In vivo production of proteins

Abstract

The present invention provides a nucleic acid compound or polynucleotide having structural and / or chemical characteristics that avoid one or more of the challenges in the present art, such as maintaining structural and functional integrity, overcoming expression thresholds, improving expression rate, half-life, and / or protein concentration, optimizing protein localization, and avoiding harmful biological responses such as immune responses and / or degradation pathways, while having characteristics useful for optimizing the formulation and delivery of nucleic acid-based therapeutics. [Solution] A method for producing a target polypeptide in vivo, comprising encoding the target polypeptide and using pseudouridine (ψ) and 5-methylcytidine (5meC, 5mc, or m) to reduce the innate immune response of cells. 5 The method includes contacting a mammalian cell, tissue, or living organism with modified mRNA (mmRNA) modified by C), etc.

Description

[Technical Field]

[0001] Sequence listing reference This application was filed electronically along with the sequence listing. The sequence listing file, titled M313SQLST.txt, was created on March 15, 2013, and is 240,793 bytes in size. The electronic information of this sequence listing is incorporated herein by reference in its entirety.

[0002] Cross-reference of related applications This application is the same as the following applications: International application PCT / US2012 / 069610 filed on December 14, 2012, titled "Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions"; U.S. Provisional Patent Application 61 / 618,862 filed on April 2, 2012, titled "Modified Polynucleotides for the Production of Biologics"; U.S. Provisional Patent Application 61 / 681,645 filed on August 10, 2012, titled "Modified Polynucleotides for the Production of Biologics"; U.S. Provisional Patent Application 61 / 737,130 filed on December 14, 2012, titled "Modified Polynucleotides for the Production of Biologics"; and U.S. Provisional Patent Application 61 / 618,866 filed on April 2, 2012, titled "Modified Polynucleotides for the Production of U.S. Provisional Patent Application No. 61 / 681,647, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Antibodies", U.S. Provisional Patent Application No. 61 / 737,134, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Antibodies", U.S. Provisional Patent Application No. 61 / 618,868, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Vaccines", U.S. Provisional Patent Application No. 61 / 681,648, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Vaccines", U.S. Provisional Patent Application No. 61 / 737,135, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Vaccines, U.S. Provisional Patent Application No. 61 / 618, filed April 2, 2012.U.S. Provisional Patent Application No. 61 / 681,649, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides", U.S. Provisional Patent Application No. 61 / 737,139, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides", U.S. Provisional Patent Application No. 61 / 618,873, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Secreted Proteins", U.S. Provisional Patent Application No. 61 / 681,650, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Secreted U.S. Provisional Patent Application No. 61 / 737,147, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Secreted Proteins", U.S. Provisional Patent Application No. 61 / 618,878, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Plasma Membrane Proteins", U.S. Provisional Patent Application No. 61 / 681,654, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Plasma Membrane Proteins", U.S. Provisional Patent Application No. 61 / 737,152, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Plasma Membrane Proteins", U.S. Provisional Patent Application No. 61 / 618, filed April 2, 2012U.S. Provisional Patent Application No. 61 / 681,658, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins", U.S. Provisional Patent Application No. 61 / 737,155, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Cytoplasmic, U.S. Provisional Patent Application No. 61 / 618,896, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins", U.S. Provisional Patent Application No. 61 / 668,157, filed July 5, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins", U.S. Provisional Patent Application No. 61 / 681,661, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins", U.S. Provisional Patent Application No. 61 / 737,160, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins" U.S. Provisional Patent Application No. 61 / 618,911, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Nuclear Proteins", U.S. Provisional Patent Application No. 61 / 681,667, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Nuclear Proteins", U.S. Provisional Patent Application No. 61 / 737,168, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Nuclear Proteins", U.S. Provisional Patent Application No. 61 / 618,922, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Proteins", U.S. Provisional Patent Application No. 61 / 681, filed August 10, 2012U.S. Provisional Patent Application No. 61 / 737,174, filed December 14, 2012, title "Modified Polynucleotides for the Production of Proteins", U.S. Provisional Patent Application No. 61 / 618,935, filed April 2, 2012, title "Modified Polynucleotides for, U.S. Provisional Patent Application No. 61 / 681,687, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; U.S. Provisional Patent Application No. 61 / 737,184, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; U.S. Provisional Patent Application No. 61 / 618,945, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; U.S. Provisional Patent Application No. 61 / 681,696, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease" U.S. Provisional Patent Application No. 61 / 737,191, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 618,953, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 681,704, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 737,203, filed December 14, 2012, titled "Modified Polynucleotides for the Production "of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 753,661, filed April 2, 2012, U.S. Provisional Patent Application No. 61 / 618,961, titled "Dosing Methods for Modified mRNA", U.S. Provisional Patent Application No. 61 / 648,286, filed May 17, 2012, titled "Dosing Methods for Modified mRNA", International Application PCT / US2013 / 030062, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Biologics and Proteins Associated with Human Disease", International Application PCT / US2013 / 030063, filed March 9, 2013, titled "Modified Polynucloetides", International Application PCT / US2013 / 030064, titled "Modified Polynucleotides for the Production of "Secreted Proteins", International Application No. PCT / US2013 / 030059, filed March 9, 2013, title "Modified Polynucleotides for the Production of Membrane Proteins", International Application No. PCT / US2013 / 030066, filed March 9, 2013, title "Modified Polynucleotides for the Prod "Auction of Cytoplasmic and Cytoskeletal Proteins," International Application No. PCT / US2013 / 030067, filed March 9, 2013, title "Modified Polynucleotides for the "Production of Nuclear Proteins," International Application No. PCT / US2013 / 030060, filed March 9, 2013, title "Modified "Polynucleotides for the Production of Proteins," International Application No. PCT / US2013 / 030061, filed March 9, 2013, title "Modified Polynucleotides for the In relation to “Production of Proteins Associated with Human Disease,” International Application PCT / US2013 / 030068, filed March 9, 2013, titled “Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides,” and International Application PCT / US2013 / 030070, filed March 9, 2013, titled “Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides,” the contents of each of these are incorporated herein by reference in their entirety.

