 2072-2073; herein incorporated by reference in its entirety). In another embodiment, the polynucleotides, primary constructs and/or mmRNA
described herein may be conjugated and/or encapsulated in gold-nanoparticles.
(Interantional Pub.
No. W0201216269 and U.S. Pub. No. 20120302940; each of which is herein incorporated by reference in its entirety).
[000642] As described in U.S. Pub. No. 20100004313, herein incorporated by reference in its entirety, a gene delivery composition may include a nucleotide sequence and a poloxamer. For example, the modified nucleic acid and mmRNA of the present inveition may be used in a gene delivery composition with the poloxamer described in U.S. Pub.
No. 20100004313.
[000643] In one embodiment, the polymer formulation of the present invention may be stabilized by contacting the polymer formulation, which may include a cationic carrier, with a cationic lipopolymer which may be covalently linked to cholesterol and polyethylene glycol groups. The polymer formulation may be contacted with a cationic lipopolymer using the methods described in U.S. Pub. No. 20090042829 herein incorporated by reference in its entirety. The cationic carrier may include, but is not limited to, polyethylenimine, poly(trimethylenimine), poly(tetramethylenimine), polypropylenimine, aminoglycoside-polyamine, dideoxy-diamino-b-cyclodextrin, spermine, spermidine, poly(2-dimethylamino)ethyl methacrylate, poly(lysine), poly(histidine), poly(arginine), cationized gelatin, dendrimers, chitosan, 1,2-Dioleoy1-3-Trimethylammonium-Propane(DOTAP), N-[1-(2,3-dioleoyloxy)propyll-N,N,N-trimethylammonium chloride (DOTMA), 142-(oleoyloxy)ethy1]-2-oley1-3-(2-hydroxyethyl)imidazolinium chloride (DOTIM), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), 3B-[N¨(N',N'-Dimethylaminoethane)-carbamoyl]Cholesterol Hydrochloride (DC-Cholesterol HC1) diheptadecylamidoglycyl spermidine (DOGS), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-y1)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride DODAC) and combinations thereof.
[000644] The polynucleotides, primary constructs and/or mmRNA of the invention may be formulated in a polyplex of one or more polymers (U.S. Pub. No. 20120237565 and 20120270927; each of which is herein incorporated by reference in its entirety). In one embodiment, the polyplex comprises two or more cationic polymers. The catioinic polymer may comprise a poly(ethylene imine) (PEI) such as linear PEI.
[000645] The polynucleotide, primary construct, and mmRNA of the invention can also be formulated as a nanoparticle using a combination of polymers, lipids, and/or other biodegradable agents, such as, but not limited to, calcium phosphate.
Components may be combined in a core-shell, hybrid, and/or layer-by-layer architecture, to allow for fine-tuning of the nanoparticle so to delivery of the polynucleotide, primary construct and mmRNA may be enhanced (Wang et al., Nat Mater. 2006 5:791-796; Fuller et al., Biomaterials. 2008 29:1526-1532; DeKoker et al., Adv Drug Deliv Rev. 2011 63:748-761; Endres et al., Biomaterials. 2011 32:7721-7731; Su et al., Mol Pharm.
2011 Jun 6;8(3):774-87; herein incorporated by reference in its entirety). As a non-limiting example, the nanoparticle may comprise a plurality of polymers such as, but not limited to hydrophilic-hydrophobic polymers (e.g., PEG-PLGA), hydrophobic polymers (e.g., PEG) and/or hydrophilic polymers (International Pub. No. W020120225129; herein incorporated by reference in its entirety).
[000646] Biodegradable calcium phosphate nanoparticles in combination with lipids and/or polymers have been shown to deliver polynucleotides, primary constructs and mmRNA in vivo. In one embodiment, a lipid coated calcium phosphate nanoparticle, which may also contain a targeting ligand such as anisamide, may be used to deliver the polynucleotide, primary construct and mmRNA of the present invention. For example, to effectively deliver siRNA in a mouse metastatic lung model a lipid coated calcium phosphate nanoparticle was used (Li et al., J Contr Rel. 2010 142: 416-421; Li et al., J
Contr Rel. 2012 158:108-114; Yang et al., Mol Ther. 2012 20:609-615; herein incorporated by reference in its entirety). This delivery system combines both a targeted nanoparticle and a component to enhance the endosomal escape, calcium phosphate, in order to improve delivery of the siRNA.
[000647] In one embodiment, calcium phosphate with a PEG-polyanion block copolymer may be used to delivery polynucleotides, primary constructs and mmRNA
(Kazikawa et al., J Contr Rel. 2004 97:345-356; Kazikawa et al., J Contr Rel.

111:368-370; herein incorporated by reference in its entirety).
[000648] In one embodiment, a PEG-charge-conversional polymer (Pitella et al., Biomaterials. 2011 32:3106-3114) may be used to form a nanoparticle to deliver the polynucleotides, primary constructs and mmRNA of the present invention. The PEG-charge-conversional polymer may improve upon the PEG-polyanion block copolymers by being cleaved into a polycation at acidic pH, thus enhancing endosomal escape.
[000649] The use of core-shell nanoparticles has additionally focused on a high-throughput approach to synthesize cationic cross-linked nanogel cores and various shells (Siegwart et al., Proc Natl Acad Sci U S A. 2011108:12996-13001). The complexation, delivery, and internalization of the polymeric nanoparticles can be precisely controlled by altering the chemical composition in both the core and shell components of the nanoparticle. For example, the core-shell nanoparticles may efficiently deliver siRNA to mouse hepatocytes after they covalently attach cholesterol to the nanoparticle.
[000650] In one embodiment, a hollow lipid core comprising a middle PLGA layer and an outer neutral lipid layer containg PEG may be used to delivery of the polynucleotide, primary construct and mmRNA of the present invention. As a non-limiting example, in mice bearing a luciferease-expressing tumor, it was determined that the lipid-polymer-lipid hybrid nanoparticle significantly suppressed luciferase expression, as compared to a conventional lipoplex (Shi et al, Angew Chem Int Ed. 2011 50:7027-7031; herein incorporated by reference in its entirety).
[000651] In one embodiment, the lipid nanoparticles may comprise a core of the modified nucleic acid molecules disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the modified nucleic acids in the core.
[000652] Core¨shell nanoparticles for use with the modified nucleic acid molecules of the present invention are described and may be formed by the methods described in U.S.
Pat. No. 8,313,777 herein incorporated by reference in its entirety.
[000653] In one embodiment, the core-shell nanoparticles may comprise a core of the modified nucleic acid molecules disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the modified nucleic acid molecules in the core. As a non-limiting example, the core-shell nanoparticle may be used to treat an eye disease or disorder (See e.g. US Publication No.
20120321719, herein incorporated by reference in its entirety).
[000654] In one embodiment, the polymer used with the formulations described herein may be a modified polymer (such as, but not limited to, a modified polyacetal) as described in International Publication No. W02011120053, herein incorporated by reference in its entirety.
Peptides and Proteins [000655] The polynucleotide, primary construct, and mmRNA of the invention can be formulated with peptides and/or proteins in order to increase transfection of cells by the polynucleotide, primary construct, or mmRNA. In one embodiment, peptides such as, but not limited to, cell penetrating peptides and proteins and peptides that enable intracellular delivery may be used to deliver pharmaceutical formulations. A non-limiting example of a cell penetrating peptide which may be used with the pharmaceutical formulations of the present invention includes a cell-penetrating peptide sequence attached to polycations that facilitates delivery to the intracellular space, e.g., HIV-derived TAT
peptide, penetratins, transportans, or hCT derived cell-penetrating peptides (see, e.g., Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton FL, 2002); El-Andaloussi et al., Curr.
Pharm. Des.
11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci. 62(16):1839-49 (2005), all of which are incorporated herein by reference in their entirety). The compositions can also be formulated to include a cell penetrating agent, e.g., liposomes, which enhance delivery of the compositions to the intracellular space. Polynucleotides, primary constructs, and mmRNA of the invention may be complexed to peptides and/or proteins such as, but not limited to, peptides and/or proteins from Aileron Therapeutics (Cambridge, MA) and Permeon Biologics (Cambridge, MA) in order to enable intracellular delivery (Cronican et al., ACS Chem. Biol. 2010 5:747-752;
McNaughton et al., Proc. Natl. Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des.
2009 73:3-6; Verdine and Hilinski, Methods Enzymol. 2012;503:3-33; all of which are herein incorporated by reference in its entirety).

[000656] In one embodiment, the cell-penetrating polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein, "protein-binding partner" includes, but are not limited to, antibodies and functional fragments thereof, scaffold proteins, or peptides. The cell-penetrating polypeptide may further comprise an intracellular binding partner for the protein-binding partner. The cell-penetrating polypeptide may be capable of being secreted from a cell where the polynucleotide, primary construct, or mmRNA may be introduced.

[000657] Formulations of the including peptides or proteins may be used to increase cell transfection by the polynucleotide, primary construct, or mmRNA, alter the biodistribution of the polynucleotide, primary construct, or mmRNA (e.g., by targeting specific tissues or cell types), and/or increase the translation of encoded protein. (See e.g., International Pub. No. W02012110636; herein incorporated by reference in its entirety).

Cells [000658] The polynucleotide, primary construct, and mmRNA of the invention can be transfected ex vivo into cells, which are subsequently transplanted into a subject. As non-limiting examples, the pharmaceutical compositions may include red blood cells to deliver modified RNA to liver and myeloid cells, virosomes to deliver modified RNA in virus-like particles (VLPs), and electroporated cells such as, but not limited to, from MAXCYTEO (Gaithersburg, MD) and from ERYTECHO (Lyon, France) to deliver modified RNA. Examples of use of red blood cells, viral particles and electroporated cells to deliver payloads other than mmRNA have been documented (Godfrin et al., Expert Opin Biol Ther. 2012 12:127-133; Fang et al., Expert Opin Biol Ther.

12:385-389; Hu et al., Proc Natl Acad Sci U S A. 2011 108:10980-10985; Lund et al., Pharm Res. 2010 27:400-420; Huckriede et al., J Liposome Res. 2007;17:39-47;
Cusi, Hum Vaccin. 2006 2:1-7; de Jonge et al., Gene Ther. 2006 13:400-411; all of which are herein incorporated by reference in its entirety).

[000659] The polynucleotides, primary constructs and mmRNA may be delivered in synthetic VLPs synthesized by the methods described in International Pub No.
W02011085231 and US Pub No. 20110171248, each of which are herein incorporated by reference in their entireties.

[000660] Cell-based formulations of the polynucleotide, primary construct, and mmRNA of the invention may be used to ensure cell transfection (e.g., in the cellular carrier), alter the biodistribution of the polynucleotide, primary construct, or mmRNA
(e.g., by targeting the cell carrier to specific tissues or cell types), and/or increase the translation of encoded protein.

[000661] A variety of methods are known in the art and suitable for introduction of nucleic acid into a cell, including viral and non-viral mediated techniques.
Examples of typical non-viral mediated techniques include, but are not limited to, electroporation, calcium phosphate mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome mediated transfer, microinjection, microprojectile mediated transfer (nanoparticles), cationic polymer mediated transfer (DEAE-dextran, polyethylenimine, polyethylene glycol (PEG) and the like) or cell fusion.

[000662] The technique of sonoporation, or cellular sonication, is the use of sound (e.g., ultrasonic frequencies) for modifying the permeability of the cell plasma membrane.
Sonoporation methods are known to those in the art and are used to deliver nucleic acids in vivo (Yoon and Park, Expert Opin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr Pharm Biotechnol. 2007 8:355-361; Newman and Bettinger, Gene Ther. 2007 14:465-475; all herein incorporated by reference in their entirety).
Sonoporation methods are known in the art and are also taught for example as it relates to bacteria in US Patent Publication 20100196983 and as it relates to other cell types in, for example, US Patent Publication 20100009424, each of which are incorporated herein by reference in their entirety.

[000663] Electroporation techniques are also well known in the art and are used to deliver nucleic acids in vivo and clinically (Andre et al., Curr Gene Ther.
2010 10:267-280; Chiarella et al., Curr Gene Ther. 2010 10:281-286; Hojman, Curr Gene Ther. 2010 10:128-138; all herein incorporated by reference in their entirety). In one embodiment, polynucleotides, primary constructs or mmRNA may be delivered by electroporation as described in Example 8.
Hyaluronidase [000664] The intramuscular or subcutaneous localized injection of polynucleotide, primary construct, or mmRNA of the invention can include hyaluronidase, which catalyzes the hydrolysis of hyaluronan. By catalyzing the hydrolysis of hyaluronan, a constituent of the interstitial barrier, hyaluronidase lowers the viscosity of hyaluronan, thereby increasing tissue permeability (Frost, Expert Opin. Drug Deliv. (2007) 4:427-440; herein incorporated by reference in its entirety). It is useful to speed their dispersion and systemic distribution of encoded proteins produced by transfected cells.
Alternatively, the hyaluronidase can be used to increase the number of cells exposed to a polynucleotide, primary construct, or mmRNA of the invention administered intramuscularly or subcutaneously.
Nanoparticle Mimics [000665] The polynucleotide, primary construct or mmRNA of the invention may be encapsulated within and/or absorbed to a nanoparticle mimic. A nanoparticle mimic can mimic the delivery function organisms or particles such as, but not limited to, pathogens, viruses, bacteria, fungus, parasites, prions and cells. As a non-limiting example the polynucleotide, primary construct or mmRNA of the invention may be encapsulated in a non-viron particle which can mimic the delivery function of a virus (see International Pub. No. W02012006376 herein incorporated by reference in its entirety).
Nanotubes [000666] The polynucleotides, primary constructs or mmRNA of the invention can be attached or otherwise bound to at least one nanotube such as, but not limited to, rosette nanotubes, rosette nanotubes having twin bases with a linker, carbon nanotubes and/or single-walled carbon nanotubes, The polynucleotides, primary constructs or mmRNA
may be bound to the nanotubes through forces such as, but not limited to, steric, ionic, covalent and/or other forces.

