Modified polynucleotides for production of proteins

Abstract

To provide nucleic acid based compounds or polynucleotides having features which are useful for optimizing formulation and delivery of nucleic acid based therapeutics.SOLUTION: An isolated polynucleotide comprises: (a) a first region of linked nucleosides, the first region encoding a polypeptide of interest, the polypeptide of interest having prescribed sequences; (b) a first flanking region located at a 5' terminus of the first region, the first flanking region comprising (i) a sequence of linked nucleosides selected from a native 5' UTR and (ii) at least one 5' terminal cap; and (c) a second flanking region located at a 3' terminus of the first region, the second flanking region comprising (i') a sequence of linked nucleosides selected from a native 3' UTR and (ii') a 3' tailing sequence of linked nucleosides.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] Sequence Listing Reference The present application includes a "long" sequence listing, which has been submitted on CD-R in lieu of a hardcopy reproduction and is incorporated herein by reference in its entirety. This includes a text file of the sequence listing as part of the originally filed subject matter, which is as follows: Filename: M309.20-2030.1309PCT_SL.txt; File Size: 149,430,208; Creation Date: March 4, 2013. These CD-Rs are referred to as "CRF", "Copy 1" and "Copy 2", respectively, and each contains only one identical file, as identified above. The machine readable format of each CD-R is IBM-PC, and the operating system of each compact disc is MS-Windows.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a joint venture between 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 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." 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," and U.S. Provisional Patent Application No. 61 / 618,870, filed April 2, 2012, entitled "Modified Polynucleotides for the Production of Therapeutic Proteins and No. 61 / 681,649, filed Aug. 10, 2012, entitled "Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides," U.S. Provisional Patent Application No. 61 / 737,139, filed Dec. 14, 2012, entitled "Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides," U.S. Provisional Patent Application No. 61 / 618,873, filed Apr. 2, 2012, entitled "Modified Polynucleotides for the Production of Secreted Proteins," U.S. Provisional Patent Application No. 61 / 681,650, filed Aug. 10, 2012, entitled "Modified Polynucleotides for the Production of Secreted Proteins," U.S. Provisional Patent Application No. 61 / 737,Issue 147, entitled "Modified Polynucleotides for the Production of Secreted, 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," and U.S. Provisional Patent Application No. 61 / 618,885, filed April 2, 2012, entitled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins." No. 61 / 681,658, filed Aug. 10, 2012, entitled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins," and U.S. Provisional Patent Application No. 61 / 737,155, filed Dec. 14, 2012, entitled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins." 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." No. 61 / 681,661, filed Aug. 10, 2012, entitled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins," U.S. Provisional Patent Application No. 61 / 737,160, filed Dec. 14, 2012, entitled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins," and U.S. Provisional Patent Application No. 61 / 618,911, filed Apr. 2, 2012, entitled "Modified Polynucleotides for the Production of Nuclear No. 61 / 681,667, filed Aug. 10, 2012, entitled "Modified Polynucleotides for the Production of Nuclear Proteins," U.S. Provisional Patent Application No. 61 / 737,168, filed Dec. 14, 2012, entitled "Modified Polynucleotides for the Production of Nuclear Proteins," U.S. Provisional Patent Application No. 61 / 618,922, filed Apr. 2, 2012, entitled "Modified Polynucleotides for the Production of Proteins," U.S. Provisional Patent Application No. 61 / 681,675, filed Aug. 10, 2012, entitled "Modified Polynucleotides for the Production of Proteins," and U.S. Provisional Patent Application No. 61 / 737,174, filed Dec. 14, 2012, entitled "Modified Polynucleotides for the Production of “Modified Polynucleotides for the Synthesis of Proteins” and U.S. Provisional Patent Application No. 61 / 618,935, filed April 2, 2012, entitled “Modified Polynucleotides for the Synthesis of Proteins” No. 61 / 681,687, filed Aug. 10, 2012, entitled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; U.S. Provisional Patent Application No. 61 / 737,184, filed Dec. 14, 2012, entitled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; U.S. Provisional Patent Application No. 61 / 618,945, filed Apr. 2, 2012, entitled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; U.S. Provisional Patent Application No. 61 / 681,696, filed Aug. 10, 2012, entitled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; No. 61 / 737,191, filed Dec. 14, 2012, entitled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; U.S. Provisional Patent Application No. 61 / 618,953, filed Apr. 2, 2012, entitled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; U.S. Provisional Patent Application No. 61 / 681,704, filed Aug. 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," filed April 2, 2012, and U.S. Provisional Patent Application No. 61 / 618,961, entitled "Dosing Methods for Modified mRNA," filed May 17, 2012, each of which is incorporated herein by reference in its entirety.

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

[0004] This application is also related to co-pending applications, each of which was filed concurrently herewith on March 9, 2013, entitled "Modified Polynucleotides for the Production of Biologics and Proteins Associated with Human Disease," Attorney Docket No. M300.20 (PCT / US13 / XXXXX), and entitled "Modified Polynucleotides for the Production of Secreted Agent Reference Number M304.20 (PCT / US13 / XXXXX) for "Modified Polynucleotides for the Production of Membrane Proteins", Agent Reference Number M305.20 (PCT / US13 / XXXXX) for "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins", Agent Reference Number M306.20 (PCT / US13 / XXXXX) for "Modified Polynucleotides for the Production of Nuclear Proteins", Agent Reference Number M308.20 (PCT / US13 / XXXXX) for "Modified Polynucleotides", Agent Reference Number M301.20 (PCT / US13 / XXXXX) for "Modified Polynucleotides", Agent Reference Number M310.20 (PCT / US13 / XXXXX) for "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", Agent Reference Number M311.20 (PCT / US13 / XXXXX) for "Modified Polynucleotides for The Production of Cosmetics No. MNC1.20 (PCT / US13 / XXXXX), entitled "Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides," and 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 entireties.

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

[0006] There are many problems with the conventional methods of protein expression.For example, introduced DNA may be frequently integrated into host cell genomic DNA, resulting in the modification and / or damage of host cell genomic DNA.Alternatively, the heterologous deoxyribonucleic acid (DNA) introduced into cells may be inherited by daughter cells (whether or not the heterologous DNA is integrated into chromosomes) or by progeny.

[0007] In addition, assuming proper delivery and no damage or integration into the host genome, there are many steps that must occur before the encoded protein is made. Once inside the cell, the DNA must be transported into the nucleus where it is transcribed into RNA. The RNA transcribed from the DNA must then enter the cytoplasm where it is translated into protein. Not only do the multiple processing steps from administered DNA to protein create a time lag before a functional protein is produced, but each step also represents an opportunity for cellular error and damage. Furthermore, it is notoriously difficult to achieve DNA expression in cells, as DNA frequently 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.

[0008] In the early 1990s, Bloom and colleagues succeeded in rescuing 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 have hindered the development of mRNA as a therapeutic agent, and efforts have since been focused rather on an alternative application that could exploit these pitfalls: immunization with mRNAs encoding cancer antigens.

[0009] 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 the immunostimulatory effect.

[0010] These studies have been reported in, for example, 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, now abandoned, published as U.S. Patent No. US20060247195; and European Patent Application National Stage Registration No. EP2004743322, filed July 9, 2004, published as European Patent No. EP1646714, now withdrawn; 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; PCT Application No. PCT / US2006 / 46120, filed December 4, 2006, published as International Publication No. WO2007064952 from the University of Rochester, and U.S. Patent Application No. 11 / 606,995, filed December 1, 2006, published as U.S. Patent No. US20070141030; BioNTech European Patent Application No. EP2007024312, filed December 14, 2007, now abandoned, to AG; PCT Application No. PCT / EP2008 / 01059, filed December 12, 2008, published as International Publication No. WO2009077134; European Patent Application National Phase Registration No. EP2008861423, filed June 2, 2010, published as European Patent No. EP2240572; U.S. Patent Application National Phase 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 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, to Shire HumanGenetic Therapeutics; U.S. Patent Application No. 12 / 957,340, filed November 20, 2010, published as U.S. Patent No. US20110244026, to Sequitur Inc.; PCT Application No. PCT / US1998 / 019492, filed September 18, 1998, published as International Publication No. WO1999014346, to 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 Maximilian University; No. 8,039,214, filed June 30, 2008, granted October 18, 2011, to Epson Corporation; 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;No. 451, as well as 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 to the Board of Regents of the University of Pennsylvania, and U.S. patent application national phase no. 11 / 990,646, filed March 27, 2009, published as U.S. Patent No. US20090286852; 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 June 5, 2002, published as International Publication No. WO2002098443, PCT application No. PCT / EP2002 / 14577, filed December 19, 2002, published as International Publication No. WO2003051401, PCT application No. PCT / EP2007 / 09469, filed December 31, 2007, published as International Publication No. WO2008052770, PCT application No. PCT / EP200 No. PCT / EP2008 / 03033, filed April 16, 2008, published as International Publication No. WO2006122828, No. PCT / EP2006 / 004784, filed May 19, 2005, published as International Publication No. WO2006122828, No. PCT / EP2008 / 00081, filed January 9, 2007, published as International Publication No. WO2008083949, as well as U.S. patent application Ser. No. 10 / 729,830, filed December 5, 2003, published as U.S. Patent No. US20050032730, Ser. No. 10 / 870, 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, U.S. Patent No. 12 / 787,755, filed May 26, 2010, published as U.S. Patent No. US20100239608, U.S. Patent No. 13 / 185,119, filed July 18, 2011, published as U.S. Patent No. US20110269950, and U.S. Patent No. 13 / 106,548, filed May 12, 2011, published as U.S. Patent No. US20110311472, all of which are incorporated herein by reference in their entireties.

[0011] 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 of barriers surrounding the effective regulation of intracellular translation and processing of nucleic acids encoding polypeptides or fragments thereof.

[0012] 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 studies are detailed 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 entirety.

[0013] 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 features that avoid one or more of the problems in the art, such as features 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

[0014] Described herein are compositions of modified mRNA (mmRNA) molecules, methods, processes, kits, and devices for the design, preparation, manufacture, and / or formulation of modified mRNA (mmRNA) molecules.

[0015] 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.

[0016] 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 description of the drawings]

[0017] [Figure 1] FIG. 1 is a schematic diagram of the primary construct of the present invention. [Figure 2-1]1 illustrates lipid structures in the prior art that are 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. [Diagram 3] Representative of a plasmid useful in the IVT reactions taught herein. This plasmid contains insert 64818 designed by the inventors. [Figure 4] 13 is a gel profile of modified mRNA encapsulated in PLGA microspheres. [Diagram 5] 1 is a histogram of Factor IX protein producing PLGA formulation Factor IX modified mRNA. [Figure 6-1] 6A-6C are histograms showing VEGF protein production in human keratinocyte cells after transfection of modified mRNA at various doses. FIG 6A shows protein production after transfection of modified mRNA containing natural nucleoside triphosphates (NTPs). [Figure 6-2] FIG. 6B shows protein production following transfection of modified mRNA fully modified with pseudouridine (pseudoU) and 5-methylcytosine (5mC). [Figure 6-3] FIG. 6C shows protein production following transfection of modified mRNA fully modified with N1-methyl-pseudouridine (N1-methyl-pseudoU) 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 following transfection of VEGF-modified mRNA in peripheral blood mononuclear cells (PBMCs). [Figure 8-2] FIG. 8B shows IFN-α induction. [Figure 9] 1 is a histogram of VEGF protein production in HeLa cells from VEGF modified mRNA. [Figure 10] 13 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 of Factor IX protein production in HeLa cell supernatants from Factor IX modified mRNA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] In the therapeutics, diagnostics, reagent fields, and in 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, for example, to cause intracellular translation of the nucleic acid and production of the encoded polypeptide of interest. Of particular importance is the delivery and function of non-integrated polynucleotides.

[0019] 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.

[0020] According to the present invention, these polynucleotides are preferably modified to avoid the deficiencies of other polypeptide-encoding molecules in the art, and therefore are referred to as modified mRNAs or mmRNAs.

[0021] The use of modified polynucleotides in the areas of antibodies, viruses, veterinary applications, and in various in vivo environments have been investigated by the present inventors, and these investigations are described, for example, in co-pending and co-owned U.S. Provisional Patent Application No. 61 / 470,451, filed March 31, 2011, which teaches in vivo applications of mmRNA; No. 61 / 517,784, filed April 26, 2011, which teaches engineered nucleic acids for the production of antibody polypeptides; and No. 61 / 520,784, filed April 26, 2011, which teaches veterinary applications of mmRNA technology. No. 61 / 519,158, filed May 17, 2011, which teaches the application of mmRNA technology in antibacterial agents; No. 61 / 533,537, filed September 12, 2011, which teaches the application of mmRNA technology in viral agents; No. 61 / 533,554, filed September 12, 2011, which teaches various chemical modifications for use in mmRNA technology; No. 61 / 542,533, filed October 3, 2011, which teaches mobile devices for use in making and using mmRNA technology; No. 61 / 570,690, filed Dec. 14, 2011, teaching the use of mmRNA in critical care settings; No. 61 / 570,708, filed Dec. 16, 2011, teaching terminal modification structures for mmRNA; No. 61 / 576,651, filed Dec. 16, 2011, teaching methods for delivering mmRNA using lipidoids; No. 61 / 576,705, filed Dec. 21, 2011, teaching methods for increasing organ or tissue viability using mmRNA; No. 1 / 578,271, filed Dec. 29, 2011, which teaches mmRNA encoding cell-penetrating peptides; No. 61 / 581,322, filed Dec. 29, 2011, which teaches the incorporation of cytotoxic nucleosides into mmRNA; and No. 61 / 631,729, filed Jan. 10, 2012, which teaches methods of using mmRNA to cross the blood-brain barrier, all of which are incorporated by reference in their entireties herein.

[0022] Provided in part herein are polynucleotides, primary constructs, and / or mmRNA encoding a polypeptide of interest designed to improve one or more of: stability and / or clearance in tissues, 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 blood circulation, protein half-life, and / or modulation of cellular state, function, and / or activity.

[0023] 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 that comprises a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the invention include, but are not limited to, ribonucleic 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-ribo configuration, α-LNA with an α-L-ribo configuration (diastereomer of LNA), 2'-amino-LNA with a 2'-amino functionalization, and 2'-amino-α-LNA with a 2'-amino functionalization), or hybrids thereof.

[0024] 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.

[0025] Traditionally, the major 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 traditional 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 where the polynucleotide is introduced. Thus, the modified mRNA molecules of the present invention are referred to as "mmRNAs." 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. Since chemical bonds are necessarily broken and reformed resulting in structural modifications, structural modifications are of a chemical nature and are therefore chemical modifications. However, structural modifications result in different nucleotide sequences. For example, the polynucleotide "ATCG" can 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.

[0026] 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 overcome existing problems in effective polypeptide production using nucleic acid-based therapeutics.

[0027] 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 permit 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.

[0028] Referring to FIG. 1, here the primary construct 100 contains a first region of bound 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 bound nucleotides that includes one or more complete or incomplete 5'UTR sequences. The flanking region 104 may also include a 5' terminal cap 108. The second flanking region 106 may include a region of bound nucleotides that includes one or more complete or incomplete 3'UTRs. The flanking region 106 may also include a 3' tail sequence 110.

[0029] Bridging the 5' end 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 may include any translation initiation sequence or signal that includes a start codon.

[0030] Bridging the 3' end of the first region 102 and the second flanking region 106 is a second operational region 107. Conventionally, this operational region includes a stop codon. Alternatively, this operational region may include any translation initiation sequence or signal that includes a stop codon. Multiple consecutive stop codons may also be used in accordance with the invention.

[0031] In general, the minimum length of the first region of the primary construct of the invention may be a length of nucleic acid sequence sufficient to code for a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In another embodiment, the length may be sufficient to code for a peptide of 2-30 amino acids, e.g., 5-30, 10-30, 2-25, 5-25, 10-25, or 10-20 amino acids. The length may be sufficient to code for a peptide of at least 11, 12, 13, 14, 15, 17, 20, 25, or 30 amino acids, or a peptide not longer than 40 amino acids, e.g., not longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids. Examples of dipeptides that the polynucleotide sequence may code for include, but are not limited to, carnosine and anserine.

[0032] 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."

[0033] In some embodiments, the polynucleotide, primary construct, or mmRNA comprises 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, 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).

[0034] In accordance with the 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).

[0035] In accordance with 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 monomer of polyA binding protein binds to a stretch of about 38 nucleotides. Thus, polyA tails of about 80 nucleotides and 160 nucleotides have been observed to be functional.

[0036] In accordance with the present invention, the capping region may comprise a single cap or a series of nucleotides forming a cap. In this embodiment, the capping region may be 1-10, e.g., 2-9, 3-8, 4-7, 1-5, 5-10, or at least 2, or no more than 10 nucleotides in length. In some embodiments, the cap is absent.

[0037] In accordance with the present invention, the first and second operational regions may be in the range of 3 to 40, e.g., 5 to 30, 10 to 20, 15, or at least 4, or up to 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.

[0038] Circular mmRNA According to the present invention, the primary construct or mmRNA can be circularized or concatemerized to generate translation competent molecules and aid in 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.

[0039] In the first pathway, the 5' and 3' ends of the nucleic acid contain chemically reactive groups that form a new covalent bond between the 5' and 3' ends of the molecule when they are in close proximity. The 5' end can contain an NHS-ester reactive group and the 3' end can 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.

[0040] In the second pathway, T4 RNA ligase can be used to enzymatically attach a 5'-phosphorylated nucleic acid molecule to the 3'-hydroxyl group of a nucleic acid to form 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 side-by-side base-pair with both the 5' and 3' regions to support the enzymatic ligation reaction.

