Modified polynucleotides for producing biologics and proteins associated with human disease

Abstract

To provide therapeutic methods for addressing numberless barriers surrounding effective regulation of intracellular translation and treatment of nucleic acid encoding polypeptide or a fragment thereof.SOLUTION: A polynucleotide comprises (a) an open reading frame encoding a target polypeptide consisting of a nucleotide of modified uridine, cytidine, adenosine, and guanosine, the modified uridine being N1-methyl-pseudouridine; (b) 5'-UTR; (c) at least one 5' cap structure; (d) 3'-UTR; and (e) a 3' tail sequence of a binding nucleoside.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing Reference This application has been filed in electronic format along with a Sequence Listing. The Sequence Listing file, entitled M300_PCTSQLST.txt, was created on March 9, 2013, and is 49,417,315 bytes in size. The information in this Sequence Listing electronic format is incorporated herein by reference in its entirety.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of 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 / US2012 / 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," and U.S. Provisional Patent Application No. 61 / 681,645, filed August 10, 2012, entitled "Modified Polynucleotides for the production of "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," and 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 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, entitled "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 / 737,139, filed December 14, 2012, entitled "Modified Polynucleotides for the Production of Secreted Proteins,"Issue 147, titled "Modified Polynucleotides for the Production of Secreted 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," and U.S. Provisional Patent Application No. 61 / 737,155, filed December 14, 2012, entitled "Modified Polynucleotides for the Production of Cytoplasmic and "Modified Polynucleotides for the Production of Intracellular Membrane Bound 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," and 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," and U.S. Provisional Patent Application No. 61 / 618,911, filed April 2, 2012, entitled "Modified Polynucleotides for the Production of Nuclear 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," and 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 "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," and 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," 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,No. 203, entitled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease," and U.S. Provisional Patent Application No. 61 / 618,961, filed April 2, 2012, entitled "Dosing Methods for Modified mRNA," and 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 incorporated herein by reference in their entireties.

[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] This application is also related to co-pending applications, each of which was filed concurrently herewith on March 9, 2013, entitled "Modified Polynucleotides," attorney docket number M301.20 (PCT / US13 / XXXXX), and entitled "Modified Polynucleotides for the Production of the Attorney Docket No. M304.20 (PCT / US13 / XXXXX) entitled "Modified Polynucleotides for the Production of Membrane Proteins" Attorney Docket No. M305.20 (PCT / US13 / XXXXX) entitled "Modified Polynucleotides for the Production of "Cytoplasmic and Cytoskeletal Proteins" attorney docket number M306.20 (PCT / US13 / XXXXX), entitled "Modified Polynucleotides for the Production of Attorney Docket No. M308.20 (PCT / US13 / XXXXX) entitled "Modified Polynucleotides for the Production of Proteins," Attorney Docket No. M309.20 (PCT / US13 / XXXXX) entitled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease," Attorney Docket No. M310.20 (PCT / US13 / XXXXX) entitled "Modified Polynucleotides for the Production of Cosmetic and Attorney Docket No. MNC2.20 (PCT / US13 / XXXXX) entitled "Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides," the contents of each of which are incorporated herein by reference in their entirety.

[0005] FIELD OF THE INVENTION The present invention relates to compositions of polynucleotides, primary constructs, and modified mRNA molecules (mmRNA), methods, processes, kits, and devices for their design, preparation, manufacturing, and / or formulation. [Background technology]

[0006] Previous methods for generating protein expression have many problems. For example, the introduced DNA may frequently be integrated into the host cell genomic DNA, resulting in the alteration and / or damage of the host cell genomic DNA. Alternatively, heterologous deoxyribonucleic acid (DNA) introduced into a cell may be inherited by daughter cells (whether the heterologous DNA is integrated into a chromosome) or by progeny. Additionally, assuming proper delivery and no damage or integration into the host genome, there are many steps that must occur before the encoded protein is produced. 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. The multiple processing steps from administered DNA to protein not only create a time lag before a functional protein is produced, but each step also presents an opportunity for cellular error and damage. Furthermore, achieving DNA expression in cells is notoriously difficult, as DNA often enters the cell but is not expressed, or is not expressed at a reasonable rate or concentration. This can be particularly problematic when DNA is introduced into primary cells or modified cell lines.

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

[0008] Others have studied the use of mRNA to deliver polypeptides of interest and have shown that certain chemical modifications of mRNA molecules, specifically pseudouridine and 5-methyl-cytosine, reduced their immunostimulatory effects.

[0009] These studies are described, for example, in UK Patent Application No. 0316089.2, filed July 9, 2003, to Ribostem Limited, now abandoned; PCT Application No. PCT / GB2004 / 002981, filed July 9, 2004, published as International Publication No. WO2005005622; U.S. Patent Application National Stage Registration No. 10 / 563,897, filed June 8, 2006, published as U.S. Patent No. US20060247195, now abandoned; and European Patent Application National Stage Registration No. EP2004743322, filed July 9, 2004, published as European Patent No. EP1646714, now withdrawn; and Novozymes, Inc., published as International Publication No. WO2008140615. PCT Application No. PCT / US2007 / 88060, filed December 19, 2007; U.S. Patent Application National Stage Registration No. 12 / 520,072, filed July 2, 2009, published as U.S. Patent No. US20100028943, and European Patent Application National Stage Registration No. EP2007874376, filed July 7, 2009, published as European Patent No. EP2104739; University of Rochester, PCT Application No. PCT / US2006 / 46120, filed December 4, 2006, published as International Publication No. WO2007064952, and U.S. Patent Application No. 11 / 606,995, filed December 1, 2006, published as U.S. Patent No. US20070141030; BioNTech AG's European Patent Application No. EP2007024312, filed December 14, 2007, now abandoned; PCT Application No. PCT / EP2008 / 01059, filed December 12, 2008, published as International Publication No. WO2009077134; European Patent Application No. EP2008861423, filed June 2, 2010, published as European Patent No. EP2240572; U.S. Patent Application No. 12 / 735, filed November 24, 2010, published as U.S. Patent No. US20110065103;060, German Patent Application No. DE 10 2005 046 490, filed September 28, 2005, PCT Application No. PCT / EP2006 / 0448, filed September 28, 2006, published as International Publication No. WO2007036366, national stage European Patent No. 1934345, filed March 21, 2012, and national stage U.S. Patent Application No. 11 / 992,638, filed August 14, 2009, published as 20100129877; Immune Disease Institute Inc., U.S. Patent Application No. 13 / 088,009, filed April 15, 2011, published as U.S. Patent No. US20120046346, and PCT Application No. PCT / US2011 / 32679, filed April 15, 2011, published as International Publication No. WO20110130624; Shire HumanGenetic Therapeutics, U.S. Patent Application No. 12 / 957,340, filed November 20, 2010, published as U.S. Patent No. US20110244026; Sequitur Inc., PCT Application No. PCT / US1998 / 019492, filed September 18, 1998, published as International Publication No. WO1999014346; The Scripps Research PCT application No. PCT / US2010 / 00567, filed February 24, 2010, published as International Publication No. WO2010098861, and U.S. patent application National Phase Registration No. 13 / 203,229, filed November 3, 2011, published as U.S. Patent No. US20120053333; PCT application No. PCT / EP2010 / 004681, filed July 30, 2010, published as International Publication No. WO2011012316, from the Ludwig-Maximilians-University; Cellscript Inc., U.S. Patent Application No. 8,039,214, filed June 30, 2008, granted October 18, 2011; U.S. Patent Application No. 12 / 962,498, filed December 7, 2010, published as U.S. Patent No. US20110143436; U.S. Patent Application No. 12 / 962,468, filed December 7, 2010, published as U.S. Patent No. US20110143397; U.S. Patent Application No. 13 / 237, filed September 20, 2011, published as U.S. Patent No. US20120009649;451, and PCT application Nos. PCT / US2010 / 59305, filed December 7, 2010, published as International Publication No. WO2011071931, and PCT / US2010 / 59317, filed December 7, 2010, published as International Publication No. WO2011071936; PCT application No. PCT / US2006 / 32372, filed August 21, 2006, published as International Publication No. WO2007024708, and U.S. patent application National Phase No. 11 / 990,646, filed March 27, 2009, published as U.S. Patent No. US20090286852, to the Board of Regents of the University of Pennsylvania; and German patent application No. DE10 2001, filed June 5, 2001, to Curevac GMBH. No. DE 10 2001 062 480.8 filed on December 19, 2001, and No. DE 20 2006 051 filed on October 31, 2006. No. 516 (all of which have since been abandoned), European Patent No. EP 1392341 granted on March 30, 2005, and European Patent No. EP 1458410 granted on January 2, 2008, PCT application No. PCT / EP2002 / 06180 filed on June 5, 2002, published as International Publication No. WO2002098443, PCT application No. PCT / EP2002 / 14577 filed on December 19, 2002, published as International Publication No. WO2003051401, PCT application No. PCT / EP2007 / 09469 filed on December 31, 2007, published as International Publication No. WO2008052770, PCT application No. PCT / EP ... No. PCT / EP2008 / 03033, filed April 16, 2008, published as International Publication No. PCT / EP2008 / 03033, filed April 16, 2008, published as International Publication No. PCT / EP2006 / 004784, filed May 19, 2005, published as International Publication No. WO2006122828, and PCT / EP2008 / 00081, filed January 9, 2007, published as International Publication No. WO2008083949; and U.S. patent application Ser. No. 10 / 729,830, filed December 5, 2003, published as U.S. Patent No. US20050032730; U.S. patent application Ser. No. 10 / 870,830, filed June 18, 2004, published as U.S. Patent No. US20050059624.No. 110, U.S. Patent No. 11 / 914,945, filed July 7, 2008, published as U.S. Patent No. US20080267873, U.S. Patent No. 12 / 446,912, filed October 27, 2009, published as U.S. Patent No. US2010047261, now abandoned, U.S. Patent No. 12 / 522,214, filed January 4, 2010, published as U.S. Patent No. US20100189729, U.S. Patent No. 12 / 522,214, filed May 26, 2010, published as U.S. Patent No. US20110077287, No. 12 / 787,566, filed May 26, 2010, published as U.S. Patent No. 20100239608, No. 12 / 787,755, filed July 18, 2011, published as U.S. Patent No. 20110269950, and No. 13 / 185,119, filed May 12, 2011, published as U.S. Patent No. 20110311472, all of which are incorporated herein by reference in their entireties.

[0010] Despite these reports being limited to a selection of chemical modifications including pseudouridine and 5-methyl-cytosine, there remains a need in the art for therapies that address the myriad barriers surrounding the effective regulation of intracellular translation and processing of nucleic acids encoding polypeptides or fragments thereof.

[0011] To this end, the inventors have shown that certain modified mRNA sequences have potential as therapeutic agents with benefits beyond simply evading, avoiding, or reducing immune responses. Such work is described in detail in published and co-pending applications, International Application Nos. PCT / US2011 / 046861, filed August 5, 2011, PCT / US2011 / 054636, filed October 3, 2011, and PCT / US2011 / 054617, filed October 3, 2011, the contents of which are incorporated herein by reference in their entireties.

[0012] The present invention addresses this need by providing nucleic acid-based compounds or polynucleotides that encode a polypeptide of interest (e.g., a modified mRNA or mmRNA) and have structural and / or chemical characteristics that avoid one or more of the problems in the art, e.g., characteristics useful for optimizing the formulation and delivery of nucleic acid-based therapeutics while retaining structural and functional integrity, overcoming expression thresholds, improving expression rate, half-life, and / or protein concentration, optimizing protein localization, and avoiding adverse biological responses, such as immune responses, and / or degradation pathways. Summary of the Invention

[0013] Described herein are compositions of modified mRNA (mmRNA) molecules, methods, processes, kits, and devices for the design, preparation, manufacturing, and / or formulation of modified mRNA (mmRNA) molecules.

[0014] The details of various embodiments of the invention are set forth in the following detailed description. Other features, objects, and advantages of the invention will be apparent from the detailed description and drawings, and from the claims.

[0015] The foregoing and other objects, features, and advantages will become apparent from the following description of specific 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 and illustrating the principles of various embodiments of the invention. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a schematic diagram of the primary construct of the present invention. [Figure 2-1]Figure 1 illustrates prior art lipid structures useful in the present invention. The structures of 98N12-5 (TETA5-LAP), DLin-DMA, DLin-K-DMA (2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane), DLin-KC2-DMA, DLin-MC3-DMA, and C12-200 are shown. [Figure 2-2] Same as above. [Figure 3]

[0049] Figure 1 is a representative plasmid useful in the IVT reactions taught herein. This plasmid contains insert 64818, designed by the inventors. [Figure 4] Gel profile of modified mRNA encapsulated in PLGA microspheres. [Figure 5] 1 is a histogram of Factor IX protein-producing PLGA formulations and Factor IX-modified mRNA. [Figure 6-1] 6A-6C are histograms showing VEGF protein production in human keratinocyte cells after transfection with modified mRNA at various doses. Figure 6A shows protein production after transfection with modified mRNA containing natural nucleoside triphosphates (NTPs). [Figure 6-2] Figure 6B is a histogram showing VEGF protein production in human keratinocyte cells after transfection with modified mRNA at various doses. Figure 6B shows protein production after transfection with modified mRNA fully modified with pseudouridine (pseudo-U) and 5-methylcytosine (5mC). [Figure 6-3] Figure 6C is a histogram showing VEGF protein production in human keratinocyte cells after transfection with various doses of modified mRNA. Figure 6C shows protein production after transfection with modified mRNA fully modified with N1-methyl-pseudouridine (N1-methyl-pseudo-U) and 5-methylcytosine (5mC). [Figure 7] 1 is a histogram of VEGF protein production in HEK293 cells. [Figure 8-1]8A shows histograms of VEGF expression and IFN-α induction after transfection of VEGF-modified mRNA in peripheral blood mononuclear cells (PBMCs). [Figure 8-2] Figure 8B shows histograms of VEGF expression and IFN-α induction after transfection of VEGF-modified mRNA in peripheral blood mononuclear cells (PBMCs). Figure 8B shows IFN-α induction. [Figure 9] 1 is a histogram of VEGF protein production in HeLa cells from VEGF-modified mRNA. [Figure 10] 1 is a histogram of VEGF protein production from lipoplexed VEGF-modified mRNA in mice. [Figure 11] 1 is a histogram of G-CSF protein production in HeLa cells from G-CSF modified mRNA. [Figure 12] 1 is a histogram showing G-CSF protein production from lipoplexed G-CSF modified mRNA in mice. [Figure 13] 1 is a histogram showing Factor IX protein production from Factor IX modified mRNA in HeLa cell supernatants. [Figure 14] 1 is a histogram showing APOA1 protein production from APOA1 wild-type modified mRNA, APOA1 Milano modified mRNA, or APOA1 Paris modified mRNA in HeLa cells. [Figure 15] 1 is a gel profile of APOA1 protein derived from APOA1 wild-type modified mRNA. [Figure 16] 1 shows a gel profile of APOA1 protein derived from APOA1 Paris-modified mRNA. [Figure 17] 1 shows a gel profile of APOA1 protein derived from APOA1 Milano-modified mRNA. [Figure 18] 1 is a gel profile of fibrinogen alpha (FGA) protein derived from FGA modified mRNA. [Figure 19]1 is a histogram showing plasminogen protein production from plasminogen-modified mRNA in HeLa cell supernatants. [Figure 20] 1 is a gel profile of plasminogen protein derived from plasminogen-modified mRNA. [Figure 21] 1 is a gel profile of galactose-1-phosphate uridylyltransferase (GALT) protein derived from GALT-modified mRNA. [Figure 22] 1 shows a gel profile of argininosuccinate lyase (ASL) protein derived from ASL-modified mRNA. [Figure 23] 1 is a gel profile of tyrosine aminotransferase (TAT) protein derived from TAT-modified mRNA. [Figure 24] Gel profile of glucan (1,4-α-) and branching enzyme 1 (GBE1) protein derived from GBE1 modified mRNA. [Figure 25] 1 is a histogram showing prothrombin protein production from prothrombin-modified mRNA in HeLa cell supernatants. [Figure 26] 1 is a histogram showing prothrombin protein production from prothrombin-modified mRNA in HeLa cell supernatants. [Figure 27] 1 is a gel profile of ceruloplasmin (CP or CLP) protein derived from CP modified mRNA. [Figure 28] 1 is a histogram showing transforming growth factor β1 (TGF-β1) protein production from TGF-β1 modified mRNA in HeLa cell supernatants. [Figure 29] 1 is a gel profile of ornithine carbamoyltransferase (OTC) protein derived from OTC-modified mRNA. [Figure 30] 1 is a flow cytometry plot of low density lipoprotein receptor (LDLR) modified mRNA. [Figure 31]Gel profile of UDP-glucuronosyltransferase 1 family, polypeptide A1 (UGT1A1) protein from UGT1A1-modified mRNA. [Figure 32] 1 is a histogram showing Factor XI protein production in HEK293 cells. [Figure 33] 1 shows a gel profile of aquaporin-5 protein derived from aquaporin-5 modified mRNA. [Figure 34] 1 is a histogram showing Factor VII protein production from Factor VII-modified mRNA in HeLa cells. [Figure 35] 1 is a histogram showing insulin glargine protein production from insulin glargine-modified mRNA in HeLa cells. [Figure 36] 1 is a histogram showing tissue factor protein production from tissue factor-modified mRNA in HeLa cells. [Figure 37] 1 is a histogram showing Factor XI protein production from Factor XI modified mRNA in HeLa cells. [Figure 38] 1 is a histogram showing Factor XI protein production from Factor XI modified mRNA in HeLa cell supernatants. [Figure 39] 1 is a histogram showing insulin aspart protein production from insulin aspart-modified mRNA. [Figure 40] 1 is a histogram showing insulin lispro protein production from insulin lispro-modified mRNA in HeLa cells. [Figure 41] 1 is a histogram showing insulin glulisine protein production from insulin glulisine-modified mRNA in HeLa cells. [Figure 42] 1 is a histogram showing human growth hormone protein production from human growth hormone modified mRNA in HeLa cells. [Figure 43]43A and 43B show gel profiles of tumor protein 53 (p53) protein derived from p53-modified mRNA. Figure 43A shows the predicted size of p53. Figure 43B shows the predicted size of p53. [Figure 44] This is a gel profile of tuftelin (TUFT1) protein derived from TUFT1-modified mRNA. [Figure 45] 45A and 45B show gel profiles of galactokinase 1 (GALK1) protein derived from GALK1 modified mRNA. Figure 45A shows the predicted size of GALK1. Figure 45B shows the predicted size of GALK1. [Figure 46] 1 is a gel profile of defensin, beta 103A (DEFB103A) protein derived from DEFB103A modified mRNA. [Figure 47] Flow cytometry plot of LDLR-modified mRNA. [Figure 48] 1 is a histogram showing vascular endothelial growth factor expression in HeLa. [Figure 49] 1 is a histogram showing cell viability of HeLa cells transfected with vascular endothelial growth factor mRNA. [Figure 50] 1 is a histogram showing insulin aspart protein expression. [Figure 51] 1 is a histogram showing insulin glargine protein expression. [Figure 52] 1 is a histogram showing insulin glulisine protein expression. [Figure 53] 1 is a histogram showing interleukin 7 (IL-7) protein expression. [Figure 54] 1 is a histogram showing erythropoietin (EPO) protein expression. [Figure 55] 1 is a gel profile of lysosomal acid lipase protein derived from lysosomal acid lipase modified mRNA. [Figure 56] 1 is a gel profile of glucocerebrosidase protein derived from glucocerebrosidase-modified mRNA. [Figure 57]1 shows a gel profile of iduronate-2-sulfatase protein derived from iduronate-2-sulfatase-modified mRNA. [Figure 58] Gel profile of luciferase protein derived from luciferase-modified mRNA. [Figure 59] 1 is a histogram showing IgG concentrations after administration of formulated Herceptin modified mRNA in mammals. [Figure 60] 1 is a histogram showing IgG concentration (ng / ml) after transfection with Herceptin-modified mRNA. [Figure 61] 1 is a gel profile of Herceptin protein derived from Herceptin modified mRNA. [Figure 62] 1 is a histogram of glucocerebrosidase enzyme activity. [Figure 63] 1 is a histogram of lysosomal acid lipase enzyme activity. [Figure 64] 1 is a histogram showing Factor VIII protein expression. [Figure 65] 1 is a histogram showing the chromogenic activity of Factor VIII. [Figure 66-1] 66A is a graph showing LDLR expression. Figure 66A shows cellular LDL receptor expression in response to the addition of LDLR mRNA. [Figure 66-2] Figure 66B is a graph showing LDL receptor expression in cells after transfection. [Figure 66-3] Figure 66C is a graph showing LDLR expression. Figure 66C shows saturation of BODIPY®-labeled LRL. [Figure 66-4] Figure 66D is a graph showing LDLR expression. Figure 66D shows the binding affinity of BODIPY-LDL to cells. [Figure 67] Graph showing the percentage of cells positive for UGT1A1 expression. [Figure 68] 1 is a graph showing the accumulation of UGT1A1 protein. [Figure 69]1 shows gel profiles of UGT1A1 protein and OTC derived from UGT1A1- or OTC-modified mRNA. [Figure 70] 1 is a flow cytometry plot of HEK293 cells transfected with PAh or UGT1A1. [Figure 71] 1 is a gel profile of UGT1A1 protein derived from UGT1A1-modified mRNA. [Figure 72] 1 is a gel profile of microsomal extracts from mice treated with LNPs containing UGT1A1. DETAILED DESCRIPTION OF THE INVENTION

[0017] In the therapeutics, diagnostics, reagent fields, and biological assays, it is of great interest to be able to deliver nucleic acids, e.g., ribonucleic acid (RNA), into cells, whether in vitro, in vivo, in situ, or ex vivo, to, for example, cause intracellular translation of the nucleic acid and production of the encoded polypeptide of interest. The delivery and function of non-integrated polynucleotides is particularly important.

[0018] Described herein are compositions (including pharmaceutical compositions) of polynucleotides encoding one or more polypeptides of interest, as well as methods for their design, preparation, manufacture, and / or formulation. Also provided are systems, processes, devices, and kits for selecting, designing, and / or utilizing the polynucleotides encoding the polypeptides of interest described herein.

[0019] According to the present invention, these polynucleotides are preferably modified to avoid the deficiencies of other polypeptide-encoding molecules in the art, and are therefore referred to as modified mRNAs or mmRNAs.

[0020] The use of modified polynucleotides in the areas of antibodies, viruses, veterinary applications, and various in vivo environments has been investigated by the present inventors, and these investigations are described, for example, in co-pending and co-owned U.S. Provisional Patent Application Nos. 61 / 470,451, filed March 31, 2011, which teaches in vivo applications of mmRNA; 61 / 517,784, filed April 26, 2011, which teaches engineered nucleic acids for the production of antibody polypeptides; and 61 / 517,784, filed April 26, 2011, which teaches veterinary applications of mmRNA technology. US Patent No. 61 / 519,158, filed May 17, 2011, which teaches the application of mmRNA technology in antibacterial agents; US Patent No. 61 / 533,537, filed September 12, 2011, which teaches the application of mmRNA technology in viral applications; US Patent No. 61 / 533,554, filed September 12, 2011, which teaches various chemical modifications for use in mmRNA technology; US Patent No. 61 / 542,533, filed October 3, 2011, which teaches mobile devices for use in the production and use of mmRNA technology; No. 61 / 570,690, filed December 14, 2011, teaching the use of mmRNA in critical care settings; No. 61 / 570,708, filed December 14, 2011, teaching terminally modified structures of mmRNA; No. 61 / 576,651, filed December 16, 2011, teaching methods for delivering mmRNA using lipidoids; No. 61 / 576,705, filed December 21, 2011, teaching methods for increasing organ or tissue viability using mmRNA; No. 1 / 578,271, filed December 29, 2011, which teaches mmRNA encoding cell-penetrating peptides; No. 61 / 581,322, filed December 29, 2011, which teaches the incorporation of cytotoxic nucleosides into mmRNA; and No. 61 / 631,729, filed January 10, 2012, which teaches methods of using mmRNA to cross the blood-brain barrier, all of which are incorporated herein by reference in their entireties.