[000667] In one embodiment, the nanotube can release one or more polynucleotides, primary constructs or mmRNA into cells. The size and/or the surface structure of at least one nanotube may be altered so as to govern the interaction of the nanotubes within the body and/or to attach or bind to the polynucleotides, primary constructs or mmRNA
disclosed herein. In one embodiment, the building block and/or the functional groups attached to the building block of the at least one nanotube may be altered to adjust the dimensions and/or properties of the nanotube. As a non-limiting example, the length of the nanotubes may be altered to hinder the nanotubes from passing through the holes in the walls of normal blood vessels but still small enough to pass through the larger holes in the blood vessels of tumor tissue.

[000668] In one embodiment, at least one nanotube may also be coated with delivery enhancing compounds including polymers, such as, but not limited to, polyethylene glycol. In another embodiment, at least one nanotube and/or the polynucleotides, primary constructs or mmRNA may be mixed with pharmaceutically acceptable excipients and/or delivery vehicles.

[000669] In one embodiment, the polynucleotides, primary constructs or mmRNA
are attached and/or otherwise bound to at least one rosette nanotube. The rosette nanotubes may be formed by a process known in the art and/or by the process described in International Publication No. W02012094304, herein incorporated by reference in its entirety. At least one polynucleotide, primary construct and/or mmRNA may be attached and/or otherwise bound to at least one rosette nanotube by a process as described in International Publication No. W02012094304, herein incorporated by reference in its entirety, where rosette nanotubes or modules forming rosette nanotubes are mixed in aqueous media with at least one polynucleotide, primary construct and/or mmRNA
under conditions which may cause at least one polynucleotide, primary construct or mmRNA to attach or otherwise bind to the rosette nanotubes.

[000670] In one embodiment, the polynucleotides, primary constructs or mmRNA
may be attached to and/or otherwise bound to at least one carbon nanotube. As a non-limiting example, the polynucleotides, primary constructs or mmRNA may be bound to a linking agent and the linked agent may be bound to the carbon nanotube (See e.g., U.S.
Pat No.
8,246,995; herein incorporated by reference in its entirety). The carbon nanotube may be a single-walled nanotube (See e.g., U.S. Pat No. 8,246,995; herein incorporated by reference in its entirety).
Conjugates [000671] The polynucleotides, primary constructs, and mmRNA of the invention include conjugates, such as a polynucleotide, primary construct, or mmRNA
covalently linked to a carrier or targeting group, or including two encoding regions that together produce a fusion protein (e.g., bearing a targeting group and therapeutic protein or peptide).

[000672] The conjugates of the invention include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid, an oligonucleotide (e.g. an aptamer). Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine.
Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

[000673] Representative U.S. patents that teach the preparation of polynucleotide conjugates, particularly to RNA, include, but are not limited to, U.S. Pat.
Nos. 4,828,979;
4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731;
5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;
5,578,718;
5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941;
4,835,263;
4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;
5,112,963;
5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;
5,317,098;
5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785;
5,565,552;
5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;
5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297;
7,037,646; each of which is herein incorporated by reference in their entireties.

[000674] In one embodiment, the conjugate of the present invention may function as a carrier for the modified nucleic acids and mmRNA of the present invention. The conjugate may comprise a cationic polymer such as, but not limited to, polyamine, polylysine, polyalkylenimine, and polyethylenimine which may be grafted to with poly(ethylene glycol). As a non-limiting example, the conjugate may be similar to the polymeric conjugate and the method of synthesizing the polymeric conjugate described in U.S. Pat. No. 6,586,524 herein incorporated by reference in its entirety.

[000675] The conjugates can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGD
peptide mimetic or an aptamer.

[000676] Targeting groups can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell.
Targeting groups may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, or aptamers. The ligand can be, for example, a lipopolysaccharide, or an activator of p38 MAP kinase.

[000677] The targeting group can be any ligand that is capable of targeting a specific receptor. Examples include, without limitation, folate, GalNAc, galactose, mannose, mannose-6P, apatamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL, and HDL ligands. In particular embodiments, the targeting group is an aptamer.
The aptamer can be unmodified or have any combination of modifications disclosed herein.

[000678] In one embodiment, pharmaceutical compositions of the present invention may include chemical modifications such as, but not limited to, modifications similar to locked nucleic acids.

[000679] Representative U.S. Patents that teach the preparation of locked nucleic acid (LNA) such as those from Santaris, include, but are not limited to, the following: U.S.
Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125;
and 7,399,845, each of which is herein incorporated by reference in its entirety.

[000680] Representative U.S. patents that teach the preparation of PNA
compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA
compounds can be found, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[000681] Some embodiments featured in the invention include polynucleotides, primary constructs or mmRNA with phosphorothioate backbones and oligonucleosides with other modified backbones, and in particular --CH2--NH¨CH2--, --CH2--N(CH3)--0--CH2--[known as a methylene (methylimino) or MMI backbone], --CH2-0--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2-- and --N(CH3)--CH2--CH2-4wherein the native phosphodiester backbone is represented as --0¨P(0)2-0--CH2--] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the polynucletotides featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No.
5,034,506.

[000682] Modifications at the 2' position may also aid in delivery.
Preferably, modifications at the 2' position are not located in a polypeptide-coding sequence, i.e., not in a translatable region. Modifications at the 2' position may be located in a 5'UTR, a 3'UTR and/or a tailing region. Modifications at the 2' position can include one of the following at the 2' position: H (i.e., 2'-deoxy); F; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl;
0-, S- or N-alkynyl; or 0-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Cio alkyl or C2 to C10 alkenyl and alkynyl.
Exemplary suitable modifications include O[(CH2)õ0] mCH3, 0(CH2).õOCH3, 0(CH2)õNH2, 0(CH2) õCH3, 0(CH2)õONH2, and 0(CH2)õONRCH2)õCH3)]2, where n and m are from 1 to about 10. In other embodiments, the polynucleotides, primary constructs or mmRNA
include one of the following at the 2' position: Ci to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, 0-alkaryl or 0-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, 502CH3, 0NO2, NO2, N35 NH25 heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties, or a group for improving the pharmacodynamic properties, and other substituents having similar properties. In some embodiments, the modification includes a 2'-methoxyethoxy (2'-0--CH2CH2OCH3, also known as 2'-0-(2-methoxyethyl) or 2'-M0E) (Martin et at., Hely.
Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., a 0(CH2)20N(CH3)2 group, also known as 2'-DMA0E, as described in examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-0-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2' -0--CH2-0--CH2--N(CH2)2, also described in examples herein below. Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2) and 2'-fluoro (2'-F). Similar modifications may also be made at other positions, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked dsRNAs and the 5' position of 5' terminal nucleotide. Polynucleotides of the invention may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos.
4,981,957;
5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;
5,519,134;
5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;
5,646,265;
5,658,873; 5,670,633; and 5,700,920 and each of which is herein incorporated by reference.

[000683] In still other embodiments, the polynucleotide, primary construct, or mmRNA
is covalently conjugated to a cell penetrating polypeptide. The cell-penetrating peptide may also include a signal sequence. The conjugates of the invention can be designed to have increased stability; increased cell transfection; and/or altered the biodistribution (e.g., targeted to specific tissues or cell types).

[000684] In one embodiment, the polynucleotides, primary constructs or mmRNA
may be conjugated to an agent to enhance delivery. As a non-limiting example, the agent may be a monomer or polymer such as a targeting monomer or a polymer having targeting blocks as described in International Publication No. W02011062965, herein incorporated by reference in its entirety. In another non-limiting example, the agent may be a transport agent covalently coupled to the polynucleotides, primary constructs or mmRNA
of the present invention (See e.g., U.S. Pat. Nos. 6,835.393 and 7,374,778, each of which is herein incorporated by reference in its entirety). In yet another non-limiting example, the agent may be a membrane barrier transport enhancing agent such as those described in U.S. Pat. Nos. 7,737,108 and 8,003,129, each of which is herein incorporated by reference in its entirety.

[000685] In another embodiment, polynucleotides, primary constructs or mmRNA
may be conjugated to SMARTT POLYMER TECHNOLOGY (PHASERXO, Inc. Seattle, WA).
Self-Assembled Nanoparticles Nucleic Acid Self-Assembled Nanoparticles [000686] Self-assembled nanoparticles have a well-defined size which may be precisely controlled as the nucleic acid strands may be easily reprogrammable. For example, the optimal particle size for a cancer-targeting nanodelivery carrier is 20-100 nm as a diameter greater than 20 nm avoids renal clearance and enhances delivery to certain tumors through enhanced permeability and retention effect. Using self-assembled nucleic acid nanoparticles a single uniform population in size and shape having a precisely controlled spatial orientation and density of cancer-targeting ligands for enhanced delivery. As a non-limiting example, oligonucleotide nanoparticles were prepared using programmable self-assembly of short DNA fragments and therapeutic siRNAs. These nanoparticles are molecularly identical with controllable particle size and target ligand location and density. The DNA fragments and siRNAs self-assembled into a one-step reaction to generate DNA/siRNA tetrahedral nanoparticles for targeted in vivo delivery. (Lee et al., Nature Nanotechnology 2012 7:389-393; herein incorporated by reference in its entirety).

[000687] In one embodiment, the polynucleotides, primary constructs and/or mmRNA
disclosed herein may be formulated as self-assembled nanoparticles. As a non-limiting example, nucleic acids may be used to make nanoparticles which may be used in a delivery system for the polynucleotides, primary constructs and/or mmRNA of the present invention (See e.g., International Pub. No. W02012125987; herein incorporated by reference in its entirety).

[000688] In one embodiment, the nucleic acid self-assembled nanoparticles may comprise a core of the polynucleotides, primary constructs or mmRNA disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the polynucleotides, primary contructs and mmRNA in the core.
Polymer-Based Self-Assembled Nanoparticles [000689] Polymers may be used to form sheets which self-assembled into nanoparticles.
These nanoparticles may be used to deliver the polynucleotides, primary constructs and mmRNA of the present invention. In one embodiment, these self-assembled nanoparticles may be microsponges formed of long polymers of RNA hairpins which form into crystalline 'pleated' sheets before self-assembling into microsponges. These microsponges are densely-packed sponge like microparticles which may function as an efficient carrier and may be able to deliver cargo to a cell. The microsponges may be from lum to 300 nm in diameter. The microsponges may be complexed with other agents known in the art to form larger microsponges. As a non-limiting example, the microsponge may be complexed with an agent to form an outer layer to promote cellular uptake such as polycation polyethyleneime (PEI). This complex can form a 250-nm diameter particle that can remain stable at high temperatures (150 C) (Grabow and Jaegar, Nature Materials 2012, 11:269-269; herein incorporated by reference in its entirety). Additionally these microsponges may be able to exhibit an extraordinary degree of protection from degradation by ribonucleases.

[000690] In another embodiment, the polymer-based self-assembled nanoparticles such as, but not limited to, microsponges, may be fully programmable nanoparticles.
The geometry, size and stoichiometry of the nanoparticle may be precisely controlled to create the optimal nanoparticle for delivery of cargo such as, but not limited to, polynucleotides, primary constructs and/or mmRNA.

[000691] In one embodiment, the polymer based nanoparticles may comprise a core of the polynucleotides, primary constructs and/or mmRNA disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the polynucleotides, primary construct and/or mmRNA in the core.

[000692] In yet another embodiment, the polymer based nanoparticle may comprise a non-nucleic acid polymer comprising a plurality of heterogenous monomers such as those described in Interantional Publication No. W02013009736, herein incorporated by reference in its entirety.
Inorganic Nanoparticles [000693] The polynucleotides, primary constructs and/or mmRNAs of the present invention may be formulated in inorganic nanoparticles (U.S. Pat. No.
8,257,745, herein incorporated by reference in its entirety). The inorganic nanoparticles may include, but are not limited to, clay substances that are water swellable. As a non-limiting example, the inorganic nanoparticle may include synthetic smectite clays which are made from simple silicates (See e.g., U.S. Pat. No. 5,585,108 and 8,257,745 each of which are herein incorporated by reference in their entirety).

[000694] In one embodiment, the inorganic nanoparticles may comprise a core of the modified nucleic acids disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the modified nucleic acids in the core.
Semi-conductive and Metallic Nanoparticles [000695] The polynucleotides, primary constructs and/or mmRNAs of the present invention may be formulated in water-dispersible nanoparticle comprising a semiconductive or metallic material (U.S. Pub. No. 20120228565; herein incorporated by reference in its entirety) or formed in a magnetic nanoparticle (U.S. Pub. No.