[0003] This application relates to International Publication PCT / US2012 / 58519, filed October 3, 2012, titled "Modified Nucleosides, Nucleotides, and Nucleic Acids, and Uses Thereof," and to International Publication PCT / US2012 / 69610, filed December 14, 2012, titled "Modified Nucleoside, Nucleotide, and Nucleic Acid Compositions," the contents of which are incorporated herein by reference in their entirety.

[0004] Field of Invention This invention relates to the composition of polynucleotides, primary constructs, and modified mRNA molecules (mmRNA), as well as methods, processes, kits, and devices for the design, preparation, manufacture, and / or formulation thereof. [Background technology]

[0005] Conventional methods for inducing protein expression have many problems. For example, introduced DNA can, at some frequency, be integrated into the host cell's genomic DNA, leading to denaturation and / or damage to the host cell's genomic DNA. Alternatively, heterologous deoxyribonucleic acid (DNA) introduced into a cell can be inherited by daughter cells (whether or not the heterologous DNA has been integrated into the chromosome) or by offspring. In addition, assuming proper delivery and no damage or integration of the host genome, there are many steps that must occur before the encoded protein can be produced. Once inside the cell, the DNA must be transported into the nucleus where it is transcribed into RNA. Subsequently, the RNA transcribed from the DNA must enter the cytoplasm where it is translated into protein. The multiple processing steps from administered DNA to protein not only create a time lag before the production of a functional protein, but each step also presents an opportunity for cellular error and damage. Furthermore, it is known that achieving DNA expression in cells is difficult because 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.

[0006] In the early 1990s, Bloom and colleagues successfully rescued vasopressin-deficient rats by injecting in vitro transcribed vasopressin mRNA into the hypothalamus (Science 255:996-998;1992). However, low levels of translational and molecular immunogenicity hindered the development of mRNA as a therapeutic agent, and since then, efforts have focused rather on alternative uses that can exploit these pitfalls, namely immunization with mRNA encoding cancer antigens.

[0007] Others have studied the use of mRNA to deliver target polypeptides and have shown that certain chemical modifications of mRNA molecules, specifically pseudouridine and 5-methylcytosine, reduced their immunostimulatory effects.