[0041] 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 may contain an active ribozyme sequence capable of ligating the 5' end of the nucleic acid molecule to the 3' end of the nucleic acid molecule. The ligase ribozyme may be derived from a group I intron, a group I intron, hepatitis delta virus, a hairpin ribozyme, or may be selected by SELEX (systematic evolution of ligands by exponential enrichment). The ribozyme ligase reaction may take 1 to 24 hours at temperatures between 0 and 37°C.

[0042] mmRNA multimers According to the present invention, multiple distinct polynucleotides, primary constructs, or mmRNAs can be linked via their 3'-ends using modified nucleotides at the 3'-ends. 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 fed to HepG2 cells in a 1:1 ratio to alter cellular fatty acid metabolism. This ratio can be controlled by chemically linking polynucleotides, primary constructs, or mmRNAs with 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, the 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.

[0043] In another example, three or more polynucleotides can be linked using a functionalized linker molecule. For example, a functionalized saccharide molecule can have multiple chemically reactive groups (SH-, NH 2 -, N 3 etc.) to react with the cognate moiety on a 3'-functionalized mRNA molecule (i.e., 3'-maleimide ester, 3'-NHS-ester, alkynyl). The number of reactive groups on this modified saccharide can be controlled in a stoichiometric manner to directly control the stoichiometry of the complexed polynucleotide, primary construct, or mmRNA.

[0044] mmRNA complexes and combinations To further enhance protein production, the primary constructs or mmRNA of the present invention may be coupled to other polynucleotides, dyes, intercalating agents (e.g., acridine), crosslinking agents (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG], 2 , polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption 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.

[0045] 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 a cell, tissue, or organism.

[0046] In accordance with the present invention, the mmRNA or primary construct may be administered together with or 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.

[0047] Bifunctional mmRNA One embodiment of the present invention is a bifunctional polynucleotide (e.g., a bifunctional primary construct or a bifunctional mmRNA). As the name suggests, a bifunctional polynucleotide is a polynucleotide that has at least two functions or is capable of at least two functions. By convention, these molecules can also be referred to as multifunctional.

[0048] The multiple functions of a bifunctional polynucleotide may be encoded by the RNA (this 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 a function that is covalently or electrostatically associated with the polynucleotide. Furthermore, these two functions may be provided in the context of a complex of mmRNA and another molecule.

[0049] The bifunctional polynucleotides may encode antiproliferative peptides. These peptides may be linear, cyclic, constrained, or random coil. They may function as aptamers, signaling molecules, ligands, or mimetics or mimetics thereof. The antiproliferative peptides, when translated, may be 3-50 amino acids long. They may be 5-40, 10-30, or about 15 amino acids long. They may be single-chain, multi-chain, or branched, and when translated, may form complexes, aggregates, or any multi-unit structure.

[0050] 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 can be the "first region" of the primary construct. Alternatively, the non-coding regions can be regions other than the first region. Such molecules are not normally translated, but can affect protein production by one or more of binding and sequestration to one or more translation machinery components, such as ribosomal proteins or transfer RNA (tRNA), thereby effectively reducing protein expression in the cell or regulating one or more pathways or cascades in the cell, which then alter protein levels. The polynucleotides or primary constructs can 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).

[0051] Polypeptide of interest In accordance with the present invention, the primary construct is designed to encode one or more polypeptides of interest or fragments thereof. A polypeptide of interest may include, but is not limited to, a whole 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 the 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. The term, as used herein, refers to proteins, polypeptides, and peptides of any size, structure, or function. In some instances, when the encoded polypeptide is smaller than about 50 amino acids, the polypeptide is referred to as a peptide. When the polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residues in length. Thus, a polypeptide includes 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 multi-molecular 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. In most cases, disulfide bonds are found in multi-chain polypeptides. The term "polypeptide" can also be applied to amino acid polymers in which one or more amino acid residues are artificial chemical analogs of the corresponding naturally occurring amino acids.

[0052] 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. Usually, variants have at least about 50% identity (homology) with the native or reference sequence, and preferably, they are at least about 80%, more preferably at least about 90% identical (homology) to the native or reference sequence.

[0053] In some embodiments, a "mutant mimic" is provided. As used herein, the term "mutant mimic" refers to one that contains one or more amino acids that mimic activation sequences. For example, glutamate can act as a mimic of phosphoro-threonine and / or phosphoro-serine. Alternatively, a mutant mimic can cause the inactivation or inactivation of the product that contains the mimic, for example, phenylalanine can act as an inactivating substitute for tyrosine, or alanine can act as an inactivating substitute for serine.

[0054] "Homology" as applied to amino acid sequences is defined as the percentage of residues in a candidate amino acid sequence that are identical to those 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 although homology depends on the calculation of percent identity, values ​​may vary due to gaps and penalties introduced in the calculation.

[0055] "Homolog" when applied to a polypeptide sequence means a corresponding sequence in another species having a significant degree of identity to a second sequence in the second species.

[0056] "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 of the properties of a parent or starting polypeptide.

[0057] The present invention contemplates several types of compositions based on polypeptides, including variants and derivatives. These include substitution, insertion, deletion, and covalent variants and derivatives. The term "derivative" is used synonymously with the term "variant," but generally refers to a molecule that has been modified and / or changed in any way relative to a reference or starting molecule.

[0058] Therefore, mmRNAs encoding polypeptides containing substitutions, insertions and / or additions, deletions, and covalent modifications to reference sequences, specifically the polypeptide sequences disclosed herein, are included 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-terminus or C-terminus).Sequence tags can be used for peptide purification or localization.Lysines are used to increase peptide solubility or allow biotinylation.Alternatively, amino acid residues located at 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-terminus or N-terminus residues) can be deleted during expression of the sequence, for example, as part of a larger soluble sequence, or can be bound to a solid support, depending on the use of the sequence.

[0059] A "substitution variant," when referring to a polypeptide, is one 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 can be single, where only one amino acid in the molecule has been substituted, or they can be multiple, where two or more amino acids in the same molecule have been substituted.

[0060] As used herein, the term "conservative amino acid substitution" refers to the replacement of an amino acid normally present in a sequence with a different amino acid having a similar size, charge, or polarity. Examples of conservative substitutions include the replacement of a non-polar (hydrophobic) residue, such as isoleucine, valine, and leucine, with another non-polar residue. Similarly, examples of conservative substitutions include the replacement of a polar (hydrophilic) residue, such as arginine with lysine, glutamine with asparagine, and glycine with a polar (hydrophilic) residue, such as serine, with another. Furthermore, the replacement of a basic residue, such as lysine, arginine, or histidine, with another basic residue, or the replacement of an acidic residue, such as aspartic acid or glutamic acid, with another acidic residue, are further examples of conservative substitutions. Examples of non-conservative substitutions include the replacement of a non-polar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, methionine, with a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, and / or the replacement of a polar residue with a non-polar residue.

[0061] An "insertion variant," when referring to a polypeptide, is one in which one or more amino acids are 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 α-carboxy or α-amino functionality of the amino acid.

[0062] A "deletion variant," when referring to a polypeptide, is one in which one or more amino acids in a native or starting amino acid sequence have been removed. Typically, deletion variants have one or more amino acids deleted in a particular region of the molecule.

[0063] "Covalent derivatives", when referring to polypeptides, include modifications of native or starting proteins with organic proteinaceous or non-proteinaceous derivatizing agents and / or post-translational modifications. Covalent modifications are traditionally introduced by reacting targeted amino acid residues of proteins with organic derivatizing agents capable of reacting with selected side chains or terminal residues, or by exploiting 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.

[0064] 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 the polypeptides produced according to the invention.

[0065] Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl groups of seryl or threonyl residues, and methylation of the α-amino groups of lysine, arginine, and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)).

[0066] A "feature," when referring to a polypeptide, is defined as a distinct amino acid sequence-based component of a molecule. Features of the polypeptides encoded by the mmRNA of the present invention include surface appearance, local conformational shape, folds, loops, semi-loops, domains, semi-domains, sites, termini, or any combination thereof.

[0067] As used herein, when referring to a polypeptide, the term "surface occurrence" refers to the appearance of a polypeptide component of the protein on the outermost surface.

[0068] 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.

[0069] As used herein, when referring to a polypeptide, the term "folding" refers to the resulting conformation of an amino acid sequence upon energy minimization. Folding can occur at the secondary or tertiary levels of the folding process. Examples of secondary level folds include β-sheets and α-helices. Examples of tertiary level folds include domains and regions formed due to the aggregation or separation of energy forces. Regions thus formed include hydrophobic and hydrophilic pockets, etc.

[0070] As used herein, the term "turn" when referring to a protein structure means a bend that changes the orientation of the peptide or polypeptide backbone and may involve one, two, three or more amino acid residues.

[0071] As used herein and 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. have identified at least five classes of protein loops (J. Mol Biol 266(4):814-830; 1997). A loop can be an open loop or a closed loop. A closed or "cyclic" loop can contain 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more amino acids between the bridging moieties. Such bridging moieties can include cysteine-cysteine ​​bridges (Cys-Cys) typical in polypeptides with disulfide bridges, or alternatively, the bridging moieties can be non-proteinaceous, such as dibromozylyl agents, as used herein.

[0072] 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 specified 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, in the case where a loop contains or is specified as containing an odd number of amino acids, a half loop of an odd number of loops includes an integer part of the loop or the next integer part (number of amino acids of loop / 2+ / -0.5 amino acids). For example, a loop specified as a 7 amino acid loop can result in a half loop of 3 amino acids or 4 amino acids (7 / 2=3.5+ / -0.5 to be 3 or 4).

[0073] 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).

[0074] As used herein and referring to a polypeptide, the term "half-domain" refers to a portion of a domain that has at least half of the amino acid residues specified 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, in cases where a domain is specified as containing or containing an odd number of amino acids, a half-domain of an odd-numbered domain includes an integer portion of the domain or the next integer portion (number of amino acids in the domain / 2+ / -0.5 amino acids). For example, a domain specified as a 7 amino acid domain may result in a half-domain of 3 amino acids or 4 amino acids (7 / 2=3.5+ / -0.5 to 3 or 4). It is also understood that sub-domains may be specified within a domain or half-domain, and these sub-domains may have structural or functional properties that are less than all of the structural or functional properties specified in the domain or half-domain from which they are derived. It is also understood that the amino acids that comprise any of the domain types herein need not be adjacent along the backbone of the polypeptide (i.e., non-adjacent amino acids may be structurally folded to result in a domain, half-domain, or sub-domain).

[0075] As used herein, when referring to polypeptides, the term "site" is used synonymously with "amino acid residue" and "amino acid side chain" when referring to amino acid-based embodiments. A site represents a position within a peptide or polypeptide that can be modified, manipulated, altered, derivatized, or changed within the polypeptide-based molecules of the invention.

[0076] As used herein, when referring to a polypeptide, the term "terminus" or "terminus" refers to the end of a peptide or polypeptide. Such ends are not limited to the first or last portion of a peptide or polypeptide, but may also include additional amino acids in the terminal region. The polypeptide-based molecules of the invention may be characterized as having both an N-terminus (terminated by an amino acid with a free amino group (NH2)) and a C-terminus (terminated by an amino acid with a free carboxyl group (COOH)). The proteins of the invention may in some cases consist of multiple polypeptide chains linked by disulfide bonds or non-covalent forces (multimers, oligomers). These types of proteins have multiple N-terminus and C-terminus. Alternatively, the ends of the polypeptides may be modified so that they begin or end with non-polypeptide-based moieties, such as organic complexes, as the case may be.

[0077] Once any of these features are identified or defined as desired components of the polypeptide encoded by the primary construct or mmRNA of the present invention, any of the manipulations and / or modifications of these features can be performed by moving, exchanging, inverting, deleting, randomizing, or duplicating.Furthermore, it is understood that the manipulation of the features will have the same result as the modification to the molecule of the present invention.For example, manipulation involving the deletion of a domain will result in the modification of the length of the molecule, such as the modification of a nucleic acid that codes for less than the full length molecule.

[0078] Modification and manipulation 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 assays known in the art.

[0079] In accordance with the present invention, a polypeptide may comprise a consensus sequence discovered by a series of experiments. As used herein, a "consensus" sequence is a single sequence that represents a collective collection of sequences that allows for variability at one or more sites.

[0080] As will be recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered within the scope of the polypeptide 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 otherwise identical) of a reference protein that is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 amino acids long is provided herein.In another example, any protein that comprises a stretch of about 20, about 30, about 40, about 50, or about 100 amino acids that are about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein can be utilized according to the present invention.In certain embodiments, the polypeptide utilized according to the present invention comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations shown in any of the sequences provided or referenced herein.

[0081] Encoded Polypeptides 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, targeting 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.