[0021] Provided herein in part are polynucleotides, primary constructs, and / or mmRNA encoding a polypeptide of interest designed to improve one or more of: stability and / or clearance in tissue, receptor uptake and / or kinetics, cellular access by the composition, engagement with the translation machinery, mRNA half-life, efficiency of translation, immune evasion, protein production capacity, efficiency of secretion (if applicable), accessibility to the blood circulation, protein half-life, and / or modulation of cellular state, function, and / or activity.

[0022] I. Compositions of the Invention (mmRNA) The present invention provides nucleic acid molecules, specifically polynucleotides, primary constructs, and / or mmRNA, that encode one or more polypeptides of interest. The term "nucleic acid," in its broadest sense, includes any compound and / or substance comprising a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present invention include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA, including LNA with a β-D-ribonucleotide configuration, α-LNA with an α-L-ribonucleotide configuration (a diastereomer of LNA), 2'-amino-LNA with a 2'-amino functionalization, and 2'-amino-α-LNA with a 2'-amino functionalization), or hybrids thereof.

[0023] In a preferred embodiment, the nucleic acid molecule is messenger RNA (mRNA). As used herein, the term "messenger RNA (mRNA)" refers to any polynucleotide that encodes a polypeptide of interest and can be translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ, or ex vivo.

[0024] Traditionally, the main components of an mRNA molecule include at least a coding region, a 5' UTR, a 3' UTR, a 5' cap, and a polyA tail. Building on this wild-type modular structure, the present invention expands the scope of functionality of conventional mRNA molecules by providing polynucleotides or primary RNA constructs that maintain the modular organization but contain one or more structural and / or chemical modifications or alterations that confer useful properties to the polynucleotide, including, in some embodiments, a lack of substantial induction of a cellular innate immune response to the site into which the polynucleotide is introduced. Thus, the modified mRNA molecules of the present invention are referred to as "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 are necessarily broken and reformed to result in the structural modification, the structural modification is chemical in nature and is therefore a chemical modification. However, the structural modification may result in a different nucleotide sequence. For example, the polynucleotide "ATCG" may be chemically modified to "AT-5meC-G." The same polynucleotide can be structurally modified from "ATCG" to "ATCCCG," where the dinucleotide "CC" is inserted, resulting in the structural modification to the polynucleotide.

[0025] mmRNA structure The mmRNAs of the present invention are distinguished from wild-type mRNAs in their functional and / or structural design features that, as demonstrated herein, help to overcome existing problems in effective polypeptide production using nucleic acid-based therapeutics.

[0026] 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 that encodes one or more polypeptides of interest and retains sufficient structural and / or chemical features to allow the polypeptides of interest encoded therein to be translated. A primary construct may be a polynucleotide of the present invention. When structurally or chemically modified, a primary construct may be referred to as an mmRNA.

[0027] Referring to FIG. 1 , a primary construct 100 contains a first region of binding nucleotides 102 flanked by a first flanking region 104 and a second flanking region 106. As used herein, the "first region" may be referred to as a "coding region" or "coding region," or simply as a "first region." This first region may include, but is not limited to, an encoded polypeptide of interest. The polypeptide of interest may include one or more signal sequences at its 5' end encoded by a signal sequence region 103. The flanking region 104 may include a region of binding nucleotides that includes one or more complete or incomplete 5' UTR sequences. The flanking region 104 may also include a 5' end cap 108. The second flanking region 106 may include a region of binding nucleotides that includes one or more complete or incomplete 3' UTRs. The flanking region 106 may also include a 3' tail sequence 110.

[0028] Bridging the 5' ends of the first region 102 and the first flanking region 104 is a first operational region 105. Conventionally, this operational region includes a start codon. Alternatively, this operational region can include any translation initiation sequence or signal that includes a start codon.

[0029] Bridging the 3' ends of the first region 102 and the second flanking region 106 is a second manipulation region 107. Conventionally, this manipulation region includes a stop codon. Alternatively, this manipulation region can include any translation initiation sequence or signal that includes a stop codon. Multiple consecutive stop codons can also be used in accordance with the present invention.

[0030] Generally, the minimum length of the first region of a primary construct of the present invention can be a length of nucleic acid sequence sufficient to encode a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In another embodiment, the length can be sufficient to encode a peptide of 2 to 30 amino acids, e.g., 5 to 30, 10 to 30, 2 to 25, 5 to 25, 10 to 25, or 10 to 20 amino acids. The length can be sufficient to encode a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or 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 sequence can encode include, but are not limited to, carnosine and anserine.

[0031] Generally, the length of the first region encoding a polypeptide of interest of the invention is greater than about 30 nucleotides in length (e.g., at least 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, and up to or exceeding 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 100,000 nucleotides in length (including 100,000). As used herein, a "first region" may be referred to as a "coding region" or "coding region," or simply as a "first region."

[0032] In some embodiments, the polynucleotide, primary construct, or mmRNA is from about 30 to about 100,000 nucleotides (e.g., 30-50, 30-100, 30-250, 30-500, 30-1,000, 30-1,500, 30-3,000, 30-5,000, 30-7,000, 30-10,000, 30-25,000, 30-50,000, 30-70,000, 100-250, 100- 500, 100-1,000, 100-1,500, 100-3,000, 100-5,000, 100-7,000, 100-10,000, 100-25,000, 100-50,000, 100-70,000, 100-100,000, 500-1,000, 500-1,500, 500-2,000, 500-3,000, 500-5,000, 500-7,000, 500-10,000, 500-25, 000, 500-50,000, 500-70,000, 500-100,000, 1,000-1,500, 1,000-2,000, 1,000-3,000, 1,000-5,000, 1,000-7,000, 1,000-10,000, 1,000-25,000, 1,000-50,000, 1,000-70,000, 1,000-100,000, 1,500-3,000, 1,500-5,000, These ranges include: 1,500-7,000, 1,500-10,000, 1,500-25,000, 1,500-50,000, 1,500-70,000, 1,500-100,000, 2,000-3,000, 2,000-5,000, 2,000-7,000, 2,000-10,000, 2,000-25,000, 2,000-50,000, 2,000-70,000, and 2,000-100,000 pieces.

[0033] In accordance with the present invention, the first and second flanking regions can independently range from 15 to 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 in length, 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 in length).

[0034] According to the present invention, the tail sequence can range from 0 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 in length). When the tail region is a polyA tail, the length can 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 four monomers of polyA binding protein. A polyA binding protein monomer binds to a stretch of approximately 38 nucleotides. Thus, polyA tails of approximately 80 nucleotides and 160 nucleotides have been observed to be functional.

[0035] According to the present invention, the capping region can comprise a single cap or a series of nucleotides forming a cap. In this embodiment, the capping region can be 1 to 10, e.g., 2 to 9, 3 to 8, 4 to 7, 1 to 5, 5 to 10, or at least 2, or no more than 10 nucleotides in length. In some embodiments, the cap is absent.

[0036] In accordance with the present invention, the first and second operational regions may be 3 to 40, e.g., in the range of 5 to 30, 10 to 20, 15, or at least 4, or no more than 30 nucleotides in length, and may include one or more signal and / or restriction sequences in addition to a start codon and / or a stop codon.

[0037] Circular mmRNA According to the present invention, the primary construct or mmRNA can be circularized or concatemerized to generate a translation-competent molecule and facilitate the interaction between polyA-binding protein and 5'-end binding protein. The mechanism of circularization or concatemerization can occur through at least three different pathways: 1) chemical pathway, 2) enzymatic pathway, and 3) ribozyme catalytic pathway. The newly formed 5' / 3' linkage can be intramolecular or intermolecular.

[0038] In the first pathway, the 5' and 3' ends of the nucleic acid contain chemically reactive groups that, when brought into close proximity, form a new covalent bond between the 5' and 3' ends of the molecule. The 5' end may contain an NHS-ester reactive group, and the 3' end may contain a 3'-amino terminal nucleotide, such that in organic solvent, the 3'-amino terminal nucleotide on the 3' end of the synthetic mRNA molecule undergoes nucleophilic attack on the 5'-NHS-ester moiety to form a new 5' / 3' amide bond.

[0039] In the second pathway, T4 RNA ligase can be used to enzymatically attach 5'-phosphorylated nucleic acid molecules to the 3'-hydroxyl group of a nucleic acid, forming a new phosphorodiester bond. In an example reaction, 1 μg of nucleic acid molecule is incubated with 1-10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) for 1 hour at 37°C according to the manufacturer's protocol. Ligation can occur in the presence of a split oligonucleotide that can base-pair side-by-side with both the 5' and 3' regions to support the enzymatic ligation reaction.

[0040] In the third pathway, either the 5' or 3' end of the cDNA template encodes a ligase ribozyme sequence, such that during in vitro transcription, the resulting nucleic acid molecule contains 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 can be derived from a group I intron, a group I intron, hepatitis delta virus, a hairpin ribozyme, or selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction can take 1 to 24 hours at temperatures between 0 and 37°C.

[0041] mmRNA multimers According to the present invention, multiple distinct polynucleotides, primary constructs, or mmRNAs can be linked via their 3' ends using 3'-modified nucleotides. Chemical complexation can be used to control the stoichiometry of delivery to cells. For example, glyoxylate cycle enzymes, isocitrate lyase, and malate synthase can be supplied to HepG2 cells at a 1:1 ratio to alter cellular fatty acid metabolism. This ratio can be controlled by chemically linking polynucleotides, primary constructs, or mmRNAs using 3'-azido-terminal nucleotides on one polynucleotide, primary construct, or mmRNA species and C5-ethynyl- or alkynyl-containing nucleotides on the other polynucleotide, primary construct, or mmRNA species. The modified nucleotides are added post-transcriptionally using terminal transferase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. After addition of the 3'-terminal modified nucleotide, these two polynucleotides, primary constructs, or mmRNA species can be combined in aqueous solution in the presence or absence of copper to form a new covalent bond via click chemistry mechanisms described in the literature.

[0042] In another example, three or more polynucleotides can be linked using a functionalized linker molecule. For example, a functionalized saccharide molecule can be chemically modified to contain multiple chemically reactive groups (SH-, NH2-, N3, etc.) to react with a cognate moiety (i.e., 3'-maleimide ester, 3'-NHS-ester, alkynyl) on a 3'-functionalized mRNA molecule. The number of reactive groups on the modified saccharide can be controlled in a stoichiometric manner to directly control the stoichiometry of the complexed polynucleotides, primary constructs, or mRNA.

[0043] mmRNA complexes and combinations To further enhance protein production, the primary constructs or mmRNA of the present invention may be combined with other polynucleotides, dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralen, 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, They can be designed to be conjugated with substituted alkyls, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases, proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for co-ligands, or antibodies, e.g., antibodies that bind to particular cell types such as cancer cells, endothelial cells, or bone cells, hormones and hormone receptors, non-peptide species, e.g., lipids, lectins, carbohydrates, vitamins, cofactors, or drugs.

[0044] Complexation may result in increased stability and / or half-life and may be particularly useful when targeting polynucleotides, primary constructs, or mmRNA to specific sites in cells, tissues, or organisms.

[0045] In accordance with the present invention, the mmRNA or primary construct may be administered together with or may further encode one or more of an RNAi agent, siRNA, shRNA, miRNA, miRNA binding site, antisense RNA, ribozyme, catalytic DNA, tRNA, RNA that induces triple helix formation, aptamer, or vector, etc.

[0046] Bifunctional mmRNA One embodiment of the present invention is a bifunctional polynucleotide (e.g., a bifunctional primary construct or bifunctional mmRNA). As the name suggests, a bifunctional polynucleotide is a polynucleotide that has or is capable of at least two functions. By convention, these molecules may also be referred to as multifunctional.

[0047] The multiple functions of a bifunctional polynucleotide may be encoded by the RNA (the function may not be apparent until the encoded product is translated) or may be a property of the polynucleotide itself. This may be structural or chemical. A bifunctional modified polynucleotide may contain functions covalently or electrostatically associated with the polynucleotide. Furthermore, these two functions may be provided in the context of a complex of the mmRNA and another molecule.

[0048] The bifunctional polynucleotide can encode an antiproliferative peptide. These peptides can be linear, cyclic, constrained, or random coil. They can function as aptamers, signaling molecules, ligands, or mimetics thereof. Antiproliferative peptides can be 3-50 amino acids long when translated. They can be 5-40, 10-30, or about 15 amino acids long. They can be single-, multi-, or branched chains, and can form complexes, aggregates, or any multi-unit structure when translated.

[0049] Non-coding polynucleotides and primary constructs As described herein, polynucleotides and primary constructs are provided that have partially or substantially non-translatable sequences, e.g., non-coding regions. Such non-coding regions may be the "first region" of the primary construct. Alternatively, the non-coding region may be a region other than the first region. Such molecules are not normally translated, but may affect protein production by one or more of binding to and sequestration by one or more components of the translation machinery, such as ribosomal proteins or transfer RNAs (tRNAs), thereby effectively reducing protein expression in cells or regulating one or more pathways or cascades in cells, which then alter protein levels. The polynucleotides or primary constructs may contain or encode one or more long non-coding RNAs (lncRNAs or lincRNAs) or portions thereof, small nuclear RNAs (sno-RNAs), microRNAs (miRNAs), small interfering RNAs (siRNAs), or Piwi interfering RNAs (piRNAs).

[0050] Target polypeptide According to the present invention, primary constructs are designed to encode one or more polypeptides of interest or fragments thereof. A polypeptide of interest can include, but is not limited to, an entire polypeptide, multiple polypeptides, or fragments of a polypeptide, which may be independently encoded by one or more nucleic acids, multiple nucleic acids, fragments of a nucleic acid, or variants of any of the foregoing. As used herein, the term "polypeptide of interest" refers to any polypeptide selected and encoded in a primary construct of the present invention. As used herein, "polypeptide" refers to a polymer of amino acid residues (natural or non-natural) most often linked by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, if the encoded polypeptide is smaller than about 50 amino acids, the polypeptide is referred to as a peptide. When a polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residues in length. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, fragments of the foregoing, and other equivalents, variants, and analogs. Polypeptides can be single molecules or multimolecular complexes such as dimers, trimers, or tetramers. They can also include single-chain or multi-chain polypeptides, such as antibodies or insulin, which can be associated or linked. Disulfide bonds are most often found in multi-chain polypeptides. The term "polypeptide" can also apply to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0051] The term "polypeptide variant" refers to molecules whose amino acid sequence differs from a native or reference sequence. Amino acid sequence variants may have substitutions, deletions, and / or insertions at certain positions within the amino acid sequence compared to the native or reference sequence. Typically, variants will have at least about 50% identity (homology) with the native or reference sequence; preferably, they will be at least about 80%, and more preferably at least about 90% identical (homological) to the native or reference sequence.

[0052] In some embodiments, a "mutant mimic" is provided. As used herein, the term "mutant mimic" refers to a mimic containing one or more amino acids that mimic an activation sequence. For example, glutamate can act as a mimic of phosphoro-threonine and / or phosphoro-serine. Alternatively, a mutant mimic can result in the inactivation or inactivation of a product containing the mimic; for example, phenylalanine can act as an inactivating substitute for tyrosine, or alanine can act as an inactivating substitute for serine.

[0053] " Homology " when applied to amino acid sequences is defined as the percentage (%) of residues in a candidate amino acid sequence that are identical to the residues in the amino acid sequence of a second sequence, after aligning the sequences and introducing gaps as necessary to obtain the maximum homology percentage. Methods and computer programs for alignment are well known in the art. It is understood that homology depends on the calculation of percent identity, but values ​​may vary due to gaps and penalties introduced in the calculation.

[0054] "Homolog" when applied to a polypeptide sequence means a corresponding sequence in another species that has a significant degree of identity to a second sequence in the second species.

[0055] "Analog" is intended to include polypeptide variants that differ by one or more amino acid modifications, e.g., substitution, addition, or deletion of an amino acid residue, that still retain one or more properties of the parent or starting polypeptide.

[0056] The present invention contemplates several types of polypeptide-based compositions, including variants and derivatives. These include substitution, insertion, deletion, and covalent variants and derivatives. The term "derivative" is used interchangeably with the term "variant," but generally refers to a molecule that has been modified and / or changed in any way relative to a reference or starting molecule.

[0057] Therefore, mmRNAs encoding polypeptides containing substitutions, insertions and / or additions, deletions, and covalent modifications relative to reference sequences, particularly the polypeptide sequences disclosed herein, are encompassed within the scope of the present invention. For example, sequence tags or amino acids, such as one or more lysines, can be added to the peptide sequences of the present invention (e.g., at the N- or C-terminus). Sequence tags can be used for peptide purification or localization. Lysines can be used to increase peptide solubility or enable biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein can be optionally deleted to provide a truncated sequence. Alternatively, certain amino acids (e.g., C- or N-terminal residues) can be deleted during expression of the sequence, for example, as part of a larger, more soluble sequence, or can be attached to a solid support, depending on the use of the sequence.

[0058] "Substitutional variants," when referring to polypeptides, are those in which at least one amino acid residue in a native or starting sequence has been removed and a different amino acid inserted in its place at the same position. These substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where more than one amino acid has been substituted in the same molecule.

[0059] As used herein, the term "conservative amino acid substitution" refers to the substitution of an amino acid normally present in a 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. Similarly, examples of conservative substitutions include the substitution of a polar (hydrophilic) residue such as arginine for lysine, glutamine for asparagine, and glycine for another. Furthermore, the substitution of a basic residue such as lysine, arginine, or histidine for another basic residue, or the substitution of an acidic residue such as aspartic acid or glutamic acid for another acidic residue, are further 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, or methionine for a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue for a non-polar residue.

[0060] An "insertion variant," when referring to a polypeptide, is one in which one or more amino acids have been inserted immediately adjacent to an amino acid at a particular position in a native or starting sequence. "Immediately adjacent to" an amino acid means linked to either the alpha-carboxy or alpha-amino functionality of the amino acid.

[0061] "Deletion variants," when referring to polypeptides, are those in which one or more amino acids in a native or starting amino acid sequence have been removed. Typically, deletion variants delete one or more amino acids in a particular region of the molecule.

[0062] "Covalent derivatives," when referring to polypeptides, include modification of the native or starting protein with an organic proteinaceous or non-proteinaceous derivatizing agent and / or post-translational modification. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of the protein with an organic derivatizing agent capable of reacting with selected side chains or terminal residues, or by utilizing post-translational modification mechanisms operative in selected recombinant host cells. The resulting covalent derivatives are useful in programs aimed at identifying residues important for biological activity, immunological assays, or preparation of anti-protein antibodies for immunoaffinity purification of recombinant glycoproteins. Such modifications are within the skill of the art and are performed without undue experimentation.

[0063] Certain post-translational modifications are the result of the action of recombinant host cells on expressed polypeptides. 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 polypeptides produced according to the present invention.

[0064] Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, and methylation of the α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)).

[0065] A "feature," when referring to a polypeptide, is defined as a distinct amino acid sequence-based component of a molecule. Features of polypeptides encoded by the mmRNA of the present invention include surface appearance, local conformational shape, fold, loop, half-loop, domain, half-domain, site, terminus, or any combination thereof.

[0066] As used herein, when referring to a polypeptide, the term "surface expression" refers to the appearance of the polypeptide component of the protein on the outermost surface.

[0067] As used herein, when referring to a polypeptide, the term "local conformational shape" refers to the appearance of a polypeptide-based structure of a protein located within a definable space of the protein.

[0068] As used herein, when referring to a polypeptide, the term "fold" refers to the resulting three-dimensional structure of an amino acid sequence upon energy minimization. Folding can 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 level folds include domains and regions formed due to the aggregation or separation of energy forces. Regions formed in this manner include hydrophobic and hydrophilic pockets, etc.

[0069] As used herein, the term "turn," when referring to protein structure, means a bend that changes the orientation of the backbone of a peptide or polypeptide and may involve one, two, three or more amino acid residues.

[0070] As used herein, when referring to a polypeptide, the term "loop" refers to a structural feature of a polypeptide that can serve to reverse the orientation of the peptide or polypeptide backbone. When a loop is found in a polypeptide and only changes the orientation of the backbone, it can contain four or more amino acid residues. Oliva et al. identified at least five classes of protein loops (J. Mol Biol 266(4):814-830; 1997). Loops can be open or closed. Closed or "cyclic" loops can contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids between bridging moieties. Such bridging moieties can include cysteine-cysteine ​​bridges (Cys-Cys), which are typical in polypeptides with disulfide bridges, or alternatively, the bridging moieties can be non-proteinaceous, such as dibromozylyl agents, as used herein.

[0071] As used herein, when referring to a polypeptide, the term "half-loop" refers to a portion of a loop that has at least half of the amino acid residues identified as the loop from which it is derived. It is understood that a loop does not necessarily contain an even number of amino acid residues. Thus, when a loop contains or is identified as containing an odd number of amino acids, a half-loop of an odd-numbered loop includes an integral or next integral portion of the loop (number of amino acids in the loop / 2 + / - 0.5 amino acids). For example, a loop identified as a 7-amino acid loop can result in a half-loop of 3 or 4 amino acids (7 / 2 = 3.5 + / - 0.5, resulting in 3 or 4).

[0072] As used herein, when referring to a polypeptide, the term "domain" refers to a motif in a polypeptide having one or more identifiable structural or functional features or characteristics (e.g., binding ability, serving as a site of interaction between proteins).

[0073] As used herein, when referring to a polypeptide, the term "half-domain" refers to a portion of a domain having at least half of the amino acid residues identified as the domain from which it is derived. It is understood that a domain does not necessarily contain an even number of amino acid residues. Thus, if a domain contains or is identified as comprising an odd number of amino acids, a half-domain of an odd-numbered domain comprises an integral or next integral portion of the domain (number of amino acids in the domain / 2 + / - 0.5 amino acids). For example, a domain identified as a 7-amino acid domain may result in a half-domain of 3 or 4 amino acids (7 / 2 = 3.5 + / - 0.5, resulting in 3 or 4). It is also understood that subdomains may be identified within a domain or half-domain, and that these subdomains may have structural or functional properties that are less than all of the structural or functional properties identified in the domain or half-domain from which they are derived. It is also understood that the amino acids comprising any of the domain types herein need not be adjacent along the polypeptide backbone (i.e., non-adjacent amino acids may structurally fold to result in a domain, half-domain, or subdomain).

[0074] As used herein, when referring to polypeptides, the term "site" is used interchangeably with "amino acid residue" and "amino acid side chain" when referring to amino acid-based embodiments. Sites represent positions within a peptide or polypeptide that can be modified, manipulated, altered, derivatized, or changed within the polypeptide-based molecules of the invention.

[0075] As used herein, when referring to a polypeptide, the term "terminus" or "terminus" refers to the end of a peptide or polypeptide. Such terminus may not only be limited to the first or final portion of a peptide or polypeptide, but may also include additional amino acids in the terminal region. The polypeptide molecules of the present invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). In some cases, proteins of the present invention may consist of multiple polypeptide chains linked by disulfide bonds or non-covalent forces (multimers, oligomers). These types of proteins have multiple N- and C-termini. Alternatively, the termini of polypeptides may be modified so that they begin or end with non-polypeptide moieties, such as organic complexes.