[0008] These studies include, for example, UK Patent Application No. 0316089.2 filed on July 9, 2003 by Ribostem Limited and now abandoned; PCT Application No. PCT / GB2004 / 002981 filed on July 9, 2004 and published as International Publication No. WO2005005622; US Patent Application National Phase Registration No. 10 / 563,897 filed on June 8, 2006 and published as US20060247195 and now abandoned; and European Patent Application National Phase Registration No. EP2004743322 filed on July 9, 2004 and published as European Patent No. EP1646714 and now withdrawn; and Novozymes, Inc.'s International Publication No. WO2008140615. PCT application PCT / US2007 / 88060 filed December 19, 2007, U.S. Patent Application National Phase Registration No. 12 / 520,072 filed July 2, 2009, published as U.S. Patent No. US20100028943, and European Patent Application National Phase Registration No. EP2007874376 filed July 7, 2009, published as European Patent No. EP2104739; PCT application PCT / US2006 / 46120 filed December 4, 2006, published as International Publication No. WO2007064952 of 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 AG's European Patent Application No. EP2007024312, filed on December 14, 2007, and now abandoned; PCT Application No. PCT / EP2008 / 01059, filed on December 12, 2008, and published as International Publication No. WO2009077134; European Patent Application National Phase Registration No. EP2008861423, filed on June 2, 2010, and published as European Patent No. EP2240572; and U.S. Patent Application National Phase No. 12 / 735, filed on November 24, 2010, and published as U.S. Patent No. US20110065103.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 Phase European Patent No. 1934345, filed March 21, 2012; and National Phase US Patent Application No. 11 / 992,638, filed August 14, 2009, published as No. 20100129877; Immune Disease Institute U.S. Patent Application No. 13 / 088,009 filed on April 15, 2011, published as U.S. Patent No. US20120046346 and PCT Application No. PCT / US2011 / 32679 filed on April 15, 2011, published as International Publication No. WO20110130624 of Shire HumanGenetic Therapeutics; U.S. Patent Application No. 12 / 957,340 filed on November 20, 2010, published as U.S. Patent No. US20110244026 of Shire HumanGenetic Therapeutics; PCT Application No. PCT / US1998 / 019492 filed on September 18, 1998, published as International Publication No. WO1999014346 of Sequitur Inc.; The Scripps Research PCT application PCT / US2010 / 00567, filed on February 24, 2010, published as International Publication No. WO2010098861 of the Institute, and U.S. Patent Application National Phase Registration No. 13 / 203,229, filed on November 3, 2011, published as U.S. Patent No. US20120053333; PCT application PCT / EP2010 / 004681, filed on July 30, 2010, published as International Publication No. WO2011012316 of Ludwig Maximilian University; Cellscript U.S. Patent No. 8,039,214, filed on June 30, 2008 and granted on October 18, 2011; U.S. Patent Application No. 12 / 962,498, filed on December 7, 2010 and published as U.S. Patent No. 20110143436; U.S. Patent Application No. 12 / 962,468, filed on December 7, 2010 and published as U.S. Patent No. 12 / 962,468, filed on September 20, 2011 and published as U.S. Patent No. 13 / 237, filed on September 20, 2011 and published as U.S. Patent No. 20120009649, filed on June 30, 2008 and granted on October 18, 2011, by Inc.PCT application PCT / US2010 / 59305, filed on December 7, 2010, and published as international publication WO2011071931, and PCT / US2010 / 59317, filed on December 7, 2010, and published as international publication WO2011071936; PCT application PCT / US2006 / 32372, filed on August 21, 2006, and published as international publication WO2007024708, and U.S. Patent application National Phase 11 / 990,646, filed on March 27, 2009, and published as U.S. U.S. US20090286852; German Patent application DE10 2001, filed on June 5, 2001, by Curevac GMBH. Patent No. 027 283.9, DE10 2001 062 480.8 filed on December 19, 2001, and DE 20 2006 051 filed on October 31, 2006. 516 (all of these have been abandoned), European Patent No. EP1392341 granted on March 30, 2005, and European Patent No. EP1458410 granted on January 2, 2008, PCT application PCT / EP2002 / 06180 filed on June 5, 2002 and published as International Publication WO2002098443, PCT / EP2002 / 14577 filed on December 19, 2002 and published as International Publication WO2003051401, PCT / EP2007 / 09469 filed on December 31, 2007 and published as International Publication WO2008052770, International Publication WO200 PCT / EP2008 / 03033, filed on April 16, 2008 and published as No. 9127230; PCT / EP2006 / 004784, filed on May 19, 2005 and published as International Publication No. WO2006122828; PCT / EP2008 / 00081, filed on January 9, 2007 and published as International Publication No. WO2008083949; and U.S. Patent Application No. 10 / 729,830, filed on December 5, 2003 and published as U.S. Patent No. US20050032730; U.S. Patent Application No. 10 / 870, filed on June 18, 2004 and published as U.S. Patent No. US20050059624.No. 110, No. 11 / 914,945 of the same application filed on July 7, 2008, published as US20080267873, No. 12 / 446,912 of the same application filed on October 27, 2009, published as US2010047261 and now abandoned, No. 12 / 522,214 of the same application filed on January 4, 2010, published as US20100189729, and No. 12 / 522,214 of the same application filed on May 26, 2010, published as US20110077287 These are described in U.S. Patent No. 12 / 787,566, U.S. Patent No. 12 / 787,755 filed on 26 May 2010 and published as U.S. Patent No. US20100239608, U.S. Patent No. 13 / 185,119 filed on 18 July 2011 and published as U.S. Patent No. US20110269950, and U.S. Patent No. 13 / 106,548 filed on 12 May 2011 and published as U.S. Patent No. US20110311472, all of which are incorporated herein by reference in their entirety.