[0082] In one embodiment, the primary construct or mmRNA can code for a mutant 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: 8922-17687 disclosed herein, for example, SEQ ID NOs: 1827, 1828, 1829, 1830, 1831, 1832, 1833, 1834, 1835, 1836, 1837, 1838, 1839, 1840, 1841, 1842, 1843, 1844, 1845, 1846, 1847, 1848, 1849, 1850, 1851, 1852, 1853, 1854, 1855, 1856, 1857, , 1858, 1859, 1860, 1861, 1862, 1863, 1864, 1865, 1866, 1867, 1868, 1869, 1870, 1871, 1872, 1873, 1874, 1875, 1876, 1877, 1878, 1879, 1880, 1881, 1882, 1883, 1884, 1885, 1886, 1887, 1888, 1889, 1890, 1891, 1892, 1893, 1894, 1895, 1896, 1897, 1898, 1899, 190 0, 1901, 1902, 1903, 1904, 1905, 1906, 1907, 1908, 1909, 1910, 1911, 1912, 1913, 1914, 1915, 1916, 1917, 1918, 1919, 1920, 1921, 1922, 1923, 1924, 1925, 1926, 1927, 1928, 1929, 1930, 1931, 1932, 1933, 1934, 1935, 1936, 1937, 1938, 1939, 1940, 1941, 1942, 1 943, 1944, 1945, 1946, 1947, 1948, 1949, 1950, 1951, 1952, 1953, 1954, 1955, 1956, 1957, 1958, 1959, 1960, 1961, 1962, 1963, 1964, 1965, 1966, 1967, 1968, 1969, 1970, 1971, 1972, 1973, 1974, 1975, 1976, 1977, 1978, 1979, 1980, 1981, 1982, 1983, 1984, 1985,1986、1987、1988、1989、1990、1991、1992、1993、1994、1995、1996、1997、1998、1999、2000、2001、2002、2003、2004、2005、2006、2007、2008、2009、2010、2011、2012、2013、2014、2015、2016、2017、2018、2019、2020、2021、2022、2023、2024、2025、2026、2027、2028、2029、2030、2031、2032、2033、2034、2035、2036、2037、2038、2039、2040、2041、2042、2043、2044、2045、2046、2047、2048、2049、2050、2051、2052、2053、2054、2055、2056、2057、2058、2059、2060、2061、2062、2063、2064、2065、2066、2067、2068、2069、2070、2071、2072、2073、2074、2075、2076、2077、2078、2079、2080、2081、2082、2083、2084、2085、2086、2087、2088、2089、2090、2091、2092、2093、2094、2095、2096、2097、2098、2099、2100、2101、2102、2103、2104、2105、2106、2107、2108、2109、2110、2111、2112、2113、2114、2115、2116、2117、2118、2119、2120、2121、2122、2123、2124、2125、2126、2127、2128、2129、2130、2131、2132、2133、2134、2135、2136、2137、2138、2139、2140、2141、2142、2143、2144、2145、2146、2147、2148、2149、2150、2151、2152、2153、2154、2155、2156、2157、2158、2159、2160、2161、2162、2163、2164、2165、2166、2167、2168、2169、2170、2171、2172、2173、2174、2175、2176、2177、2178、2179、2180、2181、2182、2183、2184、2185、2186、2187、2188、2189、2190、2191、2192、2193、2194、2195、2196、2197、2198、2199、2200、2201、2202、2203、2204、2205、2206、2207、2208、2209、2210、2211、2212、2213、2214、2215、2216、2217、2218、2219、2220、2221、2222、2223、2224、2225、2226、2227、2228、2229、2230、2231、2232、2233、2234、2235、2236、2237、2238、2239、2240、2241、2242、2243、2244、2245、2246、2247、2248、2249、2250、2251、2252、2253、2254、2255、2256、2257、2258、2259、2260、2261、2262、2263、2264、2265、2266、2267、2268、2269、2270、2271、2272、2273、2274、2275、2276、2277、2278、2279、2280、2281、2282、2283、2284、2285、2286、2287、2288、2289、2290、2291、2292、2293、2294、2295、2296、2297、2298、2299、2300、2301、2302、2303、2304、2305、2306、2307、2308、2309、2310、2311、2312、2313、2314、2315、2316、2317、2318、2319、2320、2321、2322、2323、2324、2325、2326、2327、2328、2329、2330、2331、2332、2333、2334、2335、2336、2337、2338、2339、2340、2341、2342、2343、2344、2345、2346、2347、2348、2349、2350、2351、2352、2353、2354、2355、2356、2357、2358、2359、2360、2361、2362、2363、2364、2365、2366、2367、2368、2369、2370、2371、2372、2373、2374、2375、2376、2377、2378、2379、2380、2381、2382、2383、2384、2385、2386、2387、2388、2389、2390、2391、2392、2393、2394、2395、2396、2397、2398、2399、2400、2401、2402、2403、2404、2405、2406、2407、2408、2409、2410、2411、2412、2413、2414、2415、2416、2417、2418、2419、2420、2421、2422、2423、2424、2425、2426、2427、2428、2429、2430、2431、2432、2433、2434、2435、2436、2437、2438、2439、2440、2441、2442、2443、2444、2445、2446、2447、2448、2449、2450、2451、2452、2453、2454、2455、2456、2457、2458、2459、2460、2461、2462、2463、2464、2465、2466、2467、2468、2469、2470、2471、2472、2473、2474、2475、2476、2477、2478、2479、2480、2481、2482、2483、2484、2485、2486、2487、2488、2489、2490、2491、2492、2493、2494、2495、2496、2497、2498、2499、2500、2501、2502、2503、2504、2505、2506、2507、2508、2509、2510、2511、2512、2513、2514、2515、2516、2517、2518、2519、2520、2521、2522、2523、2524、2525、2526、2527、2528、2529、2530、2531、2532、2533、2534、2535、2536、2537、2538、2539、2540、2541、2542、2543、2544、2545、2546、2547、2548、2549、2550、2551、2552、2553、2554、2555、2556、2557、2558、2559、2560、2561、2562、2563、2564、2565、2566、2567、2568、2569、2570、2571、2572、2573、2574、2575、2576、2577、2578、2579、2580、2581、2582、2583、2584、2585、2586、2587、2588、2589、2590、2591、2592、2593、2594、2595、2596、2597、2598、2599、2600、2601、2602、2603、2604、2605、2606、2607、2608、2609、2610、2611、2612、2613、2614、2615、2616、2617、2618、2619、2620、2621、2622、2623、2624、2625、2626、2627、2628、2629、2630、2631、2632、2633、2634、2635、2636、2637、2638、2639、2640、2641、2642、2643、2644、2645、2646、2647、2648、2649、2650、2651、2652、2653、2654、2655、2656、2657、2658、2659、2660、2661、2662、2663、2664、2665、2666、2667、2668、2669、2670、2671、2672、2673、2674、2675、2676、2677、2678、2679、2680、2681、2682、2683、2684、2685、2686、2687、2688、2689、2690、2691、2692、2693、2694、2695、2696、2697、2698、2699、2700、2701、2702、2703、2704、2705、2706、2707、2708、2709、2710、2711、2712、2713、2714、2715、2716、2717、2718、2719、2720、2721、2722、2723、2724、2725、2726、2727、2728、2729、2730、2731、2732、2733、2734、2735、2736、2737、2738、2739、2740、2741、2742、2743、2744、2745、2746、2747、2748、2749、2750、2751、2752、2753、2754、2755、2756、2757、2758、2759、2760、2761、2762、2763、2764、2765、2766、2767、2768、2769、2770、2771、2772、2773、2774、2775、2776、2777、2778、2779、2780、2781、2782、2783、2784、2785、2786、2787、2788、2789、2790、2791、2792、2793、2794、2795、2796、2797、2798、2799、2800、2801、2802、2803、2804、2805、2806、2807、2808、2809、 2810、2811、2812、2813、2814、2815、2816、2817、2818、2819、2820、2821、2822、2823、2824、2825、2826、2827、2828、2829、2830、2831、2832、2833、2834、2835、2836、2837、2838、2839、2840、2841、2842、2843、2844、2845、2846、2847、2848、2849、2850、2851、2852、2853、2854、2855、2856、2857、2858、2859、2860、2861、2862、2863、2864、2865、2866、2867、2868、2869、2870、2871、2872、2873、2874、2875、2876、2877、2878、2879、2880、2881、2882、2883、2884、2885、2886、2887、2888、2889、2890、2891、2892、2893、2894、2895、2896、2897、2898、2899、2900、2901、2902、2903、2904、2905、2906、2907、2908、2909、2910、2911、2912、2913、2914、2915、2916、2917、2918、2919、2920、2921、2922、2923、2924、2925、2926、2927、2928、2929、2930、2931、2932、2933、2934、2935、2936、2937、2938、2939、2940、2941、2942、2943、2944、2945、2946、2947、2948、2949、2950、2951、2952、2953、2954、2955、2956、2957、2958、2959、2960、2961、2962、2963、2964、2965、2966、2967、2968、2969、2970、2971、2972、2973、2974、2975、2976、2977、2978、2979、2980、2981、2982、2983、2984、2985、2986、2987、2988、2989、2990、2991、2992、2993、2994、2995、2996、2997、2998、2999、3000、3001、3002、3003、3004、3005、3006、3007、3008、3009、3010、3011、3012、3013、3014、3015、3016、3017、3018、3019、3020、3021、3022、3023、3024、3025、3026、3027、3028、3029、3030、3031、3032、3033、3034、3035、3036、3037、3038、3039、3040、3041、3042、3043、3044、3045、3046、3047、3048、3049、3050、3051、3052、3053、3054、3055、3056、3057、3058、3059、3060、3061、3062、3063、3064、3065、3066、3067、3068、3069、3070、3071、3072、3073、3074、3075、3076、3077、3078、3079、3080、3081、3082、3083、3084、3085、3086、3087、3088、3089、3090、3091、3092、3093、3094、3095、3096、3097、3098、3099、3100、3101、3102、3103、3104、3105、3106、3107、3108、3109、3110、3111、3112、3113、3114、3115、3116、3117、3118、3119、3120、3121、3122、3123、3124、3125、3126、3127、3128、3129、3130、3131、3132、3133、3134、3135、3136、3137、3138、3139、3140、3141、3142、3143、3144、3145、3146、3147、3148、3149、3150、3151、3152、3153、3154、3155、3156、3157、3158、3159、3160、3161、3162、3163、3164、3165、3166、3167、3168、3169、3170、3171、3172、3173、3174、3175、3176、3177、3178、3179、3180、3181、3182、3183、3184、3185、3186、3187、3188、3189、3190、3191、3192、3193、3194、3195、3196、3197、3198、3199、3200、3201、3202、3203、3204、3205、3206、3207、3208、3209、3210、3211、3212、3213、3214、3215、3216、3217、3218、3219、3220、3221、3222、3223、3224、3225、3226、3227、3228、3229、3230、3231、3232、3233、3234、3235、3236、3237、3238、3239、3240、3241、3242、3243、3244、3245、3246、3247、3248、3249、3250、3251、3252、3253、3254、3255、3256、3257、3258、3259、3260、3261、3262、3263、3264、3265、3266、3267、3268、3269、3270、3271、3272、3273、3274、3275、3276、3277、3278、3279、3280、3281、3282、3283、3284、3285、3286、3287、3288、3289、3290、3291、3292、3293、3294、3295、3296、3297、3298、3299、3300、3301、3302、3303、3304、3305、3306、3307、3308、3309、3310、3311、3312、3313、3314、3315、3316、3317、3318、3319、3320、3321、3322、3323、3324、3325、3326、3327、3328、3329、3330、3331、3332、3333、3334、3335、3336、3337、3338、3339、3340、3341、3342、3343、3344、3345、3346、3347、3348、3349、3350、3351、3352、3353、3354、3355、3356、3357、3358、3359、3360、3361、3362、3363、3364、3365、3366、3367、3368、3369、3370、3371、3372、3373、3374、3375、3376、3377、3378、3379、3380、3381、3382、3383、3384、3385、3386、3387、3388、3389、3390、3391、3392、3393、3394、3395、3396、3397、3398、3399、3400、3401、3402、3403、3404、3405、3406、3407、3408、3409、3410, 3411, 3412, 3413, 3414, 3415, 3416, 3417, 3418, 3419, 3420, 3421, 3422, 3423, 3424, 3425, 3426, 3427, 3428, 3429, 3430, 3431, 3432, 3433, 3434, 3435, 3436, 3437, 3438, 3439, 3440, 3441, 3442, 3443, 3444, 3445, 3446, 3447, 3448, 3449, 3450, 3451, 3452, 3453, 3454, 3 455, 3456, 3457, 3458, 3459, 3460, 3461, 3462, 3463, 3464, 3465, 3466, 3467, 3468, 3469, 3470, 3471, 3472, 3473, 3474, 3475, 3476, 3477, 3478, 3479, 3480, 3481, 3482, 3483, 3484, 3485, 3486, 3487, 3488, 3489, 3490, 3491, 3492, 3493, 3494, 3495, 3496, 3497.

[0083] 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 means the degree of sequence relatedness between peptides as determined by the number of matches between strings of two or more amino acid residues. Identity measures the percentage of identical matches between the smaller of two or more sequences with gap alignment (if any) as processed by a particular mathematical model or computer program (i.e., "algorithm"). The identity of related peptides can be easily calculated by known methods. Such methods include those described in 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; 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).

[0084] 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 a reference polypeptide. Generally, a variant of a particular polynucleotide or polypeptide of the invention will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, but less than 100% sequence identity with a variant of a particular reference polynucleotide or polypeptide, as determined by sequence alignment programs and parameters described herein and known to those of skill in the art. Such alignment tools include the BLAST suite of programs (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaeffer, 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".

[0085] Default parameters for the BLAST algorithm include, for example, an expectation threshold of 10, a word size of 28, a match / mismatch score of 1, -2, and a linear gap cost. Any filter can be applied, as well as a selection of species-specific repeat sequences, e.g., human-specific repeat sequences.

[0086] Biologics The polynucleotide, primary construct, or mmRNA disclosed herein may code for 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. According to 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, etc.

[0087] 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 the incorporation of polynucleotides encoding known biologics into the primary constructs or mmRNA of the present invention provides improved therapeutic efficacy, at least in part, due to the specificity, purity, and / or selectivity of construct design.

[0088] 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 with immunoglobulin Fc regions), 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 homogenous 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.

[0089] 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 of 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 of antibodies derived from another species or belonging to another antibody class or subclass, so 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.

[0090] 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.

[0091] Any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, including heavy chains designated α, δ, ε, γ, and μ, respectively, may 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 may be encoded, in part or in whole, including the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0092] 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 the mmRNA design.

[0093] Antibodies encoded in the polynucleotides, primary constructs, or mmRNA of the 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.

[0094] In one embodiment, the primary constructs and / or mmRNAs disclosed herein may encode monoclonal antibodies and / or variants thereof. Antibody variants may also 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 mmRNAs disclosed herein may encode immunoglobulin Fc regions. In another embodiment, the primary constructs and / or mmRNAs may encode variant immunoglobulin Fc regions. As a non-limiting example, the primary constructs and / or mmRNAs may encode antibodies having variant immunoglobulin Fc regions as described in U.S. Pat. No. 8,217,147, the entirety of which is incorporated herein by reference.

[0095] vaccine The primary construct or mmRNA disclosed herein may code for one or more vaccines. As used herein, a "vaccine" is a biological preparation that enhances immunity against a particular disease or infectious agent. In accordance with the present invention, one or more vaccines currently on the market or under development may be coded by the polynucleotide, primary construct, or mmRNA of the present invention. Without wishing to be bound by theory, it is believed that the incorporation of the primary construct 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.

[0096] 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.

[0097] Therapeutic Proteins or Peptides The primary constructs or mmRNA disclosed herein may encode one or more validated or "test" therapeutic proteins or peptides.

[0098] 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 the construct design.

[0099] Therapeutic proteins and peptides encoded in the polynucleotides, primary constructs, or mmRNA of the present invention can be utilized to treat conditions or diseases in many therapeutic areas, including, but not limited to, hematology, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, genetics, genitourinary, gastrointestinal, musculoskeletal, oncology, and immunology, respiratory, sensory, and anti-infective.

[0100] Cell-penetrating polypeptides The primary construct or mmRNA disclosed herein may code for 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 at all code for a detectable label. The cell-penetrating peptide may also include a signal sequence. As used herein, "signal sequence" refers to a sequence of amino acid residues that is 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.

[0101] In one embodiment, the polynucleotide, primary construct, or mmRNA may also code for a fusion protein. A fusion protein may be created by operably linking a charged protein to a therapeutic protein. As used herein, "operably linked" refers to a combination that allows for the expression of a complex when the therapeutic protein and the charged protein are introduced into a cell. As used herein, "charged protein" refers to a protein that has a positive charge, a negative charge, or an overall neutral charge. Preferably, the therapeutic protein may be covalently linked to the charged protein upon formation of the fusion protein. The ratio of surface charge to total or surface amino acids may be about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, or 0.9.

[0102] The cell-penetrating polypeptide encoded by polynucleotide, primary construct, or mmRNA can form a complex after translation. This complex can include a charged protein bound, for example, covalently bound, to the cell-penetrating polypeptide. "Therapeutic protein" refers to a protein that has therapeutic, diagnostic, and / or preventive effects and / or induces desired biological and / or pharmacological effects when administered to a cell.

[0103] 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, an antibody and its functional fragments, a scaffold protein, or a peptide. 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 the polynucleotide, the primary construct, or the mmRNA may be introduced. The cell-penetrating polypeptide may also be capable of penetrating a first cell.

[0104] In further embodiments, the cell-penetrating polypeptide can penetrate into the 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 proximal to the first cell or distal to it.

[0105] In one embodiment, the polynucleotide, primary construct, or mmRNA can code for a cell-permeable 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-permeable polypeptide that includes the protein-binding partner is introduced.

[0106] 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 function of the organelle. The cell uses "sorting signals," amino acid motifs located within proteins, to target proteins to specific organelles.

[0107] One 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).

[0108] Proteins targeted to the ER by signal sequences can be released into the extracellular space as secretory 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 take advantage of the above-mentioned cellular transport. Thus, in some embodiments of the present invention, polynucleotides, primary constructs, or mmRNAs that express secretory proteins are provided. Secretory proteins can be selected from those described herein or in US Patent Publication No. 20100255574, the contents of which are incorporated herein by reference in their entirety.

[0109] In one embodiment, they may be used in the production of large amounts of valuable human gene products.

[0110] Plasma membrane proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNA that express plasma membrane proteins are provided.

[0111] Cytoplasmic or cytoskeletal proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNA are provided that express cytoplasmic or cytoskeletal proteins.

[0112] Intracellular membrane-associated proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNA are provided that express intracellular membrane-associated proteins.

[0113] Nuclear proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNA that express nuclear proteins are provided.

[0114] Human disease-related proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNA are provided that express proteins associated with human diseases.

[0115] Various proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mmRNA are provided that express proteins with currently unknown therapeutic functions.

[0116] 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 serve to 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.

[0117] 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 a plurality of 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 polynucleotide is a primary construct 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.

[0118] In certain embodiments, multiple variants of a protein, each with a different amino acid modification(s), can be produced and tested to determine the best variant in terms of pharmacokinetics, stability, biocompatibility, and / or biological activity, or biophysical properties such as expression levels. Such libraries can be as small as 10, 100, 1000, or 2000 variants. 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 mutation (including, but not limited to, the substitution, deletion, and insertion of one or more residues).

[0119] Antibacterial and Antiviral Polypeptides The polynucleotides, primary constructs, and mmRNA of the present invention may 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 No. 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 Nos. 6,887,847, 6,906,035, 6,911,524, 6,936,432, 7,001,924, 7,071,293, 7,078,380, 7,091,185, 7,094,759, 7,166,769, 7,244,710, 7,314,858, and 7,582,301, the contents of which are incorporated by reference in their entireties.

[0120] 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., HIV-1 gp120 or gp41 transmembrane subunit. 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.

[0121] 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.

[0122] 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 homology to the corresponding sequence of the capsid binding protein.

[0123] 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%, 100% sequence homology to the corresponding viral protein sequence.

[0124] 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 a binding domain of a protease binding protein, hi some embodiments, the antimicrobial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, 100% sequence homology to the corresponding sequence of the protease binding protein.

[0125] The antimicrobial polypeptides described herein may include in vitro evolved polypeptides directed against viral pathogens.

[0126] Antimicrobial 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, which is incorporated herein by reference in its entirety). It has been shown that AMPs have a broad range of rapidly developing killing activity and may have low levels of induced resistance and a concomitant broad range of anti-inflammatory effects.

[0127] In some embodiments, the antibacterial polypeptide (e.g., antibacterial polypeptide) may be less than 10 kDa, e.g., 8 kDa, 6 kDa, 4 kDa, 2 kDa, or less than 1 kDa. In some embodiments, the antibacterial 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 antibacterial polypeptide (e.g., antibacterial polypeptide) may consist of about 15 to about 45 amino acids. In some embodiments, the antibacterial polypeptide (e.g., antibacterial polypeptide) is substantially cationic.

[0128] In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may be substantially amphipathic. In certain embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may be substantially cationic and amphipathic. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may be cytostatic to gram-positive bacteria. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may be cytotoxic to gram-positive bacteria. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may be cytostatic and cytotoxic to gram-positive bacteria. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may be cytostatic to gram-negative bacteria. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may be cytotoxic to gram-negative bacteria. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may be cytostatic and cytotoxic to gram-positive bacteria. In some embodiments, the antimicrobial polypeptide may be cytostatic to viruses, fungi, protozoa, parasites, prions, or combinations 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 anti-microbial polypeptide (eg, anti-bacterial polypeptide) can be a secreted polypeptide.

[0129] 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).

[0130] 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 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 to viruses. In some embodiments, the antiviral polypeptide is cytotoxic to viruses. In some embodiments, the antiviral polypeptide is cytostatic and cytotoxic to viruses. In some embodiments, the antiviral polypeptide is cytostatic to bacteria, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, the antiviral polypeptide is cytotoxic to bacteria, fungi, protozoa, parasites, prions, or combinations thereof. In certain embodiments, the antiviral polypeptide is cytostatic and cytotoxic to bacteria, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, the antiviral polypeptide is cytotoxic to tumor or cancer cells (e.g., human cancer cells). In some embodiments, the antiviral polypeptide is cytostatic to tumor or cancer cells (e.g., human cancer cells).In certain embodiments, the anti-viral polypeptide is cytotoxic and cytostatic to tumor or cancer cells (e.g., human cancer cells). In some embodiments, the anti-viral polypeptide is a secreted polypeptide.