20120265001 and 20120283503; each of which is herein incorporated by reference in its entirety). The water-dispersible nanoparticles may be hydrophobic nanoparticles or hydrophilic nanoparticles.

[000696] In one embodiment, the semi-conductive and/or metallic nanoparticles may comprise a core of the polynucleotides, primary constructs and/or mmRNA
disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the polynucleotides, primary constructs and/or mmRNA in the core.
Gels and Hydrogels [000697] In one embodiment, the polynucleotides, primary constructs and/or mmRNA
disclosed herein may be encapsulated into any hydrogel known in the art which may form a gel when injected into a subject. Hydrogels are a network of polymer chains that are hydrophilic, and are sometimes found as a colloidal gel in which water is the dispersion medium. Hydrogels are highly absorbent (they can contain over 99% water) natural or synthetic polymers. Hydrogels also possess a degree of flexibility very similar to natural tissue, due to their significant water content. The hydrogel described herein may used to encapsulate lipid nanoparticles which are biocompatible, biodegradable and/or porous.

[000698] As a non-limiting example, the hydrogel may be an aptamer-functionalized hydrogel. The aptamer-functionalized hydrogel may be programmed to release one or more polynucleotides, primary constructs and/or mmRNA using nucleic acid hybridization. (Battig et al., J. Am. Chem. Society. 2012 134:12410-12413;
herein incorporated by reference in its entirety).

[000699] As another non-limiting example, the hydrogel may be a shaped as an inverted opal.
The opal hydrogels exhibit higher swelling ratios and the swelling kinetics is an order of magnitude faster as well. Methods of producing opal hydrogels and description of opal hydrogels are described in International Pub. No. W02012148684, herein incorporated by reference in its entirety.

[000700] In yet another non-limiting example, the hydrogel may be an antibacterial hydrogel. The antibacterial hydrogel may comprise a pharmaceutical acceptable salt or organic material such as, but not limited to pharmaceutical grade and/or medical grade silver salt and aloe vera gel or extract. (International Pub. No.
W02012151438, herein incorporated by reference in its entirety).

[000701] In one embodiment, the modified mRNA may be encapsulated in a lipid nanoparticle and then the lipid nanoparticle may be encapsulated into a hyrdogel.

[000702] In one embodiment, the polynucleotides, primary constructs and/or mmRNA
disclosed herein may be encapsulated into any gel known in the art. As a non-limiting example the gel may be a fluorouracil injectable gel or a fluorouracil injectable gel containing a chemical compound and/or drug known in the art. As another example, the polynucleotides, primary constructs and/or mmRNA may be encapsulated in a fluorouracil gel containing epinephrine (See e.g., Smith et al. Cancer Chemotherapty and Pharmacology, 1999 44(4):267-274; herein incorporated by reference in its entirety).

[000703] In one embodiment, the polynucleotides, primary constructs and/or mmRNA
disclosed herein may be encapsulated into a fibrin gel, fibrin hydrogel or fibrin glue. In another embodiment, the polynucleotides, primary constructs and/or mmRNA may be formulated in a lipid nanoparticle or a rapidly eliminated lipid nanoparticle prior to being encapsulated into a fibrin gel, fibrin hydrogel or a fibrin glue. In yet another embodiment, the polynucleotides, primary constructs and/or mmRNA may be formulated as a lipoplex prior to being encapsulated into a fibrin gel, hydrogel or a fibrin glue.
Fibrin gels, hydrogels and glues comprise two components, a fibrinogen solution and a thrombin solution which is rich in calcium (See e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148: 49-55; Kidd et al. Journal of Controlled Release 2012.
157:80-85; each of which is herein incorporated by reference in its entirety).
The concentration of the components of the fibrin gel, hydrogel and/or glue can be altered to change the characteristics, the network mesh size, and/or the degradation characteristics of the gel, hydrogel and/or glue such as, but not limited to changing the release characteristics of the fibrin gel, hydrogel and/or glue. (See e.g., Spicer and Mikos, Journal of Controlled Release 2010. 148: 49-55; Kidd et al. Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128; each of which is herein incorporated by reference in its entirety). This feature may be advantageous when used to deliver the modified mRNA disclosed herein. (See e.g., Kidd et al.
Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008.
14:119-128;
each of which is herein incorporated by reference in its entirety).
Cations and Anions [000704] Formulations of polynucleotides, primary constructs and/or mmRNA
disclosed herein may include cations or anions. In one embodiment, the formulations include metal cations such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+ and combinations thereof As a non-limiting example, formulations may include polymers and a polynucleotides, primary constructs and/or mmRNA complexed with a metal cation (See e.g., U.S. Pat. Nos. 6,265,389 and 6,555,525, each of which is herein incorporated by reference in its entirety).
Molded Nanoparticles and Microparticles [000705] The polynucleotides, primary constructs and/or mmRNA disclosed herein may be formulated in nanoparticles and/or microparticles. These nanoparticles and/or microparticles may be molded into any size shape and chemistry. As an example, the nanoparticles and/or microparticles may be made using the PRINT technology by LIQUIDA TECHNOLOGIES (Morrisville, NC) (See e.g., International Pub. No.
W02007024323; herein incorporated by reference in its entirety).

[000706] In one embodiment, the molded nanoparticles may comprise a core of the polynucleotides, primary constructs and/or mmRNA disclosed herein and a polymer shell. The polymer shell may be any of the polymers described herein and are known in the art. In an additional embodiment, the polymer shell may be used to protect the polynucleotides, primary construct and/or mmRNA in the core.
NanoJackets and NanoLiposomes [000707] The polynucleotides, primary constructs and/or mmRNA disclosed herein may be formulated in NanoJackets and NanoLiposomes by Keystone Nano (State College, PA). NanoJackets are made of compounds that are naturally found in the body including calcium, phosphate and may also include a small amount of silicates.

Nanojackets may range in size from 5 to 50 nm and may be used to deliver hydrophilic and hydrophobic compounds such as, but not limited to, polynucleotides, primary constructs and/or mmRNA.

[000708] NanoLiposomes are made of lipids such as, but not limited to, lipids which naturally occur in the body. NanoLiposomes may range in size from 60-80 nm and may be used to deliver hydrophilic and hydrophobic compounds such as, but not limited to, polynucleotides, primary constructs and/or mmRNA. In one aspect, the polynucleotides, primary constructs and/or mmRNA disclosed herein are formulated in a NanoLiposome such as, but not limited to, Ceramide NanoLiposomes.
Excipients [000709] 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 in its entirety) 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 invention.

[000710] 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.

[000711] 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 compositions.

[000712] 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.

[000713] 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 (VEEGUM8), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.

[000714] 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 [TWEENn 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 SOLUTOL8), 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, PLUORNC F 68, POLOXAMER 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, docusate sodium, etc. and/or combinations thereof.

[000715] 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 [000716] 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 H, NEOLONETM, KATHONTm, and/or EUXYL .

[000717] 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 [000718] 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 [000719] 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.

[000720] 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.
Delivery [000721] The present disclosure encompasses the delivery of polynucleotides, primary constructs or mmRNA for any of therapeutic, pharmaceutical, diagnostic or imaging by any appropriate route taking into consideration likely advances in the sciences of drug delivery. Delivery may be naked or formulated.
Naked Delivery [000722] The polynucleotides, primary constructs or mmRNA of the present invention may be delivered to a cell naked. As used herein in, "naked" refers to delivering polynucleotides, primary constructs or mmRNA free from agents which promote transfection. For example, the polynucleotides, primary constructs or mmRNA
delivered to the cell may contain no modifications. The naked polynucleotides, primary constructs or mmRNA may be delivered to the cell using routes of administration known in the art and described herein.
Formulated Delivery [000723] The polynucleotides, primary constructs or mmRNA of the present invention may be formulated, using the methods described herein. The formulations may contain polynucleotides, primary constructs or mmRNA which may be modified and/or unmodified. The formulations may further include, but are not limited to, cell penetration agents, a pharmaceutically acceptable carrier, a delivery agent, a bioerodible or biocompatible polymer, a solvent, and a sustained-release delivery depot. The formulated polynucleotides, primary constructs or mmRNA may be delivered to the cell using routes of administration known in the art and described herein.

[000724] The compositions may also be formulated for direct delivery to an organ or tissue in any of several ways in the art including, but not limited to, direct soaking or bathing, via a catheter, by gels, powder, ointments, creams, gels, lotions, and/or drops, by using substrates such as fabric or biodegradable materials coated or impregnated with the compositions, and the like.
Administration [000725] The polynucleotides, primary constructs or mmRNA of the present invention may be administered by any route which results in a therapeutically effective outcome.
These include, but are not limited to enteral, gastroenteral, epidural, oral, transdermal, epidural (peridural), intracerebral (into the cerebrum), intracerebroventricular (into the cerebral ventricles), epicutaneous (application onto the skin), intradermal, (into the skin itself), subcutaneous (under the skin), nasal administration (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal canal), intraperitoneal, (infusion or injection into the peritoneum), intravesical infusion, intravitreal, (through the eye), intracavernous injection, ( into the base of the penis), intravaginal administration, intrauterine, extra-amniotic administration, transdermal (diffusion through the intact skin for systemic distribution), transmucosal (diffusion through a mucous membrane), insufflation (snorting), sublingual, sublabial, enema, eye drops (onto the conjunctiva), or in ear drops. In specific embodiments, compositions may be administered in a way which allows them cross the blood-brain barrier, vascular barrier, or other epithelial barrier.Non-limiting routes of administration for the polynucleotides, primary constructs or mmRNA of the present invention are described below.
Parenteral and Injecfible Administration [000726] Liquid dosage forms for 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.

[000727] 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.

[000728] 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.

[000729] 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.
Rectal and Vaginal Administration [000730] 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.
Oral Administration [000731] Liquid dosage forms for oral 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.

[000732] 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.
Topical or Transdermal Administration [000733] As described herein, compositions containing the polynucleotides, primary constructs or mmRNA of the invention may be formulated for administration topically.
The skin may be an ideal target site for delivery as it is readily accessible.
Gene expression may be restricted not only to the skin, potentially avoiding nonspecific toxicity, but also to specific layers and cell types within the skin.

[000734] The site of cutaneous expression of the delivered compositions will depend on the route of nucleic acid delivery. Three routes are commonly considered to deliver polynucleotides, primary constructs or mmRNA to the skin: (i) topical application (e.g.

for local/regional treatment and/or cosmetic applications); (ii) intradermal injection (e.g.
for local/regional treatment and/or cosmetic applications); and (iii) systemic delivery (e.g.
for treatment of dermatologic diseases that affect both cutaneous and extracutaneous regions). Polynucleotides, primary constructs or mmRNA can be delivered to the skin by several different approaches known in the art. Most topical delivery approaches have been shown to work for delivery of DNA, such as but not limited to, topical application of non-cationic liposome¨DNA complex, cationic liposome¨DNA complex, particle-mediated (gene gun), puncture-mediated gene transfections, and viral delivery approaches. After delivery of the nucleic acid, gene products have been detected in a number of different skin cell types, including, but not limited to, basal keratinocytes, sebaceous gland cells, dermal fibroblasts and dermal macrophages.

[000735] In one embodiment, the invention provides for a variety of dressings (e.g., wound dressings) or bandages (e.g., adhesive bandages) for conveniently and/or effectively carrying out methods of the present invention. Typically dressing or bandages may comprise sufficient amounts of pharmaceutical compositions and/or polynucleotides, primary constructs or mmRNA described herein to allow a user to perform multiple treatments of a subject(s).

[000736] In one embodiment, the invention provides for the polynucleotides, primary constructs or mmRNA compositions to be delivered in more than one injection.

[000737] In one embodiment, before topical and/or transdermal administration at least one area of tissue, such as skin, may be subjected to a device and/or solution which may increase permeability. In one embodiment, the tissue may be subjected to an abrasion device to increase the permeability of the skin (see U.S. Patent Publication No.
20080275468, herein incorporated by reference in its entirety). In another embodiment, the tissue may be subjected to an ultrasound enhancement device. An ultrasound enhancement device may include, but is not limited to, the devices described in U.S.
Publication No. 20040236268 and U.S. Patent Nos. 6,491,657 and 6,234,990; each of which are herein incorporated by reference in their entireties. Methods of enhancing the permeability of tissue are described in U.S. Publication Nos. 20040171980 and 20040236268 and U.S. Pat. No. 6,190,315; each of which are herein incorporated by reference in their entireties.

[000738] In one embodiment, a device may be used to increase permeability of tissue before delivering formulations of modified mRNA described herein. The permeability of skin may be measured by methods known in the art and/or described in U.S.
Patent No.
6,190,315, herein incorporated by reference in its entirety. As a non-limiting example, a modified mRNA formulation may be delivered by the drug delivery methods described in U.S. Patent No. 6,190,315, herein incorporated by reference in its entirety.