[0009] Despite these reports being limited to the selection of chemical modifications including pseudouridine and 5-methylcytosine, there is still a need in the art for therapies that address the numerous barriers surrounding the effective regulation of intracellular translation and the processing of nucleic acids encoding polypeptides or fragments thereof.

[0010] To achieve this objective, the inventors have demonstrated that certain modified mRNA sequences have potential as therapeutic agents with advantages beyond merely evading, avoiding, or reducing the immune response. Such studies are detailed in the published concurrent applications, International Application PCT / US2011 / 046861 filed August 5, 2011, and International Application PCT / US2011 / 054636 filed October 3, 2011, and International Application PCT / US2011 / 054617 filed October 3, 2011, the contents of which are incorporated herein by reference in their entirety.

[0011] The present invention addresses this need by providing nucleic acid compounds or polynucleotides that encode a target polypeptide (e.g., modified mRNA or mMRNA) and have structural and / or chemical features that avoid one or more of the problems in the art, such as maintaining structural and functional integrity, overcoming expression thresholds, improving expression rate, half-life, and / or protein concentration, optimizing protein localization, and avoiding harmful biological responses such as immune responses and / or degradation pathways, while having features useful for optimizing the formulation and delivery of nucleic acid-based therapeutics.

[0012] Summary of the Invention The composition of modified mRNA (mmRNA) molecules, the design, preparation, manufacture, and / or formulation methods, processes, kits, and devices for modified mRNA (mmRNA) molecules are described herein.

[0013] Details of various embodiments of the present invention are described in the following embodiments for carrying out the invention. Other features, purposes, and advantages of the present invention will become apparent from the embodiments for carrying out the invention, the drawings, and the claims.

[0014] The aforementioned and other purposes, features, and advantages will become apparent from the following description of specific embodiments of the invention, as illustrated in the accompanying drawings, where similar reference letters refer to the same parts throughout the different drawings. The drawings are not necessarily to scale and are rather focused on illustrating the principles of various embodiments of the invention. [Brief explanation of the drawing]