[0131] 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 polynucleotide, primary construct 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.

[0132] Several cytotoxic nucleoside analogues are in clinical use or undergoing clinical trials as anticancer drugs. Examples of such analogues include, but are not limited to, cytarabine, gemcitabine, troxacitabine, decitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), cladribine, clofarabine, 5-azacytidine, 4'-thio-aracytidine, cyclopentenylcytosine, and 1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cytosine. Another example of such a compound is fludarabine phosphate. These compounds can be administered systemically and can have typical side effects of cytotoxic drugs, including, but not limited to, little or no specificity for tumor cells over the proliferation of normal cells.

[0133] Some prodrugs of cytotoxic nucleoside analogues 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). In general, these prodrugs are mainly converted to 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" have been claimed by Fujiu et al. (U.S. Patent No. 4,966,891) and is incorporated herein by reference. The series of capecitabine analogs described by Fujiu includes N4 alkyl and aralkyl carbamates of 5'-deoxy-5-fluorocytidine, implying that these compounds are activated by hydrolysis under normal physiological conditions to yield 5'-deoxy-5-fluorocytidine.

[0134] A series of cytarabine N4-carbamates have been reported by Fadl et al. (Pharmazie. 1995, 50, 382-7, incorporated herein by reference), where the compounds were designed to convert to cytarabine in the liver and plasma. International Publication No. WO2004 / 041203, incorporated herein by reference, discloses prodrugs of gemcitabine, where some of the prodrugs are N4-carbamates. These compounds were designed to overcome the gastrointestinal toxicity of gemcitabine, and were intended to provide gemcitabine by hydrolytic release in the liver and plasma after absorbing 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-pentopharanosyl)cytosine that upon bioreduction produced intermediates that required further hydrolysis under acidic conditions to produce cytotoxic nucleoside compounds.

[0135] Cytotoxic nucleotides, which may be chemotherapeutic agents, include, but are not limited to, pyrazolo[3,4-D]-pyrimidines, allopurinol, azathioprine, capecitabine, cytosine arabinoside, fluorouracil, mercaptopurine, 6-thioguanine, acyclovir, ara-adenosine, ribavirin, 7-deaza-adenosine, 7-deaza-guanosine, 6-aza-uracil, 6-aza-cytidine, thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloro-purine, and inosine, or combinations thereof.

[0136] Flanking Regions: Untranslated Regions (UTRs) The untranslated region (UTR) of a gene is transcribed but not translated. The 5'UTR starts at the transcription initiation site and follows the initiation codon but does not include the initiation codon, while the 3'UTR starts immediately after the stop codon and continues until the transcription termination signal. There are increasing reports of the regulatory role that UTR plays in the stability of nucleic acid molecules and translation. The regulatory features of UTRs can be incorporated into the polynucleotides, primary constructs, and / or mmRNA of the present invention to increase the stability of the molecules. Certain features can also be incorporated to ensure the controlled downregulation of transcripts in case they are misdirected to undesired organ sites.

[0137] 5'UTR and translation start Natural 5'UTRs have features involved in translation initiation. They have 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.

[0138] By engineering features typically found in highly expressed genes of a particular target organ, the stability and protein production of the polynucleotides, primary constructs, or mmRNAs of the present invention can be increased. 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 increase the expression of nucleic acid molecules such as mmRNAs in hepatic cell lines or liver. Similarly, 5'UTRs from other tissue-specific mRNAs can be used to increase 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).

[0139] 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.

[0140] 3'UTR and AU-rich elements 3'UTRs are known to have stretches of adenosines and uridines embedded within them. These AU-rich features are particularly prevalent 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 that contain this type of ARE include GM-CSF and TNF-a. 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 thus message stabilization in vivo.

[0141] The introduction, removal, or modification of AU-rich elements (AREs) in the 3'UTR can be used to regulate the stability of the polynucleotides, primary constructs, or mmRNAs of the present invention. When engineering a particular polynucleotide, primary construct, or mmRNA, one or more copies of AREs can be introduced to reduce the stability of the polynucleotides, primary constructs, or mmRNAs of the present invention, thereby suppressing the translation and reducing the production of the resulting protein. Similarly, AREs can be identified and removed or mutated to improve intracellular stability, thus increasing the translation and production of the resulting protein. Transfection experiments with the polynucleotides, primary constructs, or mmRNAs of the present invention can be performed in relevant cell lines, and protein production can be assayed at various times after transfection. For example, cells can be transfected with different ARE-engineered molecules, and the proteins produced can be assayed at 6 hours, 12 hours, 24 hours, 48 ​​hours, and 7 days after transfection using ELISA kits for the relevant proteins.

[0142] 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 down-regulate gene expression by either reducing nucleic acid molecule stability or inhibiting translation. The polynucleotides, primary constructs, or mmRNAs of the 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.

[0143] The microRNA sequence comprises a "seed" region, i.e., a sequence in the region of positions 2-8 of the mature microRNA, which has perfect Watson-Crick complementarity to the miRNA target sequence. The microRNA seed may comprise positions 2-8 or positions 2-7 of the mature microRNA. In some embodiments, the microRNA seed may comprise seven nucleotides (e.g., nucleotides 2-8 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, the microRNA seed may comprise six nucleotides (e.g., nucleotides 2-7 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 bases of microRNA species have perfect complementarity with target sequence. By engineering microRNA target sequence into the 3'UTR of 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).

[0144] For example, if the nucleic acid molecule is an 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 when one or more target sites for miR-122 are engineered into the 3'UTR of a polynucleotide, primary construct, or mmRNA. The introduction of one or more binding sites for a different microRNA can be engineered to further reduce the longevity, stability, and protein translation of the polynucleotide, primary construct, or mmRNA.

[0145] 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.

[0146] Conversely, for the purpose of the polynucleotide, primary construct, or mmRNA of the present invention, naturally occurring microRNA binding sites can be engineered out (i.e., removed) from the sequence to increase protein expression in a particular tissue. For example, miR-122 binding sites 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.

[0147] 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-1d, miR-149), kidney (miR-192, miR-194, miR-204), and lung epithelial cells (let-7, miR-133, miR-126). MicroRNAs may also regulate complex biological processes such as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176, which are incorporated herein by reference in their entirety). In the polynucleotides, primary constructs, or mmRNAs of the present invention, binding sites of microRNAs involved in such processes may be removed or introduced to regulate the expression of the polynucleotides, primary constructs, or mmRNAs to 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.

[0148] Examples of the use of microRNA to drive tissue or disease-specific gene expression have been listed (Getner and Naldini, Tissue Antigens. 2012, 80:393-403, which are incorporated herein by reference in their entirety). In addition, microRNA seed sites can be incorporated into mRNA to reduce expression in certain cells, which leads to biological improvement. An example of this is the incorporation of miR-142 sites into UGT1A1-expressing lentiviral vectors. The presence of miR-142 seed sites reduces expression in hematopoietic cells, which in turn reduces expression in antigen-presenting cells, resulting in the absence of immune response to virus-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 entirety). Incorporation of miR-142 sites into modified mRNAs could not only reduce the expression of the encoded protein in hematopoietic cells, but also reduce or abolish the immune response to the protein encoded by the mRNA. Incorporation of miR-142 seed site(s) into mRNAs is important when treating patients with complete protein deficiency (e.g., UGT1A1 type I, LDLR-deficient patients, CRIM-negative Pompe patients, etc.).

[0149] 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 tissues or in association with biological conditions.

[0150] The transfection experiment using the engineered polynucleotide, primary construct or mmRNA can be carried out in the relevant cell line, and protein production can be assayed at various times after transfection.For example, cells can be transfected with different microRNA binding site engineered polynucleotides, primary constructs or mmRNA, and the protein produced can be assayed at 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours and 7 days after transfection using ELISA kits for related proteins.In vivo experiments can also be carried out with microRNA binding site engineered molecules to test the change in tissue-specific expression of formulated polynucleotides, primary constructs or mmRNA.

[0151] 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 translational competence in cells through the association of CBP with poly(A)-binding protein to form mature circular mRNA species. The cap further assists in the removal of 5'-proximal introns during mRNA splicing.

[0152] Endogenous mRNA molecules can be 5'-end capped to provide a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue and the 5'-terminal transcribed sense nucleotide of the mRNA molecule. This 5'-guanylic acid cap can then be methylated to generate an N7-methyl-guanylic acid residue. The ribose sugar of the terminal and / or pre-terminal transcribed nucleotide at the 5' end of the mRNA can also be optionally 2'-O-methylated. 5' decapping through hydrolysis and cleavage of the guanylic acid cap structure can target nucleic acid molecules, such as mRNA molecules, for degradation.

[0153] The modification of the polynucleotides, primary constructs, and mmRNA of the present invention can generate a non-hydrolyzable cap structure that prevents decapping, thereby increasing the half-life of the mRNA. Since 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 generate phosphorothioate bonds in the 5'-ppp-5' cap. Further modified guanosine nucleotides, such as α-methyl-phosphonate and seleno-phosphate nucleotides, can be used.

[0154] Further modifications include, but are not limited to, 2'-O-methylation of the ribose sugar of the 5'-terminus and / or pre-5'-terminal nucleotide of an mRNA at the 2'-hydroxyl group of the sugar ring (as described above).Several distinct 5'-cap structures can be used to generate the 5' cap of a nucleic acid molecule, such as an mRNA molecule.

[0155] 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 terms of their chemical structure but retain cap function. Cap analogs can be chemically (i.e., non-enzymatically) or enzymatically synthesized and / or attached to a nucleic acid molecule.

[0156] For example, an anti-reverse cap analog (ARCA) cap contains two guanines linked by 5'-5'-triphosphate groups, one of which has 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 contains an N7- and 3'-O-methylated guanine, which may be equivalently 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 provides the terminal portion of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0157] 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, mCAP). 7 Gm-ppp-G).

[0158] While cap analogs allow for capping of nucleic acid molecules occurring concomitantly in in vitro transcription reactions, up to 20% of the transcripts may remain uncapped. This, as well as the structural differences of the cap analog from the endogenous 5'-cap structure of nucleic acids produced by the endogenous cellular transcription machinery, may result in reduced translational competence and reduced cellular stability.

[0159] The polynucleotides, primary constructs, and mmRNA of the present invention can also be post-transcriptionally capped using enzymes to generate a more authentic 5'-cap structure. As used herein, the term "more authentic" refers to a feature that closely resembles or mimics an endogenous or wild-type feature, either structurally or functionally. That is, a "more authentic" feature better represents an endogenous, wild-type, natural, or physiological cellular function and / or structure, or outperforms the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects, compared to synthetic features or analogs of the prior art. Non-limiting examples of the more authentic 5' cap structures of the present invention include those that have, among others, enhanced cap-binding protein binding, increased half-life, decreased susceptibility to 5' endonucleases, and / or decreased 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 translational competence and cellular stability, as well as reduced activation of cellular proinflammatory cytokines, for example, compared to other 5' cap analog structures known in the art. Cap structures include, but are not limited to, 7mG(5')ppp(5')N,pN2p (Cap 0), 7mG(5')ppp(5')NlmpNp (Cap 1), and 7mG(5')-ppp(5')NlmpN2mp (Cap 2).

[0160] Because polynucleotides, primary constructs, or mmRNAs can be post-transcriptionally capped and the process is more efficient, nearly 100% of the polynucleotides, primary constructs, or mmRNAs can be capped, which is in stark contrast to the approximately 80% that occurs when a cap analog is attached to the mRNA during an in vitro transcription reaction.

[0161] According to the present invention, the 5'-end cap can include an endogenous cap or a cap analog. According to the present invention, the 5'-end cap can include 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.

[0162] Viral sequences Additional viral sequences, such as, but not limited to, the translation enhancer sequence of Barley Yellow Dwarf Virus (BYDV-PAV), Jaagsiekte Sheep Retrovirus (JSRV), and / or Enzootic Nasal Tumor Virus (see, for example, International Publication No. WO2012129648, which is incorporated by reference in its entirety), can be engineered and inserted into the 3'UTR of the polynucleotides, primary constructs, or mmRNAs of the invention to stimulate translation of the constructs 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 after transfection.

[0163] IRES sequence Additionally, polynucleotides, primary constructs, or mmRNAs are provided that may contain an internal ribosome entry site (IRES). IRES, identified as the first characteristic picornavirus RNA, plays an important role in initiating protein synthesis in the absence of a 5' cap structure. IRES may serve as the only ribosome binding site of an mRNA, or may serve as one of multiple ribosome binding sites. Polynucleotides, primary constructs, or mmRNAs that contain two or more functional ribosome binding sites may code for several peptides or polypeptides that are translated independently 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 virus (HCV), classical swine fever virus (CSFV), murine leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0164] 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, for example, about 100-250 residues long.

[0165] It has been found that particular polyA tail lengths provide certain advantages to the polynucleotides, primary constructs or mmRNA of the present invention.

[0166] Generally, the length of a polyA tail of the present invention is greater than 30 nucleotides in length. In 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, 100-1,0 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).

[0167] 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 can be based on the length of the final product expressed from the polynucleotide, primary construct, or mmRNA.

[0168] In this context, the polyA tail can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer than the polynucleotide, primary construct, or mmRNA, or features 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 full length of the construct or the full length of the construct minus the polyA tail. Additionally, engineering of binding sites and complexing of the polynucleotide, primary construct, or mmRNA to polyA binding proteins can enhance expression.

[0169] Additionally, multiple distinct polynucleotides, primary constructs, or mmRNAs can be attached to PABP (polyA binding protein) via their 3' ends using nucleotides modified 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.

[0170] In one embodiment, the polynucleotide primary constructs of the invention are designed to include 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 a protein production that is at least 75% equivalent to that produced by a 120 nucleotide polyA tail alone.

[0171] Quantification In one embodiment, the polynucleotide, primary construct, or mmRNA of the present invention can be quantified in exosomes from one or more body fluids.As used herein, "body fluid" 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, chyle, bile, interstitial fluid, leucorrhea, pus, sebum, vomit, vaginal secretions, mucosal secretions, stool, 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.

[0172] 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, immunoabsorption capture, affinity purification, microfluidic separation, or a combination thereof. In the analysis, the level or concentration of polynucleotide, primary construct, or mmRNA can be the expression level, presence, absence, truncation, 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. 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, while exosomes can 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, immunoabsorption capture, affinity purification, microfluidic separation, or a combination thereof.

[0173] 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.

[0174] II. mmRNA Design and Synthesis The polynucleotide, primary construct, or mmRNA used according to 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).

[0175] 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 clean-up process. These steps are provided in more detail below.

[0176] Genetic 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.

[0177] 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 that encodes a polypeptide of interest can be constructed with 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) that can encode a polypeptide of interest. An ORF often begins with an initiation codon ATG and ends with a nonsense or stop codon or signal.

[0178] Furthermore, the nucleotide sequence of the first region may be codon-optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of several goals. These goals include matching codon frequencies in 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 migration that may impair 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 translation rate to allow proper folding of various domains of the protein, or reducing or eliminating problematic secondary structures in the mRNA. Codon optimization tools, algorithms, and services are known in the art, non-limiting examples include GeneArt's 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]

[0179] 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 the first or second flanking region. This flanking region may be incorporated into the primary construct before and / or after optimizing the ORF. It is not necessary for the primary construct to contain both the 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, and may also include multiple cloning sites that may have XbaI recognition.

[0180] In some embodiments, 5'UTR and / or 3'UTR may be provided as flanking regions. Multiple 5' or 3'UTRs may be included in the flanking regions and may be the same or different sequences. Any portion of the flanking regions (including no such portion) may be codon optimized, any of which may independently contain one or more different structural or chemical modifications before and / or after codon optimization. A combination of features may be included in the first and second flanking regions and may be contained within other features. For example, an ORF may be flanked by a 5'UTR that may contain a strong Kozak translation initiation signal and / or a 3'UTR that may contain an oligo(dT) sequence for templated addition of a polyA tail. A 5'UTR, such as the 5'UTR described in US Patent Publication No. 20100293625, which is incorporated herein by reference in its entirety, may contain a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes.

[0181] Tables 2 and 3 provide a list of exemplary UTRs that may 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 may be utilized. Table 2.5' Untranslated area [Table 0002]

[0182] 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]

[0183] It should be understood that the ones listed in the previous table are examples, and any UTR from any gene can be incorporated into each of the first or second flanking regions of the primary construct. In addition, multiple wild-type UTRs of 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 parts thereof can be placed in the same orientation as the UTR or parts thereof of the transcript from which they can be selected, or the orientation or position can be changed. Thus, 5' or 3' UTRs can be inverted, shortened, extended, chimerized with one or more other 5' or 3' UTRs. The term "modified" as used herein, when referring to UTR sequences, means that the UTR has changed in some respect relative to a reference sequence. For example, 3' or 5' UTRs can be modified relative to wild-type or natural UTRs by changing the orientation or position as taught above, or can be modified 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.

[0184] In one embodiment, double, triple, or quadruple UTRs, such as 5' or 3' UTRs, 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' UTRs described in US Patent Publication No. 20100129877, the contents of which are incorporated herein by reference in their entirety, can be used.

[0185] It is also within the scope of the present invention to have patterned UTR.As used herein, "patterned UTR" refers to a UTR that shows a repeating or alternating pattern that repeats once, twice, or more than three times, such as ABABAB or AABBABBAABB or ABCABCABC or its variants.In these patterns, each letter A, B, or C represents a different UTR at the nucleotide level.