[000739] In another non-limiting example tissue may be treated with a eutectic mixture of local anesthetics (EMLA) cream before, during and/or after the tissue may be subjected to a device which may increase permeability. Katz et al. (Anesth Analg (2004); 98:371-76; herein incorporated by reference in its entirety) showed that using the EMLA cream in combination with a low energy, an onset of superficial cutaneous analgesia was seen as fast as 5 minutes after a pretreatment with a low energy ultrasound.

[000740] In one embodiment, enhancers may be applied to the tissue before, during, and/or after the tissue has been treated to increase permeability. Enhancers include, but are not limited to, transport enhancers, physical enhancers, and cavitation enhancers.
Non-limiting examples of enhancers are described in U.S. Patent No. 6,190,315, herein incorporated by reference in its entirety.

[000741] In one embodiment, a device may be used to increase permeability of tissue before delivering formulations of modified mRNA described herein, which may further contain a substance that invokes an immune response. In another non-limiting example, a formulation containing a substance to invoke an immune response may be delivered by the methods described in U.S. Publication Nos. 20040171980 and 20040236268;
each of which are herein incorporated by reference in their entireties.

[000742] 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.

[000743] Additionally, the present invention 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.

[000744] 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.

[000745] Topically-administrable formulations may, for example, comprise from about 0.1% 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.
Depot Administration [000746] As described herein, in some embodiments, the composition is formulated in depots for extended release. Generally, a specific organ or tissue (a "target tissue") is targeted for administration.

[000747] In some aspects of the invention, the polynucleotides, primary constructs or mmRNA are spatially retained within or proximal to a target tissue. Provided are method of providing a composition to a target tissue of a mammalian subject by contacting the target tissue (which contains one or more target cells) with the composition under conditions such that the composition, in particular the nucleic acid component(s) of the composition, is substantially retained in the target tissue, meaning that at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the composition is retained in the target tissue. Advantageously, retention is determined by measuring the amount of the nucleic acid present in the composition that enters one or more target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99 or greater than 99.99% of the nucleic acids administered to the subject are present intracellularly at a period of time following administration. For example, intramuscular injection to a mammalian subject is performed using an aqueous composition containing a ribonucleic acid and a transfection reagent, and retention of the composition is determined by measuring the amount of the ribonucleic acid present in the muscle cells.

[000748] Aspects of the invention are directed to methods of providing a composition to a target tissue of a mammalian subject, by contacting the target tissue (containing one or more target cells) with the composition under conditions such that the composition is substantially retained in the target tissue. The composition contains an effective amount of a polynucleotides, primary constructs or mmRNA such that the polypeptide of interest is produced in at least one target cell. The compositions generally contain a cell penetration agent, although "naked" nucleic acid (such as nucleic acids without a cell penetration agent or other agent) is also contemplated, and a pharmaceutically acceptable carrier.

[000749] In some circumstances, the amount of a protein produced by cells in a tissue is desirably increased. Preferably, this increase in protein production is spatially restricted to cells within the target tissue. Thus, provided are methods of increasing production of a protein of interest in a tissue of a mammalian subject. A composition is provided that contains polynucleotides, primary constructs or mmRNA characterized in that a unit quantity of composition has been determined to produce the polypeptide of interest in a substantial percentage of cells contained within a predetermined volume of the target tissue.

[000750] In some embodiments, the composition includes a plurality of different polynucleotides, primary constructs or mmRNA, where one or more than one of the polynucleotides, primary constructs or mmRNA encodes a polypeptide of interest.
Optionally, the composition also contains a cell penetration agent to assist in the intracellular delivery of the composition. A determination is made of the dose of the composition required to produce the polypeptide of interest in a substantial percentage of cells contained within the predetermined volume of the target tissue (generally, without inducing significant production of the polypeptide of interest in tissue adjacent to the predetermined volume, or distally to the target tissue). Subsequent to this determination, the determined dose is introduced directly into the tissue of the mammalian subject.

[000751] In one embodiment, the invention provides for the polynucleotides, primary constructs or mmRNA to be delivered in more than one injection or by split dose injections.

[000752] In one embodiment, the invention may be retained near target tissue using a small disposable drug reservoir, patch pump or osmotic pump. Non-limiting examples of patch pumps include those manufactured and/or sold by BD (Franklin Lakes, NJ), Insulet Corporation (Bedford, MA), SteadyMed Therapeutics (San Francisco, CA), Medtronic (Minneapolis, MN) (e.g., MiniMed), UniLife (York, PA), Valeritas (Bridgewater, NJ), and SpringLeaf Therapeutics (Boston, MA). A non-limiting example of an osmotic pump include those manufactured by DURECTO (Cupertino, CA) (e.g., DUROSO and ALZET 0).
Pulmonary Administration [000753] 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 suitably 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.

[000754] 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).

[000755] As a non-limiting example, the polynucleotides, primary constructs and/or mmRNA described herein may be formulated for pulmonary delivery by the methods described in U.S. Pat. No. 8,257,685; herein incorporated by reference in its entirety.

[000756] 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.
Intranasal, nasal and buccal Administration [000757] 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 um to 500 um. 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.

[000758] 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.
Ophthalmic Administration [000759] 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 ophthalmically-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 invention. A multilayer thin film device may be prepared to contain a pharmaceutical composition for delivery to the eye and/or surrounding tissue.
Payload Administration: Detectable Agents and Therapeutic Agents [000760] The polynucleotides, primary constructs or mmRNA described herein can be used in a number of different scenarios in which delivery of a substance (the "payload") to a biological target is desired, for example delivery of detectable substances for detection of the target, or delivery of a therapeutic agent. Detection methods can include, but are not limited to, both imaging in vitro and in vivo imaging methods, e.g., immunohistochemistry, bioluminescence imaging (BLI), Magnetic Resonance Imaging (MRI), positron emission tomography (PET), electron microscopy, X-ray computed tomography, Raman imaging, optical coherence tomography, absorption imaging, thermal imaging, fluorescence reflectance imaging, fluorescence microscopy, fluorescence molecular tomographic imaging, nuclear magnetic resonance imaging, X-ray imaging, ultrasound imaging, photoacoustic imaging, lab assays, or in any situation where tagging/staining/imaging is required.

[000761] The polynucleotides, primary constructs or mmRNA can be designed to include both a linker and a payload in any useful orientation. For example, a linker having two ends is used to attach one end to the payload and the other end to the nucleobase, such as at the C-7 or C-8 positions of the deaza-adenosine or deaza-guanosine or to the N-3 or C-5 positions of cytosine or uracil. The polynucleotide of the invention can include more than one payload (e.g., a label and a transcription inhibitor), as well as a cleavable linker. In one embodiment, the modified nucleotide is a modified 7-deaza-adenosine triphosphate, where one end of a cleavable linker is attached to the C7 position of 7-deaza-adenine, the other end of the linker is attached to an inhibitor (e.g., to the C5 position of the nucleobase on a cytidine), and a label (e.g., Cy5) is attached to the center of the linker (see, e.g., compound 1 of A*pCp C5 Parg Capless in Fig. 5 and columns 9 and 10 of U.S. Pat. No. 7,994,304, incorporated herein by reference). Upon incorporation of the modified 7-deaza-adenosine triphosphate to an encoding region, the resulting polynucleotide having a cleavable linker attached to a label and an inhibitor (e.g., a polymerase inhibitor). Upon cleavage of the linker (e.g., with reductive conditions to reduce a linker having a cleavable disulfide moiety), the label and inhibitor are released. Additional linkers and payloads (e.g., therapeutic agents, detectable labels, and cell penetrating payloads) are described herein.

[000762] Scheme 12 below depicts an exemplary modified nucleotide wherein the nucleobase, adenine, is attached to a linker at the C-7 carbon of 7-deaza adenine. In addition, Scheme 12 depicts the modified nucleotide with the linker and payload, e.g., a detectable agent, incorporated onto the 3' end of the mRNA. Disulfide cleavage and 1,2-addition of the thiol group onto the propargyl ester releases the detectable agent. The remaining structure (depicted, for example, as pApC5Parg in Scheme 12) is the inhibitor.
The rationale for the structure of the modified nucleotides is that the tethered inhibitor sterically interferes with the ability of the polymerase to incorporate a second base. Thus, it is critical that the tether be long enough to affect this function and that the inhibiter be in a stereochemical orientation that inhibits or prohibits second and follow on nucleotides into the growing polynucleotide strand.
Scheme 12 03s so, so3H
, ...- ...- ---Ne N lir NV \ 0 8 I
Llit.111H2 N N
A Capless pCpC5 Parg 1 11 II II II-OPOPOPes-q OH OH
0o ,0 ,P
-0 \

incorporation Cy5 ''.0 I\ V H
^-^i I \ 131-S-SrI
RNA^^- ..õ. m VL-i2 ./ N.
( N
I

Cleavage of S-S bond -0. 5) P.
-o, 0 N.---)....--=, 0 r 0 ..' 1-rSH P'0 RNA-1n ,....

0 (3)/
OH OH I

NV i \ = OH
RNA-1-1 .._, 0 I N N

_________ 0) + S\) /
OH OH

[000763] For example, the polynucleotides, primary constructs or mmRNA
described herein can be used in reprogramming induced pluripotent stem cells (iPS
cells), which can directly track cells that are transfected compared to total cells in the cluster. In another example, a drug that may be attached to the polynucleotides, primary constructs or mmRNA via a linker and may be fluorescently labeled can be used to track the drug in vivo, e.g. intracellularly. Other examples include, but are not limited to, the use of a polynucleotides, primary constructs or mmRNA in reversible drug delivery into cells.

[000764] The polynucleotides, primary constructs or mmRNA described herein can be used in intracellular targeting of a payload, e.g., detectable or therapeutic agent, to specific organelle. Exemplary intracellular targets can include, but are not limited to, the nuclear localization for advanced mRNA processing, or a nuclear localization sequence (NLS) linked to the mRNA containing an inhibitor.

[000765] In addition, the polynucleotides, primary constructs or mmRNA
described herein can be used to deliver therapeutic agents to cells or tissues, e.g., in living animals.
For example, the polynucleotides, primary constructs or mmRNA described herein can be used to deliver highly polar chemotherapeutics agents to kill cancer cells.
The polynucleotides, primary constructs or mmRNA attached to the therapeutic agent through a linker can facilitate member permeation allowing the therapeutic agent to travel into a cell to reach an intracellular target.

[000766] In one example, the linker is attached at the 2'-position of the ribose ring and/or at the 3' and/or 5' positionof the polynucleotides, primary constructs mmRNA
(See e.g., International Pub. No. W02012030683, herein incorporated by reference in its entirety). The linker may be any linker disclosed herein, known in the art and/or disclosed in International Pub. No. W02012030683, herein incorporated by reference in its entirety.

[000767] In another example, the polynucleotides, primary constructs or mmRNA
can be attached to the polynucleotides, primary constructs or mmRNA a viral inhibitory peptide (VIP) through a cleavable linker. The cleavable linker can release the VIP and dye into the cell. In another example, the polynucleotides, primary constructs or mmRNA can be attached through the linker to an ADP-ribosylate, which is responsible for the actions of some bacterial toxins, such as cholera toxin, diphtheria toxin, and pertussis toxin. These toxin proteins are ADP-ribosyltransferases that modify target proteins in human cells. For example, cholera toxin ADP-ribosylates G proteins modifies human cells by causing massive fluid secretion from the lining of the small intestine, which results in life-threatening diarrhea.

[000768] In some embodiments, the payload may be a therapeutic agent such as a cytotoxin, radioactive ion, chemotherapeutic, or other therapeutic agent. A
cytotoxin or cytotoxic agent includes any agent that may be detrimental to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No. 5,208,020 incorporated herein in its entirety), rachelmycin (CC-1065, see U.S. Pat. Nos. 5,475,092, 5,585,499, and 5,846,545, all of which are incorporated herein by reference), and analogs or homologs thereof Radioactive ions include, but are not limited to iodine (e.g., iodine 125 or iodine 131), strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Other therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thiotepa chlorambucil, rachelmycin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g., vincristine, vinblastine, taxol and maytansinoids).