[0076] Once any of these features have been identified or defined as desired components of the polypeptide encoded by the primary construct or mmRNA of the present invention, any of these features can be manipulated and / or modified by moving, exchanging, inverting, deleting, randomizing, or duplicating. Furthermore, it is understood that manipulation of features will produce the same results as modifications to the molecules of the present invention. For example, manipulation involving the deletion of domains will result in altering the length of the molecule, such as modifying a nucleic acid that encodes less than the full-length molecule.

[0077] 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 can 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.

[0078] In accordance with the present invention, a polypeptide can comprise a consensus sequence discovered through a series of experiments. As used herein, a "consensus" sequence is a single sequence that represents a collective population of sequences that allows for variability at one or more sites.

[0079] As will be appreciated by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered within the scope of the polypeptides of interest of the present invention. For example, any protein fragment (meaning a polypeptide sequence that is at least one amino acid residue shorter than the reference polypeptide sequence but is otherwise identical) of a reference protein that is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acids in length is provided herein. In another example, any protein that contains a stretch of about 20, 30, 40, 50, or 100 amino acids that is 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 used in accordance with the present invention. In certain embodiments, the polypeptides used in accordance with the present invention contain 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations as shown in any of the sequences provided or referenced herein.

[0080] Encoded Polypeptide The primary constructs or mmRNA of the present invention may be designed to encode a polypeptide 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-associated proteins, nuclear proteins, proteins associated with human diseases, target moieties, or proteins encoded by the human genome that have utility in the fields of research and discovery despite not having any identified therapeutic indication.

[0081] In one embodiment, the primary construct or mmRNA can encode a variant polypeptide that has a certain identity with a reference polypeptide sequence.As used herein, "reference polypeptide sequence" refers to the starting polypeptide sequence.The reference sequence can be a wild-type sequence or any sequence that is referenced in the design of another sequence. A "reference polypeptide sequence" can be, for example, any one of SEQ ID NOs: 769-1392 disclosed herein, e.g., 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, 900, 901, 902, 903, 904, 905, 906, 907, 908, 909, 910, 911, 912, 913, 914, 915, 916, 917, 918, 61, 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, 91 4, 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,、It can be any of 1374, 1375, 1376, 1377, 1378, 1379, 1380, 1381, 1382, 1383, 1384, 1385, 1386, 1387, 1388, 1389, 1390, 1391, 1392.

[0082] The term "identity," as known in the art, refers to the relationship between the sequences of two or more peptides, as determined by comparing the sequences. In the art, identity also refers to 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 percentage of identical matches between the smaller of two or more sequences, with gap alignment (if any), as processed by a specific mathematical model or computer program (i.e., "algorithm"). The identity of related peptides can be easily calculated by known methods. Such methods include "Computational Molecular Biology," Lesk, AM, ed., Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects," Smith, DW, ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data," Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; and "Sequence Analysis in Molecular Biology," von These include, but are not limited to, the methods described in 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).

[0083] In some embodiments, a polypeptide variant may have the same or similar activity as a reference polypeptide. Alternatively, a variant may have altered (e.g., increased or decreased) activity compared to the reference polypeptide. Generally, a particular polynucleotide or polypeptide variant 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 with a particular reference polynucleotide or polypeptide variant, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. Tools for such alignments include the BLAST suite of programs (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schön, 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."

[0084] Default parameters for the BLAST algorithm include, for example, a prediction threshold of 10, a word size of 28, a match / mismatch score of 1, -2, and a linear gap cost. Optional filters can be applied, as well as selection of species-specific repeat sequences, e.g., human-specific repeat sequences.

[0085] Biologics The polynucleotides, primary constructs, or mmRNA disclosed herein may encode one or more biologics. As used herein, a "biologic" is a polypeptide-based molecule that can be produced by the methods provided herein and used to treat, cure, alleviate, prevent, or diagnose serious or life-threatening diseases or conditions. In accordance with the present invention, biologics include, but are not limited to, allergen extracts (e.g., for allergy injections and testing), blood components, gene therapy products, human tissue or cell products used for transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytic agents, and immunomodulators.

[0086] In accordance with the present invention, one or more biologics currently on the market or in development may be encoded by the polynucleotides, primary constructs, or mmRNA of the present invention. Without wishing to be bound by theory, it is believed that incorporation of polynucleotides encoding known biologics into the primary constructs or mmRNA of the present invention will result in improved therapeutic efficacy, at least in part, due to specificity, purity, and / or selectivity of construct design.

[0087] antibody 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 having an immunoglobulin Fc region), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments. The term "immunoglobulin (Ig)" is used synonymously with "antibody" herein. As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., an individual antibody comprising that population that is identical except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site.

[0088] The monoclonal antibodies herein expressly include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical 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 identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as long as they exhibit the desired biological activity, as well as fragments of such antibodies. 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.

[0089] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; bispecific antibodies; linear antibodies; nanobodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0090] Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, which contain heavy chains designated α, δ, ε, γ, and μ, respectively, can be encoded by the mmRNA of the present invention. Polynucleotide sequences encoding the subclasses γ and μ are also included. Thus, any of the subclasses of antibodies can be encoded, in part or in whole, including the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0091] In accordance with the present invention, one or more antibodies or fragments currently on the market or in development may be encoded by the polynucleotides, primary constructs, or mmRNA of the present invention. Without wishing to be bound by theory, it is believed that incorporation into the primary constructs of the present invention results in improved therapeutic efficacy, at least in part, due to the specificity, purity, and selectivity of mmRNA design.

[0092] Antibodies encoded in the polynucleotides, primary constructs, or mmRNA of the present invention can be used to treat conditions or diseases in many therapeutic areas, including, but not limited to, hematology, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, gastrointestinal, medical imaging, musculoskeletal, oncology, immunology, respiratory, sensory, and anti-infective.

[0093] In one embodiment, the primary constructs and / or mmRNA disclosed herein may encode monoclonal antibodies and / or variants thereof. Antibody variants may include, but are not limited to, substitution variants, conservative amino acid substitutions, insertion variants, deletion variants, and / or covalent derivatives. In one embodiment, the primary constructs 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. Patent No. 8,217,147, the entire contents of which are incorporated herein by reference.

[0094] vaccine The primary constructs or mmRNA disclosed herein may encode one or more vaccines. As used herein, a "vaccine" is a biological preparation that enhances immunity against a specific disease or infectious agent. In accordance with the present invention, one or more vaccines currently on the market or under development may be encoded by the polynucleotides, primary constructs, or mmRNA of the present invention. Without wishing to be bound by theory, it is believed that incorporation into the primary constructs or mmRNA of the present invention results in improved therapeutic efficacy, at least in part, due to the specificity, purity, and selectivity of the construct design.

[0095] Vaccines encoded in the polynucleotides, primary constructs, or mmRNA of the present invention can be used to treat conditions or diseases in many therapeutic areas, including, but not limited to, cardiovascular, CNS, dermatology, endocrinology, oncology, immunology, respiratory, and anti-infective.

[0096] Therapeutic proteins or peptides The primary constructs or mmRNA disclosed herein may encode one or more validated or "test" therapeutic proteins or peptides.

[0097] In accordance with the present invention, one or more therapeutic proteins or peptides currently on the market or in development may be encoded by the polynucleotides, primary constructs, or mmRNA of the present invention. Without wishing to be bound by theory, it is believed that incorporation into the primary constructs or mmRNA of the present invention results in improved therapeutic efficacy, at least in part, due to the specificity, purity, and selectivity of construct design.

[0098] Therapeutic proteins and peptides encoded in the polynucleotides, primary constructs, or mmRNA of the present invention can be used to treat conditions or diseases in many therapeutic areas, including, but not limited to, hematological, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, genetics, genitourinary, gastrointestinal, musculoskeletal, oncology, and immunology, respiratory, sensory, and anti-infective.

[0099] Cell-penetrating polypeptides The primary construct or mmRNA disclosed herein may encode one or more cell-penetrating polypeptides. As used herein, "cell-penetrating polypeptide" or CPP refers to a polypeptide that can facilitate cellular uptake of a molecule. The cell-penetrating polypeptide of the present invention may contain one or more detectable labels. The polypeptide may be partially labeled or completely labeled throughout. The polynucleotide, primary construct, or mmRNA may completely, partially, or not encode a detectable label at all. The cell-penetrating peptide may also include a signal sequence. As used herein, "signal sequence" refers to a sequence of amino acid residues attached to the amino terminus of a nascent protein during protein translation. A signal sequence can be used to signal the secretion of a cell-penetrating polypeptide.

[0100] In one embodiment, the polynucleotide, primary construct, or mmRNA may also encode a fusion protein. Fusion proteins can 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 linked in a manner that allows for the expression of a complex when introduced into a cell. As used herein, "charged protein" refers to a protein that has a positive, negative, or overall neutral charge. Preferably, the therapeutic protein is covalently linked to the charged protein when forming the fusion protein. The ratio of surface charge to total amino acids or surface amino acids can be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.

[0101] The cell-penetrating polypeptide encoded by the polynucleotide, primary construct, or mmRNA may form a complex after translation. This complex may include a charged protein bound, for example, covalently bound, to the cell-penetrating polypeptide. A "therapeutic protein" refers to a protein that has a therapeutic, diagnostic, and / or preventive effect and / or induces a desired biological and / or pharmacological effect when administered to a cell.

[0102] 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 of the protein-binding partner. The cell-penetrating polypeptide may be capable of being secreted from a cell into which a polynucleotide, primary construct, or mmRNA may be introduced. The cell-penetrating polypeptide may also be capable of penetrating a first cell.

[0103] In another embodiment, the cell-penetrating polypeptide can penetrate into a second cell. The second cell can be from the same region as the first cell or from a different region. This region can include, but is not limited to, tissues and organs. The second cell can be located proximal to or distal from the first cell.

[0104] In one embodiment, the polynucleotide, primary construct, or mmRNA can encode a cell-penetrating polypeptide that can include a protein-binding partner. The protein-binding partner can include, but is not limited to, an antibody, a supercharged antibody, or a functional fragment. The polynucleotide, primary construct, or mmRNA can be introduced into a cell into which the cell-penetrating polypeptide that includes the protein-binding partner is introduced.

[0105] secreted proteins Human and other eukaryotic cells are subdivided by membranes into many functionally distinct compartments. Each membrane-bound compartment, or organelle, contains different proteins essential for the organelle's function. The cell uses "sorting signals," amino acid motifs located within proteins, to target proteins to specific organelles.

[0106] A type of sorting signal, called a signal sequence, signal peptide, or leader sequence, directs a class of proteins to an organelle called the endoplasmic reticulum (ER).

[0107] Proteins targeted to the ER by a signal sequence can be released into the extracellular space as secreted proteins. Similarly, proteins present on the cell membrane can be secreted into the extracellular space by proteolytic cleavage of the "linker" that holds the protein to the membrane. Without wishing to be bound by theory, the molecules of the present invention can be used to utilize the above-mentioned cellular transport. Thus, in some embodiments of the present invention, polynucleotides, primary constructs, or mmRNAs that express secreted proteins are provided. Secreted proteins can be selected from those described herein or in U.S. Patent Publication No. 20100255574, the contents of which are incorporated herein by reference in their entirety.

[0108] In one embodiment, they can be used in the production of large amounts of valuable human gene products.

[0109] Plasma membrane proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNA that express plasma membrane proteins are provided.

[0110] cytoplasmic or cytoskeletal proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNAs that express cytoplasmic or cytoskeletal proteins are provided.

[0111] Intracellular membrane-associated proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNAs that express intracellular membrane-associated proteins are provided.

[0112] nuclear proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNA that express nuclear proteins are provided.

[0113] Human disease-associated proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNAs that express proteins associated with human diseases are provided.

[0114] Various proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNA are provided that express proteins with currently unknown therapeutic functions.

[0115] target area In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNAs are provided that express targeting moieties. These include protein binding partners or receptors on the surface of cells that target cells to specific tissue spaces or interact with specific moieties, either in vivo or in vitro. Suitable protein binding partners include, but are not limited to, antibodies and their functional fragments, scaffold proteins, or peptides. Furthermore, polynucleotides, primary constructs, or mmRNAs can be used to direct the synthesis and extracellular localization of lipids, carbohydrates, or other biological moieties or biomolecules.

[0116] Polypeptide Library In one embodiment, polynucleotides, primary constructs, or mmRNAs can be used to generate polypeptide libraries. These libraries can result from the generation of a population of polynucleotides, primary constructs, or mmRNAs, each with a variety of structures or chemical modification designs. In this embodiment, the population of polynucleotides, primary constructs, or mmRNAs can contain multiple encoded polypeptides, including, but not limited to, antibodies or antibody fragments, protein-binding partners, scaffold proteins, and other polypeptides taught herein or known in the art. In a preferred embodiment, the polynucleotides are primary constructs of the present invention, including mmRNAs, which can be suitable for direct introduction into target cells or cultures that can then synthesize the encoded polypeptides.

[0117] In certain embodiments, multiple variants of a protein, each with a different amino acid modification(s), can be generated and tested to determine the best variant in terms of biophysical properties such as pharmacokinetics, stability, biocompatibility, and / or biological activity, or expression levels. Such libraries can be as small as 10, 10 2 , 10 3 , 10 4 , 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , or 10 9 The polypeptide may contain more than one possible variant (including, but not limited to, substitution, deletion, and insertion of one or more residues).

[0118] Antibacterial and Antiviral Polypeptides The polynucleotides, primary constructs, and mmRNA of the present invention can be designed to encode one or more antimicrobial peptides (AMPs) or antiviral peptides (AVPs). AMPs and AVPs have been isolated and described from a variety of animals, including, but not limited to, microorganisms, invertebrates, plants, amphibians, birds, fish, and mammals (Wang et al., Nucleic Acids Res. 2009; 37 (Database Publication): D933-7). For example, antimicrobial polypeptides can be found in the Antimicrobial Peptide Database (http: / / aps.unmc.edu / AP / main.php; Wang et al., Nucleic Acids Res. 2009; 37 (Database Publication): D933-7), CAMP: Collection of Anti-Microbial Peptides (http: / / www.bicnirrh.res.in / antimicrobial / ); Thomas et al., Nucleic Acids Res.2010;38(Database Publication):D774-80), U.S. Patent Nos. US5221732, US5447914, US5519115, US5607914, US5714577, US5734015, US5798336, US5821224, US5849490, US5856127, US5905187, US5994308, US5998374, US61074 60, US6191254, US6211148, US6300489, US6329504, US6399370, US6476189, US6478825, US64923 28, US6514701, US6573361, US6573361, US6576755, US6605698, US6624140, US6638531, US664220 3, US6653280, US6696238, US6727066, US6730659, US6743598, US6743769, US6747007, US679083 3, US6794490, US6818407, US6835536, US6835713, US6838435, US6872705, US6875907, US6884776 No. 6,887,847, No. 6,906,035, No. 6,911,524, No. 6,936,432, No. 7,001,924, No. 7,071,293, No. 7,078,380, No. 7,091,185, No. 7,094,759, No. 7,166,769, No. 7,244,710, No. 7,314,858, and No. 7,582,301, the contents of which are incorporated by reference in their entireties.

[0119] The antimicrobial polypeptides described herein can block cell fusion and / or viral entry by one or more enveloped viruses (e.g., HIV, HCV). For example, the antimicrobial polypeptides can comprise or consist of a region, e.g., a synthetic peptide corresponding to a contiguous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids, of a viral envelope protein, e.g., the transmembrane subunit of HIV-1 gp120 or gp41. The amino acid and nucleotide sequences of gp120 or gp41 are described, for example, in Kuiken et al., (2008) "HIV Sequence Compendium," Los Alamos National Laboratory.

[0120] In some embodiments, the antimicrobial polypeptides may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding viral protein sequence. In some embodiments, the antimicrobial polypeptides may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding viral protein sequence.

[0121] In other embodiments, the antimicrobial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, e.g., a contiguous 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 the capsid-binding protein. In some embodiments, the antimicrobial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the corresponding sequence of the capsid-binding protein.

[0122] The antimicrobial polypeptides described herein can block protease dimerization and inhibit cleavage of viral proproteins (e.g., HIV Gag-pol processing) into functional proteins, thereby preventing the release of one or more enveloped viruses (e.g., HIV, HCV). In some embodiments, the antimicrobial polypeptides can have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the corresponding viral protein sequence.

[0123] In other embodiments, the antimicrobial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, e.g., a contiguous 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 antimicrobial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to the corresponding sequence of the protease-binding protein.

[0124] The antimicrobial polypeptides described herein may include in vitro evolved polypeptides directed against viral pathogens.

[0125] Antibacterial Polypeptides Antimicrobial polypeptides (AMPs) are small peptides of variable length, sequence, and structure that have broad-spectrum activity against a variety 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, incorporated herein by reference in its entirety). AMPs have been shown to have broad-spectrum, rapidly developing killing activity, potentially with low levels of induced resistance and concomitant broad-spectrum anti-inflammatory effects.

[0126] In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) can be less than 10 kDa, e.g., less than 8 kDa, 6 kDa, 4 kDa, 2 kDa, or 1 kDa. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) consists of about 6 to about 100 amino acids, e.g., 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 antimicrobial polypeptide (e.g., antibacterial polypeptide) can consist of about 15 to about 45 amino acids. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) is substantially cationic.

[0127] In some embodiments, an antimicrobial polypeptide (e.g., an antibacterial polypeptide) may be substantially amphipathic. In certain embodiments, an antimicrobial polypeptide (e.g., an antibacterial polypeptide) may be substantially cationic and amphipathic. In some embodiments, an antimicrobial polypeptide (e.g., an antibacterial polypeptide) may be cytostatic against Gram-positive bacteria. In some embodiments, an antimicrobial polypeptide (e.g., an antibacterial polypeptide) may be cytotoxic against Gram-positive bacteria. In some embodiments, an antimicrobial polypeptide (e.g., an antibacterial polypeptide) may be cytostatic and cytotoxic against Gram-positive bacteria. In some embodiments, an antimicrobial polypeptide (e.g., an antibacterial polypeptide) may be cytostatic against Gram-negative bacteria. In some embodiments, an antimicrobial polypeptide (e.g., an antibacterial polypeptide) may be cytotoxic against Gram-negative bacteria. In some embodiments, an antimicrobial polypeptide (e.g., an antibacterial polypeptide) may be cytostatic and cytotoxic against Gram-positive bacteria. In some embodiments, an antimicrobial polypeptide may be cytostatic against a virus, a fungus, a protozoan, a parasite, a prion, or a combination thereof. In some embodiments, the antimicrobial polypeptide may be cytotoxic to viruses, fungi, protozoans, parasites, prions, or combinations thereof. In certain embodiments, the antimicrobial polypeptide may be cytostatic and cytotoxic to viruses, fungi, protozoans, parasites, prions, or combinations thereof. In some embodiments, the antimicrobial polypeptide may be cytotoxic to tumor or cancer cells (e.g., human tumor and / or cancer cells). In some embodiments, the antimicrobial polypeptide may be cytostatic to tumor or cancer cells (e.g., human tumor and / or cancer cells). In certain embodiments, the antimicrobial polypeptide may be cytotoxic and cytostatic to tumor or cancer cells (e.g., human tumor or cancer cells).In some embodiments, the antimicrobial polypeptide (eg, antibacterial polypeptide) can be a secreted polypeptide.

[0128] In some embodiments, the antimicrobial polypeptide comprises or consists of a defensin. Exemplary defensins include, but are not limited to, α-defensins (e.g., neutrophil defensin 1, defensin α1, neutrophil defensin 3, neutrophil defensin 4, defensin 5, defensin 6), β-defensins (e.g., β-defensin 1, β-defensin 2, β-defensin 103, β-defensin 107, β-defensin 110, β-defensin 136), and θ-defensin. In other embodiments, the antimicrobial polypeptide comprises or consists of a cathelicidin (e.g., hCAP18).

[0129] Antiviral Polypeptides Antiviral polypeptides (AVPs) are small peptides of variable length, sequence, and structure that have broad activity against a variety of viruses. See, e.g., Zaiou, J Mol See Med, 2007;85:317. AVP has been shown to have broad-spectrum, rapidly developing killing activity, potentially with low levels of induced resistance and concomitant broad-spectrum anti-inflammatory activity. In some embodiments, the antiviral polypeptide is less than 10 kDa, e.g., less than 8 kDa, 6 kDa, 4 kDa, 2 kDa, or 1 kDa. In some embodiments, the antiviral polypeptide comprises or consists of about 6 to about 100 amino acids, e.g., 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 antiviral polypeptide comprises or consists of about 15 to about 45 amino acids. In some embodiments, the antiviral polypeptide is substantially cationic. In some embodiments, the antiviral polypeptide is substantially amphipathic. In certain embodiments, the antiviral polypeptide is substantially cationic and amphipathic. In some embodiments, the antiviral polypeptide is cytostatic against viruses. In some embodiments, the antiviral polypeptide is cytotoxic against viruses. In some embodiments, the antiviral polypeptide is cytostatic and cytotoxic against viruses. In some embodiments, the antiviral polypeptide is cytostatic against bacteria, fungi, protozoans, parasites, prions, or combinations thereof. In some embodiments, the antiviral polypeptide is cytotoxic against bacteria, fungi, protozoans, parasites, prions, or combinations thereof. In certain embodiments, the antiviral polypeptide is cytostatic and cytotoxic against bacteria, fungi, protozoans, parasites, prions, or combinations thereof. In some embodiments, the antiviral polypeptide is cytotoxic against tumor or cancer cells (e.g., human cancer cells). In some embodiments, the antiviral polypeptide is cytostatic against tumor or cancer cells (e.g., human cancer cells).In certain embodiments, the antiviral polypeptide is cytotoxic and cytostatic to tumor or cancer cells (e.g., human cancer cells). In some embodiments, the antiviral polypeptide is a secreted polypeptide.

[0130] Cytotoxic Nucleosides In one embodiment, the polynucleotide, primary construct, or mmRNA of the present invention can incorporate one or more cytotoxic nucleosides.For example, cytotoxic nucleosides can be incorporated into polynucleotides, primary constructs, or mmRNA, such as bifunctional modified RNA or mRNA.Cytotoxic nucleoside anticancer drugs include, but are not limited to, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, floxuridine, FTORAFUR® (a combination of tegafur and uracil), tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), and 6-mercaptopurine.

[0131] Several cytotoxic nucleoside analogs are in clinical use or undergoing clinical trials as anticancer drugs. Examples of such analogs 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-β-D-arabino-pentofuranosyl)-cytosine. Another example of such a compound is fludarabine phosphate. These compounds can be administered systemically and may have typical side effects of cytotoxic drugs, including, but not limited to, little or no specificity for tumor cells over normal cell proliferation.

[0132] Some prodrugs of cytotoxic nucleoside analogs have also been reported in the art.Examples include, but are not limited to, N4-behenoyl-1-β-D-arabinofuranosylcytosine, N4-octadecyl-1-β-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidic acid ester).Generally, these prodrugs are mainly converted into active drugs in the liver and systemic circulation, and exhibit little or no selective release of active drugs in tumor tissue.For example, capecitabine, a prodrug of 5'-deoxy-5-fluorocytidine (ultimately, 5-fluorouracil), is metabolized in the liver and tumor tissue. A series of capecitabine analogs containing "radicals readily hydrolyzable under physiological conditions" has been claimed by Fujiu et al. (U.S. Pat. No. 4,966,891) and is incorporated herein by reference. The series of capecitabine analogs described by Fujiu includes N-alkyl and aralkyl carbamates of 5'-deoxy-5-fluorocytidine, implying that these compounds are activated by hydrolysis to yield 5'-deoxy-5-fluorocytidine under normal physiological conditions.