[0186] In one embodiment, the flanking region is selected from a family of transcripts whose proteins share common function, structure, characteristic features.For example, the polypeptide of interest may belong to a family of proteins that are expressed in a particular cell, tissue, or at a certain time during development. Any UTR of these genes may be exchanged with any other UTR of the same or different protein family to create a new chimeric primary transcript.As used herein, "family of proteins" is used in the broadest sense to refer to a group of two or more polypeptides of interest that share at least one function, structure, characteristic, localization, origin, or expression pattern.

[0187] 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 are generated by the methods described herein.

[0188] 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 Publication No. 20090226470, the entirety of which is incorporated herein by reference, and TEEs known in the art.

[0189] Stop codon In one embodiment, the primary construct of the present invention may include 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 includes 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 includes three stop codons.

[0190] 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, Calif.).

[0191] Plasmid linearization The plasmid may then be linearized using methods known in the art, such as, but not limited to, the use of restriction enzymes and buffers. The linearized reaction may be purified using methods including, 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, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), as well as Invitrogen's standard PURELINK™ PCR Kit (Carlsbad, CA). Purification methods may 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.

[0192] cDNA template synthesis The cDNA template can be synthesized by subjecting the linearized plasmid to polymerase chain reaction (PCR). Table 4 is a list of primers and probes that can be useful in the PCR reaction 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 can also contain chemically modified bases to enhance base pairing fidelity and strength to the target molecule. Such modifications can include 5-methyl-cytidine, 2,6-di-amino-purine, 2'-fluoro, phosphoro-thioate, or locked nucleic acid. Table 4. Primers and probes [Table 0004] *UFP is the universal forward primer and URP is the universal reverse primer.

[0193] In one embodiment, the cDNA may be submitted for sequencing analysis prior to undergoing transcription.

[0194] 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.

[0195] 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), RNase inhibitors, and a polymerase. The NTPs can be made 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 non-natural (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 that can incorporate modified nucleic acids.

[0196] RNA polymerase Any number of RNA polymerases or variants may be used in designing the primary constructs of the present invention.

[0197] RNA polymerase can be modified by inserting or deleting amino acids of 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).

[0198] The 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, which is incorporated herein by reference in its entirety), to produce T7 The RNA polymerase clones contained the following mutations: lysine at position 93 substituted 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 K416I. 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, the 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.

[0199] In one embodiment, the primary constructs can be designed to be recognized by a wild-type or mutant RNA polymerase, and thus can be modified to contain sites or regions of sequence variation from the wild-type or parent primary constructs.

[0200] In one embodiment, a 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.

[0201] In one embodiment, the 5'UTR of the primary construct may be replaced by the insertion of at least one region and / or string of nucleotides of the same base. The region and / or string of nucleotides may include, but is not limited to, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 nucleotides, which may be natural and / or non-natural. As a non-limiting example, the nucleotide group may include a string of 5-8 adenines, cytosines, thymines, any of the other nucleotides disclosed herein, and / or combinations thereof.

[0202] 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.

[0203] In one embodiment, the primary construct may include at least one substitution and / or insertion downstream of the transcription start site that may be recognized by an RNA polymerase. As a non-limiting example, the at least one substitution and / or insertion may occur downstream of the transcription start site by substituting at least one nucleic acid in 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 may affect the initiation rate, increase the apparent nucleotide triphosphate (NTP) reaction constant, and enhance 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). Modification, substitution, and / or insertion of at least one nucleic acid may cause silent mutations in the nucleic acid sequence or may cause mutations in the amino acid sequence.

[0204] 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.

[0205] In one embodiment, the primary construct may include at least one, at least two, at least three, at least four, at least five, or at least six guanine base substitutions in the region immediately downstream of the transcription start site. As a non-limiting example, when the nucleotides in the region are GGGAGA, the guanine bases may be replaced by at least one, at least two, at least three, or at least four adenine nucleotides. In another non-limiting example, when the nucleotides in the region are GGGAGA, the guanine bases may be replaced by at least one, at least two, at least three, or at least four cytosine bases. In another non-limiting example, when the nucleotides in the region are GGGAGA, the guanine bases may be replaced by at least one, at least two, at least three, or at least four thymines, and / or any of the nucleotides described herein.

[0206] 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 of 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 or all C or all T or all G), two different bases (e.g., A and C, A and T, or C and T), three different bases (e.g., A, C, and T, or A, C, and T), or at least four different bases. As a non-limiting example, the guanine base upstream of the coding region in the primary construct can be replaced with adenine, cytosine, thymine, or any of the nucleotides described herein.In another non-limiting example, the replacement of the guanine base in the primary construct can be designed to leave one guanine base in the region downstream of the transcription start site and 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.

[0207] 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, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC).

[0208] Capping and / or tailing reactions The primary construct or mmRNA may also undergo a capping and / or tailing reaction. The capping reaction may be carried out using methods known in the art to add a 5' cap to the 5' end of the primary construct. Methods for capping include, but are not limited to, the use of vaccinia capping enzyme (New England Biolabs, Ipswich, MA).

[0209] 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.

[0210] mRNA purification Primary construct or mmRNA purification may include, but is not limited to, mRNA or mmRNA purification, quality assurance, and quality control. mRNA or mmRNA purification may 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, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term "purified," such as "purified mRNA or mmRNA," when used in relation 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 it is found in nature or that is different from the form or environment in which it existed prior to being subjected to a treatment or purification method.

[0211] 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.

[0212] In another embodiment, mRNA or mmRNA may be sequenced using methods including, but not limited to, reverse transcription PCR.

[0213] In one embodiment, the mRNA or mmRNA may 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). The quantified mRNA or mmRNA may be analyzed to determine if the mRNA or mmRNA may be of appropriate size and to ensure that no degradation of the mRNA or mmRNA has occurred. Degradation of the mRNA and / or mmRNA may 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, reversed-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC); liquid chromatography mass spectrometry (LCMS); capillary electrophoresis (CE); and capillary gel electrophoresis (CGE).

[0214] 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 9-200 nucleotides in length (3-60 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.

[0215] Table 5 is a representative list of protein signal sequences that may be incorporated to encode the polynucleotides, primary constructs, 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]

[0216] In this table, SS is a secretion signal and MLS is a mitochondrial leader signal. The primary constructs or mmRNA of the invention may be designed to encode any of the signal sequences of SEQ ID NOs: 94-155, or fragments or variants thereof. These sequences may be included at the beginning, middle, or end of the polypeptide coding region, or alternatively in a flanking region. Additionally, any of the polynucleotide primary constructs of the invention may also include one or more of the sequences defined by SEQ ID NOs: 32-93. These may be present in the first region or in any of the flanking regions.

[0217] Additional signal sequences that may be utilized in the present invention include, for example, signal sequences 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.

[0218] Target Selection In accordance with the present invention, the primary construct comprises at least one first region of linked nucleosides that encodes 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 (target description), the ENSEMBL transcript sequence number (ENST), the ENSEMBL protein sequence number (ENSP), and, where available, the optimized open reading frame sequence number (optimized ORF sequence number) are also shown in Table 6. For any particular gene, one or more variants or isoforms may exist. If these exist, they are also shown in the table. Those skilled in the art will understand that what is disclosed in the table are possible flanking regions. These are encoded either 5' (upstream) or 3' (downstream) of the ORF or coding region in each ENST transcript. The coding regions are categorically and specifically disclosed by teaching the ENSP sequence. As a result, the adjacent sequences of the teachings that encode proteins are considered flanking regions. The 5' and 3' flanking regions can also 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]

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[0219] Protein cleavage signals and sites In one embodiment, the polypeptide of the present invention may comprise at least one proteolytic signal containing at least one proteolytic cleavage site. The proteolytic cleavage site 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 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.

[0220] The 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 called subtilisin kexin isozyme 1 (SKI-1) and proprotein convertase subtilisin kexin 9 (PCSK9). Non-limiting examples of protein cleavage signal amino acid sequences are 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 protein cleavage site. In Table 7, SEQ ID NO: 61636 refers to when n is 4, and SEQ ID NO: 61637 refers to when n is 6. Table 7. Protein cleavage site sequences [Table 0007]

[0221] In one embodiment, the primary constructs and mmRNA of the 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, such as, 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.

[0222] In one embodiment, the primary construct or mmRNA of the present invention may include at least one encoded protein cleavage signal that contains 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. Those skilled in the art can use Table 1 above or other known methods to determine the appropriate encoded protein cleavage signal to include in the primary construct or mmRNA of the present invention. For example, starting with the signals in Table 7, one can design a signal in the primary construct that can generate a protein signal in the resulting polypeptide, taking into account the codons in Table 1.

[0223] In one embodiment, a polypeptide of the invention comprises at least one proteolytic signal and / or site.

[0224] 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 includes at least one protein cleavage signal and / or site, with the proviso that the polypeptide is not GLP-1.

[0225] In one embodiment, the primary construct or mmRNA of the invention comprises at least one encoded protein cleavage signal and / or site.

[0226] 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.

[0227] In one embodiment, the primary construct or mmRNA of the present invention may contain two or more coding regions. When multiple coding regions are present in the primary construct or mmRNA of the present invention, the multiple coding regions may be separated by encoded protein cleavage sites. As a non-limiting example, the primary construct or mmRNA may be written in an ordered pattern. Such a pattern follows the AXBY format, where A and B are coding regions that may be the same or different coding regions and / or may code for the same or different polypeptides, and X and Y are encoded protein cleavage signals that may code for the same or different protein cleavage signals. A second such pattern follows the AXYBZ format, where A and B are coding regions that may be the same or different coding regions and / or may code for the same or different polypeptides, and X, Y, and Z are encoded protein cleavage signals that may code for 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.

[0228] 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 may be released from the carrier domain or fusion partner by treatment with a protease specific for the protein cleavage site.

[0229] In one embodiment, the polypeptides, primary constructs, and mmRNA of the present invention may include a sequence encoding a 2A peptide. In one embodiment, this sequence may be used to separate the coding regions of two or more polypeptides of interest. As a non-limiting example, a sequence encoding a 2A peptide may be present between coding region A and coding region B (A-2Apep-B). The presence of the 2A peptide results in cleavage of one long protein into protein A, protein B, and the 2A peptide. Protein A and protein B may be the same or different polypeptides of interest. In another embodiment, the 2A peptide may be used in the polynucleotides, primary constructs, and / or mmRNA of the present invention to produce 2, 3, 4, 5, 6, 7, 8, 9, 10, or more proteins.

[0230] Incorporation of post-transcriptional regulators In one embodiment, the polynucleotides, primary constructs, and / or mmRNA of the present invention may include at least one post-transcriptional regulatory regulator. These post-transcriptional regulatory regulators 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.

[0231] The post-transcriptional regulatory factor can be a gene expression regulator 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.

[0232] 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.

[0233] In another embodiment, the polynucleotide, primary construct, and / or mmRNA of the present invention can comprise 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 method 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, each of which is incorporated herein by reference in its entirety).

[0234] In one embodiment, the polynucleotide, primary construct, and / or mmRNA of the present invention can include at least one post-transcriptional regulatory element to change protein expression.As a non-limiting example, the expression of VEGF can 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.

[0235] 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.

[0236] 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, these terms are not intended herein to refer to ribonucleotide modifications in the naturally occurring 5'-terminal mRNA cap moiety. In the context of polypeptides, the term "modification" refers to modifications compared to a reference set of 20 amino acids (moieties).

[0237] This modification 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 than one (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.

[0238] Polynucleotides, primary constructs, and mmRNAs may include any useful modifications to the sugar, nucleobase, or internucleoside linkage (e.g., to the phosphate / phosphodiester linkage / phosphodiester backbone), etc. One or more atoms of the pyrimidine nucleobase may be replaced 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, a modification (e.g., one or more modifications) is present in each of the sugar and internucleoside linkage. The modification according to the present invention may be a modification of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or a hybrid thereof). Further modifications are described herein.

[0239] As described herein, the polynucleotides, primary constructs, and mmRNA of the present invention do not substantially induce an innate immune response in a cell 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 (RIG-I, MDA5, etc.), and / or 3) termination or reduction of protein translation.

[0240] In certain embodiments, it may be desirable to degrade modified nucleic acid molecules introduced into cells in cells.For example, degradation of modified nucleic acid molecules may be preferred when precise timing of protein production is desired.Thus, in some embodiments, the present invention provides modified nucleic acid molecules that contain degradation domains, which can be acted on in 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).

[0241] The polynucleotides, primary constructs, and mmRNAs may optionally include other agents (e.g., RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, tRNAs, RNAs that induce triple helix formation, aptamers, vectors, etc.). In some embodiments, the 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.

[0242] 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.

[0243] 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 pharma- ceutically acceptable salt or stereoisomer thereof,

[0244] During the ceremony,

[0245] U is O, S, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer from 0 to 2, and each R U is independently H, halo, or optionally substituted alkyl;

[0246] --- is a single bond or is absent,

[0247] 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, 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 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); 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), 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);

[0248] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -NR N1 -, optionally substituted alkylene, or optionally substituted heteroalkylene, N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent;

[0249] 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;

[0250] Each Y 5 is independently O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;

[0251] n is an integer from 1 to 100,000;

[0252] 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” or B and R 2”can optionally form a bicyclic group (e.g., a bicyclic heterocyclyl) together with the carbons to which they are attached, or B, R 1” , and R 3 or B, R 2” , and R 3 may optionally form a tricyclic or tetracyclic group (e.g., a tricyclic or tetracyclic heterocyclyl such as 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, the first flanking region, or the second flanking region) comprises n linked nucleosides having formula (Ia-2)-(Ia-5), or a pharma- ceutically acceptable salt or stereoisomer thereof. [ka]

[0253] 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 pharma- ceutically acceptable salt or stereoisomer thereof,

[0254] During the ceremony,

[0255] U is O, S, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer from 0 to 2, and each R U is independently H, halo, or optionally substituted alkyl;

[0256] --- is a single bond or is absent,

[0257] 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; 1 and R 3’ combination of 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),

[0258] 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;

[0259] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -NR N1 -, optionally substituted alkylene, or optionally substituted heteroalkylene, N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;

[0260] 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;

[0261] n is an integer from 1 to 100,000;

[0262] B is a nucleobase.

[0263] 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 pharma- ceutically acceptable salt or stereoisomer thereof,

[0264] During the ceremony,

[0265] U is O, S, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer from 0 to 2, and each R U is independently H, halo, or optionally substituted alkyl;

[0266] --- is a single bond or is absent,

[0267] B 1 , B 2 , and B. 3each is independently a nucleobase (e.g., a purine, pyrimidine, or derivative thereof as described herein), H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl; B 1 , B 2 , and B. 3 Only one of the bases is a nucleobase,

[0268] 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;

[0269] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -NR N1 -, optionally substituted alkylene, or optionally substituted heteroalkylene, N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;

[0270] 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;

[0271] Each Y 5 is independently O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;

[0272] n is an integer from 1 to 100,000;

[0273] The ring containing U may contain one or more double bonds.

[0274] 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.

[0275] 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 pharma- ceutically acceptable salt or stereoisomer thereof,

[0276] During the ceremony,

[0277] U is O, S, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer from 0 to 2, and each R Uis independently H, halo, or optionally substituted alkyl;

[0278] 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;

[0279] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -NR N1 -, optionally substituted alkylene, or optionally substituted heteroalkylene, N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl;

[0280] 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;

[0281] Each Y 5 is independently O, S, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;

[0282] n is an integer from 1 to 100,000;

[0283] B is a nucleobase (eg, a purine, pyrimidine, or derivative thereof).

[0284] 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 pharma- ceutically acceptable salt or stereoisomer thereof,

[0285] During the ceremony,

[0286] Each of U' and U" is independently O, S, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer from 0 to 2, and each R U is independently H, halo, or optionally substituted alkyl;

[0287] 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;

[0288] Each Y 5’ is independently O, S, optionally substituted alkylene (e.g., methylene or ethylene), or optionally substituted heteroalkylene;

[0289] n is an integer from 1 to 100,000;

[0290] B is a nucleobase (eg, a purine, pyrimidine, or derivative thereof).

[0291] 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 (If) or (If-1): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof,

[0292] During the ceremony,

[0293] Each of U' and U" is independently O, S, N, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer from 0 to 2, and each R U is independently H, halo, or optionally substituted alkyl (e.g., U' is O and U" is N);

[0294] --- is a single bond or is absent,

[0295] 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; 1’ and R 3Combination of R 1” and R 3 Combination of R 2’ and R 3 combination of, 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), 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);

[0296] Y 1 , Y 2 , and Y 3 each independently represents O, S, Se, -NR N1 -, optionally substituted alkylene, or optionally substituted heteroalkylene, N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent;

[0297] 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;

[0298] Each Y 5 is independently O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene;

[0299] n is an integer from 1 to 100,000;

[0300] B is a nucleobase (eg, a purine, pyrimidine, or derivative thereof).

[0301] 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.

[0302] 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)), R 1 , R 1’ , and R 1” Each, when present, is H. In a further embodiment, R 2 , R 2’ , and R 2” Each, 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 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 OR', in which 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 an alkyl.

[0303] 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)), R 2 , R 2’ , and R 2” Each, when present, is H. In a further embodiment, R 1 , R 1’ , and R 1” Each, 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 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 OR', in which 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 an alkyl.

[0304] 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)), R 3 , R 4 , and R 5Each 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 H. In certain embodiments, R 3 C 1~6 is alkyl, R 4 C 1~6 is alkyl, R 5 C 1~6 alkyl or R 3 , R 4 , and R 5 All of the above are C 1~6 In certain embodiments, R 3 Also 4 are both H, and R 5 is C 1~6 It is an alkyl.