[000769] In some embodiments, the payload may be a detectable agent, such as various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin), chemiluminescent materials, radioactive materials (e.g., 18F5 67Ga, 8imKr5 82Rb, 111In5 12315 133xe5 201115 12515 35, 14-5 3H, or 99mTc (e.g., as pertechnetate (technetate(VII), Tc04-)), and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), monocrystalline iron oxide nanoparticles (MIONs), and ultrasmall superparamagnetic iron oxide (USPIO)), manganese chelates (e.g., Mn-DPDP), barium sulfate, iodinated contrast media (iohexol), microbubbles, or perfluorocarbons). Such optically-detectable labels include for example, without limitation, 4-acetamido-4'-isothiocyanatostilbene-2,2'disulfonic acid;
acridine and derivatives (e.g., acridine and acridine isothiocyanate); 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS); 4-amino-N-[3-vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate; N-(4-anilino-l-naphthyl)maleimide;
anthranilamide; BODIPY; Brilliant Yellow; coumarin and derivatives (e.g., coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), and 7-amino-4-trifluoromethylcoumarin (Coumarin 151)); cyanine dyes; cyanosine; 4',6-diaminidino-2-phenylindole (DAPI); 5' 5"-dibromopyrogallol-sulfonaphthalein (Bromopyrogallol Red);
7-diethylamino-3-(4'-isothiocyanatopheny1)-4-methylcoumarin;
diethylenetriamine pentaacetate; 4,4'-diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid; 5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansylchloride); 4-dimethylaminophenylazopheny1-4'-isothiocyanate (DABITC); eosin and derivatives (e.g., eosin and eosin isothiocyanate);
erythrosin and derivatives (e.g., erythrosin B and erythrosin isothiocyanate); ethidium;
fluorescein and derivatives (e.g., 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein, fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITC
or XRITC), and fluorescamine); 2-[2-[3-[[1,3-dihydro-1,1-dimethy1-3-(3-sulfopropy1)-2H-benz[e]indo1-2-ylidene]ethylidene]-2-[4-(ethoxycarbony1)-1-piperazinyl]-1-cyclopenten-1-yl]etheny1]-1,1-dimethyl-3-(3-sulforpropy1)-1H-benz[e]indolium hydroxide, inner salt, compound with n,n-diethylethanamine(1:1) (IR144); 5-chloro-2-[2-[3-[(5-chloro-3-ethy1-2(3H)-benzothiazol- ylidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1-yl]etheny1]-3-ethyl benzothiazolium perchlorate (IR140);
Malachite Green isothiocyanate; 4-methylumbelliferone orthocresolphthalein; nitrotyrosine;
pararosaniline; Phenol Red; B-phycoerythrin; o-phthaldialdehyde; pyrene and derivatives(e.g., pyrene, pyrene butyrate, and succinimidyl 1-pyrene);
butyrate quantum dots; Reactive Red 4 (CIBACRONTM Brilliant Red 3B-A); rhodamine and derivatives (e.g., 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodarnine (Rhod), rhodamine B, rhodamine 123, rhodamine X

isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N ',N 'tetramethyl-6-carboxyrhodamine (TAMRA) tetramethyl rhodamine, and tetramethyl rhodamine isothiocyanate (TRITC));
riboflavin;
rosolic acid; terbium chelate derivatives; Cyanine-3 (Cy3); Cyanine-5 (Cy5);
cyanine-5.5 (Cy5.5), Cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalo cyanine; and naphthalo cyanine.

[000770] In some embodiments, the detectable agent may be a non-detectable pre-cursor that becomes detectable upon activation (e.g., fluorogenic tetrazine-fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X) or enzyme activatable fluorogenic agents (e.g., PROSENSEO (VisEn Medical))). In vitro assays in which the enzyme labeled compositions can be used include, but are not limited to, enzyme linked immunosorbent assays (ELISAs), immunoprecipitation assays, immunofluorescence, enzyme immunoassays (EIA), radioimmunoassays (RIA), and Western blot analysis.
Combinations [000771] The polynucleotides, primary constructs or mmRNA 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 may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body. As a non-limiting example, the nucleic acids or mmRNA
may be used in combination with a pharmaceutical agent for the treatment of cancer or to control hyperproliferative cells. In U.S. Pat. No. 7,964,571, herein incorporated by reference in its entirety, a combination therapy for the treatment of solid primary or metastasized tumor is described using a pharmaceutical composition including a DNA
plasmid encoding for interleukin-12 with a lipopolymer and also administering at least one anticancer agent or chemotherapeutic. Further, the nucleic acids and mmRNA
of the present invention that encodes anti-proliferative molecules may be in a pharmaceutical composition with a lipopolymer (see e.g., U.S. Pub. No. 20110218231, herein incorporated by reference in its entirety, claiming a pharmaceutical composition comprising a DNA plasmid encoding an anti-proliferative molecule and a lipopolymer) which may be administered with at least one chemotherapeutic or anticancer agent.

[000772] 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.
In one embodiment, the combinations, each or together may be administered according to the split dosing regimens described herein.
Dosing [000773] The present invention provides methods comprising administering modified mRNAs and their encoded proteins or complexes in accordance with the invention to a subject in need thereof. Nucleic acids, 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 invention 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 invention may be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective, prophylactically 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.

[000774] In certain embodiments, compositions in accordance with the present invention may be administered at dosage levels sufficient to deliver from about 0.0001 mg/kg to about 100 mg/kg, from about 0.001 mg/kg to about 0.05 mg/kg, from about 0.005 mg/kg to about 0.05 mg/kg, from about 0.001 mg/kg to about 0.005 mg/kg, from about 0.05 mg/kg to about 0.5 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). When multiple administrations are employed, split dosing regimens such as those described herein may be used.

[000775] According to the present invention, it has been discovered that administration of mmRNA in split-dose regimens produce higher levels of proteins in mammalian subjects. As used herein, a "split dose" is the division of single unit dose or total daily dose into two or more doses, e.g, two or more administrations of the single unit dose. As used herein, a "single unit dose" is a dose of any therapeutic administed in one dose/at one time/single route/single point of contact, i.e., single administration event. As used herein, a "total daily dose" is an amount given or prescribed in 24 hr period.
It may be administered as a single unit dose. In one embodiment, the mmRNA of the present invention are administed to a subject in split doses. The mmRNA may be formulated in buffer only or in a formulation described herein.
Dosage Forms [000776] A pharmaceutical composition described herein can be formulated into a dosage form described herein, such as a topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, subcutaneous).
Liquid dosage forms [000777] Liquid dosage forms for 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 including, but not limited to, 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 In certain embodiments for parenteral administration, compositions may be mixed with solubilizing agents such as CREMOPHOR , alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof.
Injectable [000778] Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art and may include 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, a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed include, but are not limited to, 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 may be 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 polynucleotide, primary construct or mmRNA 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 polynucleotide, primary construct or mmRNA may be accomplished by dissolving or suspending the polynucleotide, primary construct or mmRNA in an oil vehicle. Injectable depot forms are made by forming microencapsule matrices of the polynucleotide, primary construct or mmRNA in biodegradable polymers such as polylactide-polyglycolide. Depending upon the ratio of polynucleotide, primary construct or mmRNA to polymer and the nature of the particular polymer employed, the rate of polynucleotide, primary construct or mmRNA release can be controlled.
Examples of other biodegradable polymers include, but are not limited to, poly(orthoesters) and poly(anhydrides). Depot injectable formulations may be prepared by entrapping the polynucleotide, primary construct or mmRNA in liposomes or microemulsions which are compatible with body tissues.
Pulmonary [000779] Formulations described herein as being useful for pulmonary delivery may also be used for intranasal delivery of a pharmaceutical composition. Another formulation suitable for intranasal administration may be a coarse powder comprising the active ingredient and having an average particle from about 0.2 ilm to 500 pm.
Such a formulation may be 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.

[000780] 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, contain about 0.1% to 20% (w/w) active ingredient, where the balance may comprise 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.

[000781] 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 in its entirety).
Coatings or Shells [000782] 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.
Properties of Pharmaceutical Compositions [000783] The pharmaceutical compositions described herein can be characterized by one or more of bioavailability, therapeutic window and/or volume of distribution.

Bioavailability [000784] The polynucleotides, primary constructs or mmRNA, when formulated into a composition with a delivery agent as described herein, can exhibit an increase in bioavailability as compared to a composition lacking a delivery agent as described herein.
As used herein, the term "bioavailability" refers to the systemic availability of a given amount of polynucleotides, primary constructs or mmRNA administered to a mammal.
Bioavailability can be assessed by measuring the area under the curve (AUC) or the maximum serum or plasma concentration (C.) of the unchanged form of a compound following administration of the compound to a mammal. AUC is a determination of the area under the curve plotting the serum or plasma concentration of a compound along the ordinate (Y-axis) against time along the abscissa (X-axis). Generally, the AUC
for a particular compound can be calculated using methods known to those of ordinary skill in the art and as described in G. S. Banker, Modern Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, New York, Inc., 1996, herein incorporated by reference in its entirety.

[000785] The C. value is the maximum concentration of the compound achieved in the serum or plasma of a mammal following administration of the compound to the mammal. The C. value of a particular compound can be measured using methods known to those of ordinary skill in the art. The phrases "increasing bioavailability" or "improving the pharmacokinetics," as used herein mean that the systemic availability of a first polynucleotide, primary construct or mmRNA, measured as AUC, Cmax, Or Cmin in a mammal is greater, when co-administered with a delivery agent as described herein, than when such co-administration does not take place. In some embodiments, the bioavailability of the polynucleotide, primary construct or mmRNA can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
Therapeutic Window [000786] The polynucleotides, primary constructs or mmRNA, when formulated into a composition with a delivery agent as described herein, can exhibit an increase in the therapeutic window of the administered polynucleotide, primary construct or mmRNA
composition as compared to the therapeutic window of the administered polynucleotide, primary construct or mmRNA composition lacking a delivery agent as described herein.
As used herein "therapeutic window" refers to the range of plasma concentrations, or the range of levels of therapeutically active substance at the site of action, with a high probability of eliciting a therapeutic effect. In some embodiments, the therapeutic window of the polynucleotide, primary construct or mmRNA when co-administered with a delivery agent as described herein can increase by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or about 100%.
Volume of Distribution [000787] The polynucleotides, primary constructs or mmRNA, when formulated into a composition with a delivery agent as described herein, can exhibit an improved volume of distribution (Vdist), e.g., reduced or targeted, relative to a composition lacking a delivery agent as described herein. The volume of distribution (Vdist) relates the amount of the drug in the body to the concentration of the drug in the blood or plasma. As used herein, the term "volume of distribution" refers to the fluid volume that would be required to contain the total amount of the drug in the body at the same concentration as in the blood or plasma: Vdist equals the amount of drug in the body/concentration of drug in blood or plasma. For example, for a 10 mg dose and a plasma concentration of 10 mg/L, the volume of distribution would be 1 liter. The volume of distribution reflects the extent to which the drug is present in the extravascular tissue. A large volume of distribution reflects the tendency of a compound to bind to the tissue components compared with plasma protein binding. In a clinical setting, Vdist can be used to determine a loading dose to achieve a steady state concentration. In some embodiments, the volume of distribution of the polynucleotide, primary construct or mmRNA
when co-administered with a delivery agent as described herein can decrease at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%.
Biological Effect [000788] In one embodiment, the biological effect of the modified mRNA
delivered to the animals may be categorized by analyzing the protein expression in the animals. The protein expression may be determined from analyzing a biological sample collected from a mammal administered the modified mRNA of the present invention. In one embodiment, the expression protein encoded by the modified mRNA administered to the mammal of at least 50 pg/ml may be preferred. For example, a protein expression of 50-200 pg/ml for the protein encoded by the modified mRNA delivered to the mammal may be seen as a therapeutically effective amount of protein in the mammal.
Detection of Modified Nucleic Acids by Mass Spectrometry [000789] Mass spectrometry (MS) is an analytical technique that can provide structural and molecular mass/concentration information on molecules after their conversion to ions. The molecules are first ionized to acquire positive or negative charges and then they travel through the mass analyzer to arrive at different areas of the detector according to their mass/charge (m/z) ratio.

[000790] Mass spectrometry is performed using a mass spectrometer which includes an ion source for ionizing the fractionated sample and creating charged molecules for further analysis. For example ionization of the sample may be performed by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. The skilled artisan will understand that the choice of ionization method can be determined based on the analyte to be measured, type of sample, the type of detector, the choice of positive versus negative mode, etc.

[000791] After the sample has been ionized, the positively charged or negatively charged ions thereby created may be analyzed to determine a mass-to-charge ratio (i.e., m/z). Suitable analyzers for determining mass-to-charge ratios include quadropole analyzers, ion traps analyzers, and time-of-flight analyzers. The ions may be detected using several detection modes. For example, selected ions may be detected (i.e., using a selective ion monitoring mode (SIM)), or alternatively, ions may be detected using a scanning mode, e.g., multiple reaction monitoring (MRM) or selected reaction monitoring (SRM).

[000792] Liquid chromatography-multiple reaction monitoring (LC-MS/MRM) coupled with stable isotope labeled dilution of peptide standards has been shown to be an effective method for protein verification (e.g., Keshishian et al., Mol Cell Proteomics 2009 8: 2339-2349; Kuhn et al., Clin Chem 2009 55:1108-1117; Lopez et al., Clin Chem 2010 56:281-290; each of which are herein incorporated by reference in its entirety).
Unlike untargeted mass spectrometry frequently used in biomarker discovery studies, targeted MS methods are peptide sequence¨based modes of MS that focus the full analytical capacity of the instrument on tens to hundreds of selected peptides in a complex mixture. By restricting detection and fragmentation to only those peptides derived from proteins of interest, sensitivity and reproducibility are improved dramatically compared to discovery-mode MS methods. This method of mass spectrometry-based multiple reaction monitoring (MRM) quantitation of proteins can dramatically impact the discovery and quantitation of biomarkers via rapid, targeted, multiplexed protein expression profiling of clinical samples.

[000793] In one embodiment, a biological sample which may contain at least one protein encoded by at least one modified mRNA of the present invention may be analyzed by the method of MRM-MS. The quantification of the biological sample may further include, but is not limited to, isotopically labeled peptides or proteins as internal standards.