[0133] A series of cytarabine N4-carbamates has been reported by Fadl et al. (Pharmazie. 1995, 50, 382-7, incorporated herein by reference), in which the compounds were designed to be converted to cytarabine in the liver and plasma. International Publication No. WO 2004 / 041203, incorporated herein by reference, discloses prodrugs of gemcitabine, some of which 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, incorporated herein by reference) described acetal derivatives of 1-(3-C-ethynyl-β-D-ribo-pentofaranosyl)cytosine that upon bioreduction produced intermediates that required further hydrolysis under acidic conditions to produce cytotoxic nucleoside compounds.

[0134] Cytotoxic nucleotides that may be chemotherapeutic agents also include, but are not limited to, pyrazolo[3,4-D]-pyrimidine, 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.

[0135] Flanking region: untranslated region (UTR) The untranslated region (UTR) of a gene is transcribed but not translated. The 5'UTR begins at the transcription initiation site and follows but does not include the initiation codon, while the 3'UTR begins immediately after the termination codon and continues until the transcription termination signal. A growing number of studies have reported on the regulatory role that UTRs play in the stability of nucleic acid molecules and translation. Regulatory features of UTRs can be incorporated into the polynucleotides, primary constructs, and / or mmRNA of the present invention to enhance the stability of the molecules. Specific features can also be incorporated to ensure controlled downregulation of transcripts in the event that they are misdirected to undesirable organ sites.

[0136] 5'UTR and translation start Natural 5'UTRs have features involved in translation initiation. They contain features such as the Kozak sequence, which is commonly known to be involved in the process by which ribosomes initiate the translation of many genes. The Kozak sequence has the consensus CCR(A / G)CCAUGG, where R is a purine (adenine or guanine) three bases upstream of the start codon (AUG), followed by another "G." 5'UTRs are also known to form secondary structures involved in elongation factor binding.

[0137] The stability and protein production of polynucleotides, primary constructs, or mmRNA of the present invention can be enhanced by manipulating features typically found in highly expressed genes of specific target organs. For example, introduction of the 5'UTR of liver-expressed mRNAs such as albumin, serum amyloid A, apolipoprotein A / B / E, transferrin, alpha-fetoprotein, erythropoietin, or factor VIII can be used to enhance expression of nucleic acid molecules such as mmRNA in hepatic cell lines or the liver. Similarly, 5'UTRs from other tissue-specific mRNAs can be used to enhance expression in that tissue in muscle (MyoD, myosin, myoglobin, myogenin, herculin), endothelial cells (Tie-1, CD36), bone marrow cells (C / EBP, AML1, G-CSF, GM-CSF, CD11b, MSR, Fr-1, i-NOS), leukocytes (CD45, CD18), adipose tissue (CD36, GLUT4, ACRP30, adiponectin), and lung epithelial cells (SP-A / B / C / D).

[0138] Other non-UTR sequences can be incorporated into the 5'UTR (or 3'UTR). For example, introns or parts of intron sequences can be incorporated into the adjacent regions of the polynucleotides, primary constructs, or mmRNA of the present invention. The incorporation of intron sequences can increase protein production as well as mRNA levels.

[0139] 3'UTR and AU-rich elements 3'UTRs are known to have stretches of adenosine and uridine embedded within them. These AU-rich features are particularly common in genes with high turnover rates. Based on their sequence characteristics and functional properties, AU-rich elements (AREs) can be classified into three classes (Chen et al., 1995): Class I AREs contain several dispersed copies of the AUUUA motif within the U-rich region. C-Myc and MyoD contain class I AREs. Class II AREs have two or more overlapping UUAUUUA(U / A)(U / A) nonamers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are less well defined. These U-rich regions do not contain the AUUUA motif. C-Jun and myogenin are two well-studied examples of this class. While most proteins that bind to AREs are known to destabilize messengers, members of the ELAV family, most notably HuR, have been demonstrated to enhance mRNA stability. HuR binds to these three classes of AREs. Engineering a HuR-specific binding site into the 3'UTR of a nucleic acid molecule leads to HuR binding and, therefore, to message stabilization in vivo.

[0140] The introduction, removal, or modification of AU-rich elements (AREs) in the 3'UTR can be used to regulate the stability of polynucleotides, primary constructs, or mmRNA of the present invention. When manipulating a particular polynucleotide, primary construct, or mmRNA, one or more copies of AREs can be introduced to reduce the stability of the polynucleotide, primary construct, or mmRNA of the present invention, thereby suppressing translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to improve intracellular stability, thereby increasing the translation and production of the resulting protein. Transfection experiments using the polynucleotides, primary constructs, or mmRNA of the present invention can be performed in relevant cell lines, and protein production can be assayed at various time points after transfection. For example, cells can be transfected with different ARE-engineered molecules, and the proteins produced can be assayed using an ELISA kit for the relevant protein at 6 hours, 12 hours, 24 hours, 48 ​​hours, and 7 days after transfection.

[0141] Incorporation of microRNA binding sites MicroRNAs (or miRNAs) are 19-25 nucleotide-long non-coding RNAs that bind to the 3'UTR of nucleic acid molecules and downregulate gene expression by either reducing nucleic acid molecule stability or inhibiting translation. Polynucleotides, primary constructs, or mmRNAs of the present invention may contain one or more microRNA target sequences, microRNA sequences, or microRNA species. Such sequences may correspond to any known microRNA, such as those taught in U.S. Patent Publication Nos. US2005 / 0261218 and US2005 / 0059005, the contents of which are incorporated herein by reference in their entireties.

[0142] A microRNA sequence comprises a "seed" region, i.e., a region from positions 2 to 8 of the mature microRNA, which has perfect Watson-Crick complementarity to the miRNA target sequence. A microRNA seed may comprise positions 2 to 8 or positions 2 to 7 of the mature microRNA. In some embodiments, a microRNA seed may comprise seven nucleotides (e.g., nucleotides 2 to 8 of the mature microRNA), and the seed-complementary site in the corresponding miRNA target is adjacent to an adenine (A) opposite microRNA position 1. In some embodiments, a microRNA seed may comprise six nucleotides (e.g., nucleotides 2 to 7 of the mature microRNA), and the seed-complementary site in the corresponding miRNA target is adjacent to an adenine (A) opposite microRNA position 1. For example, see 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 incorporated herein by reference in its entirety. The base of the microRNA species has perfect complementarity with the target sequence. By engineering the microRNA target sequence into the 3'UTR of the polynucleotide, primary construct, or mmRNA of the present invention, this molecule can be targeted for degradation or translation reduction, provided that the microRNA in question is available. This process reduces the risk of off-target effects during nucleic acid molecule delivery. The identification of microRNAs, microRNA target regions, and their expression patterns, as well as their roles in biology, have been reported (Bonauer et al., Curr Drug Targets 2010 11:943-949; Anand and Cheresh Curr Opin Hematol 2011 18:171-176; Contreras and Rao Leukemia 2012 26:404-413 (2011 Dec 20. doi:10.1038 / leu.2011.356); Bartel Cell 2009 136:215-233; Landgraf et al., Cell, 2007, each of which is incorporated by reference in its entirety). 129:1401-1414).

[0143] For example, if the nucleic acid molecule is mRNA and is not intended to be delivered to the liver but settles there, miR-122, a microRNA abundant in the liver, can inhibit expression of a gene of interest if one or more target sites for miR-122 are engineered into the 3'UTR of a polynucleotide, primary construct, or mmRNA. Introduction of one or more binding sites for different microRNAs can be engineered to further reduce the longevity, stability, and protein translation of the polynucleotide, primary construct, or mmRNA.

[0144] As used herein, the term "microRNA site" refers to a microRNA target site or microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. It is understood that "binding" may follow conventional Watson-Crick hybridization rules or may refer to any stable association of a microRNA with a target sequence at or adjacent to the microRNA site.

[0145] Conversely, for the purpose of the polynucleotide, primary construct, or mmRNA of the present invention, naturally occurring microRNA binding sites can be engineered out of the sequence (i.e., removed from the sequence) to increase protein expression in specific tissues. For example, the miR-122 binding site can be removed to improve protein expression in the liver. Control of expression in multiple tissues can be achieved by introducing or removing one or several microRNA binding sites.

[0146] Examples of tissues in which microRNAs are known to regulate mRNA and therefore 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), bone marrow 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-ld, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). MicroRNAs can also regulate complex biological processes, such as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176, incorporated herein by reference in their entireties). In the polynucleotides, primary constructs, or mmRNAs of the present invention, binding sites for microRNAs involved in such processes can be removed or introduced to regulate the expression of the polynucleotides, primary constructs, or mmRNAs in biologically relevant cell types or in relation to relevant biological processes. Lists of microRNAs, miR sequences, and miR binding sites are listed in Table 9 of U.S. Provisional Patent Application No. 61 / 753,661, filed January 17, 2013; Table 9 of U.S. Provisional Patent Application No. 61 / 754,159, filed January 18, 2013; and Table 7 of U.S. Provisional Patent Application No. 61 / 758,921, filed January 31, 2013, each of which is incorporated herein by reference in its entirety.

[0147] Finally, through understanding the expression patterns of microRNAs in different cell types, polynucleotides, primary constructs, or mmRNAs can be engineered for more targeted expression in specific cell types or only under specific biological conditions. By introducing tissue-specific microRNA binding sites, polynucleotides, primary constructs, or mmRNAs can be designed that are optimal for protein expression in a tissue or in relation to a biological condition. Examples of the use of microRNAs to drive tissue- or disease-specific gene expression are listed (Getner, 2004, pp. 111-114, which are incorporated herein by reference in their entirety). and Naldini, Tissue Antigens. 2012, 80:393-403). In addition, microRNA seed sites can be incorporated into mRNA to reduce expression in certain cells, resulting in biological improvements. An example of this is the incorporation of miR-142 sites into UGT1A1-expressing lentiviral vectors. The presence of miR-142 seed sites reduced expression in hematopoietic cells, resulting in reduced expression in antigen-presenting cells, and the absence of an immune response to the virally expressed UGT1A1 (Schmitt et al., Gastroenterology 2010;139:999-1007; Gonzalez-Asequinolaza et al. Gastroenterology 2010,139:726-729, both of which are incorporated herein by reference in their entireties). Incorporation of miR-142 sites into modified mRNA not only reduced the expression of the encoded protein in hematopoietic cells, but also reduced or abolished the immune response to the mRNA-encoded protein. Incorporation of miR-142 site(s) into mRNA is important when treating patients with complete protein deficiency (e.g., UGT1A1 type I, LDLR-deficient patients, CRIM-negative Pompe patients, etc.).

[0148] The transfection experiment using engineered polynucleotide, primary construct or mmRNA can be carried out in relevant cell lines, and protein production can be measured at various time points after transfection.For example, cells can be transfected with different microRNA binding site engineered polynucleotide, primary construct or mmRNA, and the protein produced can be measured at 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours and 7 days after transfection using ELISA kit for relevant protein.In vivo experiments can also be carried out using microRNA binding site engineered molecules to test the tissue-specific expression changes of formulated polynucleotide, primary construct or mmRNA.

[0149] 5' Capping The 5' cap structure of mRNA is involved in nuclear export, improves mRNA stability, and binds to mRNA cap-binding protein (CBP), which is involved in mRNA stability and translation competence in cells through its association with poly(A)-binding protein to form mature circular mRNA species. This cap also assists in the removal of 5'-proximal introns during mRNA splicing.

[0150] Endogenous mRNA molecules can be 5'-end capped, resulting in a 5'-ppp-5'-triphosphate linkage between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylate cap can then be methylated to generate an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or pre-terminal transcribed nucleotides at the 5' end of the mRNA can also optionally be 2'-O-methylated. 5' decapping, via hydrolysis and cleavage of the guanylate cap structure, can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0151] The modification of the polynucleotides, primary constructs, and mRNA of the present invention can generate a non-hydrolyzable cap structure that prevents decapping, thereby increasing the half-life of the mRNA. Because hydrolysis of the cap structure requires cleavage of the 5'-ppp-5' phosphorodiester bond, modified nucleotides can be used during the capping reaction. For example, vaccinia capping enzyme from New England Biolabs (Ipswich, MA) can be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to create a phosphorothioate bond in the 5'-ppp-5' cap. Additional modified guanosine nucleotides, such as α-methyl-phosphonate and seleno-phosphate nucleotides, can be used.

[0152] Further modifications include, but are not limited to, 2'-O-methylation (as described above) of the ribose sugar of the 5'-terminal and / or pre-5' terminal nucleotide of an mRNA at the 2'-hydroxyl group of the sugar ring. Several distinct 5'-cap structures can be used to generate the 5' cap of a nucleic acid molecule, such as an mRNA molecule.

[0153] Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ from the natural (i.e., endogenous, wild-type, or physiological) 5' cap in their chemical structure but retain cap function. Cap analogs can be synthesized and / or attached to nucleic acid molecules chemically (i.e., non-enzymatically) or enzymatically.

[0154] For example, the anti-reverse cap analog (ARCA) cap contains two guanines joined by 5'-5'-triphosphate groups, one guanine having an N7 methyl group as well as a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine (m 7The capped nucleic acid molecule (e.g., mRNA or mmRNA) contains the N7- and 3'-O-methylated guanine (G-3'mppp-G, which may equivalently be designated 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanine is attached to the 5'-terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA or mmRNA). The N7- and 3'-O-methylated guanine provide the terminal portion of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0155] Another exemplary cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on the guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 Gm-ppp-G).

[0156] While cap analogs allow for the capping of nucleic acid molecules concomitantly in in vitro transcription reactions, up to 20% of the transcripts may remain uncapped. This, as well as structural differences between the cap analog and the endogenous 5'-cap structure of nucleic acids produced by the endogenous cellular transcription machinery, can result in reduced translational competence and reduced cellular stability.

[0157] The polynucleotides, primary constructs, and mRNAs of the present invention can also be post-transcriptionally capped using enzymes to generate more authentic 5'-cap structures. As used herein, the term "more authentic" refers to features that closely resemble or mimic endogenous or wild-type features, either structurally or functionally. That is, a "more authentic" feature better represents an endogenous, wild-type, natural, or physiological cellular function and / or structure compared to prior art synthetic features or analogs, or surpasses 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 include those that have, among others, enhanced cap-binding protein binding, increased half-life, reduced susceptibility to 5' endonucleases, and / or reduced 5'-decapping compared to synthetic 5'-cap structures (or wild-type, natural, or physiological 5'-cap structures) known in the art. For example, recombinant vaccinia virus capping enzyme and recombinant 2'-O-methyltransferase enzyme can generate a standard 5'-5'-triphosphate bond between the 5'-terminal nucleotide of an mRNA and a guanine cap nucleotide, where the cap guanine contains an N7 methylation and the 5'-terminal nucleotide of the mRNA contains a 2'-O-methyl. Such a structure is referred to as the Cap 1 structure. This cap results in greater translation competence and cellular stability, as well as reduced activation of cellular proinflammatory cytokines, compared to, for example, 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).

[0158] Because polynucleotides, primary constructs, or mmRNA can be capped post-transcriptionally and this process is more efficient, nearly 100% of polynucleotides, primary constructs, or mmRNA can be capped, compared to approximately 80% when a cap analog is attached to the mRNA during an in vitro transcription reaction.

[0159] According to the present invention, the 5'-end cap can comprise an endogenous cap or a cap analog. According to the present invention, the 5'-end cap can 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.

[0160] Viral sequence Additional viral sequences, such as, but not limited to, the translational enhancer sequence of Barley Yellow Dwarf Virus (BYDV-PAV), Jaagsiekte Sheep Retrovirus (JSRV), and / or Enzootic Nasal Tumor Virus (see, e.g., International Publication No. WO2012129648, incorporated herein by reference in its entirety), can be engineered and inserted into the 3'UTR of the polynucleotides, primary constructs, or mmRNA of the invention to stimulate translation of the construct in vitro and in vivo. Transfection experiments can be performed in relevant cell lines, and protein production can be assayed by ELISA at 12 hours, 24 hours, 48 ​​hours, 72 hours, and 7 days post-transfection.

[0161] IRES sequence Additionally, polynucleotides, primary constructs, or mmRNAs are provided that may contain an internal ribosome entry site (IRES). IRESs, first identified in characteristic picornavirus RNAs, play an important role in initiating protein synthesis in the absence of a 5' cap structure. An IRES may serve as the sole ribosome binding site for an mRNA, or may serve as one of multiple ribosome binding sites. Polynucleotides, primary constructs, or mmRNAs containing two or more functional ribosome binding sites may encode several peptides or polypeptides that are independently translated by ribosomes ("polycistronic nucleic acid molecules"). When an IRES is provided in a polynucleotide, primary construct, or mmRNA, a second translatable region is optionally further provided. Examples of IRES sequences that may be used in accordance with the present invention include, without limitation, those derived from picornaviruses (e.g., FMDV), plague viruses (CFFV), polioviruses (PV), encephalomyocarditis viruses (ECMV), foot-and-mouth disease viruses (FMDV), hepatitis C viruses (HCV), classical swine fever viruses (CSFV), murine leukemia viruses (MLV), simian immunodeficiency viruses (SIV), or cricket paralysis viruses (CrPV).

[0162] Poly A tail During RNA processing, long chains of adenine nucleotides (poly-A tails) can be added to polynucleotides, such as mRNA molecules, to improve stability. Immediately after transcription, the 3' end of the transcript can be cleaved to free a 3' hydroxyl. Poly-A polymerase then adds chains of adenine nucleotides to the RNA. This process, called polyadenylation, adds a poly-A tail that can be approximately 100-250 residues long.

[0163] It has been found that particular polyA tail lengths provide certain advantages to the polynucleotides, primary constructs or mmRNA of the present invention.

[0164] Generally, the length of a polyA tail of the present invention is greater than 30 nucleotides in length, hi another embodiment, the polyA 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 length). In some embodiments, the polynucleotide, primary construct, or mmRNA is from about 30 to about 3,000 nucleotides (e.g., 30-50, 30-100, 30-250, 30-500, 30-750, 30-1,000, 30-1,500, 30-2,000, 30-2,500, 50-100, 50-250, 50-500, 50-750, 50-1,000, 50-1,500, 50-2,000, 50-2,500, 50-3,000, 100-500, 100-750, 100-1,000, 00, 100-1,500, 100-2,000, 100-2,500, 100-3,000, 500-750, 500-1,000, 500-1,500, 500-2,000, 500-2,500, 500-3,000, 1,000-1,500, 1,000-2,000, 1,000-2,500, 1,000-3,000, 1,500-2,000, 1,500-2,500, 1,500-3,000, 2,000-3,000, 2,000-2,500, and 2,500-3,000 pieces).

[0165] In one embodiment, the polyA tail is designed into the length of the entire polynucleotide, primary construct, or mmRNA. This design can be based on the length of the coding region, the length of a particular feature or region (such as the first or adjacent region), or the length of the final product expressed from the polynucleotide, primary construct, or mmRNA.

[0166] In this context, the polyA tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer than the length of the polynucleotide, primary construct, or mmRNA, or a feature thereof. The polyA tail can also be designated as a fraction of the polynucleotide, primary construct, or mmRNA to which it belongs. In this context, the polyA tail can 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 polyA tail. Furthermore, engineering binding sites and conjugating the polynucleotide, primary construct, or mmRNA to polyA-binding proteins can enhance expression.

[0167] Additionally, multiple distinct polynucleotides, primary constructs, or mmRNA can be attached to PABP (polyA binding protein) via their 3' ends using modified nucleotides at the 3' end of the polyA tail. Transfection experiments can be performed in relevant cell lines, and protein production can be assayed by ELISA at 12 hours, 24 hours, 48 ​​hours, 72 hours, and 7 days post-transfection.

[0168] In one embodiment, polynucleotide primary constructs of the invention are designed to contain a polyA-G quartet. A 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, a G quartet is incorporated at the end of a polyA tail. The resulting mmRNA constructs are assayed for stability, protein production, and half-life and other parameters at various time points. It has been found that a polyA-G quartet results in protein production that is at least 75% of the protein production that results from using a 120-nucleotide polyA tail alone.

[0169] Quantification In one embodiment, the polynucleotides, primary constructs, or mmRNA of the present invention can be quantified in exosomes derived from one or more bodily fluids. As used herein, "bodily fluids" includes peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cooper's gland fluid or pre-ejaculatory fluid, sweat, feces, hair, tears, cyst fluid, pleural and peritoneal fluid, pericardial fluid, lymph, chyme, chyle, bile, interstitial fluid, leucorrhea, pus, sebum, vomit, vaginal secretions, mucosal secretions, stool water, pancreatic juice, nasal lavage fluid, bronchopulmonary aspirate, blastocyst cavity fluid, and umbilical cord blood. Alternatively, exosomes may be recovered from an organ selected from the group consisting of lung, heart, pancreas, stomach, intestine, bladder, kidney, ovary, testicle, skin, colon, breast, prostate, brain, esophagus, liver, and placenta.

[0170] In the quantification method, a sample of less than 2 mL is obtained from a subject, and exosomes are isolated by size-exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or a combination thereof. In the analysis, the level or concentration of polynucleotides, primary constructs, or mmRNA can be the expression level, presence, absence, cleavage, or modification of the administered construct. It is advantageous to correlate this level with an assay for one or more clinical phenotypes or human disease biomarkers. While this assay can be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or a combination thereof, exosomes can also be isolated using immunohistochemistry, such as enzyme-linked immunosorbent assay (ELISA). Exosomes can also be isolated by size-exclusion chromatography, density gradient centrifugation, differential centrifugation, nanomembrane ultrafiltration, immunosorbent capture, affinity purification, microfluidic separation, or a combination thereof.

[0171] These methods provide researchers with the ability to monitor the levels of remaining or delivered polynucleotides, primary constructs, or mmRNA in real time. This is possible because the polynucleotides, primary constructs, or mmRNA of the present invention differ from endogenous forms due to structural or chemical modifications.

[0172] II. mmRNA Design and Synthesis Polynucleotides, primary constructs, or mmRNA used in accordance with the present invention can be prepared according to any available technique, including, but not limited to, chemical synthesis, enzymatic synthesis, commonly referred to as in vitro transcription (IVT), or enzymatic or chemical cleavage of longer precursors. Methods for synthesizing RNA are known in the art (see, for example, Gait, MJ (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, NJ) Totowa, NJ: Humana Press, 2005, both of which are incorporated herein by reference).

[0173] The process of designing and synthesizing the primary construct of the present invention generally includes a gene construction step, an mRNA production step (with or without modification), and a purification step. In the enzymatic synthesis method, a target polynucleotide sequence encoding a polypeptide of interest is first selected for incorporation into a vector that is amplified to produce a cDNA template. Optionally, the target polynucleotide sequence and / or any adjacent sequences can be codon-optimized. The cDNA template is then used to produce mRNA by in vitro transcription (IVT). After production, the mRNA can undergo a purification and cleanup process. These steps are provided in more detail below.

[0174] Gene construction Gene construction steps may include, but are not limited to, gene synthesis, vector amplification, plasmid purification, plasmid linearization and purification, and cDNA template synthesis and purification.