[0305] 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)), R 3 and R 5 taken together form an optionally substituted alkylene or an optionally substituted heteroalkylene and taken together with the carbons to which they are attached provide an optionally substituted heterocyclyl (e.g., a bicyclic, tricyclic, or tetracyclic heterocyclyl), such as the trans-3',4' analogs, and R 3 and R 5 taken together form a heteroalkylene (e.g., -(CH 2 ) b1 O(CH 2 ) b2 O(CH2 ) b3 wherein each of b1, b2, and b3 is independently an integer of 0 to 3.

[0306] 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)), R 3 And, R 1’ , R 1” , R 2’ , R 2” , or R 5 one or more of, taken together, form an optionally substituted alkylene or an optionally substituted heteroalkylene, which, 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 one or more of the following may be taken together to form a heteroalkylene (e.g., -(CH 2 ) b1 O(CH 2 ) b2 O(CH 2 ) b3 wherein each of b1, b2, and b3 is independently an integer of 0 to 3.

[0307] 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)), R 5 And, R 1’ , R 1” , R 2’ , or R2” one or more of, taken together, form an optionally substituted alkylene or an optionally substituted heteroalkylene, which, 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” one or more of the following may be taken together to form a heteroalkylene (e.g., -(CH 2 ) b1 O(CH 2 ) b2 O(CH 2 ) b3 wherein each of b1, b2, and b3 is independently an integer of 0 to 3.

[0308] 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)), 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).

[0309] 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)), each Y 3 is independently O or S.

[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)), R 1 is H, and each R 2 are independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g., -(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 OR', in which 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 the formula R N1 is H or optionally substituted alkyl (e.g., C 1~6alkyl, for example, methyl, ethyl, isopropyl, or n-propyl, 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 the formula R N1 is H or optionally substituted alkyl (e.g., C 1~6 alkyl, for example, methyl, ethyl, isopropyl, or n-propyl, 4 is independently H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.

[0311] In some embodiments of the polynucleotides, primary constructs, 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 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3OR', in which 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 the formula R N1 is H or optionally substituted alkyl (e.g., C 1~6 alkyl, for example, methyl, ethyl, isopropyl, or n-propyl, 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 the formula R N1 is H or optionally substituted alkyl (e.g., C 1~6 alkyl, for example, methyl, ethyl, isopropyl, or n-propyl, 4 is independently H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.

[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)), the ring containing the U is in the β-D (e.g., β-D-ribo) configuration.

[0313] 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.

[0314] 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 not ψ or m 5 Not C (for example, 10%-20%, 10%-35%, 10%-50%, 10%-60%, 10%-75%, 10%-90%, 10%-95%, 10%-98%, 10%-99%, 20%-35%, 20%-50%, 20%-60%, 20%-75%, 20%-90%, 20%-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 5In some embodiments, B is neither ψ nor m 5 It's not C either.

[0315] 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)), 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.

[0316] 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 pharma- ceutically acceptable salt or stereoisomer thereof. In certain embodiments, U is O or C(R U ) nu In the formula, 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 2 - or -CH-. In another embodiment, R 1 , R 2 , R 3 , R 4 , and R 5each 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; each R 3 and R 4 are independently H or optionally substituted alkyl, R 5 is H or hydroxy), and --- is a single or double bond.

[0317] In certain embodiments, the polynucleotide or mmRNA has the formula (IIb-1) to (IIb-2): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof. In some embodiments, U is O or C(R U ) nu In the formula, 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 2 - or -CH-. In another embodiment, R 1 and R 2each 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. In certain embodiments, R 2 is hydroxy or optionally substituted alkoxy (e.g., methoxy, ethoxy, or any described herein).

[0318] In certain embodiments, the polynucleotide, primary construct, or mmRNA has the formula (IIc-1) to (IIc-4): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof. In some embodiments, U is O or C(R U ) nu In the formula, 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 2 - or -CH-. In some embodiments, R 1 , R 2 , and R 3each 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 is independently H or optionally substituted alkyl. In certain embodiments, R 2 is an optionally substituted alkoxy (e.g., methoxy or ethoxy, or any of those described herein). In certain embodiments, R 1 is optionally substituted alkyl, 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.

[0319] 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) comprises a ribose having the structure represented by Formula (IId)-(IIf): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0320] 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) comprises a hexitol having the formula (IIg)-(IIj): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof.

[0321] In some embodiments, the polynucleotide, primary construct, or mmRNA comprises a sugar moiety having 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 pharma- ceutically 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; 2’ and R 3 combination of or R 2” and R 3 combinations of may be taken together to form an optionally substituted alkylene or an optionally substituted heteroalkylene.

[0322] 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 the formula (IIn): [ka] or a pharma- ceutically 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; R 3” is an optionally substituted alkylene (e.g., -CH 2 -, -CH 2 CH 2 - or -CH 2 CH 2 CH 2 -) or optionally substituted heteroalkylene (e.g., -CH 2 NH-, -CH 2 CH 2 NH-, -CH 2 OCH 2 - or -CH 2 CH 2 OCH 2 -) (e.g., R 3’ is O and R 3” is an optionally substituted alkylene (e.g., -CH 2 -, -CH 2 CH 2 - or -CH 2 CH 2 CH 2 -) is).

[0323] In some embodiments, the polynucleotide, primary construct, or mmRNA has the formula (IIn-1) to (II-n2): [ka] or a pharma- ceutically 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; R 3” is an optionally substituted alkylene (e.g., -CH 2 -, -CH 2 CH 2 - or -CH 2 CH 2 CH 2 -) or optionally substituted heteroalkylene (e.g., -CH 2 NH-, -CH 2 CH 2 NH-, -CH 2 OCH 2 - or -CH 2 CH 2 OCH 2 -) (e.g., R 3’ is O and R 3” is an optionally substituted alkylene (e.g., -CH 2 -, -CH 2 CH 2 - or -CH 2 CH 2 CH 2 -) is).

[0324] 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 the formula (IIo): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein R12a , R 12c , T 1’ , T 1” , T 2’ , T 2” , V 1 , and V 3 is as described herein.

[0325] Any of the polynucleotide, primary construct, or mmRNA formulas may include one or more of the nucleobases described herein (eg, formulas (b1)-(b43)).

[0326] In one embodiment, the present invention provides a method for preparing a polynucleotide, primary construct, or mmRNA, the polynucleotide having formula (Ia): [ka] The method comprises the step of: [ka] The method includes reacting the compound of the formula (I) with an RNA polymerase and a cDNA template.

[0327] In a further embodiment, the present invention provides a method of 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.

[0328] 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 the formula (Ia): [ka] The method comprises the step of: [ka] The method includes reacting the compound of the formula (I) with an RNA polymerase and a cDNA template.

[0329] In a further embodiment, the 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:

[0330] It involves reacting a compound of formula (IIIa-1) as defined herein with a primer, a cDNA template, and an RNA polymerase.

[0331] In one embodiment, the present invention provides a method for preparing a modified mRNA (e.g., an mmRNA molecule) comprising at least one polynucleotide having the formula (Ia-2): [ka] The method comprises n nucleosides having formula (IIIa-2): [ka] The method includes reacting the compound of the formula (I) with an RNA polymerase and a cDNA template.

[0332] In a further embodiment, the present invention provides a method for amplifying a modified mRNA comprising at least one nucleotide (e.g., an mmRNA molecule), the method comprising:

[0333] It involves reacting a compound of formula (IIIa-2) as defined herein with a primer, a cDNA template, and an RNA polymerase.

[0334] In some embodiments, the reaction may be repeated from 1 to about 7,000 times.In any of the embodiments herein, B may be a nucleobase of formula (b1)-(b43).

[0335] The polynucleotides, primary constructs, and mmRNA may optionally include 5' and / or 3' flanking regions as described herein.

[0336] Modified RNA (mRNA) molecules The present invention also includes building blocks of modified RNA (mmRNA) molecules, such as modified ribonucleosides, modified ribonucleotides. For example, these building blocks may be useful in preparing polynucleotides, primary constructs, or mmRNA of the present invention. In some embodiments, the building block molecules are represented by formula (IIIa) or (IIIa-1): [ka] or a pharma- ceutically 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, Y 1 , Y 2 , or Y 3 At least one of them is not O.

[0337] 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 pharma- ceutically 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)).

[0338] In some embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA has the formula (IVc)-(IVk): [ka] or a pharma- ceutically 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)).

[0339] 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 pharma- ceutically acceptable salt or stereoisomer thereof, wherein B is as described herein (e.g., any one of (b1)-(b43)).

[0340] 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 pharma- ceutically 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)).

[0341] 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 pharma- ceutically 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)).

[0342] In other embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA has the formula (IXh)-(IXk): [ka] or a pharma- ceutically 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)).

[0343] In other embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA has the formula (IXl)-(IXr): [ka] or a pharma- ceutically 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)).

[0344] In some embodiments, the building block molecules that can be incorporated into the polynucleotides, primary constructs, or mmRNA are: [ka] [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof, where each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0345] In some embodiments, the building block molecules that can be incorporated into the polynucleotides, primary constructs, or mmRNA are: [ka] or a pharma- ceutically 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), and s1 is as described herein.

[0346] 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., one selected from the group consisting of: 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., 0 to 3, 1 to 3, or 1 to 5): [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0347] In some embodiments, a building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA is a modified cytidine (e.g., one selected from the group consisting of: 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., 0 to 3, 1 to 3, or 1 to 5): [ka] [ka] [ka] [ka] [ka] [ka] For example, building block molecules that can be incorporated into a polynucleotide, primary construct, or mmRNA include: [ka] or a pharma- ceutically acceptable salt, or stereoisomer thereof, where each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0348] 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: 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., 0 to 3, 1 to 3, or 1 to 5): [ka] [ka] [ka] [ka] [ka] [ka] [ka]

[0349] 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: 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., 0 to 3, 1 to 3, or 1 to 5): [ka] [ka] [ka] [ka] [ka] [ka]

[0350] In some embodiments, the chemical modification is the replacement 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 >NR N1 Substitution with a group (wherein R N For example, a building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA can include: [ka] or a pharma- ceutically acceptable salt, or stereoisomer thereof, where each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0351] 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 pharma- ceutically acceptable salt, or stereoisomer thereof, where each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0352] In yet a further embodiment, the chemical modification is an NH 2 For example, a building block molecule that can be incorporated into a polynucleotide, primary construct, or mmRNA can include a fused ring formed by: [ka] or a pharma- ceutically acceptable salt, or stereoisomer thereof, where each r is independently an integer from 0 to 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0353] Sugar Modifications Modified nucleosides and nucleotides (e.g., building block molecules) that can be incorporated into a polynucleotide, primary construct, or mmRNA (e.g., an 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 a number of different substituents. Exemplary substitutions at the 2' position include H, halo, optionally substituted C, OH ... 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 of those described herein), polyethylene glycol (PEG), -O(CH 2 CH 2 O) n CH 2 CH 2 OR, where R is H or an optionally substituted alkyl, 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 C 1~6These include, but are not limited to, "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), aminoalkyl as defined herein, aminoalkoxy as defined herein, amino as defined herein, and amino acid as defined herein. Generally, RNA comprises the sugar ribose, a five-membered ring bearing an oxygen. Non-limiting exemplary modified nucleotides include substitution of the ribose oxygen (e.g., with S, Se, or an 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 six- or seven-membered rings with additional carbon or heteroatoms, such as anhydrohexitol, altritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also have phosphoramidate backbones), polycyclic forms (e.g., tricyclic, and "unlocked" forms, e.g., For example, 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 (PNAs, where a 2-amino-ethyl-glycine bond replaces the ribose and phosphodiester backbone). The sugar group may also contain one or more carbons that have the opposite stereochemical configuration to that of the corresponding carbon of ribose. Thus, a polynucleotide, primary construct, or mmRNA molecule may include nucleotides that contain, for example, arabinose as the sugar.

[0354] Modifications in Nucleobases The present disclosure provides modified nucleosides and nucleotides. A "nucleoside" as described herein is defined as a compound that contains 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"). A "nucleotide" as described herein is defined as a nucleoside that contains a phosphate group. Modified nucleotides can be synthesized by any useful method as described herein (e.g., chemically, enzymatically, or recombinantly to include one or more modified or non-natural nucleosides).

[0355] 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 non-standard bases, where the arrangement of hydrogen bond donors and hydrogen bond acceptors allows for 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.

[0356] Modified nucleosides and nucleotides may 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 may 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. The circles identify the atoms that comprise each chemical region. [Table 0008]

[0357] In some embodiments, B is a modified uracil. Exemplary modified uracils are represented by formulas (b1)-(b5): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof,

[0358] During the ceremony,

[0359] [ka] is a single or double bond,

[0360] 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 of or T 2’ and T 2” Combinations of these together (e.g., T 2 (as in Figure 1), O (oxo), S (thio), or Se (seleno),

[0361] V 1 and V 2 Each of the groups is independently O, S, N(R Vb ) nv , or C(R Vb ) nv In the formula, nv is an integer from 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);

[0362] R 10 is H, halo, an optionally substituted amino acid, hydroxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aminoalkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl;

[0363] R 11 is H or optionally substituted alkyl;

[0364] 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;

[0365] 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.

[0366] Other exemplary modified uracils are represented by formulas (b6)-(b9): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof,

[0367] During the ceremony,

[0368] [ka] is a single or double bond,

[0369] 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” Combinations of these together (e.g., T 1 ), or T 2’ and T 2” Combinations of these together (e.g., T 2 ), O (oxo), S (thio), or Se (seleno), or each T 1 and T 2 are independently O (oxo), S (thio), or Se (seleno);

[0370] W 1 and W 2 Each of N(R Wa ) nw or C(R Wa ) nw In the formula, nw is an integer of 0 to 2, and each R Wa is independently H, optionally substituted alkyl, or optionally substituted alkoxy;

[0371] Each V 3 However, independently, O, S, N(R Va ) nv , or C(R Va ) nv In the formula, nv is an integer from 0 to 2, and each R Vaare independently H, halo, an 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., For example, 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 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 selected from (1) to (21); 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);

[0372] 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;

[0373] 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;

[0374] R 12b and T 1’ combination of or R 12b and R 12c may be combined to form an optionally substituted heterocyclyl,

[0375] 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.

[0376] Further exemplary modified uracils are represented by formulae (b28)-(b31): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof,

[0377] During the ceremony,

[0378] T 1 and T 2 each is independently O (oxo), S (thio), or Se (seleno);

[0379] Each R Vb’ and R Vb”are independently H, halo, an 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., trifluoroacetyl, or sulfoalkyl), 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, selected from (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;

[0380] 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;

[0381] 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);

[0382] optionally substituted alkoxycarbonyl acyl, optionally substituted alkoxycarbonyl alkoxy, optionally substituted alkoxycarbonyl alkyl, optionally substituted alkoxycarbonyl alkenyl, optionally substituted alkoxycarbonyl alkynyl, optionally substituted alkoxycarbonyl alkoxy, optionally substituted carboxy alkoxy, optionally substituted carboxy alkyl, or optionally substituted carbamoyl alkyl.

[0383] 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.

[0384] 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 Also 12b Both are H.

[0385] 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 of those 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 of those 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 Also Vb” are both H. In certain embodiments, T 1 is O (oxo) and T 2 is S (thio) or Se (seleno).

[0386] In some embodiments, R Vb’ is an optionally substituted alkoxycarbonylalkyl or an optionally substituted carbamoylalkyl.

[0387] In certain embodiments, R 12a , R 12b , R 12c , or R Va The optional substituents may be a polyethylene glycol group (e.g., -(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3 OR', in which 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 (CH 2 ) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1 In the formula, s1 is an integer from 1 to 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are each independently an integer from 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).

[0388] In some embodiments, B is a modified cytosine. Exemplary modified cytosines are represented by formulas (b10)-(b14): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof, During the ceremony,

[0389] 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” Combinations of these together (e.g., T 3 (as in Figure 1), O (oxo), S (thio), or Se (seleno),

[0390] Each V 4 However, independently, O, S, N(R Vc ) nv , or C(R Vc ) nv In the formula, nv is an integer from 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 of the substituents described herein, e.g., alkyl, selected from (1)-(21)); R 13b and R Vc may be combined to form an optionally substituted heterocyclyl,

[0391] Each V 5 But independently, N(R Vd ) nv , or C(R Vd ) nv In the formula, nv is an integer from 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 of the substituents described herein, e.g., alkyl, selected from (1)-(21)) (e.g., V 5is -CH or N;

[0392] R 13a and R 13b is independently H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy; 13b and R 14 may be combined to form an optionally substituted heterocyclyl,

[0393] 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;

[0394] R 15 and R 16 Each of is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0395] Further exemplary modified cytosines are represented by formulas (b32)-(b35): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof,

[0396] During the ceremony,

[0397] T 1 and T 3 each is independently O (oxo), S (thio), or Se (seleno);

[0398] R 13a and R 13b is independently H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy; 13b and R 14 may be combined to form an optionally substituted heterocyclyl,

[0399] 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;

[0400] R 15 and R 16is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl (e.g., R 15 is H and R 16 is H or optionally substituted alkyl.

[0401] 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 Also 15 are both H. In some embodiments, R 15 Also 16 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.

[0402] Further non-limiting examples of modified cytosines include those of formula (b36): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof,

[0403] During the ceremony,

[0404] Each R 13b is independently H, optionally substituted acyl, optionally substituted acyloxyalkyl, optionally substituted alkyl, or optionally substituted alkoxy; R 13b and R 14b may be combined to form an optionally substituted heterocyclyl,

[0405] Each R 14a and R 14b 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 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;

[0406] R 15 Each of is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

[0407] In certain embodiments, R 14b is an optionally substituted amino acid (e.g., an optionally substituted lysine). In some embodiments, R 14a is H.