[000794] According to the present invention, the biological sample, once obtained from the subject, may be subjected to enzyme digestion. As used herein, the term "digest"
means to break apart into shorter peptides. As used herein, the phrase "treating a sample to digest proteins" means manipulating a sample in such a way as to break down proteins in a sample. These enzymes include, but are not limited to, trypsin, endoproteinase Glu-C and chymotrypsin. In one embodiment, a biological sample which may contain at least one protein encoded by at least one modified mRNA of the present invention may be digested using enzymes.

[000795] In one embodiment, a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed for protein using electrospray ionization. Electrospray ionization (ESI) mass spectrometry (ESIMS) uses electrical energy to aid in the transfer of ions from the solution to the gaseous phase before they are analyzed by mass spectrometry. Samples may be analyzed using methods known in the art (e.g., Ho et al., Clin Biochem Rev. 2003 24(1):3-12; herein incorporated by reference in its entirety). The ionic species contained in solution may be transferred into the gas phase by dispersing a fine spray of charge droplets, evaporating the solvent and ejecting the ions from the charged droplets to generate a mist of highly charged droplets. The mist of highly charged droplets may be analyzed using at least 1, at least 2, at least 3 or at least 4 mass analyzers such as, but not limited to, a quadropole mass analyzer. Further, the mass spectrometry method may include a purification step. As a non-limiting example, the first quadrapole may be set to select a single m/z ratio so it may filter out other molecular ions having a different m/z ratio which may eliminate complicated and time-consuming sample purification procedures prior to MS
analysis.

[000796] In one embodiment, a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed for protein in a tandem ESIMS system (e.g., MS/MS). As non-limiting examples, the droplets may be analyzed using a product scan (or daughter scan) a precursor scan (parent scan) a neutral loss or a multiple reaction monitoring.

[000797] In one embodiment, a biological sample which may contain protein encoded by modified mRNA of the present invention may be analyzed using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MALDIMS). MALDI provides for the nondestructive vaporization and ionization of both large and small molecules, such as proteins. In MALDI analysis, the analyte is first co-crystallized with a large molar excess of a matrix compound, which may also include, but is not limited to, an ultraviolet absorbing weak organic acid. Non-limiting examples of matrices used in MALDI
are a-cyano-4-hydroxycinnamic acid, 3,5-dimethoxy-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid. Laser radiation of the analyte-matrix mixture may result in the vaporization of the matrix and the analyte. The laser induced desorption provides high ion yields of the intact analyte and allows for measurement of compounds with high accuracy. Samples may be analyzed using methods known in the art (e.g., Lewis, Wei and Siuzdak, Encyclopedia of Analytical Chemistry 2000:5880-5894; herein incorporated by reference in its entirety). As non-limiting examples, mass analyzers used in the MALDI analysis may include a linear time-of-flight (TOF), a TOF reflectron or a Fourier transform mass analyzer.

[000798] In one embodiment, the analyte-matrix mixture may be formed using the dried-droplet method. A biologic sample is mixed with a matrix to create a saturated matrix solution where the matrix-to-sample ratio is approximately 5000:1. An aliquot (approximately 0.5-2.0 uL) of the saturated matrix solution is then allowed to dry to form the analyte-matrix mixture.

[000799] In one embodiment, the analyte-matrix mixture may be formed using the thin-layer method. A matrix homogeneous film is first formed and then the sample is then applied and may be absorbed by the matrix to form the analyte-matrix mixture.

[000800] In one embodiment, the analyte-matrix mixture may be formed using the thick-layer method. A matrix homogeneous film is formed with a nitro-cellulose matrix additive. Once the uniform nitro-cellulose matrix layer is obtained the sample is applied and absorbed into the matrix to form the analyte-matrix mixture.

[000801] In one embodiment, the analyte-matrix mixture may be formed using the sandwich method. A thin layer of matrix crystals is prepared as in the thin-layer method followed by the addition of droplets of aqueous trifluoroacetic acid, the sample and matrix. The sample is then absorbed into the matrix to form the analyte-matrix mixture.
V. Uses of Polynucleotides, primary constructs and mmRNA of the Invention [000802] The polynucleotides, primary constructs and mmRNA of the present invention are designed, in preferred embodiments, to provide for avoidance or evasion of deleterious bio-responses such as the immune response and/or degradation pathways, overcoming the threshold of expression and/or improving protein production capacity, improved expression rates or translation efficiency, improved drug or protein half life and/or protein concentrations, optimized protein localization, to improve one or more of the stability and/or clearance in tissues, receptor uptake and/or kinetics, cellular access by the compositions, engagement with translational machinery, secretion efficiency (when applicable), accessibility to circulation, and/or modulation of a cell's status, function and/or activity.
Therapeutics Therapeutic Agents [000803] The polynucleotides, primary constructs or mmRNA of the present invention, such as modified nucleic acids and modified RNAs, and the proteins translated from them described herein can be used as therapeutic or prophylactic agents. They are provided for use in medicine. For example, a polynucleotide, primary construct or mmRNA
described herein can be administered to a subject, wherein the polynucleotide, primary construct or mmRNA is translated in vivo to produce a therapeutic or prophylactic polypeptide in the subject. Provided are compositions, methods, kits, and reagents for diagnosis, treatment or prevention of a disease or condition in humans and other mammals. The active therapeutic agents of the invention include polynucleotides, primary constructs or mmRNA, cells containing polynucleotides, primary constructs or mmRNA or polypeptides translated from the polynucleotides, primary constructs or mmRNA.

[000804] In certain embodiments, provided herein are combination therapeutics containing one or more polynucleotide, primary construct or mmRNA 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 toxicity. For example, provided herein 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)).

[000805] Provided herein are methods of inducing translation of a recombinant polypeptide in a cell population using the polynucleotide, primary construct or mmRNA
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.

[000806] 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.

[000807] Aspects of the invention 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 structural or chemical 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.

[000808] In certain embodiments, the administered polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that provide a functional activity which is substantially absent in the cell, tissue or organism in which the recombinant polypeptide is translated. For example, the missing functional activity may be enzymatic, structural, or gene regulatory in nature. In related embodiments, the administered polynucleotide, primary construct or mmRNA directs production of one or more recombinant polypeptides that increases (e.g., synergistically) a functional activity which is present but substantially deficient in the cell in which the recombinant polypeptide is translated.

[000809] In other embodiments, the administered polynucleotide, primary construct or mmRNA 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 In some embodiments, the recombinant polypeptide increases the level of an endogenous protein in the cell to a desirable level;
such an increase may bring the level of the endogenous protein from a subnormal level to a normal level or from a normal level to a super-normal level.

[000810] 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, due 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, a protein toxin such as shiga and tetanus toxins, or a small molecule toxin such as botulinum, cholera, and diphtheria toxins. Additionally, the antagonized biological molecule may be an endogenous protein that exhibits an undesirable activity, such as a cytotoxic or cytostatic activity.

[000811] The recombinant proteins described herein may be 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.

[000812] In some embodiments, modified mRNAs and their encoded polypeptides in accordance with the present invention may be used for treatment of any of a variety of diseases, disorders, and/or conditions, including but not limited to one or more of the following: autoimmune disorders (e.g. diabetes, lupus, multiple sclerosis, psoriasis, rheumatoid arthritis); inflammatory disorders (e.g. arthritis, pelvic inflammatory disease);
infectious diseases (e.g. viral infections (e.g., HIV, HCV, RSV), bacterial infections, fungal infections, sepsis); neurological disorders (e.g. Alzheimer's disease, Huntington's disease; autism; Duchenne muscular dystrophy); cardiovascular disorders (e.g.
atherosclerosis, hypercholesterolemia, thrombosis, clotting disorders, angiogenic disorders such as macular degeneration); proliferative disorders (e.g. cancer, benign neoplasms); respiratory disorders (e.g. chronic obstructive pulmonary disease); digestive disorders (e.g. inflammatory bowel disease, ulcers); musculoskeletal disorders (e.g.
fibromyalgia, arthritis); endocrine, metabolic, and nutritional disorders (e.g. diabetes, osteoporosis); urological disorders (e.g. renal disease); psychological disorders (e.g.
depression, schizophrenia); skin disorders (e.g. wounds, eczema); blood and lymphatic disorders (e.g. anemia, hemophilia); etc.

[000813] Diseases characterized by dysfunctional or aberrant protein activity include cystic fibrosis, sickle cell anemia, epidermolysis bullosa, amyotrophic lateral sclerosis, and glucose-6-phosphate dehydrogenase deficiency. The present invention provides a method for treating such conditions or diseases in a subject by introducing nucleic acid or cell-based therapeutics containing the polynucleotide, primary construct or mmRNA
provided herein, wherein the polynucleotide, primary construct or mmRNA 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.

[000814] Diseases characterized by missing (or substantially diminished such that proper (normal or physiological protein function does not occur) protein activity include cystic fibrosis, Niemann-Pick type C, 0 thalassemia major, Duchenne muscular dystrophy, Hurler Syndrome, Hunter Syndrome, and Hemophilia A. Such proteins may not be present, or are essentially non-functional. The present invention provides a method for treating such conditions or diseases in a subject by introducing nucleic acid or cell-based therapeutics containing the polynucleotide, primary construct or mmRNA
provided herein, wherein the polynucleotide, primary construct or mmRNA 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.

[000815] Thus, provided are methods of treating cystic fibrosis in a mammalian subject by contacting a cell of the subject with a polynucleotide, primary construct or mmRNA
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, endothelial and mesothelial 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.

[000816] In another embodiment, the present invention 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 SORT] 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, rs12740374, in the 1p13 locus of the SORT1 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 SORT] in mouse liver tissue led to significantly lower LDL-cholesterol levels, as much as 80% lower, and that silencing SORT1 increased LDL
cholesterol approximately 200% (Musunuru K et al. From noncoding variant to phenotype via SORT] at the 1p13 cholesterol locus. Nature 2010; 466: 714-721).

[000817] In another embodiment, the present invention provides a method for treating hematopoietic disorders, cardiovascular disease, oncology, diabetes, cystic fibrosis, neurological diseases, inborn errors of metabolism, skin and systemic disorders, and blindness. The identity of molecular targets to treat these specific diseases has been described (Templeton ed., Gene and Cell Therapy: Therapeutic Mechanisms and Strategies, 3rd Edition, Bota Raton, FL:CRC Press; herein incorporated by reference in its entirety).

[000818] Provided herein, are methods to prevent infection and/or sepsis in a subject at risk of developing infection and/or sepsis, the method comprising administering to a subject in need of such prevention a composition comprising a polynucleotide, primary construct or mmRNA precursor encoding an anti-microbial polypeptide (e.g., an anti-bacterial polypeptide), or a partially or fully processed form thereof in an amount sufficient to prevent infection and/or sepsis. In certain embodiments, the subject at risk of developing infection and/or sepsis may be a cancer patient. In certain embodiments, the cancer patient may have undergone a conditioning regimen. In some embodiments, the conditioning regiment may include, but is not limited to, chemotherapy, radiation therapy, or both. As a non-limiting example, a polynucleotide, primary construct or mmRNA can encode Protein C, its zymogen or prepro-protein, the activated form of Protein C (APC) or variants of Protein C which are known in the art. The polynucleotides, primary constructs or mmRNA may be chemically modified and delivered to cells. Non-limiting examples of polypeptides which may be encoded within the chemically modified mRNAs of the present invention include those taught in US
Patents 7,226,999; 7,498,305; 6,630,138 each of which is incorporated herein by reference in its entirety. These patents teach Protein C like molecules, variants and derivatives, any of which may be encoded within the chemically modified molecules of the present invention.

[000819] Further provided herein, are methods to treat infection and/or sepsis in a subject, the method comprising administering to a subject in need of such treatment a composition comprising a polynucleotide, primary construct or mmRNA precursor encoding an anti-microbial polypeptide (e.g., an anti-bacterial polypeptide), e.g., an anti-microbial polypeptide described herein, or a partially or fully processed form thereof in an amount sufficient to treat an infection and/or sepsis. In certain embodiments, the subject in need of treatment is a cancer patient. In certain embodiments, the cancer patient has undergone a conditioning regimen. In some embodiments, the conditioning regiment may include, but is not limited to, chemotherapy, radiation therapy, or both.

[000820] In certain embodiments, the subject may exhibits acute or chronic microbial infections (e.g., bacterial infections). In certain embodiments, the subject may have received or may be receiving a therapy. In certain embodiments, the therapy may include, but is not limited to, radiotherapy, chemotherapy, steroids, ultraviolet radiation, or a combination thereof In certain embodiments, the patient may suffer from a microvascular disorder. In some embodiments, the microvascular disorder may be diabetes. In certain embodiments, the patient may have a wound. In some embodiments, the wound may be an ulcer. In a specific embodiment, the wound may be a diabetic foot ulcer. In certain embodiments, the subject may have one or more burn wounds.
In certain embodiments, the administration may be local or systemic. In certain embodiments, the administration may be subcutaneous. In certain embodiments, the administration may be intravenous. In certain embodiments, the administration may be oral. In certain embodiments, the administration may be topical. In certain embodiments, the administration may be by inhalation. In certain embodiments, the administration may be rectal. In certain embodiments, the administration may be vaginal.

[000821] Other aspects of the present disclosure relate to transplantation of cells containing polynucleotide, primary construct, or mmRNA to a mammalian subject.