[0175] Gene synthesis Once a polypeptide or target of interest is selected for production, a primary construct is designed. Within the primary construct, a first region of linked nucleosides encoding the polypeptide of interest can be constructed using the open reading frame (ORF) of a selected nucleic acid (DNA or RNA) transcript. The ORF can include a wild-type ORF, an isoform, a mutant, or a fragment thereof. As used herein, "open reading frame" or "ORF" is intended to refer to a nucleic acid sequence (DNA or RNA) capable of encoding a polypeptide of interest. The ORF often begins with an initiation codon ATG and ends with a nonsense or stop codon or signal.

[0176] Furthermore, the nucleotide sequence of the first region can be codon-optimized. Codon optimization methods are known in the art and can be useful in efforts to achieve one or more of several goals. These goals include: matching the codon frequency in the target and host organisms to ensure proper folding; biasing GC content to increase mRNA stability or reduce secondary structure; minimizing tandem repeat codons or base pairs that may interfere with gene assembly or expression; customizing transcriptional and translational control regions; inserting or removing protein transport sequences; removing / adding post-translational modification sites (e.g., glycosylation sites) in the encoded protein; adding, removing, or recombining protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; adjusting the translation rate to allow the various domains of the protein to fold properly; or reducing or eliminating problematic secondary structures in mRNA. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include GeneArt services (Life Technologies), DNA2.0 (Menlo Park, CA), and / or proprietary methods. In one embodiment, the ORF sequence is optimized using an optimization algorithm. Codon choices for each amino acid are provided in Table 1. Table 1. Codon choices [Table 0001]

[0177] Features that may be considered beneficial in some embodiments of the present invention may be encoded by the primary construct and may flank the ORF as first or second flanking regions. These flanking regions may be incorporated into the primary construct before and / or after ORF optimization. It is not necessary for the primary construct to contain both 5' and 3' flanking regions. Examples of such features include, but are not limited to, untranslated regions (UTRs), Kozak sequences, oligo(dT) sequences, and detectable tags, as well as multiple cloning sites that may have XbaI recognition.

[0178] In some embodiments, a 5'UTR and / or a 3'UTR can be provided as a flanking region. Multiple 5' or 3'UTRs can be included in a flanking region and can be the same or different sequences. Any portion of a flanking region (including none) can be codon-optimized, any of which can independently contain one or more different structural or chemical modifications before and / or after codon optimization. A combination of features can be included in the first and second flanking regions, and can be contained within other features. For example, an ORF can be flanked by a 5'UTR that can contain a strong Kozak translation initiation signal and / or a 3'UTR that can contain an oligo(dT) sequence for templated addition of a polyA tail. A 5'UTR, such as the 5'UTR described in U.S. Patent Application Publication No. 20100293625, incorporated herein by reference in its entirety, can comprise a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes.

[0179] Tables 2 and 3 provide a list of exemplary UTRs that can be utilized in the primary constructs of the present invention as flanking regions. A list of 5' untranslated regions of the present invention is shown in Table 2. Variants of the 5' UTR in which one or more nucleotides, including A, T, C, or G, are added to or removed from the end can be utilized. Table 2.5' Untranslated area [Table 0002]

[0180] A representative list of 3' untranslated regions of the present invention is shown in Table 3. Variants of the 3' UTR in which one or more nucleotides, including A, T, C, or G, are added to or removed from the end may be utilized. Table 3.3'Untranslated area [Table 0003-1] [Table 0003-2] [Table 0003-3] [Table 0003-4] [Table 0003-5] [Table 0003-6] [Table 0003-7] [Table 0003-8]

[0181] It should be understood that the UTRs listed in the previous table are examples, and any UTR from any gene can be incorporated into the first or second flanking region of each primary construct. Furthermore, multiple wild-type UTRs from any known gene can be utilized. It is also within the scope of the present invention to provide artificial UTRs that are not mutants of wild-type genes. These UTRs or portions thereof can be positioned in the same orientation as the UTRs or portions thereof of the transcripts from which they can be selected, or their orientation or position can be changed. Thus, 5' or 3' UTRs can be inverted, shortened, extended, or chimerized with one or more other 5' or 3' UTRs. As used herein, the term "altered" when referring to a UTR sequence means that the UTR has changed in some respect relative to the reference sequence. For example, the 3' or 5' UTR can be altered relative to the wild-type or natural UTR by changing the orientation or position as taught above, or by including additional nucleotides, deleting nucleotides, exchanging or transposing nucleotides. Any of these changes that result in an "altered" UTR (whether 3' or 5') includes a mutant UTR.

[0182] In one embodiment, a double, triple, or quadruple UTR, such as a 5' or 3' UTR, can be used. As used herein, a "double" UTR is one in which two copies of the same UTR are encoded either contiguously or substantially contiguously. For example, the double β-globin 3' UTR described in U.S. Patent Publication No. 20100129877, the contents of which are incorporated herein by reference in its entirety, can be used.

[0183] It is also within the scope of the present invention to have patterned UTR.As used herein, " patterned UTR " refers to the UTR that shows a repeat or alternating pattern that repeats once, twice, or three or more times, such as ABABAB or AABBAABBAABB or ABCABCABC or its variants.In these patterns, each letter A, B or C represents different UTR at nucleotide level.

[0184] In one embodiment, the flanking region is selected from a family of transcripts whose proteins share common function, structure, and characteristic features.For example, the target polypeptide may belong to a family of proteins that are expressed in a specific cell, tissue, or at a certain time during development.Any UTR of these genes can be exchanged with any other UTR of the same or different protein family to create a new chimeric primary transcript.As used herein, the term "protein family" is used in the broadest sense to refer to a group of two or more target polypeptides that share at least one function, structure, characteristic, localization, origin, or expression pattern.

[0185] After optimization (if desired), the primary construct components can be reconstructed and transformed into vectors, such as, but not limited to, plasmids, viruses, cosmids, and artificial chromosomes. For example, optimized constructs can be reconstructed and transformed into chemically competent E. coli, yeast, Neurospora crassa, maize, Drosophila, etc., and high copy number, plasmid-like or chromosomal structures generated by the methods described herein.

[0186] The untranslated region may also include a translational enhancer element (TEE). By way of non-limiting example, the TEE may include those described in U.S. Patent Application No. 20090226470, which is incorporated herein by reference in its entirety, and TEEs known in the art.

[0187] stop codon In one embodiment, the primary construct of the present invention may comprise at least two stop codons before the 3' untranslated region (UTR). The stop codons may be selected from TGA, TAA, and TAG. In one embodiment, the primary construct of the present invention comprises the stop codon TGA and another stop codon. In a further embodiment, the other stop codon may be TAA. In another embodiment, the primary construct of the present invention comprises three stop codons.

[0188] Vector amplification 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, maxiprep using the Invitrogen PURELINK™ HiPure Maxiprep kit (Carlsbad, CA).

[0189] Plasmid linearization The plasmid can then be linearized using methods known in the art, such as, but not limited to, the use of restriction enzymes and buffers. The linearized reaction can be purified using, for example, Invitrogen's PURELINK™ PCR Micro Kit (Carlsbad, CA), as well as 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), as well as Invitrogen's standard PURELINK™ PCR Kit (Carlsbad, CA). Purification methods can be modified depending on the size of the resulting linearized reaction. The linearized plasmid is then used to generate cDNA for in vitro transcription (IVT) reactions.

[0190] cDNA template synthesis The cDNA template can be synthesized by subjecting the linearized plasmid to polymerase chain reaction (PCR). Table 4 lists primers and probes that may be useful in the PCR reactions of the present invention. It should be understood that this list is not exhaustive, and primer-probe design for any amplification is within the skill of the art. The probe may also contain chemically modified bases to enhance base-pairing fidelity and strength to the target molecule. Such modifications may include 5-methyl-cytidine, 2,6-diamino-purine, 2'-fluoro, phosphoro-thioate, or locked nucleic acid. Table 4. Primers and probes [Table 0004] *UFP is a universal forward primer and URP is a universal reverse primer.

[0191] In one embodiment, the cDNA may be submitted for sequencing analysis before undergoing transcription.

[0192] mRNA production The process of mRNA or mmRNA production may include, but is not limited to, in vitro transcription, cDNA template removal and RNA purification, and mRNA capping and / or tailing reactions.

[0193] In vitro transcription The cDNA produced in the previous step can be transcribed using an in vitro transcription (IVT) system. This system typically includes a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor, and a polymerase. The NTPs can be produced in-house, selected from a supplier, or synthesized as described herein. The NTPs can be selected from, but are not limited to, the NTPs described herein, including natural and unnatural (modified) NTPs. The polymerase can be selected from, but is not limited to, mutant polymerases, such as, but not limited to, T7 RNA polymerase, T3 RNA polymerase, and polymerases capable of incorporating modified nucleic acids.

[0194] RNA polymerase Any number of RNA polymerases or variants may be used in designing the primary constructs of the present invention.

[0195] RNA polymerase can be modified by inserting or deleting amino acids in the RNA polymerase sequence.As a non-limiting example, RNA polymerase can be modified to show improved ability to incorporate 2'-modified nucleotide triphosphates compared to unmodified RNA polymerase (see International Publication No. WO2008078180 and U.S. Patent No. 8,101,385, which are incorporated herein by reference in their entirety).

[0196] Variants can be obtained by evolving RNA polymerases, optimizing RNA polymerase amino acid and / or nucleic acid sequences, and / or using other methods known in the art. As a non-limiting example, T7 RNA polymerase variants can be evolved using the continuous directed evolution system designed by Esvelt et al. (Nature (2011) 472(7344):499-503, incorporated herein by reference in its entirety), to generate T7 The RNA polymerase clones contained mutations that resulted in a substitution of lysine at position 93 with threonine (K93T), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, and β161M. It may encode at least one mutation such as, but not limited to, E356D, L360P, A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A, H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E, N748D, Q754R, E775K, A827V, D851N, or L864F. As another non-limiting example, a T7 RNA polymerase variant may encode at least one mutation described in U.S. Patent Publication Nos. 20100120024 and 20070117112, which are incorporated by reference in their entireties. Mutant forms of RNA polymerase can also include, but are not limited to, substitution mutants, conservative amino acid substitutions, insertion mutants, deletion mutants, and / or covalent derivatives.

[0197] In one embodiment, the primary construct may be designed to be recognized by a wild-type or mutant RNA polymerase, and in doing so, the primary construct may be modified to contain sites or regions of sequence variation from the wild-type or parent primary construct.

[0198] In one embodiment, the primary construct may be designed to contain at least one substitution and / or insertion within the 5'UTR, before the 5'UTR, and / or after the 5'UTR upstream of the RNA polymerase binding or recognition site of the primary construct, downstream of the RNA polymerase binding or recognition site, upstream of the TATA box sequence, downstream of the TATA box sequence but upstream of the coding region of the primary construct.

[0199] In one embodiment, the 5'UTR of the primary construct can 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 can 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 can be natural and / or non-natural. As a non-limiting example, the nucleotide group can include a string of 5-8 adenines, cytosines, thymines, any of the other nucleotides disclosed herein, and / or combinations thereof.

[0200] 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 5-8 cytosine bases. In another example, the 5'UTR may be replaced by inserting 5-8 cytosine bases followed by 5-8 adenine bases.

[0201] In one embodiment, the primary construct may contain at least one substitution and / or insertion downstream of the transcription start site that can be recognized by RNA polymerase. As a non-limiting example, at least one substitution and / or insertion may occur downstream of the transcription start site by substituting at least one nucleic acid within the region immediately downstream of the transcription start site (such as, but not limited to, +1 to +6). Altering the nucleotide region immediately downstream of the transcription start site can affect the initiation rate, increase the apparent nucleotide triphosphate (NTP) reaction constant, and enhance the dissociation of short transcripts from the transcription complex by stiffening the initial transcript (Brieba et al., Biochemistry (2002) 41:5144-5149, incorporated herein by reference in its entirety). The modification, substitution, and / or insertion of at least one nucleic acid may result in a silent mutation in the nucleic acid sequence or in a mutation in the amino acid sequence.

[0202] In one embodiment, the primary construct may include substitutions 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.

[0203] In one embodiment, the primary construct may contain substitutions of at least one, at least two, at least three, at least four, at least five, or at least six guanine bases in the region immediately downstream of the transcription start site. As a non-limiting example, when the nucleotides in that region are GGGAGA, the guanine bases may be substituted with at least one, at least two, at least three, or at least four adenine nucleotides. In another non-limiting example, when the nucleotides in that region are GGGAGA, the guanine bases may be substituted with at least one, at least two, at least three, or at least four cytosine bases. In another non-limiting example, when the nucleotides in that region are GGGAGA, the guanine bases may be substituted with at least one, at least two, at least three, or at least four thymines and / or any of the nucleotides described herein.

[0204] In one embodiment, the primary construct may contain at least one substitution and / or insertion upstream of the start codon. For clarity, those skilled in the art will understand that the start codon is the first codon in a protein-coding region, while the transcription start site is the site where transcription begins. The primary construct may contain, but is not limited to, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, or at least eight substitutions and / or insertions of nucleotide bases. Nucleotide bases may be inserted or substituted at one, at least one, at least two, at least three, at least four, or at least five positions upstream of the start codon. The inserted and / or substituted nucleotides may be the same base (e.g., all A's or all C's or all T's or all G's), 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 can be replaced with adenine, cytosine, thymine, or any of the nucleotides described herein. In another non-limiting example, the guanine base replacement in the primary construct can be designed to leave one guanine base downstream of the transcription start site and in the region before the start codon (see Esvelt et al. Nature (2011) 472 (7344): 499-503, which is incorporated herein by reference in its entirety). As a non-limiting example, at least five nucleotides can be inserted into one position downstream of the transcription start site and upstream of the start codon, and at least five nucleotides can be of the same base type.

[0205] cDNA template removal and purification 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 purification may also include purification methods such as, but not limited to, the AGENCOURT® CLEANSEQ® 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).

[0206] Capping and / or tailing reactions The primary construct or mmRNA can also undergo capping and / or tailing reaction. The capping reaction can be carried out using a method 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, the use of vaccinia capping enzyme (New England Biolabs, Ipswich, MA).

[0207] The polyA tailing reaction can be performed using methods known in the art, such as, but not limited to, 2'O-methyltransferase and the methods described herein. If the primary construct generated from cDNA does not contain polyT, it may be beneficial to perform the polyA tailing reaction before the primary construct is purified.

[0208] mRNA purification Primary construct or mmRNA purification can include, but is not limited to, mRNA or mmRNA purification, quality assurance, and quality control. mRNA or mmRNA purification can be performed using methods known in the art, such as, but not limited to, AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA); poly-T beads; LNA™ Oligo-T capture probes (EXIQON® 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," as in "purified mRNA or mmRNA," when used in reference to a polynucleotide, refers to something that is separated from at least one contaminant. As used herein, a "contaminant" is any substance that renders another substance unsuitable, impure, or inferior. Thus, purified polynucleotides (e.g., DNA and RNA) are present in a form or environment that is different from that in which they are found in nature or that is different from the form or environment in which they existed prior to being subjected to a treatment or purification method.

[0209] Quality assurance and / or quality control testing may be performed using methods such as, but not limited to, gel electrophoresis, ultraviolet absorbance, or analytical HPLC.

[0210] In another embodiment, mRNA or mmRNA can be sequenced using methods including, but not limited to, reverse transcription PCR.

[0211] In one embodiment, mRNA or mmRNA can be quantified using methods such as, but not limited to, ultraviolet-visible spectroscopy (UV / Vis). A non-limiting example of a UV / Vis spectrometer is the NANODROP® spectrometer (ThermoFisher, Waltham, MA). Quantified mRNA or mmRNA can be analyzed to determine whether the mRNA or mmRNA may be of the appropriate size and to confirm that mRNA or mmRNA degradation has not occurred. Degradation of mRNA and / or mmRNA can be confirmed using 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).

[0212] signal sequence The primary construct or mmRNA may also encode additional features that facilitate transport of the polypeptide to a therapeutically relevant site. One such feature that aids in protein transport is a signal sequence. As used herein, a "signal sequence" or "signal peptide" is a polynucleotide or polypeptide, respectively, approximately 9 to 200 nucleotides in length (3 to 60 amino acids in length), that is incorporated into the 5' (or N-terminus) of a coding region or encoded polypeptide. The addition of these sequences results in transport of the encoded polypeptide through one or more secretory pathways to the endoplasmic reticulum. Some signal peptides are cleaved from the protein by a signal peptidase after the protein has been transported.

[0213] Table 5 is a representative list of protein signal sequences that can be incorporated to encode a polynucleotide, primary construct, or mmRNA of the present invention. Table 5. Signal sequences [Table 0005-1] [Table 0005-2] [Table 0005-3] [Table 0005-4] [Table 0005-5] [Table 0005-6]

[0214] In this table, SS is a secretion signal and MLS is a mitochondrial leader signal. Primary constructs or mmRNA of the invention can be designed to encode any of the signal sequences of SEQ ID NOs: 94-155, or fragments or variants thereof. These sequences can be included at the beginning, middle, or end of the polypeptide coding region, or alternatively, in flanking regions. Additionally, any of the polynucleotide primary constructs of the invention can also include one or more of the sequences defined by SEQ ID NOs: 32-93, which can be present in the first region or any flanking region.

[0215] Additional signal sequences that can be utilized in the present invention include, for example, those taught in databases such as those found at http: / / www.signalpeptide.de / or http: / / proline.bic.nus.edu.sg / spdb / . Also within the scope of the present invention are those described in U.S. Patent Nos. 8,124,379, 7,413,875, and 7,385,034, the contents of each of which are incorporated herein by reference in their entirety.

[0216] Target Selection In accordance with the present invention, a primary construct comprises at least one first region of linked nucleosides encoding at least one polypeptide of interest. Polypeptides of interest or "targets" of the present invention are listed in Table 6. In addition to the name and description of the gene encoding the polypeptide of interest, Table 6 also provides the ENSEMBL transcript sequence number (ENST), the ENSEMBL protein sequence number (ENSP), and, where available, the optimized transcript sequence number (OPtim Trans sequence number) or the optimized open reading frame sequence number (OptimORF sequence number). For any particular gene, one or more variants or isoforms may exist. If present, they are also listed in the table. Those skilled in the art will appreciate that the disclosed sequences are potential flanking regions. These are encoded either 5' (upstream) or 3' (downstream) of the ORF or coding region in each ENST transcript. Coding regions are categorically and specifically disclosed by teaching the ENSP sequence. Consequently, the adjacent sequences of the teachings that encode proteins are considered flanking regions. The 5' and 3' flanking regions may be further characterized by utilizing one or more available databases or algorithms. Databases annotating features contained in the flanking regions of ENST transcripts are available in the art. Table 6. Target [Table 0006-1] [Table 0006-2] [Table 0006-3] [Table 0006-4]

Table 0006-5

Table 0006-6

Table 0006-7

Table 0006-8

Table 0006-9

Table 0006-10

Table 0006-11

Table 0006-12

Table 0006-13

Table 0006-14

Table 0006-15

Table 0006-16

Table 0006-17

Table 0006-18

Table 0006-19

Table 0006-20

Table 0006-21

Table 0006-22

Table 0006-23

Table 0006-24

Table 0006-25

Table 0006-26

Table 0006-27

Table 0006-28

Table 0006-29

Table 0006-30

Table 0006-31

Table 0006-32

Table 0006-33

Table 0006-34

Table 0006-35

Table 0006-36

Table 0006-37

Table 0006-38

Table 0006-39

Table 0006-40

Table 0006-41

Table 0006-42

Table 0006-43

Table 0006-44

Table 0006-45

Table 0006-46

Table 0006-47

Table 0006-48

Table 0006-49

Table 0006-50

Table 0006-51

Table 0006-52

Table 0006-53

Table 0006-54

Table 0006-55

Table 0006-56

Table 0006-57

Table 0006-58

[0217] Protein cleavage signals and sites In one embodiment, the polypeptide of the present invention may comprise at least one proteolytic cleavage signal containing at least one proteolytic cleavage site, which may be located at the N-terminus, the C-terminus, in any space between the N-terminus and the C-terminus, including, but not limited to, in the middle of the N-terminus and the C-terminus, between the N-terminus and the midpoint, between the midpoint and the C-terminus, and combinations thereof.

[0218] Polypeptides of the present invention may include, but are not limited to, proprotein convertase (or prohormone convertase), thrombin, or factor Xa protein cleavage signals. Proprotein convertases are a family of nine proteinases, including 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, termed subtilisin kexin isozyme 1 (SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9). Non-limiting examples of protein cleavage signal amino acid sequences are listed in Table 7. In Table 7, "X" refers to any amino acid, "n" can be 0, 2, 4, or 6 amino acids, and "n" can be 0, 2, 4, or 6 amino acids. * " refers to the proteolytic cleavage site. In Table 7, SEQ ID NO: 21426 refers to when n is 4, and SEQ ID NO: 21427 refers to when n is 6. Table 7. Protein cleavage site sequences [Table 0007]

[0219] In one embodiment, the primary constructs and mmRNA of the present invention can be engineered such that the primary construct or mmRNA contains at least one encoded protein cleavage signal. The encoded protein cleavage signal can be located before the start codon, after the start codon, before the coding region, within the coding region, for example, but not limited to, in the middle of the coding region, between the start codon and the midpoint, between the midpoint and the stop codon, after the coding region, after the stop codon, between two stop codons, after the stop codon, and combinations thereof.

[0220] In one embodiment, a primary construct or mmRNA of the present invention may contain 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 can use Table 1 above or other known methods to determine an appropriate encoded protein cleavage signal to include in a primary construct or mmRNA of the present invention. For example, starting with a signal in Table 7, one can design a signal in a primary construct that can generate a protein signal in the resulting polypeptide, taking into account the codons in Table 1.

[0221] In one embodiment, the polypeptide of the invention comprises at least one protein cleavage signal and / or site.

[0222] As a non-limiting example, U.S. Patent No. 7,374,930 and U.S. Patent Publication No. 20090227660, which are 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 cell. In one embodiment, a polypeptide of the invention comprises at least one protein cleavage signal and / or site, provided that the polypeptide is not GLP-1.

[0223] In one embodiment, the primary construct or mmRNA of the invention comprises at least one encoded protein cleavage signal and / or site.

[0224] In one embodiment, the primary construct or mmRNA of the present invention comprises at least one encoded protein cleavage signal and / or site, provided that the primary construct or mmRNA does not encode GLP-1.

[0225] In one embodiment, a primary construct or mmRNA of the present invention may contain two or more coding regions. When multiple coding regions are present in a 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, a primary construct or mmRNA may be written in an ordered pattern. Such a pattern follows the format AXBY, where A and B are coding regions that may be the same or different and / or may encode the same or different polypeptides, and X and Y are encoded protein cleavage signals that may encode the same or different protein cleavage signals. A second such pattern follows the format AXYBZ, where A and B are coding regions that may be the same or different and / or may encode the same or different polypeptides, and X, Y, and Z are encoded protein cleavage signals that may encode the same or different protein cleavage signals. The third pattern follows the ABXCY format, where A, B, and C are coding regions that 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 that may encode the same or different protein cleavage signals.

[0226] In one embodiment, the polypeptides, primary constructs and mmRNA may also contain sequences encoding the aforementioned protein cleavage sites, such that the polypeptides, primary constructs and mmRNA can be released from the carrier domain or fusion partner by treatment with a protease specific for the protein cleavage site.

[0227] In one embodiment, the polypeptides, primary constructs, and mmRNA of the present invention may contain a sequence encoding a 2A peptide. In one embodiment, this sequence can be used to separate the coding regions for two or more polypeptides of interest. As a non-limiting example, a sequence encoding a 2A peptide can be present between coding region A and coding region B (A-2Apep-B). The presence of the 2A peptide results in cleavage of a single long protein into protein A, protein B, and the 2A peptide. Protein A and protein B can be the same or different polypeptides of interest. In another embodiment, the 2A peptide can 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.