[0408] In some embodiments, B is a modified guanine. Exemplary modified guanines are represented by formulas (b15)-(b17): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof,

[0409] During the ceremony,

[0410] T 4’ , T 4” , T 5’ , T 5” , T 6’ , and T 6” each is independently H, optionally substituted alkyl, or optionally substituted alkoxy; T 4’ and T 4” Combinations of (e.g., T 4 (as in 5’ and T 5” Combinations of (e.g., T 5 (as in 6’ and T 6” Combinations of (e.g., T 6 ) together to form O (oxo), S (thio), or Se (seleno),

[0411] V 5 and V 6 Each of the groups is independently O, S, N(R Vd ) nv , or C(R Vd ) nv In the formula, nv is an integer from 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 of the substituents described herein, e.g., alkyl, selected from (1)-(21), optionally substituted thioalkoxy, or optionally substituted amino;

[0412] R 17 , R 18 , R 19a , R 19b , R 21 , R 22, R 23 , and R 24 each 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.

[0413] Exemplary modified guanosines include those of formulas (b37)-(b40): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof,

[0414] During the ceremony,

[0415] T 4’ is independently H, optionally substituted alkyl, or optionally substituted alkoxy; 4 are independently O (oxo), S (thio), or Se (seleno);

[0416] R 18 , R 19a , R 19b , and R 21 each 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.

[0417] In some embodiments, R 18 is H or optionally substituted alkyl. In a further embodiment, T 4 In some embodiments, R 19a and R 19b Each of is independently H or optionally substituted alkyl.

[0418] In some embodiments, B is a modified adenine. Exemplary modified adenines are represented by formulas (b18)-(b20): [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein each V 7 However, independently, O, S, N(R Ve ) nv , or C(R Ve ) nv In the formula, nv is an integer from 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 of the substituents described herein, e.g., alkyl, selected from (1)-(21);

[0419] Each R 25 are independently H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino;

[0420] R 26a and R 26b each of which is independently H, an optionally substituted acyl, an optionally substituted amino acid, an optionally substituted carbamoyl alkyl, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted hydroxy alkyl, an optionally substituted hydroxy alkenyl, an optionally substituted hydroxy alkynyl, an optionally substituted alkoxy, or a polyethylene glycol group (e.g., -(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) s3OR' (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 aminopolyethylene glycol group (e.g., -NR N1 (CH 2 ) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1 (In the formula, 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)

[0421] 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;

[0422] Each R 28 is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl;

[0423] 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.

[0424] Exemplary modified adenines include those of formulae (b41)-(b43):

[0425] [ka] or a pharma- ceutically acceptable salt or stereoisomer thereof,

[0426] During the ceremony,

[0427] Each R 25 are independently H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino;

[0428] R 26a and R 26b each of which is independently H, an optionally substituted acyl, an optionally substituted amino acid, an optionally substituted carbamoyl alkyl, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted hydroxy alkyl, an optionally substituted hydroxy alkenyl, an optionally substituted hydroxy alkynyl, an optionally substituted alkoxy, or a polyethylene glycol group (e.g., -(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) 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 aminopolyethylene glycol group (e.g., -NR N1 (CH 2 ) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1(In the formula, 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)

[0429] Each R 27 is independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy, or optionally substituted amino.

[0430] 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. 27 is an optionally substituted alkyl, an optionally substituted alkoxy, or an optionally substituted thioalkoxy. In other embodiments, R 25 is optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy.

[0431] In certain embodiments, R 26a , R 26b , or R 29 The optional substituents may be a polyethylene glycol group (e.g., -(CH 2 ) s2 (OCH 2 CH 2 ) s1 (CH 2 ) 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 aminopolyethylene glycol group (e.g., -NR N1 (CH 2 ) s2 (CH 2 CH 2 O) s1 (CH 2 ) s3 NR N1 (In the formula, 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 is alkyl).

[0432] In some embodiments, B is represented by the 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.

[0433] In some embodiments, B is of 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.

[0434] In some embodiments, B is of formula (b23): [ka] wherein R 10 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 of the substituents described herein (e.g., R 10 (those of R 11 (e.g., H or any of the substituents described herein), R 12a (e.g., H or any substituent described herein), T 1 (e.g., oxo or any of the substituents described herein), and T 2 (eg, oxo or any of the substituents described herein) are as described herein. In some embodiments, B is of 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.

[0435] In some embodiments, B is of 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 of the substituents described herein (e.g., R 14 or R 14’ (those of R 13a (e.g., H or any of the substituents described herein), R 13b (e.g., H or any of the substituents described herein), R 15 (e.g., H or any of the substituents described herein), and T 3 (eg, oxo or any of the substituents described herein) are as described herein.

[0436] In some embodiments, B is a nucleobase selected from the group consisting of cytosine, guanine, adenine, and uracil. [ka] It could be.

[0437] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine (ψ), pyridin-4-one ribonucleosides, 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 5 U), uridine 5-oxyacetic acid (cmo 5 U), uridine 5-oxyacetic 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-thio-uridine (mcm 5 s 2 U), 5-aminomethyl-2-thio-uridine (nm 5 s 2 U), 5-methylaminomethyl-uridine (mnm 5 U), 5-methylaminomethyl-2-thio-uridine (mnm 5 s 2U), 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-thio-uridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (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-thio-uridine (m 5 s 2 U), 1-methyl-4-thio-pseudouridine (m 1 s 4 ψ), 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 3ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thio-uridine (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 5 Um), 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.

[0438] 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 2C), 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 (k 2 C), 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 4 2 Cm), 1-thio-cytidine, 2'-F-ara-cytidine, 2'-F-cytidine, and 2'-OH-ara-cytidine.

[0439] 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 6A), 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 2 A), N6-hydroxynorvalylcarbamoyl-adenosine (hn 6 A), 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 2 Am), 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.

[0440] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine (I), 1-methyl-inosine (m 1 I), wyosine (imG), methylwyosine (mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wyosine (yW), peroxywyosine (o 2 yW), hydroxywybutosine (OHyW), unmodified hydroxywybutosine (OHyW*), 7-deaza-guanosine, queosine (Q), epoxyqueosine (oQ), galactosyl-queosine (galQ), mannosyl-queosine (manQ), 7-cyano-7-deaza-guanosine (preQ 0 ), 7-aminomethyl-7-deaza-guanosine (preQ 1 ), Archaeosin (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 (m 1 G), N2-methyl-guanosine (m 2 G), N2,N2-dimethyl-guanosine (m 2 2 G), 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 2 Gm), 1-methyl-2'-O-methyl-guanosine (m 1Gm), 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)).

[0441] 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, the 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 uracils. Also included may be naturally occurring and synthetic derivatives of bases including substituted adenine and guanine, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and adenine, 8-azaguanine and adenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidines, imidazo[1,5-a]1,3,5 triazinones, 9-deazapurines, imidazo[4,5-d]pyrazines, thiazolo[4,5-d]pyrimidines, pyrazin-2-ones, 1,2,4-triazines, pyridazines, and 1,3,5 triazines. 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, for example, 7-deazaadenine.

[0442] Modifications in internucleoside linkages Modified nucleotides that may be incorporated into polynucleotides, primary constructs, or mmRNA molecules may be modified in the internucleoside bond (e.g., phosphate backbone). In the present specification, in the context of polynucleotide backbones, the terms "phosphate" and "phosphodiester" are used interchangeably. The backbone phosphate group may be modified by replacing one or more of the oxygen atoms with different substituents. In addition, modified nucleosides and nucleotides may include extensive 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 at the nitrogen (bridging phosphoramidates), sulfur (bridging phosphorothioates), and carbon (bridging methylene phosphonates) of the linking oxygen.

[0443] The α-thio-substituted phosphate moiety is provided to provide stability to RNA and DNA polymers through non-natural phosphorothioate backbone linkages. Phosphorothioate DNA and RNA have increased nuclease resistance and subsequently 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.

[0444] 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).

[0445] 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.

[0446] Combinations of modified sugars, nucleobases, and internucleoside linkages The polynucleotides, primary constructs, and mmRNA of the present invention may include combinations of modifications to the sugar, nucleobase, and / or internucleoside linkage. These combinations may include any one or more modifications described herein. For example, any of the nucleotides described herein in formulae (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) may be combined with any of the nucleobases described herein (e.g., formulae (b1) to (b43) or any others described herein).

[0447] Synthesis of Polypeptides, Primary Constructs, and mmRNA Molecules The polypeptides, primary constructs, and mmRNA molecules used according to 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. When typical or preferred process conditions (e.g., reaction temperature, time, molar ratio of reactants, solvent, pressure, etc.) are provided, those skilled in the art will be able to optimize and develop further process conditions. Although optimal reaction conditions may vary depending on the specific reactants or solvents used, such conditions can be determined by those skilled in the art using routine optimization procedures.

[0448] 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 or 13 C), may be monitored by spectroscopic means, such as infrared spectroscopy, spectrophotometry (eg, ultraviolet-visible), or mass spectrometry, or by chromatography, such as high performance liquid chromatography (HPLC) or thin layer chromatography.

[0449] The preparation of the polypeptide, primary construct and mmRNA 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 suitable 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.

[0450] 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. Depending on the particular reaction step, a suitable solvent for a particular reaction step can be selected.

[0451] The 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" that is an optically active, salt-forming organic acid. Suitable resolving acids for fractional recrystallization methods include optically active acids such as, for example, tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or the D and L forms of various optically active camphorsulfonic acids. The 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 one skilled in the art. Modified nucleosides and nucleotides (e.g., building block molecules) can be prepared according to the synthetic methods described in Ogata et al., J. Org. Chem. 74:2585-2588 (2009); Purmal et al., Nucl. Acids Res. 22(1):72-78, (1994); Fukuhara et al., Biochemistry, 1(4):563-568 (1962); and Xu et al., Tetrahedron, 48(9):1729-1740 (1992), each of which is incorporated by reference in its entirety.

[0452] The polypeptides, primary constructs, and mmRNA 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, C) may or may not be uniformly modified in the polynucleotide of the present invention or in a given, predefined sequence region thereof (e.g., one or more of the sequence regions depicted in Figure 1). In some embodiments, all nucleotides X in the 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, 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.

[0453] Different sugar modifications, nucleotide modifications, and / or internucleoside linkages (e.g., backbone structures) can be present at various positions of a polynucleotide, primary construct, or mmRNA. Those skilled in the art will recognize that nucleotide analogs or other modification(s) can be located at any position(s) of a polynucleotide, primary construct, or mmRNA such that the function of the polynucleotide, primary construct, or mmRNA is 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 of the following 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%-8 ...%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%-70%, 10%-80%, 1%-95%, 1%-95%, 10%-20%, 10%-25%, 10%-50%, 10%-60%, 10%- 0%-90%, 10%-95%, 10%-100%, 20%-25%, 20%-50%, 20%-60%, 20%-70%, 20%-80%, 20%-90%, 20%-95%, 20%-100%, 50%-60%, 50%-70%, 50%-80%, 50%-90%, 50%-95%, 50%-100%, 70%-80%, 70%-90%, 70%-95%, 70%-100%, 80%-90%, 80%-95%, 80%-100%, 90%-95%, 90%-100%, and 95%-100%).

[0454] In some embodiments, the 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 the 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 50%, 30% to 60%, 30% to 70%, 30% to 80%, 30% to 90%, 30% to 95%, 30% to 100%, 30% to 50%, 30% to 10 ... % 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 modified cytidines (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%, 10%-90%, 10%-95%, 10%-100%, 20%-30%, 30%-40%, 30%-50%, 30%-60%, 30%-70%, 30%-80%, 30%-90%, 30%-95%, 30%-100%, 30%-100%, 30%-20%, 30%-35 ...20%, 30%-35%, 30%-40%, 30%-50%, 30%-60%, 30%-70%, 30%-80%, 30%-9 In one embodiment, the cytosine or cytidine may be substituted with 0% 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 cytosines or cytidines 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 present disclosure provides a compound of formula (Ia-1): [ka] 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) that includes 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 is independently a suitable protecting group; [ka] indicates a solid support), Providing a polynucleotide, primary construct, or mmRNA of formula (VI-1); [ka] and b) oxidizing or sulfurizing a 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 mmRNA of formula (Ia).

[0455] 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.

[0456] Other components of polynucleotides, primary constructs, and mmRNAs are optional and are useful in some embodiments.For example, 5' untranslated region (UTR) and / or 3'UTR are provided, either or both of which 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.

[0457] Exemplary syntheses of modified nucleic acids or mRNA, such as modified nucleotides incorporated into RNA or mRNA, are provided below in Schemes 1-11. Scheme 1 provides a general method for phosphorylating nucleosides, including modified nucleosides. Scheme 1 [ka]

[0458] A variety of protecting groups can be used to control the reaction. For example, Scheme 2 provides the use of multiple protecting and deprotecting steps to facilitate phosphorylation of the 5' position of the sugar, rather than the 2' and 3' hydroxyl groups. Scheme 2 [ka]

[0459] Modified nucleotide can be synthesized by any useful method.Scheme 3, 4 and 7 provide exemplary methods for synthesizing modified nucleotide with modified purine nucleobase, and scheme 5 and 6 provide exemplary methods for synthesizing modified nucleotide with modified pseudouridine or pseudoisocytidine, respectively. Scheme 3 [ka] Scheme 4 [ka] Scheme 5 [ka] Scheme 6 [ka] Scheme 7 [ka]

[0460] 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]

[0461] Scheme 11 provides an exemplary synthesis of modified uracils, in which the N1 position is R 12b and 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 alkyl...

Claims

1. 1. An isolated polynucleotide comprising: (a) a first region of linked nucleosides, said first region encoding a polypeptide of interest, said polypeptide of interest being selected from the group consisting of SEQ ID NOs: 8922-17687; (b) a first flanking region located at the 5′ end of the first region, (i) a sequence of linked nucleosides selected from the group consisting of the naturally occurring 5'UTR of any of the nucleic acids encoding any of SEQ ID NOs: 8922-17687, SEQ ID NOs: 1-4, and functional variants thereof; (ii) a first flanking region comprising at least one 5′ end cap; (c) a second flanking region located at the 3' end of the first region, (i') a sequence of linked nucleosides selected from the group consisting of the naturally occurring 3'UTR of any of the nucleic acids encoding any of SEQ ID NOs: 8922-17687, SEQ ID NOs: 5-21, and functional variants thereof; (ii') a 3' tail sequence of the linked nucleoside; and a second flanking region comprising the linked nucleoside.

2. 2. The isolated polynucleotide of claim 1, wherein the first region of linked nucleosides comprises at least one open reading frame of a nucleic acid sequence, the nucleic acid sequence being selected from the group consisting of SEQ ID NOs: 17688-61633.

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

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

5. 5. The isolated polynucleotide of claim 1, wherein the at least one 5' end cap is selected from the group consisting of Cap 0, Cap 1, ARCA, inosine, N1-methyl-guanosine, 2'fluoro-guanosine, 7-deaza-guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine.

6. 6. The isolated polynucleotide of any one of claims 1 to 5, wherein at least one of the linked nucleosides comprises at least one modification compared to the chemical structure of an A, G, U, or C ribonucleotide.

7. 7. The isolated polynucleotide of claim 6, wherein at least one of said modifications is located in the nucleoside base and / or sugar moiety.

8. The first region has formula (Ia): 【Chemistry 1-1】 or a pharma- ceutically acceptable salt or stereoisomer thereof, During the ceremony, U is O, S, N (R Ｕ ) ｎｕ , or C(R Ｕ ) ｎｕ where nu is an integer from 0 to 2, and each R Ｕ is independently H, halo, or optionally substituted alkyl; 【Chemistry 1-2】 is a single or double bond, --- is a single bond or is absent, R １’ , R ２’ , 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, or absent; ３ And, R １’ , R １” , R ２’ , R ２” , or R ５ may 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; R ５ And, R １’ , R １” , R ２’ , or R ２” may 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; R ４ And, R １’ , R １” , R ２’ , R ２” , R ３ , or R ５ may be taken together to form an optionally substituted alkylene or an optionally substituted heteroalkylene and, together with the carbon to which they are attached, provide an optionally substituted heterocyclyl; Y １ , Y ２ , and Y ３ each independently represents O, S, -NR Ｎ１ -, optionally substituted alkylene, or optionally substituted heteroalkylene, where R Ｎ１ is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent; Each Y ４ are independently H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino; Each Y ５ is independently O, S, optionally substituted alkylene, or optionally substituted heteroalkylene; n is an integer from 1 to 100,000; B is a nucleobase, B and R １’ Combination of B and R ２’ Combination of B and R １” or B and R ２” may optionally form a bicyclic group together with the carbons to which they are attached, or B and R １” and R ３ or combination of B and R ２” and R ３ 8. The isolated polynucleotide of claim 1, wherein the combination of may optionally form a tricyclic or tetracyclic group.

9. B is either pseudouridine (ψ) or 5-methyl-cytidine (m ５ The isolated polynucleotide of claim 8, which is not C).

10. U is O or C(R Ｕ ) ｎｕ where nu is an integer from 1 to 2, and each R Ｕ is independently H, halo, or optionally substituted alkyl; R １ , R １’ , R １” , R ２ , R ２’ , and R ２” each, if present, is independently H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, or optionally substituted aminoalkyl; R ３ and R ４ each is independently H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxyalkoxy; Y １ , Y ２ , and Y ３ each independently represents O, S, -NR Ｎ１ -, optionally substituted alkylene, or optionally substituted heteroalkylene, where R Ｎ１ is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; Each Y ４ are independently H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, or optionally substituted amino; Each Y ５ is independently O or an optionally substituted alkylene; 10. The isolated polynucleotide of claim 8, wherein n is an integer between 10 and 10,000.