Administration of cells to mammalian subjects is known to those of ordinary skill in the art, and include, but is not limited to, local implantation (e.g., topical or subcutaneous administration), organ delivery or systemic injection (e.g., intravenous injection or inhalation), and the formulation of cells in pharmaceutically acceptable carrier. Such compositions containing polynucleotide, primary construct, or mmRNA can be formulated for administration intramuscularly, transarterially, intraperitoneally, intravenously, intranasally, subcutaneously, endoscopically, transdermally, or intrathecally. In some embodiments, the composition may be formulated for extended release.

[000822] The subject to whom the therapeutic agent may be administered suffers from or may be 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.

Rare Liver Diseases or Disorders Progressive Familial Intrahepatic Cholestasis (PFIC) [000823] In one embodiment, the rare liver disease or disorder polynucleotide, primary construct, or mmRNA of the present invention may be used to treat progressive familial intrahepatic cholestasis (PFIC). The term "progressive familial intrahepatic cholestasis"
or "PFIC" as used herein refers to a liver disorder that can lead to liver failure. PFIC is characterized by autosomal recessive defects in bile formation and hepatocellular cholestasis. As used herein, "hepatocellular" is a term used to describe something that affects or pertains to liver cells (also referred to as hepatocytes). As used herein, the term "cholestasis" refers to a condition characterized by slowed or disrupted flow of bile from the liver. As used herein, the term "bile" refers to a liquid substance produced by the liver comprising water, bile salts, mucus, fats inorganic salts and cholesterol, which aids in the emulsification and digestion of dietary fats.

[000824] There are three known types of PFIC (PFIC-1, PFIC-2 and PFIC-3), and they have been traced to mutations in genes encoding proteins involved in the hepatocellular transport system and in the formation of bile.

[000825] PFIC-1 and PFIC-2 are typically diagnosed in infancy but diagnosis may occur during the prenatal period or the neonatal period. As used herein, the term "prenatal" refers to the period of time in the life of an organism that occurs before birth.
As used herein, the term "neonatal" refers to a period of time in the life of an organism that occurs after birth. In humans, the neonatal period may include a period of time from birth to about 1 month, to about 3 months or to about 6 months after birth. As used herein, the term "infancy" refers to a period in the life of an organism that occurs between birth and childhood. In humans, infancy may include the period of time from birth to about 1 year of life, to about 2 years of life, to about 3 years of life or to about 4 years of life.

[000826] PFIC-3 may be diagnosed during the prenatal period, during the neonatal period or during infancy. In some cases, PFIC-3 may be able evade diagnosis until childhood or young adulthood. As used herein, the term "childhood" refers to the period of time in the life of an organism that occurs after infancy and before young adulthood. In humans, childhood may include the period of time from about 2 years of age to about 10 DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.

NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME

NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:

Claims (92)

Claims We claim:

1. An isolated polynucleotide comprising;
(a) a first region of linked nucleosides, said first region encoding a polypeptide of interest, said polypeptide of interest selected from the group consisting of SEQ ID NOs 769-1392;
(b) a first flanking region located at the 5' terminus of said first region comprising;
(i) a sequence of linked nucleosides selected from the group consisting of the native 5' UTR of any of the nucleic acids that encode any of SEQ ID NOs 769-1392, SEQ ID NOs: 1-4 and functional variants thereof; and (ii) at least one 5' terminal cap;
(c) a second flanking region located at the 3' terminus of said first region comprising;
(i) a sequence of linked nucleosides selected from the group consisting of the native 3' UTR of any of the nucleic acids that encode any of SEQ ID NOs 769-1392, SEQ ID NOs 5-21 and functional variants thereof; and (ii') a 3' tailing sequence of linked nucleosides.

2. The isolated polynucleotide of claim 1 wherein the first region of linked nucleosides comprises at least an open reading frame of a nucleic acid sequence, wherein the nucleic acid sequence selected from the group consisting of SEQ ID NOs:

21423, 21435-21438, 21442-21443, 21449, 21451-21473, 21521-21542, 21639-21642, 21671-21679, 21681, 21682, 21684, 21686, 21687, 21724-21726, 21728 and 21730-21738.

3. The isolated polynucleotide of claim 1, wherein the 3'tailing sequence of linked nucleosides is selected from the group consisting of a poly-A tail of approximately 160 nucleotides and a polyA-G quartet.

4. The isolated polynucleotide of any one of claims 1-3 which is purified.

5. The isolated polynucleotide of any one of claims 1-4, wherein the at least one 5' terminal cap is selected from the group consisting of Cap0, Cap1, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

6. The isolated polynucleotide of any preceding claim, wherein at least one of said linked nucleosides comprises at least one modification as compared to the chemical structure of an A, G, U or C ribonucleotide.

7. The isolated polynucleotide of claim 6, wherein at least one said modification is located in a nucleoside base and/or sugar portion.

8. The isolated polynucleotide of any one of claims 1-7, wherein said first region comprises n number of linked nucleosides having Formula (Ia):
(Ia), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein U is O, S, N(R U)nu, or C(R U)nu, wherein nu is an integer from 0 to 2 and each R U
is, independently, H, halo, or optionally substituted alkyl;
is a single or double bond;
- - - is a single bond or absent;
each of R1', R2', R1", R2", R3, R4, and R5 is, independently, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, or absent; wherein the combination of R3 with one or more of R1', R1", R2', R2", or R5 can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl; wherein the combination of R5 with one or more of R1', R1", R2', or R2" can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl; and wherein the combination of R4 and one or more of R1', R1", R2', R2", R3, or R5 can join together to form optionally substituted alkylene or optionally substituted heteroalkylene and, taken together with the carbons to which they are attached, provide an optionally substituted heterocyclyl;
each of Y1, Y2, and Y3, is, independently, O, S, -NR N1-, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent;
each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino;
each Y5 is, independently, O, S, optionally substituted alkylene, or optionally substituted heteroalkylene;
n is an integer from 1 to 100,000; and B is a nucleobase, wherein the combination of B and R1', the combination of B
and R2', the combination of B and R1", or the combination of B and R2" can, taken together with the carbons to which they are attached, optionally form a bicyclic group or wherein the combination of B, R1", and R3 or the combination of B, R2", and R3 can optionally form a tricyclic or tetracyclic group.

9. The isolated polynucleotide of claim 8, wherein B is not pseudouridine (.PSI.) or 5-methyl-cytidine (m5C).

10. The isolated polynucleotide of any one of claims 8-9, wherein U is O or C(R U)nu, wherein nu is an integer from 1 to 2 and each R U is, independently, H, halo, or optionally substituted alkyl;
each of R1, R1', R1", R2, R2', and R2", if present, is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, or optionally substituted aminoalkyl;
each of R3 and R4 is, independently, H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxyalkoxy;
each of Y1, Y2, and Y3, is, independently, O, S, -NR N1-, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;
each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, or optionally substituted amino;
each Y5 is, independently, O or optionally substituted alkylene; and n is an integer from 10 to 10,000.

11. The isolated polynucleotide of claim 10, wherein each of R1, R1', and R1", if present, is H.

12. The isolated polynucleotide of claim 11, wherein each of R2, R2', and R2", if present, is, independently, H, halo, hydroxy, optionally substituted alkoxy, or optionally substituted alkoxyalkoxy.

13. The isolated polynucleotide of claim 10, wherein each of R2, R2', and R2", if present, is H.

14. The isolated polynucleotide of claim 13, wherein each of R1, R1', and R1", if present, is, independently, H, halo, hydroxy, optionally substituted alkoxy, or optionally substituted alkoxyalkoxy.

15. The isolated polynucleotide of claim 8, wherein said first region comprises n number of linked nucleotides having Formula (IIa):
(IIa), or a pharmaceutically acceptable salt or stereoisomer thereof.

16. isolated The polypeptide of claim 15, wherein said first region comprises n number of linked nucleosides having Formula (IIb) or (IIc), or a pharmaceutically acceptable salt thereof.

17. The isolated polynucleotide of claim 8, wherein said first region comprises n number of linked nucleosides having Formula (IId):
(IId), or a pharmaceutically acceptable salt or stereoisomer thereof.

18. The isolated polypeptide of claim 17, wherein said first region comprises n number of linked nucleosides having Formula (IIe) or (IIf), or a pharmaceutically acceptable salt thereof.

19. The isolated polynucleotide of claim 8, wherein said first region comprises n number of linked nucleotides, each of said linked nucleotides independently having one of Formulas (IIg)-(IIj):
pharmaceutically acceptable salt or stereoisomer thereof

20. The isolated polynucleotide of claim 8, wherein said first region comprises n number of linked nucleosides having Formula (IIk):
(IIk) , or a pharmaceutically acceptable salt or stereoisomer thereof.

21. The isolated polynucleotide of claim 20, wherein said first region comprises n number of linked nucleosides having Formula (IIl):
(II1), or a pharmaceutically acceptable salt or stereoisomer thereof

22. The isolated polynucleotide of claim 20, wherein said first region comprises n number of linked nucleosides having Formula (IIm):
(IIm), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each of R1', R1", R2', and R2" is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, or absent; and wherein the combination of R2' and R3 or the combination of R2" and R3 can be taken together to form optionally substituted alkylene or optionally substituted heteroalkylene.

23. The isolated polynucleotide of any one of claims 11-22, wherein U is O or C(R U)nu, wherein nu is an integer from 1 to 2 and each R U is, independently, H, halo, or optionally substituted alkyl;

each of R1 and R2 is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, or optionally substituted aminoalkyl;
each of R3 and R4 is, independently, H or optionally substituted alkyl;
each of Y1, Y2, and Y3, is, independently, O, S, -NR N1-, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;
each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, or optionally substituted amino;
each Y5 is, independently, O or optionally substituted alkylene; and n is an integer from 10 to 10,000.

24. The isolated polynucleotide of claim 8, wherein said first region comprises n number of linked nucleosides having Formula (IIn):
(IIn), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein U is O or C(R U)nu, wherein nu is an integer from 1 to 2 and each R U is, independently, H, halo, or optionally substituted alkyl;
each of R1 and R4 is, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, or optionally substituted aminoalkyl;
R3' is O, S, or -NR N1-, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;
R3" is optionally substituted alkylene or optionally substituted heteroalkylene;
each of Y1, Y2, and Y3, is, independently, O, S, -NR N1-, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;
each Y4 is, independently, H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, or optionally substituted amino;
each Y5 is, independently, O, S, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene; and n is an integer from 10 to 10,000.

25. The isolated polynucleotide of any one of claims 8-24, wherein in said n number of B has, each B independently has a formula selected from Formula (b1)-(b5):
or (b5), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein connecting V1 and V2 in formula (b1) is a single or double bond;

each of T1', T1", T2', and T2" is, independently, H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or the combination of T1' and T1" or the combination of T2' and T2" join together to form O (oxo), S
(thio), or Se (seleno);
each of V1 and V2 is, independently, O, S, N(R Vb), or C(R Vb), wherein nv is an integer from 0 to 2 and each R Vb is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy;
R10 is H, halo, optionally substituted amino acid, hydroxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aminoalkyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl;
R11 is H or optionally substituted alkyl;
R12a is H, optionally substituted alkyl, or optionally substituted aminoalkyl;
and R12c is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted aminoalkyl.

26. The isolated polynucleotide of any one of claims 8-25, wherein n number of B has Formula (b6)-(b9):
(b9), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein is a single or double bond;

each of T1', T1", T2', and T2" is, independently, H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or the combination of T1' and T1" or the combination of T2' and T2" join together to form O (oxo), S
(thio), or Se (seleno);
each of W1 and W2 is, independently, N(R Wa)nw or c(R Wa)nw, wherein nw is an integer from 0 to 2 and each R Wa is, independently, H, optionally substituted alkyl, or optionally substituted alkoxy;
each V3 is, independently, O, S, N(R Va)nv, or C(R Va), wherein nv is an integer from 0 to 2 and each R Va is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy, and wherein R Va and R12C taken together with the carbon atoms to which they are attached can form optionally substituted cycloalkyl, optionally substituted aryl, or optionally substituted heterocyclyl;
R12a is H, optionally substituted alkyl, optionally substituted aminoalkyl, or absent;
R12b is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkaryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, or optionally substituted amino acid, wherein the combination of R12b and T1' or the combination of R12b and R12C can join together to form optionally substituted heterocyclyl; and R12c is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted aminoalkyl.

27. The isolated polynucleotide of claim 26, wherein R12a, R12b, R12c, or R
Va is substituted with -(CH2)s2(OCH2CH2)s1(CH2)OR', wherein s1 is an integer from 1 to 10, each of s2 and s3, independently, is an integer from 0 to 10, and R' is H or C1-20 alkyl); or -NR N1(CH2)s2(CH2CH2O)s1(CH2)s3NR N1, wherein s1 is an integer from 1 to 10, each of s2 and s3, independently, is an integer from 0 to 10, and each R N1 is, independently, hydrogen or optionally substituted C1-6 alkyl.