[0228] Incorporation of post-transcriptional regulatory factors In one embodiment, the polynucleotides, primary constructs, and / or mmRNA of the present invention may comprise at least one post-transcriptional regulatory factor. These post-transcriptional regulatory factors may be, but are not limited to, small molecules, chemical compounds, and regulatory sequences. As a non-limiting example, post-transcriptional regulation may be achieved using small molecules identified by PTC Therapeutics Inc. (South Plainfield, NJ) using GEMS™ (Gene Expression Modulation by Small-Molecules) screening technology.

[0229] The post-transcriptional regulatory factor may be a gene expression regulatory factor screened by or described in the method detailed in International Publication No. WO2006022712, which is incorporated herein by reference in its entirety. A method for identifying RNA regulatory sequences involved in translational control is described in International Publication No. WO2004067728, which is incorporated herein by reference in its entirety, and a method for identifying compounds that regulate the untranslated region-dependent expression of genes is described in International Publication No. WO2004065561, which is incorporated herein by reference in its entirety.

[0230] In one embodiment, the polynucleotides, primary constructs, and / or mmRNA of the present invention may comprise at least one post-transcriptional regulatory element located in the 5' and / or 3' untranslated region of the polynucleotides, primary constructs, and / or mmRNA of the present invention.

[0231] In another embodiment, the polynucleotide, primary construct, and / or mmRNA of the present invention can contain at least one post-transcriptional regulatory factor to regulate premature translation termination.The post-transcriptional regulatory factor can be a compound described in International Publication Nos. WO2004010106, WO2006044456, WO2006044682, WO2006044503, and WO2006044505, each of which is incorporated herein by reference in its entirety, or a compound found by the methods outlined therein.As a non-limiting example, the compound can bind to a region of 28S ribosomal RNA to regulate premature translation termination (see, for example, International Publication No. WO2004010106, which is incorporated herein by reference in its entirety).

[0232] In one embodiment, the polynucleotides, primary constructs, and / or mmRNA of the present invention may contain at least one post-transcriptional regulatory element to alter protein expression. As a non-limiting example, VEGF expression may be controlled using compounds described in International Publication Nos. WO2005118857, WO2006065480, WO2006065479, and WO2006058088, each of which is incorporated herein by reference in its entirety, or compounds that can be found by the methods described therein.

[0233] The polynucleotide, primary construct, and / or mmRNA of the present invention can comprise at least one post-transcriptional regulatory element to control translation.In one embodiment, the post-transcriptional regulatory element can be an RNA regulatory sequence.As a non-limiting example, the RNA regulatory sequence can be identified by the method described in International Publication No. WO2006071903, the entirety of which is incorporated herein by reference.

[0234] III. Qualification In the context of polynucleotides (such as primary constructs or mRNA molecules) herein, the term "modification" or, where appropriate, "modified" refers to modifications to A, G, U, or C ribonucleotides. Generally, as used herein, these terms are not intended to refer to ribonucleotide modifications in the naturally occurring 5'-terminal mRNA cap moiety. In the context of polypeptides, the term "modification" refers to a modification compared to a reference set of 20 amino acids (moieties).

[0235] The modifications can be a variety of distinct modifications. In some embodiments, the coding region, the flanking region, and / or the terminal region can contain one, two, or more (optionally different) nucleoside or nucleotide modifications. In some embodiments, the modified polynucleotide, primary construct, or mmRNA introduced into a cell can exhibit reduced degradation in the cell compared to the unmodified polynucleotide, primary construct, or mmRNA.

[0236] Polynucleotides, primary constructs, and mRNAs can include any useful modifications to the sugar, nucleobase, or internucleoside linkage (e.g., to the phosphate / phosphodiester linkage / phosphodiester backbone). One or more atoms of a pyrimidine nucleobase can be replaced with or substituted with an optionally substituted amino, an optionally substituted thiol, an optionally substituted alkyl (e.g., methyl or ethyl), or a halo (e.g., chloro or fluoro). In certain embodiments, modifications (e.g., one or more modifications) are present in each of the sugar and internucleoside linkage. Modifications according to the present invention can be modifications of ribonucleic acid (RNA) relative to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof. Additional modifications are described herein.

[0237] As described herein, the polynucleotides, primary constructs, and mmRNA of the present invention do not substantially induce an innate immune response in cells into which the mRNA is introduced. Characteristics of an induced innate immune response include: 1) increased expression of proinflammatory cytokines, 2) activation of intracellular PRRs (e.g., RIG-I, MDA5), and / or 3) termination or reduction of protein translation.

[0238] In certain embodiments, it may be desirable to degrade modified nucleic acid molecules introduced into cells within the cell. For example, degradation of modified nucleic acid molecules may be preferable when precise timing of protein production is desired. Thus, in some embodiments, the present invention provides modified nucleic acid molecules that contain degradation domains that can be acted on within cells in a directed manner. In another aspect, the present disclosure provides polynucleotides that include nucleosides or nucleotides that can disrupt the binding of major groove interaction (e.g., binding) partners with polynucleotides (e.g., when modified nucleotides have reduced binding affinity for major groove interaction partners compared to unmodified nucleotides).

[0239] Polynucleotides, primary constructs, and mmRNAs may optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, tRNA, RNA that induces triple helix formation, aptamers, vectors, etc.). In some embodiments, polynucleotides, primary constructs, or mmRNAs may include one or more messenger RNAs (mRNAs) and one or more modified nucleosides or nucleotides (e.g., mmRNA molecules). More details about these polynucleotides, primary constructs, and mmRNAs follow below.

[0240] Polynucleotides and primary constructs The polynucleotides, primary constructs, and mmRNA of the present invention comprise a first region of linked nucleosides encoding a polypeptide of interest, a first flanking region located at the 5' end of the first region, and a second flanking region located at the 3' end of the first region.

[0241] In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) has the structure of Formula (Ia) or Formula (Ia-1): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0242] During the ceremony,

[0243] 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;

[0244] --- is a single bond or is absent,

[0245] R 1’ , R 2’ , R 1” , R 2” , R 1 , R 2 , R 3 , R 4 , and R 5 each, when present, 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 R3 in combination with one or more of R1', R1", R2', R2", or R5 (e.g., a combination of R1' and R3, a combination of R1" and R3, a combination of R2' and R3, a combination of R2" and R3, or The combination of R and R) together can form an optionally substituted alkylene or an optionally substituted heteroalkylene and, together with the carbons to which they are attached, can provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); the combination of R and one or more of R, R", R, or R (e.g., a combination of R and R, a combination of R and R, a combination of R and R, or a combination of R and R) can together form an optionally substituted alkylene or an optionally substituted heteroalkylene and, together with the carbons to which they are attached, can provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl); R 4 and R 1’ , R 1” , R 2’ , R 2” , R 3 , or R 5may be combined together to form an optionally substituted alkylene or an optionally substituted heteroalkylene, and together with the carbons to which they are attached, provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl), wherein each of m' and m" is independently an integer from 0 to 3 (e.g., 0 to 2, 0 to 1, 1 to 3, or 1 to 2);

[0246] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -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;

[0247] Each Y 4 are 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;

[0248] Each Y 5 is independently O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;

[0249] n is an integer from 1 to 100,000,

[0250] B is a nucleobase (e.g., a purine, a pyrimidine, or a derivative thereof), and B and R 1’ Combination of B and R 2’ Combination of B and R 1” combination of B and R 2”together with the carbons to which they are attached can optionally form a bicyclic group (e.g., a bicyclic heterocyclyl), or B, R 1” , and R 3 A combination of B and R 2” , and R 3 may optionally form a tricyclic or tetracyclic group (e.g., a tricyclic or tetracyclic heterocyclyl such as those of formula (IIo)-(IIp) herein). In some embodiments, the polynucleotide, primary construct, or mmRNA comprises a modified ribose. In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, first flanking region, or second flanking region) comprises n linked nucleosides having formula (Ia-2) through (Ia-5), or a pharmaceutically acceptable salt or stereoisomer thereof. [ka]

[0251] In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) has the structure of Formula (Ib) or Formula (Ib-1): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0252] During the ceremony,

[0253] 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;

[0254] --- is a single bond or is absent,

[0255] R 1 , R 3’ , R 3” , and R 4 each 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 R 1 and R 3’ combination or R 1 and R 3” can be taken together to form an optionally substituted alkylene or an optionally substituted heteroalkylene (e.g., to produce a locked nucleic acid),

[0256] Each R 5 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;

[0257] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -NR N1 -, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;

[0258] Each Y 4are 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;

[0259] n is an integer from 1 to 100,000,

[0260] B is a nucleobase.

[0261] In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) has the formula (Ic): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0262] During the ceremony,

[0263] 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;

[0264] --- is a single bond or is absent,

[0265] B 1 , B 2 , and B 3each is independently a nucleobase (e.g., a purine, pyrimidine, or derivative thereof 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; and B 1 , B 2 , and B 3 only one of the is a nucleobase,

[0266] R b1 , R b2 , R b3 , R 3 , and R 5 each 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;

[0267] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -NR N1 -, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;

[0268] Each Y 4are 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;

[0269] Each Y 5 is independently O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;

[0270] n is an integer from 1 to 100,000,

[0271] The ring containing U may contain one or more double bonds.

[0272] In certain embodiments, the ring containing U is U-CB 3 R b3 Between or CB 3 R b3 -C B2 R b2 There is no double bond between them.

[0273] In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) has the formula (Id): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0274] During the ceremony,

[0275] 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 Uis independently H, halo, or optionally substituted alkyl;

[0276] Each R 3 are 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;

[0277] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -NR N1 -, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;

[0278] Each Y 4 are 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;

[0279] Each Y 5 is independently O, S, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;

[0280] n is an integer from 1 to 100,000,

[0281] B is a nucleobase (eg, a purine, pyrimidine, or derivative thereof).

[0282] In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) has the formula (Ie): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0283] During the ceremony,

[0284] Each of U' and U" is independently 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;

[0285] Each R 6 are 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;

[0286] Each Y 5’ is independently O, S, optionally substituted alkylene (e.g., methylene or ethylene), or optionally substituted heteroalkylene;

[0287] n is an integer from 1 to 100,000,

[0288] B is a nucleobase (eg, a purine, pyrimidine, or derivative thereof).

[0289] In some embodiments, the polynucleotide, primary construct, or mmRNA (e.g., the first region, the first flanking region, or the second flanking region) has a structure represented by formula (If) or (If-1): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0290] During the ceremony,

[0291] Each of U' and U" is independently O, S, N, N(R U ) nu , or C(R U ) nu wherein nu is an integer from 0 to 2, and each R U are independently H, halo, or optionally substituted alkyl (e.g., U' is O and U" is N);

[0292] --- is a single bond or is absent,

[0293] R 1’ , R 2’ , R 1” , R 2” , R 3 , and R 4 each 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 R 1’ and R 3Combination of R 1” and R 3 Combination of R 2’ and R 3 combination, or R 2” and R 3 can be taken together to form an optionally substituted alkylene or an optionally substituted heteroalkylene (e.g., to produce a locked nucleic acid), and each of m' and m" is independently an integer from 0 to 3 (e.g., 0 to 2, 0 to 1, 1 to 3, or 1 to 2);

[0294] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -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;

[0295] Each Y 4 are 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;

[0296] Each Y 5 is independently O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;

[0297] n is an integer from 1 to 100,000,

[0298] B is a nucleobase (eg, a purine, pyrimidine, or derivative thereof).

[0299] In some embodiments of the polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia), (Ia-1) through (Ia-3), (Ib) through (If), and (IIa) through (IIp)), the ring containing U has one or two double bonds.

[0300] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), R 1 , R 1’ , and R 1” Each of, when present, is H. In a further embodiment, R 2 , R 2’ , and R 2” Each of, when present, is independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In certain embodiments, alkoxyalkoxy is —(CH) s2 (OCH2CH2) s1 (CH2) s3 OR', wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R' is C 1~6 It is alkyl.

[0301] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), R2 , R 2’ , and R 2” Each of, when present, is H. In a further embodiment, R 1 , R 1’ , and R 1” Each of, when present, is independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In certain embodiments, alkoxyalkoxy is —(CH) s2 (OCH2CH2) s1 (CH2) s3 OR', wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R' is C 1~6 It is alkyl.

[0302] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), R 3 , R 4 , and R 5 Each of R is independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In certain embodiments, R 3 is H and R 4 is H and R 5 is H or R 3 , R 4 , and R 5 are all H. In certain embodiments, R 3 C1~6 alkyl, and R 4 C 1~6 alkyl, and R 5 C 1~6 alkyl or R 3 , R 4 , and R 5 All of them are C 1~6 In certain embodiments, R 3 MoR 4 are both H, and R 5 is C 1~6 It is alkyl.

[0303] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), R 3 and R 5 taken together form an optionally substituted alkylene or an optionally substituted heteroalkylene, and together with the carbons to which they are attached provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl), such as the trans-3',4' analogs, and R 3 and R 5 together form a heteroalkylene (e.g., -(CH2) b1 O(CH2) b2 O(CH2) b3 wherein each of b1, b2, and b3 is independently an integer of 0 to 3.

[0304] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), R 3 and R 1’ , R1” , R 2’ , R 2” , or R 5 taken together form an optionally substituted alkylene or an optionally substituted heteroalkylene, and together with the carbons to which they are attached provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl), and R 3 and R 1’ , R 1” , R 2’ , R 2” , or R 5 taken together form a heteroalkylene (e.g., -(CH2) b1 O(CH2) b2 O(CH2) b3 wherein each of b1, b2, and b3 is independently an integer of 0 to 3.

[0305] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), R 5 and R 1’ , R 1” , R 2’ , or R 2” taken together form an optionally substituted alkylene or an optionally substituted heteroalkylene, and together with the carbons to which they are attached provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl), and R 5 and R 1’ , R 1” , R 2’ , or R 2” taken together form a heteroalkylene (e.g., -(CH2) b1 O(CH2) b2 O(CH2) b3wherein each of b1, b2, and b3 is independently an integer of 0 to 3.

[0306] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), each Y 2 are independently O, S, or -NR N1 -, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl. In certain embodiments, Y 2 is NR N1 -, wherein R N1 is H or optionally substituted alkyl (e.g., C 1~6 alkyl, for example, methyl, ethyl, isopropyl, or n-propyl).

[0307] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), each Y 3 are independently O or S.

[0308] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), R 1 is H, and each R 2are independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g., —(CH) s2 (OCH2CH2) s1 (CH2) s3 OR', wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl, for example, in which s2 is 0, s1 is 1 or 2, and s3 is 0 or 1 (e.g., R' is C 1~6 alkyl), and each Y 2 are independently -O or -NR N1 -, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., in formula R N1 is H or optionally substituted alkyl (e.g., C 1~6 alkyl, for example, methyl, ethyl, isopropyl, or n-propyl), and each Y 3 is independently O or S (e.g., S). In a further embodiment, R 3 is H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In still further embodiments, each Y 1 are independently -O or -NR N1 -, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., in formula R N1 is H or optionally substituted alkyl (e.g., C 1~6 alkyl, for example, methyl, ethyl, isopropyl, or n-propyl), and each Y 4are independently H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.

[0309] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia) through (Ia-5), (Ib) through (If-1), (IIa) through (IIp), (IIb-1), (IIb-2), (IIc-1) through (IIc-2), (IIn-1), (IIn-2), (IVa) through (IVl), and (IXa) through (IXr)), each R 1 are independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g., —(CH) s2 (OCH2CH2) s1 (CH2) s3 OR', wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl (e.g., where s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R' is C 1~6 alkyl), R 2 is H, and each Y 2 are independently -O or -NR N1 -, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., in formula R N1 is H or optionally substituted alkyl (e.g., C 1~6 alkyl, for example, methyl, ethyl, isopropyl, or n-propyl), and each Y 3 is independently O or S (e.g., S). In a further embodiment, R 3is H, halo (e.g., fluoro), hydroxy, optionally substituted alkyl, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In still further embodiments, each Y 1 are independently -O or -NR N1 -, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl (e.g., in formula R N1 is H or optionally substituted alkyl (e.g., C 1~6 alkyl, for example, methyl, ethyl, isopropyl, or n-propyl), and each Y 4 are independently H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.

[0310] In some embodiments of the polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), the ring containing the U is in the β-D (e.g., β-D-ribo) configuration.

[0311] In some embodiments of the polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), the ring containing the U is in the α-L (e.g., α-L-ribo) configuration.

[0312] In some embodiments of the polynucleotides, primary constructs, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), one or more B is pseudouridine (ψ) or 5-methyl-cytidine (m 5 In some embodiments, about 10% to about 100% of the n B nucleobases are neither ψ nor m 5 Neither C (for example, 10% to 20%, 10% to 35%, 10% to 50%, 10% to 60%, 10% to 75%, 10% to 90%, 10% to 95%, 10% to 98%, 10% to 99%, 20% to 35%, 20% to 50%, 20% to 60%, 20% to 75%, 20% to 90%, 20% to 95% of n Bs) %, 20%~98%, 20%~99%, 20%~100%, 50%~60%, 50%~75%, 50%~90%, 50%~95%, 50%~98%, 50%~99%, 50%~100%, 75%~90%, 75%~95%, 75%~98%, 75%~99%, and 75%~100% are also m 5 C). In some embodiments, B is neither ψ nor m 5 It's not C either.

[0313] In some embodiments of the polynucleotide, primary construct, or mmRNA (e.g., Formulas (Ia)-(Ia-5), (Ib)-(If-1), (IIa)-(IIp), (IIb-1), (IIb-2), (IIc-1)-(IIc-2), (IIn-1), (IIn-2), (IVa)-(IVl), and (IXa)-(IXr)), when B is an unmodified nucleobase selected from cytosine, guanine, uracil, and adenine, Y 1 , Y 2 , or Y 3 At least one of them is not O.

[0314] In some embodiments, the polynucleotide, primary construct, or mmRNA comprises 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) has a structure represented by Formula (IIa)-(IIc): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof. In certain embodiments, U is O 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 (e.g., U is -CH- or -CH-). In other embodiments, R 1 , R 2 , R 3 , R 4 , and R 5 each 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 R 1 and R 2 are independently H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, and each R 3 and R 4 are independently H or optionally substituted alkyl, and R 5 is H or hydroxy), and --- is a single or double bond.

[0315] In certain embodiments, the polynucleotide or mRNA has the formula (IIb-1) to (IIb-2): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, U is O 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 (e.g., U is -CH- or -CH-). In other embodiments, R 1 and R 2 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 R 1 and R 2 are independently H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, e.g., H, halo, hydroxy, alkyl, or alkoxy). In certain embodiments, R 2 is hydroxy or optionally substituted alkoxy (e.g., methoxy, ethoxy, or any described herein).

[0316] In certain embodiments, the polynucleotide, primary construct, or mmRNA has the formula (IIc-1) to (IIc-4): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, U is O 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 (e.g., U is -CH- or -CH-). In some embodiments, R 1 , R 2 , and R 3 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 R 1 and R 2 is independently H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, e.g., H, halo, hydroxy, alkyl, or alkoxy, and each R 3 are independently H or optionally substituted alkyl). In certain embodiments, R 2 is an optionally substituted alkoxy (e.g., methoxy or ethoxy, or any described herein). In certain embodiments, R 1 is optionally substituted alkyl, and R 2 is hydroxy. In other embodiments, R 1 is hydroxy and R 2 is optionally substituted alkyl. In a further embodiment, R 3 is optionally substituted alkyl.

[0317] In some embodiments, the polynucleotide, primary construct, or mmRNA comprises 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) has a structure represented by Formula (IId)-(IIf): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0318] In some embodiments, the polynucleotide, primary construct, or mmRNA comprises 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) has a structure represented by Formula (IIg)-(IIj): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof.

[0319] In some embodiments, the polynucleotide, primary construct, or mmRNA comprises a sugar moiety with a shortened or extended 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) has the formula (IIk)~(IIm): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 1’ , R 1” , R 2’ , and R 2” each 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 R 2’ and R 3 combination or R 2” and R 3may be taken together to form an optionally substituted alkylene or an optionally substituted heteroalkylene.

[0320] In some embodiments, the polynucleotide, primary construct, or mmRNA comprises 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) has a structure represented by Formula (IIn): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 3’ is O, S, or -NR N1 -, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl, and R 3” is an optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-) or an optionally substituted heteroalkylene (e.g., -CH2NH-, -CH2CH2NH-, -CH2OCH2-, or -CH2CH2OCH2-) (e.g., R 3’ is O and R 3” is optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-).

[0321] In some embodiments, the polynucleotide, primary construct, or mmRNA has the formula (IIn-1) to (II-n2): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 3’ is O, S, or -NR N1 -, wherein R N1is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl, and R 3” is an optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-) or an optionally substituted heteroalkylene (e.g., -CH2NH-, -CH2CH2NH-, -CH2OCH2-, or -CH2CH2OCH2-) (e.g., R 3’ is O and R 3” is optionally substituted alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-).

[0322] In some embodiments, the polynucleotide, primary construct, or mmRNA comprises 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) has a structure represented by Formula (IIo): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein R 12a , R 12c , T 1’ , T 1” , T 2’ , T 2” , V 1 , and V 3 is as described herein.

[0323] Any of the polynucleotide, primary construct, or mmRNA formulas may include one or more of the nucleobases described herein (eg, formulas (b1)-(b43)).

[0324] In one embodiment, the present invention provides a method for preparing a polynucleotide, primary construct, or mmRNA, wherein the polynucleotide has formula (Ia) as defined herein: [ka] and the method comprises n nucleosides having formula (IIIa) as defined herein: [ka] with an RNA polymerase and a cDNA template.

[0325] In a further embodiment, the present invention provides a method for amplifying a polynucleotide, primary construct, or mmRNA comprising at least one nucleotide (e.g., an mmRNA molecule), the method comprising reacting a compound of formula (IIIa) as defined herein with a primer, a cDNA template, and an RNA polymerase.

[0326] In one embodiment, the present invention provides a method for preparing a polynucleotide, primary construct, or mmRNA comprising at least one nucleotide (e.g., an mmRNA molecule), the polynucleotide having a structure represented by formula (Ia), as defined herein: [ka] and the method comprises n nucleosides having formula (IIIa-1) as defined herein: [ka] with an RNA polymerase and a cDNA template.

[0327] In a further embodiment, the present invention provides a method for amplifying a polynucleotide, primary construct, or mmRNA comprising at least one nucleotide (e.g., an mmRNA molecule), the method comprising:

[0328] It involves reacting a compound of formula (IIIa-1) as defined herein with a primer, a cDNA template, and an RNA polymerase.

[0329] In one embodiment, the present invention provides a method for preparing a modified mRNA (e.g., an mmRNA molecule) comprising at least one nucleotide, the polynucleotide having the formula (Ia-2) as defined herein: [ka] and the method comprises n nucleosides having formula (IIIa-2): [ka] with an RNA polymerase and a cDNA template.

[0330] In a further embodiment, the present invention provides a method for amplifying modified mRNA comprising at least one nucleotide (e.g., an mRNA molecule), the method comprising:

[0331] It involves reacting a compound of formula (IIIa-2) as defined herein with a primer, a cDNA template, and an RNA polymerase.

[0332] In some embodiments, the reaction can be repeated from 1 to about 7,000 times.In any of the embodiments herein, B can be a nucleobase of formula (b1)-(b43).

[0333] The polynucleotides, primary constructs, and mmRNA may optionally include 5' and / or 3' flanking regions as described herein.