11. R １ , R １’ , and R １” 11. The isolated polynucleotide of claim 10, wherein each of, if present, is H.

12. R ２ , R ２’ , and R ２” 12. The isolated polynucleotide of claim 11, wherein each of, if present, is independently H, halo, hydroxy, optionally substituted alkoxy, or optionally substituted alkoxyalkoxy.

13. R ２ , R ２’ , and R ２” 11. The isolated polynucleotide of claim 10, wherein each of, if present, is H.

14. R １ , R １’ , and R １” 14. The isolated polynucleotide of claim 13, wherein each of, if present, is independently H, halo, hydroxy, optionally substituted alkoxy, or optionally substituted alkoxyalkoxy.

15. The first region has formula (IIa): 【Chemistry 2】 9. The isolated polynucleotide of claim 8, comprising n linked nucleotides having the formula:

16. 16. The isolated polypeptide of claim 15, wherein the first region comprises n linked nucleosides having the formula (IIb) or (IIc), or a pharma- ceutically acceptable salt thereof.

17. The first region has formula (IId): 【Chemistry 3】 9. The isolated polynucleotide of claim 8, comprising n linked nucleosides having the formula:

18. 18. The isolated polypeptide of claim 17, wherein the first region comprises n linked nucleosides having the formula (IIe) or (IIf), or a pharma- ceutically acceptable salt thereof.

19. The first region comprises n linked nucleotides, each of the linked nucleotides independently having the formula (IIg)-(IIj): 【Chemistry 4】 or a pharma- ceutically acceptable salt or stereoisomer thereof.

20. The first region has the formula (IIk): 【Chemistry 5】 9. The isolated polynucleotide of claim 8, comprising n linked nucleosides having the formula:

21. The first region has formula (IIl): 【Chemistry 6】 or a pharma- ceutically acceptable salt or stereoisomer thereof.

22. The first region has the formula (IIm): 【Chemistry 7】 or a pharma- ceutically acceptable salt or stereoisomer thereof, During the ceremony, 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; ２’ and R ３ or a combination of R ２” and R ３ 21. The isolated polynucleotide of claim 20, wherein the combination of can be taken together to form an optionally substituted alkylene or an optionally substituted heteroalkylene.

23. U is O or C(R Ｕ ) ｎｕ where nu is an integer from 1 to 2, and each R Ｕ is independently H, halo, or optionally substituted alkyl; R １ 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, optionally substituted amino, azido, optionally substituted aryl, or optionally substituted aminoalkyl; R ３ and R ４ is independently H or optionally substituted alkyl; Y １ , Y ２ , and Y ３ each independently represents O, S, -NR Ｎ１ -, optionally substituted alkylene, or optionally substituted heteroalkylene, where R Ｎ１ is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; Each Y ４ are independently H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, or optionally substituted amino; Each Y ５ is independently O or an optionally substituted alkylene; 23. The isolated polynucleotide of any one of claims 11 to 22, wherein n is an integer between 10 and 10,000.

24. The first region has the formula (IIn): 【Chemistry 8】 or a pharma- ceutically acceptable salt or stereoisomer thereof, During the ceremony, U is O or C(R Ｕ ) ｎｕ where nu is an integer from 1 to 2, and each R Ｕ is independently H, halo, or optionally substituted alkyl; R １ 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 amino, azido, optionally substituted aryl, or optionally substituted aminoalkyl; 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; R ３” is optionally substituted alkylene or optionally substituted heteroalkylene; Y １ , Y ２ , and Y ３ each independently represents O, S, -NR Ｎ１ -, optionally substituted alkylene, or optionally substituted heteroalkylene, where R Ｎ１ is H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; Each Y ４ are independently H, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, or optionally substituted amino; Each Y ５ is independently O, S, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene; 9. The isolated polynucleotide of claim 8, wherein n is an integer between 10 and 10,000.

25. In the n number of B, each B is independently represented by the formulas (b1) to (b5): 【Chemistry 9-1】 or a pharma- ceutically acceptable salt or stereoisomer thereof; During the ceremony, In formula (b1), V １ and V ２ Concatenate 【Chemistry 9-2】 is a single or double bond, 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 of ２’ and T ２” taken together to form O (oxo), S (thio), or Se (seleno), V １ and V ２ Each of R Ｖｂ ) ｎｖ , or C(R Ｖｂ ) ｎｖ where nv is an integer from 0 to 2, and each R Ｖｂ is 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; R １０ is H, halo, an optionally substituted amino acid, hydroxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aminoalkyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl; R １１ is H or optionally substituted alkyl; R １２ａ is H, optionally substituted alkyl, or optionally substituted aminoalkyl; R １２ｃ 25. The isolated polynucleotide of any one of claims 8-24, wherein is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted aminoalkyl.

26. n B's are represented by the formulas (b6) to (b9): 【Chemistry 10-1】 or a pharma- ceutically acceptable salt or stereoisomer thereof; During the ceremony, 【Chemistry 10-2】 is a single or double bond, 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 of ２’ and T ２” taken together to form O (oxo), S (thio), or Se (seleno), W １ and W. ２ Each of N(R Ｗａ ) ｎｗ or C(R Ｗａ ) ｎｗ where nw is an integer from 0 to 2, and each R Ｗａ is independently H, optionally substituted alkyl, or optionally substituted alkoxy; Each V ３ are independently O, S, N (R Ｖａ ) ｎｖ , or C(R Ｖａ ) ｎｖ where nv is an integer from 0 to 2, and each R Ｖａ are independently H, halo, an optionally substituted amino acid, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted heterocyclyl, an optionally substituted alkylheterocyclyl, an optionally substituted alkoxy, an optionally substituted alkenyloxy, or an optionally substituted alkynyloxy; 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; R １２ａ is H, optionally substituted alkyl, optionally substituted aminoalkyl, or absent; R １２ｂ is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkylaryl, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, or an optionally substituted amino acid; R １２ｂ and T １’ or a combination of R １２ｂ and R １２ｃ may be combined to form an optionally substituted heterocyclyl, R １２ｃ 26. The isolated polynucleotide of any one of claims 8-25, wherein is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted aminoalkyl.

27. R １２ａ , R １２ｂ , R １２ｃ , or R Ｖａ But -(CH ２ ) ｓ２ (O.C.H. ２ CH ２ ) ｓ１ (CH ２ ) ｓ３ OR', where s1 is an integer from 1 to 10, s2 and s3 are each independently an integer from 0 to 10, and R' is H or C １～２０ alkyl or -NR Ｎ１ (CH ２ ) ｓ２ (CH ２ CH ２ O) ｓ１ (CH ２ ) ｓ３ N.R. Ｎ１ wherein s1 is an integer from 1 to 10, each of s2 and s3 is independently an integer from 0 to 10, and each R Ｎ１ are independently hydrogen or optionally substituted C １～６ 27. The isolated polynucleotide of claim 26, wherein said polynucleotide is alkyl.

28. n B's are represented by the formulas (b10) to (b14): 【Chemistry 11】 or a pharma- ceutically acceptable salt or stereoisomer thereof; During the ceremony, 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” taken together to form O (oxo), S (thio), or Se (seleno), Each V ４ are independently O, S, N (R Ｖｃ ) ｎｖ , or C(R Ｖｃ ) ｎｖ where nv is an integer from 0 to 2, and each R Ｖｃ is 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; R １３ｂ and R Ｖｃ may be combined to form an optionally substituted heterocyclyl, R １３ａ and R １３ｂ is independently H, optionally substituted acyl, optionally substituted alkyl, or optionally substituted alkoxy; １３ｂ and R １４ may be combined to form an optionally substituted heterocyclyl, Each R １４ are independently H, halo, hydroxy, thiol, optionally substituted acyl, optionally substituted amino acid, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted hydroxyalkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted amino, azido, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, or optionally substituted aminoalkyl; R １５ and R １６ 28. The isolated polynucleotide of any one of claims 8-27, wherein each of is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

29. n B's are represented by the formulas (b15) to (b17): 【Chemistry 12】 or a pharma- ceutically acceptable salt or stereoisomer thereof; During the ceremony, T ４’ , T ４” , T ５’ , T ５” , T ６’ , and T ６” is independently H, optionally substituted alkyl, or optionally substituted alkoxy; T ４’ and T ４” or combination of T ５’ and T ５” or combination of T ６’ and T ６” together to form O, S, or Se; V ５ and V ６ Each of R Ｖｄ ) ｎｖ , or C(R Ｖｄ ) ｎｖ where nv is an integer from 0 to 2, and each R Ｖｄ are independently H, halo, an optionally substituted amino acid, cyano, amidine, optionally substituted aminoalkyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy; R １７ , R １８ , R １９ａ , R １９ｂ , R ２１ , R ２２ , R ２３ , and R ２４ 28. The isolated polynucleotide of any one of claims 7-27, wherein each of is independently H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted amino acid.

30. n B's are represented by the formulas (b18) to (b20): 【Chemistry 13】 or a pharma- ceutically acceptable salt or stereoisomer thereof; During the ceremony, Each V ７ are independently O, S, N (R Ｖｅ ) ｎｖ , or C(R Ｖｅ ) ｎｖ where nv is an integer from 0 to 2, and each R Ｖｅ is 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; Each R ２５ is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; R ２６ａ and R ２６ｂ each is independently H, optionally substituted acyl, an optionally substituted amino acid, an optionally substituted carbamoylalkyl, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted hydroxyalkyl, an optionally substituted hydroxyalkenyl, or an optionally substituted alkoxy; Each R ２７ is independently H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, or optionally substituted amino; Each R ２８ is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl; Each R ２９ is independently H, optionally substituted acyl, an optionally substituted amino acid, an optionally substituted carbamoylalkyl, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, an optionally substituted hydroxyalkyl, an optionally substituted hydroxyalkenyl, an optionally substituted alkoxy, or an optionally substituted amino.

31. R ２６ａ , R ２６ｂ , or R ２９ But -(CH ２ ) ｓ２ (O.C.H. ２ CH ２ ) ｓ１ (CH ２ ) ｓ３ OR', where s1 is an integer from 1 to 10, s2 and s3 are each independently an integer from 0 to 10, and R' is H or C １～２０ alkyl) or -NR Ｎ１ (CH ２ ) ｓ２ (CH ２ CH ２ O) ｓ１ (CH ２ ) ｓ３ N.R. Ｎ１ wherein s1 is an integer from 1 to 10, each of s2 and s3 is independently an integer from 0 to 10, and each R Ｎ１ are independently hydrogen or optionally substituted C １～６ 31. The isolated polynucleotide of claim 30, wherein said polynucleotide is alkyl.

32. n B's are represented by the formula (b21): 【Chemistry 14】 or a pharma- ceutically acceptable salt or stereoisomer thereof; In the formula, X １２ is independently O, S, an optionally substituted alkylene, or an optionally substituted heteroalkylene; xa is an integer from 0 to 3; R １２ａ is H, optionally substituted alkyl, optionally substituted aminoalkyl, or absent; T ２ The isolated polynucleotide of any one of claims 8 to 31, wherein is O, S, or Se.

33. n B's are represented by the formula (b22): 【Chemistry 15】 or a pharma- ceutically acceptable salt or stereoisomer thereof, wherein R １０’ is independently optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted aminoalkyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl; R １１ is H or optionally substituted alkyl, R １２ａ is H, optionally substituted alkyl, optionally substituted aminoalkyl, or absent; T １ and T ２ The isolated polynucleotide of any one of claims 8 to 32, wherein each of is independently O, S, or Se.

34. n B's are represented by the formula (b23): 【Chemistry 16】 wherein R １０ is optionally substituted heterocyclyl or optionally substituted aryl; R １１ is H or optionally substituted alkyl, R １２ａ is H, optionally substituted alkyl, optionally substituted aminoalkyl, or absent; T １ and T ２ The isolated polynucleotide of any one of claims 8 to 33, wherein each of is independently O, S, or Se.

35. n B's are represented by the formula (b24): 【Chemistry 17】 wherein T ３ is O, S, or Se; R １３ａ and R １３ｂ is independently H, optionally substituted acyl, optionally substituted alkyl, or optionally substituted alkoxy; １３ｂ and R １４ may be combined to form an optionally substituted heterocyclyl, R １４’ are independently optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, optionally substituted alkylaryl, optionally substituted aminoalkyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl; Each R １５ 35. The isolated polynucleotide of any one of claims 8-34, wherein is independently H, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted alkynyl.

36. 36. The isolated polynucleotide of any one of claims 1 to 35, further comprising a targeting moiety, said targeting moiety being covalently attached to said polynucleotide.

37. 37. The isolated polynucleotide of claim 36, wherein the targeting moiety is an antibody, thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, a mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine multivalent mannose, multivalent fucose, glycosylated polyamino acids, multivalent galactose, transferrin, bisphosphonates, polyglutamates, polyaspartates, lipids, cholesterol, steroids, bile acids, folate, vitamin B12, biotin, an RGD peptide, an RGD peptide mimetic, or an aptamer.

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

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

40. 40. The pharmaceutical composition of claim 39, wherein the excipient is selected from a solvent, an aqueous solvent, a non-aqueous solvent, a dispersion medium, a diluent, a dispersant, a suspension aid, a surfactant, an isotonicity agent, a thickening agent or an emulsifier, a preservative, a lipid, a lipidoid, a liposome, a lipid nanoparticle, a core-shell nanoparticle, a polymer, a lipoplex peptide, a protein, a cell, a hyaluronidase, and a mixture thereof.

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

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

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

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

45. 45. The method of claim 44, wherein the isolated polynucleotide is administered in a total daily dose of 1 μg to 150 μg.

46. 46. ​​The method of claim 45, wherein the administration is by injection.

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

48. 46. ​​The method of claim 45, wherein the concentration of the desired polypeptide in the serum of the mammal is at least 50 pg / mL at least 2 hours after administration.

49. 46. ​​The method of claim 45, wherein the concentration of the desired polypeptide in the serum of the mammal remains above 50 pg / mL at least 72 hours after administration.

50. 50. The method of claim 49, wherein the concentration of the desired polypeptide in the serum of the mammal remains above 60 pg / mL at least 72 hours after administration.

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

52. 42. A method for producing increased concentrations of a polypeptide of interest selected from the group consisting of SEQ ID NOs: 8922-17687 in a mammalian cell, tissue, or organism, comprising administering to said cell, tissue, or organism a total daily dose of the isolated polynucleotide of any one of claims 4-37 or the pharmaceutical composition of any one of claims 38-41 in two or more evenly or unequally divided doses.

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

54. 53. The method of claim 52, wherein the mammalian organism is a human patient in need of increased concentrations of the desired polypeptide.

55. 55. The method of claim 54, wherein the increased concentration of the target polypeptide is detectable in a body fluid of the patient.

56. 56. The method of claim 55, wherein the bodily fluid is selected from the group consisting of peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, 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, chyle, bile, interstitial fluid, leucorrhea, pus, sebum, vomit, vaginal secretions, mucosal secretions, stool water, pancreatic juice, nasal washings, bronchopulmonary aspirate, blastocyst cavity fluid, and umbilical cord blood.

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

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

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

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

61. 60. The method of claim 59, wherein the administration follows a dosing regimen that occurs over hours, days, weeks, months, or years.

62. 53. The method of claim 52, wherein the two or more divided doses comprise a first dose of the polynucleotide or pharmaceutical composition administered at a time T1 and a second dose of the polynucleotide or pharmaceutical composition administered at a subsequent time T2, wherein the T1 and T2 time points are not separated by more than one minute, and the first and second doses are administered to the subject in amounts that result in a higher concentration of the target polypeptide than if that amount of polynucleotide or pharmaceutical composition was administered all at once in a single unit dose.

63. 63. The method of claim 62, further comprising administering multiple doses of said polynucleotide or pharmaceutical composition Nx times at Tn time points, where x and n are independently selected from 3 to about 1000, and the time between Tn and Tn+1 is not separated by an increment of more than 10 seconds.

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

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

66. 65. The method of claim 64, wherein the first dose is administered proximally to the second dose or doses.

67. 65. The method of claim 64, wherein the first dose is administered distally to the second dose or doses.

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

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

70. 66. The method of claim 65, wherein the direct injection is accomplished by using one or more devices selected from a multi-needle injection system, a catheter or luminal system, and an ultrasound, electrical, or radiation-based system.

71. 64. The method of claim 63, wherein the amounts of said polynucleotide or pharmaceutical composition administered in any dose are substantially equal.

72. 64. The method of claim 63, wherein the T1 and T2 time points are not separated by more than 30 seconds.

73. 64. The method of claim 63, wherein the T1 and T2 time points are not separated by more than 10 seconds.

74. 64. The method of claim 63, wherein any of the first dose, the second dose, and the multiple doses are administered substantially simultaneously.

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

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

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

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

1.

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

80. 80. The method of claim 79, further comprising dialysing the resulting suspension against phosphate buffered saline (PBS) at pH 7.

4.

81. 81. The method of claim 80, wherein dialysis is performed two or more times.

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

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

84. The method according to any one of claims 78 to 83, wherein the polynucleotide is selected from the polynucleotides according to any one of claims 1 to 37.

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

86. The lipid nanoparticle formulation of claim 85, wherein the polynucleotide is selected from the polynucleotides of any one of claims 1 to 37.

87. A reLNP formulation of a polynucleotide, wherein the polynucleotide encodes a polypeptide of interest.

88. The reLNP formulation of claim 87, wherein the polynucleotide is selected from the polynucleotides of any one of claims 1 to 36.

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

90. The sustained release formulation of claim 89, wherein the polynucleotide is selected from the polynucleotides of any one of claims 1 to 36.

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

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

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