28. The isolated polynucleotide of any one of claims 8-27, wherein n number of B has Formula (b 10)-(b 14):
, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each of T3' and T3" is, independently, H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or the combination of T3' and T3"
join together to form O (oxo), S (thio), or Se (seleno);
each V4 is, independently, O, S, N(R Vc)nv, or C(R Vc)nv, wherein nv is an integer from 0 to 2 and each R Vc is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, or optionally substituted alkynyloxy, wherein the combination of R13b and R Vc can be taken together to form optionally substituted heterocyclyl;
each of R13a and R13b is, independently, H, optionally substituted acyl, optionally substituted alkyl, or optionally substituted alkoxy, wherein the combination of R13b and R14 can be taken together to form optionally substituted heterocyclyl;

each R14 is, independently, H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, or optionally substituted aminoalkyl;
and each of R15 and R16 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

29. The isolated polynucleotide of any one of claims 7-27, wherein n number of B has Formula (b15)-(b17):
, or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each of T4', T4", T5', T5", T6', and T6" is, independently, H, optionally substituted alkyl, or optionally substituted alkoxy, and wherein the combination of T4' and T4" or the combination of T5' and T5" or the combination of T6' and T6" together form O, S, or Se;
each of V5 and V6 is, independently, O, S, N(R Vd), or C(R Vd), wherein nv is an integer from 0 to 2 and each R Vd is, independently, H, halo, optionally substituted amino acid, cyano, amidine, optionally substituted aminoalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy; and each of R17, R18, R19a R19b, R21, R22, R23, and R24 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted amino acid.

30. The polynucleotide of any one of claims 8-29, wherein n number of B has Formula (b18)-(b20):
(b20), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each V7 is, independently, O, S, N(R Ve)nv, or C(R Ve)nv, wherein nv is an integer from 0 to 2 and each R Ve is, independently, H, halo, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy;
each R25 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;
each of R26a and R26b is, independently, H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoylalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, or optionally substituted alkoxy;
each R27 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, or optionally substituted amino;

each R28 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; and each R29 is, independently, H, optionally substituted acyl, optionally substituted amino acid, optionally substituted carbamoylalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted alkoxy, or optionally substituted amino.

31. The isolated polynucleotide of claim 30, wherein R26a, R26b, or R29 is substituted with -(CH2)s2(OCH2CH2)s1(CH2)s3OR', wherein s1 is an integer from 1 to 10, each of s2 and s3, independently, is an integer from 0 to 10, and R' is H or C1-20 alkyl); or -NR Ni(CH2)s2(CH2CH2O)s1(CH2)s3NR N1, wherein s1 is an integer from 1 to 10, each of s2 and s3, independently, is an integer from 0 to 10, and each R N1 is, independently, hydrogen or optionally substituted C1-6 alkyl.

32. The isolated polynucleotide of any one of claims 8-3 1, wherein n number of B has Formula (b21):
(b21), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein X12 is, independently, O, S, optionally substituted alkylene, or optionally substituted heteroalkylene; xa is an integer from 0 to 3; R12a is H, optionally substituted alkyl, optionally substituted aminoalkyl, or absent; and T2 is O, S, or Se.

33. The isolated polynucleotide of any one of claims 8-32, wherein n number of B has Formula (b22):
(b22), or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R10' is, independently, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aminoalkyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl; R11 is H or optionally substituted alkyl; R12a is H, optionally substituted alkyl, optionally substituted aminoalkyl, or absent;
and each of T1 and T2 is, independently, O, S, or Se.

34. The isolated polynucleotide of any one of claims 8-33, wherein n number of B has Formula (b23):
, wherein R10 is optionally substituted heterocyclyl or optionally substituted aryl; R11 is H or optionally substituted alkyl; R12a is H, optionally substituted alkyl, optionally substituted aminoalkyl, or absent; and each of T1 and T2 is, independently, O, S, or Se.

35. The isolated polynucleotide of any one of claims 8-34, wherein n number of B has Formula (b24):
T3 is O, S, or Se;
each of R13a and R13b is, independently, H, optionally substituted acyl, optionally substituted alkyl, or optionally substituted alkoxy, wherein the combination of R13b and R14 can be taken together to form optionally substituted heterocyclyl;
R14' is, independently, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkheterocyclyl, optionally substituted alkaryl, optionally substituted aminoalkyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl; and each R15 is, independently, H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

36. The isolated polynucleotide of any one of claims 1-35, further comprising a targeting moiety, wherein said targeting moiety is covalently bound to said polynucleotide.

37. The isolated polynucleotide of claim 36, wherein said targeting moiety is an antibody, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, an RGD
peptide, an RGD peptide mimetic, or an aptamer.

38. A pharmaceutical composition comprising the isolated polynucleotide of any one of claims 1-37.

39. A pharmaceutical composition comprising the isolated polynucleotide of any one of claims 1-37 and a pharmaceutically acceptable excipient.

40. The pharmaceutical composition of claim 39, wherein the excipient is selected from a solvent, aqueous solvent, non-aqueous solvent, dispersion media, diluent, dispersion, suspension aid, surface active agent, isotonic agent, thickening or emulsifying agent, preservative, lipid, lipidoids liposome, lipid nanoparticle, core-shell nanoparticles, polymer, lipoplexe peptide, protein, cell, hyaluronidase, and mixtures thereof.

41. The pharmaceutical composition of claim 40, where the pharmaceutical composition comprises a lipid and wherein said lipid is selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA and PEGylated lipids and mixtures thereof.

42. A method of producing a polypeptide of interest in a mammalian cell, tissue or organism comprising administering to said cell, tissue or organism the isolated polynucleotide of any one of claims 1-37 or the pharmaceutical composition of any of claims 38-41.

43. The method of claim 42, wherein the isolated polynucleotide is formulated.

44. The method of claim 43, wherein the formulation comprises a lipid which is selected from one of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA, PEGylated lipids and mixtures or combinations thereof.

45. The method of claim 44, wherein the isolated polynucleotide is administered at a total daily dose of between 1 ug and 150 ug.

46. The method of claim 45, wheriein administration is by injection.

47. The method of claim 46, wherein administration is intradermal or subcutaneous or intramuscular or intravitreal.

48. The method of claim 45, wherein levels of the polypeptide of interest in the serum of the mammal are at least 50 pg/mL at least two hours after administration.

49. The method of claim 45, wherein the levels of the polypeptide of interest in the serum of the mammal remain above 50 pg/mL for at least 72 hours after administration.

50. The method of claim 49, wherein the levels of the polypeptide of interest in the serum of the mammal remain above 60 pg/mL for at least 72 hours after administration.

51. The method of claim 44, wherein the resulting polynucleotide formulation has a mean particle size of 80 nm-160 nm, a PDI of between 0.02 and 0.20 and a lipid to polnucleotide ratio (wt/wt) of between 10-20.

52. A method for producing an increased level of a polypeptide of interest selected from the group consisting of SEQ ID NOs 769-1392 in a mammalian cell, tissue or organism, comprising administering to said cell, tissue or organism a total daily dose of the isolated polynucleotide of any one of claims 4-37 or the pharmaceutical composition of any one of claims 38-41 in two or more equal or unequal split doses.

53. The method of claim 52, wherein the level of the polypeptide produced in response to said administration is greater than the levels produced by administering the same total daily dose of the isolated polynucleotide or pharmaceutical composition as a single administration.

54. The method of claim 52, wherein the mammalian organism is a human patient in need of an increased level of the polypeptide of interest.

55. The method of claim 54, wherein the increased level of the polypeptide of interest is detectable in a bodily fluid of said patient.

56. The method of claim 55, wherein the bodily fluid is selected from the group consisting of peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, cerumen, breast milk, broncheoalveolar lavage fluid, semen, prostatic fluid, cowper's fluid or pre-ejaculatory fluid, sweat, fecal matter, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, menses, pus, sebum, vomit, vaginal secretions, mucosal secretion, stool water, pancreatic juice, lavage fluids from sinus cavities, bronchopulmonary aspirates, blastocyl cavity fluid, and umbilical cord blood.

57. The method of claim 56, wherein the bodily fluid is serum and the polpeptide per unit drug (PUD) is greater than 1.

58. The method of claim 57, wherein the dose splitting factor (DSF) is greater than 4.

59. The method of claim 55, wherein administration is transdermal.

60. The method of claim 59, wherein transdermal administration comprises utilization of one or more members selected from the group consisting of a patch, cream, ointment, mechanical device, needle, sponge, depot and fabric.

61. The method of claim 59, wherein administration is according to a dosing regimen which occurs over the course of hours, days, weeks, months, or years.

62. The method of claim 52, wherein said two or more split doses comprise a first dose of the polynucleotide or pharmaceutical composition administered at a time T1 followed by a second dose of the polynucleotide or pharmaceutical composition administered at a time T2, wherein said time T1 and said time T2 are separated by no more than 1 minute and wherein said first dose and said second dose are administered in amounts that result in higher levels of the polypeptide of interest in said subject than if the amounts of polynucleotide or pharmaceutical composition were administered together in a single unit dose.

63. The method of claim 62, further comprising administering a plurality of doses of said polynucleotide or pharmaceutical composition, Nx at times Tn, wherein x and n are independently selected from 3 to about 1000 and where the time between Tn and Tn+1 is separated by increments of no more than 10 seconds.

64. The method of claim 63, wherein administration occurs by direct injection.

65. The method of claim 64, wherein direct injection is selected from the group consisting of intravenous, intradermal, subcutaneous, intramuscular and intravitreal.

66. The method of claim 64, wherein said first dose is administered proximal to said second or plurality of doses.

67. The method of claim 64, wherein said first dose is administered distal to said second or plurality of doses.

68. The method of claim 64, wherein the distance between the site of injection of said first dose and the site of injection of any second or plurality of doses is from about 1mm to about 10 cm.

69. The method of claim 64, wherein injection is made at a depth of from 0.1mm to about 1cm.

70. The method of claim 65, wherein direct injection is achieved by using one or more devices selected from multineedle injection systems, catheter or lumen systems, and ultrasound, electrical or radiation based systems.

71. The method of claim 63, wherein the amount polynucleotide or pharmaceutical composition administered in any dose is substantially equal.

72. The method of claim 63, wherein time T1 and time T2 are separated by no more than 30 seconds.

73. The method of claim 63, wherein time T1 and time T2 are separated by no more than 10 seconds.

74. The method of claim 63, wherein the first dose, the second dose and any of a plurality of doses are administered at substantially the same time.

75. The method of claim 63, wherein the single unit dose is between about 10 mg/kg and about 500 mg/kg.

76. The method of claim 63, wherein the single unit dose is between about 1.0 mg/kg and about 10 mg/kg.

77. The method of claim 63, wherein the single unit dose is between about 0.001 mg/kg and about 1.0 mg/kg.

78. A method of preparing a lipid nanopartical formulation of a polynucleotide encoding a polypeptide of interest comprising rapidly injecting a first ethanolic solution into a second aqueous solution wherein, (a) said first ethanolic solution comprises a mixture of lipid:DSPC:
Cholesterol:
PEG-c-DOMG to yield a molar ratio of 50:10:38.5:1.5 and having a final lipid concentration of approximately 25mM, and (b) said second aqueous solution comprises a sodium citrate buffered solution of the polynucleotide encoding the polypeptide of interest having a concentration of 1-2 mg/mL and a pH of approximately 3, wherein the rapid injection results in a suspension containing 33% ethanol and a total lipid to polynucleotide weight ratio of at least 10:1.

79. The method of claim 78, wherein the rapid injection is performed either manually (MI) or by the aid of a syringe pump (SP).

80. The method of claim 79, further comprising dialyzing the resultant suspension against phosphate buffered saline (PBS) at pH 7.4.

81. The method of claim 80, wherein dialysis is performed more than once.

82. The method of claim 81, further comprising filtering the dialyzed suspension through a 0.2 µm sterile filter.

83. The method of any of claims 78-82, wherein the lipid is selected from the group consisting of DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA and PEGylated lipids.

84. The method of any of claims 78-83, wherein the polynucleotide is selected from the polynucleotide of any of claims 1-37.

85. A lipid nanopartical formulation of a polynucleotide encoding a polypeptide of interest produced by the method of any of claims 78-84 and having a particle size of 80 nm-160 nm, a PDI of between 0.02 and 0.20 and a lipid to polnucleotide ratio (wt/wt) of between 10-30.

86. A lipid nanopartical formulation of claim 85, wherein the polynucleotide is selected from the polynucleotide of any of claims 1-37.

87. A reLNP formulation of a polynucleotide said polynucleotide encoding a polypeptide of interest.

88. A reLNP formulation of claim 87, wherein the polynucleotide is selected from the polynucleotide of any of claims 1-36.

89. A sustained release formulation of a polynucleotide said polynucleotide encoding a polypeptide of interest.

90. A sustained release formulation of claim 89, wherein the polynucleotide is selected from the polynucleotide of any of claims 1-36.

91. A polynucleotide encoding a fusion protein said fusion protein comprising a first polypeptide and a second polypeptide.

92. The polynucleotide of claim 91 where the first polypeptide is selected from the group consisting of Fc receptor, Fab fragment, Fab' fragment, F(ab')2 fragment, Fv fragment, IgA domain, IgD domain, IgE domain, IgD domain, IgM domain, IgV
domain, IgC1 domain, IgC2 domain and IgI domain and the second polypeptide is a polypeptide of interest.