[0334] Modified RNA (mRNA) molecules The present invention also includes building blocks of modified RNA (mmRNA) molecules, such as modified ribonucleosides and modified ribonucleotides. For example, these building blocks can be useful in preparing polynucleotides, primary constructs, or mmRNA of the present invention. In some embodiments, the building block molecules have the formula (IIIa) or (IIIa-1): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein the substituents are as described herein (e.g., Formulas (Ia) and (Ia-1)), and when B is an unmodified nucleobase selected from cytosine, guanine, uracil, and adenine, then Y 1 , Y 2 , or Y 3 At least one of them is not O.

[0335] In some embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA has the formula (IVa)-(IVb): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In certain embodiments, Formula (IVa) or (IVb) is combined with a modified uracil (e.g., any one of Formulas (b1)-(b9), (b21)-(b23), and (b28)-(b31), e.g., Formula (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, Formula (IVa) or (IVb) is combined with a modified cytosine (e.g., any one of Formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), e.g., Formula (b10) or (b32)). In certain embodiments, Formula (IVa) or (IVb) is combined with a modified guanine (e.g., any one of Formulas (b15)-(b17) and (b37)-(b40)). In certain embodiments, Formula (IVa) or (IVb) is combined with a modified adenine (e.g., any one of Formulas (b18)-(b20) and (b41)-(b43)).

[0336] In some embodiments, the building block molecules that can be incorporated into polynucleotides, primary constructs, or mmRNA have the formula (IVc)-(IVk): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In certain 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), e.g., Formula (b1), (b8), (b28), (b29), or (b30)). In certain 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), e.g., Formula (b10) or (b32)). In certain embodiments, one of Formulas (IVc)-(IVk) is combined with a modified guanine (e.g., any one of Formulas (b15)-(b17) and (b37)-(b40)). In certain embodiments, one of Formulas (IVc)-(IVk) is combined with a modified adenine (e.g., any one of Formulas (b18)-(b20) and (b41)-(b43)).

[0337] In other embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA has the formula (Va) or (Vb): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1) to (b43)).

[0338] In other embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA has the formula (IXa)-(IXd): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In certain 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), e.g., Formula (b1), (b8), (b28), (b29), or (b30)). In certain 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), e.g., Formula (b10) or (b32)). In certain embodiments, one of formulas (IXa)-(IXd) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In certain embodiments, one of formulas (IXa)-(IXd) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).

[0339] In other embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA has the formula (IXe)-(IXg): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In certain 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), e.g., Formula (b1), (b8), (b28), (b29), or (b30)). In certain 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), e.g., Formula (b10) or (b32)). In certain embodiments, one of Formulas (IXe)-(IXg) is combined with a modified guanine (e.g., any one of Formulas (b15)-(b17) and (b37)-(b40)). In certain embodiments, one of Formulas (IXe)-(IXg) is combined with a modified adenine (e.g., any one of Formulas (b18)-(b20) and (b41)-(b43)).

[0340] In other embodiments, the building block molecules that can be incorporated into polynucleotides, primary constructs, or mmRNA are represented by formulas (IXh)-(IXk): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)). In certain 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), e.g., Formula (b1), (b8), (b28), (b29), or (b30)). In certain 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), e.g., Formula (b10) or (b32)). In certain embodiments, one of formulas (IXh)-(IXk) is combined with a modified guanine (e.g., any one of formulas (b15)-(b17) and (b37)-(b40)). In certain embodiments, one of formulas (IXh)-(IXk) is combined with a modified adenine (e.g., any one of formulas (b18)-(b20) and (b41)-(b43)).

[0341] In other embodiments, the building block molecules that can be incorporated into polynucleotides, primary constructs, or mmRNA have the formula (IXl)-(IXr): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r1 and r2 is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5), and B is as described herein (e.g., any one of (b1) to (b43)). In certain embodiments, one of Formulas (IXl) to (IXr) is combined with a modified uracil (e.g., any one of Formulas (b1) to (b9), (b21) to (b23), and (b28) to (b31), e.g., Formula (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of Formulas (IXl)-(IXr) is combined with a modified cytosine (e.g., any one of Formulas (b10)-(b14), (b24), (b25), and (b32)-(b36), e.g., Formula (b10) or (b32)). In certain embodiments, one of Formulas (IXl)-(IXr) is combined with a modified guanine (e.g., any one of Formulas (b15)-(b17) and (b37)-(b40)). In certain embodiments, one of Formulas (IXl)-(IXr) is combined with a modified adenine (e.g., any one of Formulas (b18)-(b20) and (b41)-(b43)).

[0342] In some embodiments, the building block molecules that can be incorporated into the polynucleotide, primary construct, or mmRNA are: [ka] TIFF2025169942000109.tif138170 or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0343] In some embodiments, the building block molecules that can be incorporated into the polynucleotide, primary construct, or mmRNA are: [ka] 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 s1 is as described herein.

[0344] In some embodiments, a building block molecule that can be incorporated into a nucleic acid (e.g., an RNA, mRNA, polynucleotide, primary construct, or mmRNA) is a modified uridine (e.g., selected from the group consisting of: or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y 1 , Y 3 , Y 4 , Y 6 and r is as described herein (e.g., 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): [ka] TIFF2025169942000112.tif231170 TIFF2025169942000113.tif209170 TIFF2025169942000114.tif226152 TIFF2025169942000115.tif214170 TIFF2025169942000116.tif240170 TIFF2025169942000117.tif214170 TIFF2025169942000118.tif214170 TIFF2025169942000119.tif251170 TIFF2025169942000120.tif251170 TIFF2025169942000121.tif209170 TIFF2025169942000122.tif209170 TIFF2025169942000123.tif209170 TIFF2025169942000124.tif209170 TIFF2025169942000125.tif199170 TIFF2025169942000126.tif212170 TIFF2025169942000127.tif210170

[0345] In some embodiments, a building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA is a modified cytidine (e.g., selected from the group consisting of: or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y 1 , Y 3 , Y 4 , Y 6 and r is as described herein (e.g., 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): [ka] TIFF2025169942000129.tif229170 TIFF2025169942000130.tif215170 TIFF2025169942000131.tif215170 TIFF2025169942000132.tif215170 TIFF2025169942000133.tif143170 For example, building block molecules that can be incorporated into polynucleotides, primary constructs, or mmRNA include: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0346] In some embodiments, a building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA is a modified adenosine (e.g., selected from the group consisting of: or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y 1 , Y 3 , Y 4 , Y 6 and r is as described herein (e.g., 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): [ka] TIFF2025169942000136.tif221169 TIFF2025169942000137.tif250169 TIFF2025169942000138.tif241168 TIFF2025169942000139.tif250168 TIFF2025169942000140.tif111167

[0347] In some embodiments, a building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA is a modified guanosine (e.g., selected from the group consisting of: or a pharmaceutically acceptable salt or stereoisomer thereof, wherein Y 1 , Y 3 , Y 4 , Y 6 and r is as described herein (e.g., 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): [ka] TIFF2025169942000142.tif249167 TIFF2025169942000143.tif246170 TIFF2025169942000144.tif213170 TIFF2025169942000145.tif83170

[0348] In some embodiments, the chemical modification is a substitution of a C group at C-5 of the ring (e.g., of a pyrimidine nucleoside, e.g., cytosine or uracil) with an N (e.g., a >CH group at C-5 with an >NR N1 Substitution with a group (wherein R N 1 is H or optionally substituted alkyl). For example, a building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA can include: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0349] In another embodiment, the chemical modification can include replacing 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, building block molecules that can be incorporated into polynucleotides, primary constructs, or mmRNA include: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0350] In yet further embodiments, the chemical modification may include a fused ring formed by NH2 at the C-4 position and a carbon atom at the C-5 position. For example, a building block molecule that may be incorporated into a polynucleotide, primary construct, or mmRNA may be: [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0351] Sugar modifications Modified nucleosides and nucleotides (e.g., building block molecules) that can be incorporated into polynucleotides, primary constructs, or mmRNA (e.g., RNA or mRNA described herein) can be modified in the sugar of the ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or replaced with several different substituents. Exemplary substitutions at the 2' position include H, halo, optionally substituted C, hydroxyl ... 1~6 Alkyl, optionally substituted C 1~6 Alkoxy, optionally substituted C 6~10 Aryloxy, optionally substituted C 3~8 Cycloalkyl, optionally substituted C 3~8 Cycloalkoxy, optionally substituted C 6~10 Aryloxy, optionally substituted C 6~10 Aryl-C 1~6 Alkoxy, optionally substituted C 1~12 (heterocyclyl)oxy, sugar (e.g., ribose, pentose, or any described herein), polyethylene glycol (PEG), -O(CH2CHO) n CH2CH2OR, where R is H or optionally substituted alkyl and n is an integer from 0 to 20 (e.g., 0 to 4, 0 to 8, 0 to 10, 0 to 16, 1 to 4, 1 to 8, 1 to 10, 1 to 16, 1 to 20, 2 to 4, 2 to 8, 2 to 10, 2 to 16, 2 to 20, 4 to 8, 4 to 10, 4 to 16, and 4 to 20), and the 2'-hydroxyl is C 1~6 Alkylene or C1~6 "Locked" nucleic acids (LNAs) linked to the 4'-carbon of the same ribose sugar by a heteroalkylene bridge (exemplary bridges include methylene, propylene, ether, or amino bridges), include, but are not limited to, aminoalkyl as defined herein, aminoalkoxy as defined herein, amino as defined herein, and amino acid as defined herein. Generally, RNA comprises a five-membered ring of the sugar ribose bearing an oxygen atom. Non-limiting exemplary modified nucleotides include substitution of the ribose oxygen (e.g., with S, Se, or alkylene, e.g., 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 cyclobutane or oxetane four-membered ring), ring expansion of ribose (e.g., to form a six- or seven-membered ring with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also have a phosphoramidate backbone), polycyclic forms (e.g., tricyclic, and "unlocked" forms, e.g., Examples include glycol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, where the ribose is replaced with a glycol unit linked to a phosphodiester bond), threose nucleic acids (TNA, where the ribose is replaced with α-L-threofuranosyl-(3'→2')), and peptide nucleic acids (PNA, where a 2-amino-ethyl-glycine bond replaces the ribose and phosphodiester backbone). The sugar group may also contain one or more carbons with the opposite stereochemical configuration to that of the corresponding carbon in ribose. Thus, a polynucleotide, primary construct, or mmRNA molecule may include nucleotides containing, for example, arabinose as the sugar.

[0352] Modifications in Nucleobases The present disclosure provides modified nucleosides and nucleotides. As described herein, a "nucleoside" is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). As described herein, a "nucleotide" is defined as a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides).

[0353] Modified nucleotide base pairing encompasses not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides containing and / or modified bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows hydrogen bonding between the non-standard and standard bases or between two complementary non-standard base structures. One example of such non-standard base pairing is base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil.

[0354] Modified nucleosides and nucleotides can contain modified nucleobases. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobases found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases can be modified or completely replaced to provide polynucleotides, primary constructs, or mmRNA molecules with enhanced properties, such as resistance to nucleases by disrupting the binding of major groove binding partners. Table 8 below identifies the chemical appearance of each standard nucleotide. Circles identify the atoms that comprise each chemical region. Table 8 [Table 0008]

[0355] In some embodiments, B is a modified uracil. Exemplary modified uracils have the formulas (b1)-(b5): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0356] During the ceremony,

[0357] [ka] is a single or double bond,

[0358] T 1’ , T 1” , T 2’ , and T 2” each is independently H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or T 1’ and T 1” combination or T 2’ and T 2” The combination of these together (e.g., T 2 (as in Figure 1), O (oxo), S (thio), or Se (seleno),

[0359] V 1 and V 2 each independently selected from O, S, N(R Vb ) nv , or C(R Vb ) nv wherein nv is an integer of 0 to 2, and each R Vbare independently H, halo, an 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, e.g., any of those described herein, e.g., trifluoroacetyl), optionally a substituted aminoalkenyl, an optionally substituted aminoalkynyl, an optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, e.g., any described herein, e.g., trifluoroacetyl), an optionally substituted alkoxycarbonylalkyl, an optionally substituted alkoxycarbonylalkenyl, an optionally substituted alkoxycarbonylalkynyl, or an optionally substituted alkynyloxy (e.g., optionally substituted with any of the substituents described herein, e.g., alkyl selected from (1) to (21));

[0360] R 10 is H, halo, optionally substituted amino, 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;

[0361] R 11 is H or optionally substituted alkyl;

[0362] R 12a 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;

[0363] R 12c 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.

[0364] Other exemplary modified uracils are represented by formulas (b6)-(b9): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0365] During the ceremony,

[0366] [ka] is a single or double bond,

[0367] T 1’ , T 1” , T 2’ , and T 2”each is independently H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or T 1’ and T 1” The combination of these together (e.g., T 1 ), or T 2’ and T 2” The combination of these together (e.g., T 2 (as in 1 and T 2 are independently O (oxo), S (thio), or Se (seleno);

[0368] W 1 and W 2 each independently represents N(R Wa ) nw or C(R Wa ) nw wherein nw is an integer of 0 to 2, and each R Wa is independently H, optionally substituted alkyl, or optionally substituted alkoxy;

[0369] Each V 3 However, independently, O, S, N(R Va ) nv , or C(R Va ) nv wherein nv is an integer of 0 to 2, and each R Vaare 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 alkylheterocyclyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy, optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, e.g., any of those described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g., and optionally substituted with an N-protecting group, e.g., any of those 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 a hydroxy and / or O-protecting group), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g., optionally substituted with any of the substituents described herein, e.g., alkyl (1) to (21)), and R Va and R 12c may be taken together with the carbon atom to which they are attached to form an optionally substituted cycloalkyl, an optionally substituted aryl, or an optionally substituted heterocyclyl (e.g., a 5- or 6-membered ring);

[0370] R 12ais 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 O-protecting groups), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, optionally substituted carbamoylalkyl, or absent;

[0371] R 12b 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 alkylaryl, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, 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 O-protecting groups), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl;

[0372] R 12b and T 1’ combination or R 12b and R 12c can be combined to form an optionally substituted heterocyclyl,

[0373] R 12cis H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl.

[0374] Further exemplary modified uracils are represented by formulae (b28)-(b31): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0375] During the ceremony,

[0376] T 1 and T 2 each is independently O (oxo), S (thio), or Se (seleno);

[0377] Each R Vb’ and R Vb”are 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, e.g., any of those described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, e.g., any of those described herein, e.g., any of those described herein, e.g., substituted with 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 O-protecting groups), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl (e.g., optionally substituted with any of the substituents described herein, e.g., alkyl (1) to (21)) (e.g., R Vb’ is optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted aminoalkyl, e.g., substituted with an N-protecting group, e.g., any of those described herein, e.g., trifluoroacetyl, or sulfoalkyl;

[0378] R 12ais H, optionally substituted alkyl, optionally substituted carboxyaminoalkyl, optionally substituted aminoalkyl (e.g., substituted with, for example, an N-protecting group, e.g., any of those described herein, e.g., trifluoroacetyl, or sulfoalkyl), optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl;

[0379] R 12b 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., substituted with, for example, an N-protecting group, e.g., any of those described herein, e.g., trifluoroacetyl, or sulfoalkyl);

[0380] 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.

[0381] In certain embodiments, T 1 is O (oxo) and T 2 is S (thio) or Se (seleno). 1 is S(thio) and T 2 is O (oxo) or Se (seleno). In some embodiments, R Vb’ is H, optionally substituted alkyl, or optionally substituted alkoxy.

[0382] In other embodiments, each R 12a and R 12bis independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted hydroxyalkyl. In certain embodiments, R 12a is H. In other embodiments, R 12a MoR 12b Both are H.

[0383] In some embodiments, R 12b Each R Vb’ are independently an optionally substituted aminoalkyl (e.g., substituted with an N-protecting group, e.g., any described herein, e.g., trifluoroacetyl, or sulfoalkyl), an optionally substituted aminoalkenyl, an optionally substituted aminoalkynyl, or an optionally substituted acylaminoalkyl (e.g., substituted with an N-protecting group, e.g., any described herein, e.g., trifluoroacetyl). In some embodiments, the amino and / or alkyl of the optionally substituted aminoalkyl are substituted with one or more of an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted sulfoalkyl, an optionally substituted carboxy (e.g., substituted with an O-protecting group), an optionally substituted hydroxy (e.g., substituted with an O-protecting group), an optionally substituted carboxyalkyl (e.g., substituted with an O-protecting group), an optionally substituted alkoxycarbonylalkyl (e.g., substituted with an O-protecting group), or an N-protecting group. In some embodiments, the optionally substituted aminoalkyl is substituted with an optionally substituted sulfoalkyl or an optionally substituted alkenyl. In certain embodiments, R 12a MoR Vb” are both H. In certain embodiments, T 1 is O (oxo) and T 2 is S (thio) or Se (seleno).

[0384] In some embodiments, R Vb’ is an optionally substituted alkoxycarbonylalkyl or an optionally substituted carbamoylalkyl.

[0385] In certain embodiments, R 12a , R 12b , R 12c , or R Va The optional substituents may be polyethylene glycol groups (e.g., -(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl), or an amino-polyethylene glycol group (e.g., —NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 alkyl).

[0386] In some embodiments, B is a modified cytosine. Exemplary modified cytosines are represented by formulas (b10)-(b14): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony,

[0387] T 3’ and T 3” each is independently H, optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy, or T 3’ and T 3”The combination of these together (e.g., T 3 (as in Figure 1), O (oxo), S (thio), or Se (seleno),

[0388] Each V 4 However, independently, O, S, N(R Vc ) nv , or C(R Vc ) nv wherein nv is an integer of 0 to 2, and each R Vc are independently H, halo, an optionally substituted amino acid, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted alkoxy, an optionally substituted alkenyloxy, an optionally substituted heterocyclyl, an optionally substituted alkylheterocyclyl, or an optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, e.g., a substituent selected from alkyl (1)-(21)), and R 13b and R Vc can be combined to form an optionally substituted heterocyclyl,

[0389] Each V 5 However, independently, N(R Vd ) nv , or C(R Vd ) nv wherein nv is an integer of 0 to 2, and each R Vd are independently H, halo, an optionally substituted amino acid, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted alkoxy, an optionally substituted alkenyloxy, an optionally substituted heterocyclyl, an optionally substituted alkylheterocyclyl, or an optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, e.g., alkyl, selected from (1)-(21)) (e.g., V 5 is -CH or N),

[0390] R 13a and R 13beach is independently H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy; R 13b and R 14 can be combined to form an optionally substituted heterocyclyl,

[0391] Each R 14 are 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 O-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 where R is H, alkyl, aryl, or phosphoryl), azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkyl;

[0392] R 15 and R 16 is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0393] Further exemplary modified cytosines are represented by formulas (b32)-(b35): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0394] During the ceremony,

[0395] T 1 and T 3 each is independently O (oxo), S (thio), or Se (seleno);

[0396] R 13a and R 13b each is independently H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy; R 13b and R 14 can be combined to form an optionally substituted heterocyclyl,

[0397] Each R 14 are 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 O-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 where R is H, alkyl, aryl, or phosphoryl), azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, optionally substituted aminoalkyl (e.g., hydroxyalkyl, alkyl, alkenyl, or alkynyl), optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl;

[0398] R 15 and R 16 is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl (e.g., R 15is H and R 16 is H or optionally substituted alkyl).

[0399] In some embodiments, R 15 is H and R 16 is H or optionally substituted alkyl. In certain embodiments, R 14 is H, acyl, or hydroxyalkyl. In some embodiments, R 14 is halo. In some embodiments, R 14 MoR 15 and R are both H. In some embodiments, R 15 MoR 16 and R are both H. In some embodiments, R 14 and R 15 and R 16 Each of is H. In a further embodiment, R 13a and R 13b Each of is independently H or optionally substituted alkyl.

[0400] Further non-limiting examples of modified cytosines include those of formula (b36): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0401] During the ceremony,

[0402] Each R 13b are independently H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy; R 13b and R 14b can be combined to form an optionally substituted heterocyclyl,

[0403] Each R 14a and R 14bare 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 O-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 where R is H, alkyl, aryl, phosphoryl, optionally substituted aminoalkyl, or optionally substituted carboxyaminoalkyl), azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl;

[0404] R 15 is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0405] In certain embodiments, R 14b is an optionally substituted amino acid (e.g., an optionally substituted lysine). In some embodiments, R 14a is H.

[0406] In some embodiments, B is a modified guanine. Exemplary modified guanines are represented by formulas (b15)-(b17): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0407] During the ceremony,

[0408] T4’ , T 4” , T 5’ , T 5” , T 6’ , and T 6” is independently H, optionally substituted alkyl, or optionally substituted alkoxy; 4’ and T 4” combinations (e.g., T 4 (as in) or T 5’ and T 5” combinations (e.g., T 5 (as in) or T 6’ and T 6” combinations (e.g., T 6 ) together to form O (oxo), S (thio), or Se (seleno),

[0409] V 5 and V 6 each independently selected from O, S, N(R Vd ) nv , or C(R Vd ) nv wherein nv is an integer of 0 to 2, and each R Vd are independently H, halo, thiol, an 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, e.g., alkyl, selected from (1) to (21)), optionally substituted thioalkoxy, or optionally substituted amino;

[0410] R 17 , R 18 , R 19a , R 19b , R 21 , R 22 , R 23 , and R 24is independently H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or an optionally substituted amino acid.

[0411] Exemplary modified guanosines are represented by formulas (b37)-(b40): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0412] During the ceremony,

[0413] T 4’ is independently H, optionally substituted alkyl, or optionally substituted alkoxy; and each T 4 are independently O (oxo), S (thio), or Se (seleno);

[0414] R 18 , R 19a , R 19b , and R 21 is independently H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or an optionally substituted amino acid.

[0415] In some embodiments, R 18 is H or optionally substituted alkyl. In a further embodiment, T 4 is oxo. In some embodiments, R 19a and R 19b Each of is independently H or optionally substituted alkyl.

[0416] In some embodiments, B is a modified adenine. Exemplary modified adenines are represented by formulas (b18)-(b20): [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, wherein each V 7 However, independently, O, S, N(R Ve ) nv , or C(R Ve ) nv wherein nv is an integer of 0 to 2, and each R Ve are independently H, halo, an optionally substituted amino acid, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted alkoxy, an optionally substituted alkenyloxy, or an optionally substituted alkynyloxy (e.g., optionally substituted with any substituent described herein, e.g., an alkyl substituent selected from (1)-(21));

[0417] Each R 25 are independently H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino;

[0418] R 26a and R 26b each independently is 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 a polyethylene glycol group (e.g., —(CH) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20alkyl)), or an amino polyethylene glycol group (e.g., —NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 alkyl)

[0419] Each R 27 are independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy, or optionally substituted amino;

[0420] Each R 28 are independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0421] Each R 29 are 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.

[0422] Exemplary modified adenines are represented by formulas (b41)-(b43):

[0423] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof,

[0424] During the ceremony,

[0425] Each R 25 are independently H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino;

[0426] R 26a and R 26b each independently is 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 a polyethylene glycol group (e.g., —(CH) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl), or an amino polyethylene glycol group (e.g., —NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 alkyl)

[0427] Each R 27are independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy, or optionally substituted amino.

[0428] In some embodiments, R 26a is H and R 26b is optionally substituted alkyl. In some embodiments, R 26a and R 26b Each of R is independently an optionally substituted alkyl. In certain embodiments, R 27 is optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy. In other embodiments, R 25 is optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy.

[0429] In certain embodiments, R 26a , R 26b , or R 29 The optional substituents may be polyethylene glycol groups (e.g., -(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR' (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is H or C 1~20 alkyl), or an amino polyethylene glycol group (e.g., —NR N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (wherein s1 is an integer of 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer of 0 to 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 are independently hydrogen or optionally substituted C 1~6 It is alkyl).

[0430] In some embodiments, B is a group represented by formula (b21): [ka] wherein X 12 are independently O, S, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene; xa is an integer from 0 to 3; R 12a and T 2 is as described herein.

[0431] In some embodiments, B is a group represented by formula (b22): [ka] wherein R 10’ are 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; R 11 , R 12a , T 1 , and T 2 is as described herein.

[0432] In some embodiments, B is a group represented by formula (b23): [ka] wherein R 10is an optionally substituted heterocyclyl (e.g., optionally substituted furyl, optionally substituted thienyl, or optionally substituted pyrrolyl), an optionally substituted aryl (e.g., optionally substituted phenyl or optionally substituted naphthyl), or any substituent described herein (e.g., R 10 (those of R 11 (e.g., H or any substituent described herein), R 12a (e.g., H or any substituent described herein), T 1 (e.g., oxo or any substituent described herein), and T 2 (eg, oxo or any substituent described herein) is as defined herein. In some embodiments, B is a group represented by formula (b24): [ka] wherein R 14’ are independently optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkylaryl, optionally substituted alkylheterocyclyl, 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; R 13a , R 13b , R 15 , and T 3 is as described herein.

[0433] In some embodiments, B is a group represented by formula (b25): [ka] wherein R 14’ is an optionally substituted heterocyclyl (e.g., optionally substituted furyl, optionally substituted thienyl, or optionally substituted pyrrolyl), an optionally substituted aryl (e.g., optionally substituted phenyl or optionally substituted naphthyl), or any substituent described herein (e.g., R 14 or R 14’ (those of R 13a (e.g., H or any substituent described herein), R 13b (e.g., H or any substituent described herein), R 15 (e.g., H or any substituent described herein), and T 3 (eg, oxo or any substituent described herein) is as defined herein.

[0434] In some embodiments, B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil. [ka] It could be.

[0435] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5-hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5U), uridine 5-hydroxyacetic acid (cmo 5 U), uridine 5-hydroxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbonylmethyl-uridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-carbamoylmethyl-uridine (ncm 5 U), 5-carboxymethylaminomethyl-uridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thiouridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m 5 U, i.e., those with the nucleobase deoxythymine), 1-methylpseudouridine (m 1 ψ), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4ψ), 4-thio-1-methyl-pseudouridine, 3-methyl-pseudouridine (m 3 ψ), 2-thio-1-methyl-pseudouridine, 1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m 5 D), 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 (1-methylpseudouridine (m 1 ψ), 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine (inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudouridine (ψm), 2-thio-2'-O-methyl-uridine (s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm 5 Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm 5 Um), 3,2'-O-dimethyl-uridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm 5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine].

[0436] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosines include 5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine (m 3 C), N4-acetyl-cytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methyl-cytidine (m 4 C), 5-methyl-cytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm 5 C), 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine (s 2 C), 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), α-thio-cytidine, 2'-O-methyl-cytidine (Cm), 5,2'-O-dimethyl-cytidine (m 5 Cm), N4-acetyl-2'-O-methyl-cytidine (ac 4 Cm), N4,2'-O-dimethyl-cytidine (m 4 Cm), 5-formyl-2'-O-methyl-cytidine (f 5 Cm), N4,N4,2'-O-trimethyl-cytidine (m 42Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0437] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenines 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 (m 1 A), 2-methyl-adenine (m 2 A), N6-methyl-adenosine (m 6 A), 2-methylthio-N6-methyl-adenosine (ms 2 m 6 A), N6-isopentenyl-adenosine (i 6 A), 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A), N6-(cis-hydroxyisopentenyl)adenosine (io 6 A), 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A), N6-glycinylcarbamoyl-adenosine (g 6 A), N6-threonylcarbamoyl-adenosine (t 6 A), N6-methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A), 2-methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6 A), N6,N6-dimethyl-adenosine (m 6 2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn 6A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms 2 hn 6 A), N6-acetyl-adenosine (ac 6 A), 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m 6 2Am), 1,2'-O-dimethyl-adenosine (m 1 Am), 2'-O-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-pentaoxanonadecyl)-adenosine.

[0438] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having a modified guanine include inosine (I), 1-methyl-inosine (m), and 1-methyl-inosine (m). 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyobutosine (yW), peroxywyobutosine (o2yW), hydroxywyobutosine (OHyW), unmodified hydroxywyobutosine (OHyW*), 7-deaza-guanosine, queosine (Q), epoxyqueosine (oQ), galactosyl-queosine (galQ), mannosyl-queosine (manQ), 7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine (preQ1), and archaeosine (G + ), 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m 7 G), 6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine, 1-methyl-guanosine (m1 G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2G), N2,7-dimethyl-guanosine (m 2,7 G), N2, N2, 7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Im), and 2'-O-ribosylguanosine(phosphate) (Gr(p)).

[0439] The nucleobases of the nucleotides can be independently selected from purines, pyrimidines, purine, or pyrimidine analogs. For example, each nucleobase can be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, nucleobases include, for example, pyrazolo[3,4-d]pyrimidine, 5-methylcytosine (5-me-C), 5-hydroxymethylcytosine, 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-propynyluracil and cytosine, 6-azouracil, 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 nucleobases. Naturally occurring and synthetic derivatives of bases may also be included, including substituted adenine and guanine, 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 triazinone, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine, and 1,3,5 triazine. When nucleotides are designated using the abbreviations A, G, C, T, or U, each letter refers to a representative base and / or its derivatives, e.g., A includes adenine or an adenine analog such as, e.g., 7-deazaadenine.

[0440] Modifications in internucleoside linkages Modified nucleotides that can be incorporated into polynucleotides, primary constructs, or mmRNA molecules can be modified in the internucleoside linkage (e.g., phosphate backbone). Herein, the terms "phosphate" and "phosphodiester" are used interchangeably in the context of polynucleotide backbones. The backbone phosphate group can be modified by replacing one or more of the oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides can contain large-scale replacement of unmodified phosphate moieties with other internucleoside linkages described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphororamidates, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotriesters. Phosphorodithioates have both non-linked oxygens replaced with sulfur. Phosphate linkers can also be modified by substitution of the linking oxygen at nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates).

[0441] The α-thio-substituted phosphate moiety is provided to provide stability to RNA and DNA polymers through non-natural phosphorothioate backbone bonds.Phosphorothioate DNA and RNA have increased nuclease resistance, and subsequently have a longer half-life in cellular environments.Phosphorothioate-linked polynucleotides, primary constructs, or mmRNA molecules are also expected to reduce innate immune response by weaker binding / activation of cellular innate immune molecules.

[0442] In certain embodiments, the modified nucleoside comprises an α-thio-nucleoside (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (α-thio-cytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudouridine).

[0443] Other internucleoside linkages that can be used in accordance with the present invention, including internucleoside linkages that do not contain a phosphorus atom, are described herein below.

[0444] Combinations of modified sugars, nucleobases, and internucleoside linkages The polynucleotides, primary constructs, and mmRNA of the present invention can include combinations of modifications to the sugar, nucleobase, and / or internucleoside linkage. These combinations can include any one or more of the modifications described herein. For example, any of the nucleotides described herein in formulas (Ia), (Ia-1) to (Ia-3), (Ib) to (If), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr) can be combined with any of the nucleobases described herein (e.g., formulas (b1) to (b43) or any others described herein).

[0445] Synthesis of polypeptides, primary constructs, and mmRNA molecules The polypeptides, primary constructs, and mmRNA molecules used in accordance with the present invention can be prepared according to any useful technique described herein. The modified nucleosides and nucleotides used in the synthesis of the polynucleotides, primary constructs, and mmRNA molecules disclosed herein can be prepared from readily available starting materials using the following general methods and procedures. Given the typical or preferred process conditions (e.g., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.), those skilled in the art will be able to optimize and develop further process conditions. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art using routine optimization procedures.

[0446] The processes described herein can be monitored according to any suitable method known in the art. For example, product formation can be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or13 C), can be monitored by spectroscopic means such as infrared spectroscopy, spectrophotometry (e.g., ultraviolet-visible), or mass spectrometry, or by chromatography such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0447] The preparation of polypeptides, primary constructs, and mRNA molecules of the present invention may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, and the selection of appropriate protecting groups, can be easily determined by those 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.

[0448] The reactions of the processes described herein can be carried out in a suitable solvent that can be easily selected by one skilled in the art of organic synthesis. A suitable solvent can be substantially non-reactive with the starting materials (reactants), intermediates, or products at the temperature at which the reaction is carried out, i.e., a temperature that can range from the freezing temperature of the solvent to the boiling point of the solvent. A given reaction can be carried out in one solvent or a mixture of two or more solvents. A suitable solvent for a particular reaction step can be selected depending on the particular reaction step.

[0449] Resolution of racemic mixtures of modified nucleosides and nucleotides can be carried out by any of a number of methods known in the art. Exemplary methods include fractional recrystallization using a "chiral resolving acid," which is an optically active, salt-forming organic acid. Suitable resolving acids for fractional recrystallization include optically active acids such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or the D and L forms of 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 compositions can be determined by those 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):1729-1740 (1992), each of which is incorporated by reference in its entirety.

[0450] The polypeptides, primary constructs, and mRNA of the present invention may or may not be uniformly modified along the entire length of the molecule. For example, one or more or all types of nucleotides (e.g., purines or pyrimidines, or any one or more or all of A, G, U, and C) may or may not be uniformly modified in a polynucleotide of the present invention or in a given, predetermined sequence region thereof (e.g., one or more of the sequence regions depicted in Figure 1). In some embodiments, all nucleotides X in a polynucleotide of the present invention (or in a given sequence region thereof) are modified, where X can be any one of the nucleotides A, G, U, and 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, or A+G+C.

[0451] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at various positions in a polynucleotide, primary construct, or mmRNA. Those skilled in the art will recognize that nucleotide analogs or other modification(s) can be placed at any position(s) in a polynucleotide, primary construct, or mmRNA such that the function of the polynucleotide, primary construct, or mmRNA is substantially reduced. Modifications can also be 5' or 3' terminal modifications. A polynucleotide, primary construct, or mmRNA may contain from about 1% to about 100% modified nucleotides (either the total nucleotide content or associated with any one or more types of nucleotides, i.e., A, G, U, or C), or any intermediate percentage (e.g., 1%-20%, 1%-25%, 1%-50%, 1%-60%, 1%-70%, 1%-80%, 1%-90%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 1%-95%, 10%-10 ... It may contain 0% to 90%, 10% to 95%, 10% to 100%, 20% to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100%.

[0452] In some embodiments, a polynucleotide, primary construct, or mmRNA comprises modified pyrimidines (e.g., modified uracil / uridine / U or modified cytosine / cytidine / C). In some embodiments, the uracil or uridine (generally, U) in a polynucleotide, primary construct, or mmRNA molecule is about 1% to about 100% modified uracil or uridine (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 30%, 30% to 40%, 30% to 40%. % to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100% modified uracil or modified uridine). The modified uracil or uridine can be substituted with a compound having a single unique structure or with multiple compounds having different structures (eg, two, three, four or more unique structures as described herein).In some embodiments, the cytosines or cytidines (generally, C) in the polynucleotide, primary construct, or mmRNA molecule are from about 1% to about 100% modified cytosines or cytidines (e.g., 1% to 20%, 1% to 25%, 1% to 50%, 1% to 60%, 1% to 70%, 1% to 80%, 1% to 90%, 1% to 95%, 10% to 20%, 10% to 25%, 10% to 50%, 10% to 60%, 10% to 70%, 10% to 80%, 10% to 90%, 10% to 95%, 10% to 100%, 20% to 300%, 30% to 400%, 30% to 400%, 30% to 500%, 30% to 600%, 30% to 700%, 30% to 800%, 30% to 900%, 30% to 950%, 30% to 1000%, 30% to 4000, 30% to 5000, 30% to 6000, 30% to 7000, 30% to 8000, 30% to 9000, 30% to 9500, 30% to 10000, 30% to 4000, 30% to 5000, 30% to 6 ... % to 25%, 20% to 50%, 20% to 60%, 20% to 70%, 20% to 80%, 20% to 90%, 20% to 95%, 20% to 100%, 50% to 60%, 50% to 70%, 50% to 80%, 50% to 90%, 50% to 95%, 50% to 100%, 70% to 80%, 70% to 90%, 70% to 95%, 70% to 100%, 80% to 90%, 80% to 95%, 80% to 100%, 90% to 95%, 90% to 100%, and 95% to 100% modified cytosine or modified cytidine). The modified cytosine or cytidine can be substituted with a compound having a single unique structure or with multiple compounds having different structures (eg, two, three, four or more unique structures described herein). In some embodiments, the present disclosure provides a compound of formula (Ia-1): [ka]

[0013] The present invention provides a method for synthesizing a polynucleotide, primary construct, or mmRNA (e.g., a first region, a first flanking region, or a second flanking region) comprising n linked nucleosides having a) Formula (IV-1): [ka] A nucleotide of formula (V-1): [ka] (wherein Y 9is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino, thiol, an optionally substituted amino acid, or a peptide (e.g., containing 2 to 12 amino acids), and each P 1 , P 2 , and P 3 are independently suitable protecting groups; [ka] indicates a solid support), Providing a polynucleotide, primary construct, or mmRNA of formula (VI-1); [ka] , and b) oxidizing or sulfurizing the polynucleotide, primary construct, or mmRNA of formula (V) to produce a polynucleotide, primary construct, or mmRNA of formula (VII-1); [ka] , and and c) removing the protecting group to yield the polynucleotide, primary construct, or mRNA of formula (Ia).

[0453] In some embodiments, steps a) and b) are repeated 1 to about 10,000 times. In some embodiments, the method further comprises a nucleotide (e.g., an 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 a derivative thereof. In some embodiments, the polynucleotide, primary construct, or mmRNA may be translatable.

[0454] Other components of polynucleotides, primary constructs, and mmRNAs are optional and useful in some embodiments. For example, a 5' untranslated region (UTR) and / or a 3' UTR can be provided, and either or both of these can independently contain one or more different nucleotide modifications. In such embodiments, nucleotide modifications can also be present in translatable regions. Polynucleotides, primary constructs, and mmRNAs containing Kozak sequences are also provided.

[0455] Exemplary syntheses of modified nucleic acids or mRNA, e.g., modified nucleotides to be incorporated into RNA or mRNA, are provided below in Schemes 1 through 11. Scheme 1 provides a general method for the phosphorylation of nucleosides, including modified nucleosides. Scheme 1 [ka]

[0456] Various protecting groups can be used to control the reaction. For example, Scheme 2 provides the use of multiple protection and deprotection steps to facilitate phosphorylation of the 5' position of the sugar, rather than the 2' and 3' hydroxyl groups. Scheme 2 [ka]

[0457] Modified nucleotide can be synthesized by any useful method.Schemes 3, 4 and 7 provide exemplary methods for synthesizing modified nucleotides having modified purine nucleobases, and schemes 5 and 6 provide exemplary methods for synthesizing modified nucleotides having modified pseudouridine or pseudoisocytidine, respectively. Scheme 3 [ka] Scheme 4 [ka] Scheme 5 [ka] Scheme 6 [ka] Scheme 7 [ka]

[0458] Schemes 8 and 9 provide exemplary syntheses of modified nucleotides. Scheme 10 provides a non-limiting biocatalytic method for producing nucleotides. Scheme 8 [ka] Scheme 9 [ka] Scheme 10 [ka]

[0459] Scheme 11 provides an exemplary synthesis of modified uracils, where the N1 position is substituted with R 12b The 5' position of the ribose is phosphorylated. 1 , T 2 , R 12a , R 12b, and r are as provided herein. This synthesis, as well as optimized versions thereof, can be used to modify other pyrimidine and purine nucleobases (see, e.g., formulas (b1)-(b43)) and / or introduce one or more phosphate groups (e.g., at the 5' position of the sugar). This alkylation reaction can also be used to include one or more optionally substituted alkyl groups at any reactive group (e.g., amino group) in any nucleobase described herein (e.g., amino groups at the Watson-Crick base pairing interface of cytosine, uracil, adenine, and guanine). Scheme 11 [ka]

[0460] Nucleotide combinations in mmRNA Further examples of modified nucleotides and combinations of modified nucleotides are provided in Table 9 below. These combinations of modified nucleotides can be used to form the polypeptides, primary constructs, or mRNA of the present invention. Unless otherwise stated, modified nucleotides can be completely substituted for natural nucleotides in the modified nucleic acids or mRNA of the present invention. As a non-limiting example, the natural nucleotide uridine can be substituted with a modified nucleoside described herein. In another non-limiting example, the natural nucleotide uridine can 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 nucleosides disclosed herein. Table 9 [Table 0009-1] [Table 0009-2]

[0461] Further examples of combinations of modified nucleotides are provided below in Table 10. These combinations of modified nucleotides can be used to form the polypeptides, primary constructs, or mRNA of the present invention. Table 10 [Table 0010]

[0462] In some embodiments, at least 25% of the cytosines are substituted with compounds of formulas (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%).

[0463] In some embodiments, at least 25% of the uracils are substituted with 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%).

[0464] In some embodiments, at least 25% of the cytosines are substituted with compounds of Formulae (b10)-(b14) and at least 25% of the uracils are substituted with compounds of Formulae (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%).

[0465] IV. Pharmaceutical Compositions Formulation, Administration, Delivery, and Dosing The present invention provides compositions and complexes of polynucleotides, primary constructs, and mmRNA in combination with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions may optionally contain one or more additional active agents, e.g., therapeutically and / or prophylactically active agents. General considerations in the formulation and / or manufacture of pharmaceuticals can be found, for example, in Remington: The Science and Practice of Pharmacy 21, incorporated herein by reference. st ed., Lippincott Williams & Wilkins, 2005.

[0466] In some embodiments, the compositions are administered to a human, i.e., a human patient or subject. For purposes of this disclosure, the term "active ingredient" generally refers to the polynucleotides, primary constructs, and mmRNA to be delivered as described herein.

[0467] Although the description of pharmaceutical compositions provided herein is directed primarily to pharmaceutical compositions suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, e.g., non-human animals, e.g., non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans to make them suitable for administration to various animals are well understood, and veterinary pharmacologists can design and / or implement such modifications using no more than routine experimentation, if at all. Subjects to which the pharmaceutical compositions are intended for administration include, but are not limited to, humans and / or other primates; mammals, including commercially relevant mammals such as cows, pigs, horses, sheep, cats, dogs, mice, and / or rats; and / or birds, including commercially suitable birds such as poultry, chickens, ducks, geese, and / or turkeys.

[0468] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such preparation methods include the step of combining the active ingredient 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 desired single or multiple dose units.

[0469] Pharmaceutical compositions according to the present invention can be prepared, packaged, and / or sold in bulk, as a single unit dose, and / or as multiple single unit doses.As used herein, a "unit dose" is a discrete amount of pharmaceutical composition containing a predetermined amount of active ingredient.The amount of active ingredient is generally equal to the dosage of the active ingredient that 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.

[0470] The relative amounts of active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the invention will vary depending on the identity, size, and / or condition of the subject being treated, and will further vary depending on the route by which the composition is to be administered. By way of example, the composition may contain 0.1% to 100% w / w, e.g., 0.5 to 50% w / w, 1 to 30% w / w, 5 to 80% w / w, or at least 80% w / w of the active ingredient.

[0471] Formulation The polynucleotides, primary constructs, and mmRNA of the present invention can be formulated with one or more excipients to: (1) increase stability, (2) increase cell transfection, (3) allow for sustained or delayed release (e.g., from a depot formulation of the polynucleotide, primary construct, or mmRNA), (4) alter biodistribution (e.g., targeting the polynucleotide, primary construct, or mmRNA to a particular tissue or cell type), (5) increase translation of the encoded protein in vivo, and / or (6) alter the release profile of the encoded protein in vivo. In addition to traditional excipients such as any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, excipients of the present invention can include, without limitation, lipidoids, liposomes, lipid nanoparticles, polymers, lipoplexes, core-shell nanoparticles, cells transfected with peptides, proteins, polynucleotides, primary constructs, or mmRNA (e.g., for transplantation into a subject), hyaluronidase, nanoparticle mimics, and combinations thereof. Thus, the formulations of the present invention can contain one or more excipients, each in an amount that together increase the stability of the polynucleotide, primary construct, or mmRNA, increase cellular transfection with the polynucleotide, primary construct, or mmRNA, increase expression of the protein encoded by the polynucleotide, primary construct, or mmRNA, and / or alter the release profile of the protein encoded by the polynucleotide, primary construct, or mmRNA. Furthermore, the primary constructs and mmRNA of the present invention may be formulated using self-assembling nucleic acid nanoparticles.

[0472] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. Generally, such methods include combining the active ingredient with an excipient and / or one or more other accessory ingredients.

[0473] Pharmaceutical compositions according to the present disclosure may be prepared, packaged, and / or sold in bulk, as one single unit dose, and / or as multiple single unit doses. As used herein, a "unit dose" refers to a discrete amount of pharmaceutical composition containing a predetermined amount of active ingredient. The amount of active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject, and / or a convenient fraction of such a dosage, including, but not limited to, for example, one-half or one-third of such a dosage.

[0474] The relative amounts of the active ingredient, pharmaceutically acceptable excipient, and / or any additional ingredients in a pharmaceutical composition according to the present disclosure will vary depending on the identity, size, and / or condition of the subject being treated, and will further vary depending on the route by which the composition is to be administered. For example, the composition may contain from 0.1% w / w to 99% active ingredient.

[0475] In some embodiments, the formulations described herein may contain at least one mmRNA. As a non-limiting example, the formulation may contain one, two, three, four, or five mmRNAs. In one embodiment, the formulation may contain modified mRNAs encoding proteins selected from categories including, 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, intracellular membrane-associated proteins, nuclear proteins, proteins associated with human diseases, and / or proteins associated with non-human diseases. In one embodiment, the formulation contains proteins encoded by at least three modified mRNAs. In one embodiment, the formulation contains proteins encoded by at least five modified mRNAs.

[0476] Pharmaceutical formulations may further comprise pharmaceutically acceptable excipients, including, but not limited to, any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersing or suspending aids, surfactants, isotonicity agents, thickening or emulsifying agents, preservatives, and the like, as used herein, that are appropriate for the particular dosage form desired. Various excipients for formulating pharmaceutical compositions and techniques for preparing compositions are known in the art (see, for example, Remington: The Science and Practice of Pharmacy, 21, incorporated herein by reference in its entirety). st (See, Edition, A.R. Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006.) Except insofar as any conventional excipient vehicle is incompatible with the substance or its derivatives, such as causing any undesirable biological effects or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use may be contemplated within the scope of the present disclosure.

[0477] In some embodiments, the particle size of the lipid nanoparticles may be increased and / or decreased. The change in particle size may help combat biological reactions, such as, but not limited to, inflammation, or may increase the biological effectiveness of the modified mRNA delivered to a mammal.

[0478] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, surfactants and / or emulsifiers, preservatives, buffers, lubricants, and / or oils. Such excipients may optionally be included in the pharmaceutical formulations of the present invention.

[0479] Lipidoid The synthesis of lipidoids has been described in detail, and formulations containing these compounds are particularly suitable 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 S...

Claims

[Claim 1] The invention as shown in the drawings.

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