[0015] [Figure 1] This is a schematic diagram of the primary structure of the present invention. [Figure 2]The lipid structures of prior art useful in the present invention are illustrated. 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 3] This plasmid is a representative plasmid useful in the IVT reaction taught herein. This plasmid contains insert 64818, designed by the inventors. [Figure 4] This is a gel profile of modified mRNA encapsulated in PLGA microspheres. [Figure 5] This is a histogram of factor IX modified mRNA in a factor IX protein-producing PLGA preparation. [Figure 6-1] These are histograms showing VEGF protein production in human keratinocyte cells after transfection with modified mRNA at various doses. Figure 6A shows protein production after transfection with modified mRNA containing natural nucleoside triphodes (NTPs). Figure 6B shows protein production after transfection with modified mRNA fully modified with pseudouridine (pseudoU) and 5-methylcytosine (5mC). Figure 6C shows protein production after transfection with modified mRNA fully modified with N1-methyl-pseudridine (N1-methyl-pseudoU) and 5-methylcytosine (5mC). [Figure 6-2] Same as above. [Figure 7] This is a histogram of VEGF protein production in HEK293 cells. [Figure 8] This is a gel profile of GLA protein production in mammals. Figure 8A shows the predicted size of GLA. Figure 8B shows the predicted size of GLA. [Figure 9] This is a gel profile of ARSB protein production in mammals. Figure 9A shows the predicted size of ARSB. Figure 9B shows the predicted size of ARSB. [Figure 10] This is a gel profile of IFNB1 protein production in mammals. Figure 10A shows the predicted size of IFNB1. Figure 10B shows the predicted size of IFNB1. [Figure 11] This is a gel profile of factor XI protein production in mammals. Figure 11A shows the predicted size of factor XI. Figure 11B shows the predicted size of factor XI. [Figure 12] This is a gel profile of TP53 protein production in mammals. Figure 11A shows the predicted size of TP53. Figure 11B shows the predicted size of TP53. [Figure 13] This is a gel profile of TGFβ protein production in mammals. Figure 13A shows the predicted size of TGFβ. Figure 11B shows the predicted size of TGFβ. [Figure 14] This is a gel profile of SIRT6 protein production in mammals. Figure 16A shows the predicted size of SIRT6. Figure 16B shows the predicted size of SIRT6. [Figure 15] This is a gel profile of NAGS protein production in mammals. Figure 15A shows the predicted size of NAGS. Figure 15B shows the predicted size of NAGS. [Figure 16] This is a gel profile of SORT1 protein production in mammals. Figure 16A shows the predicted size of SORT1. Figure 16B shows the predicted size of SORT1. [Figure 17] This is a gel profile of GM-CSF protein production in mammals. Figure 17A shows the predicted size of GM-CSF. Figure 17B shows the predicted size of GM-CSF. [Figure 18] This is a gel profile of Klotho protein production in mammals. Figure 18A shows the predicted size of Klotho. Figure 18B shows the predicted size of Klotho. [Figure 19]This is a gel profile of GALK1 protein production in mammals. Figure 19A shows the predicted size of GALK1. Figure 19B shows the predicted size of GALK1. [Figure 20] This is a gel profile of SERPINF2 protein production in mammals. Figure 20A shows the predicted size of SERPINF2. Figure 20B shows the predicted size of SERPINF2. [Figure 21] This is a gel profile of ALDOA protein production in mammals. [Figure 22] This is a gel profile of TYR protein production in mammals. Figure 22A shows the predicted size of TYR. Figure 22B shows the predicted size of TYR. [Figure 23] This is a gel profile of BMP7 protein production in mammals. Figure 23A shows the predicted size of BMP7. Figure 23B shows the predicted size of BMP7. [Figure 24] This is a gel profile of NRG1 protein production in mammals. Figure 24A shows the predicted size of NRG1. Figure 24B shows the predicted size of NRG1. [Figure 25] This is a gel profile of APCS protein production in mammals. Figure 25A shows the predicted size of APCS. Figure 25B shows the predicted size of APCS. [Figure 26] This is a gel profile of LCAT protein production in mammals. Figure 26A shows the predicted size of LCAT. Figure 26B shows the predicted size of LCAT. [Figure 27] This is a gel profile of ARTN protein production in mammals. Figure 27A shows the predicted size of ARTN. Figure 27B shows the predicted size of ARTN. [Figure 28] This is a gel profile of HGF protein production in mammals. Figure 28A shows the predicted size of HGF. Figure 28B shows the predicted size of HGF. [Figure 29]This is a gel profile of EPO protein production in mammals. Figure 29A shows the predicted size of EPO. Figure 29B shows the predicted size of EPO. [Figure 30] This is a gel profile of IL-7 protein production in mammals. Figure 30A shows the predicted size of IL-7. Figure 30B shows the predicted size of IL-7. [Figure 31] This is a gel profile of LIPA protein production in mammals. Figure 31A shows the predicted size of LIPA. Figure 31B shows the predicted size of LIPA. [Figure 32] This is a gel profile of DNAse1 protein production in mammals. Figure 32A shows the predicted size of DNAse1. Figure 32B shows the predicted size of DNAse1. [Figure 33] This is a gel profile of APOA1 Milano protein production in mammals. Figure 33A shows the predicted size of APOA1 Milano. Figure 33B shows the predicted size of APOA1 Milano. [Figure 34] This is a gel profile of TUFT1 protein production in mammals. Figure 34A shows the predicted size of TUFT1. Figure 34B shows the predicted size of TUFT1. [Figure 35] This is a gel profile of APOA1 Paris protein production in mammals. Figure 35A shows the predicted size of APOA1 Paris. Figure 35B shows the predicted size of APOA1 Paris. [Figure 36] This is a gel profile of APOA1 protein production in mammals. Figure 36A shows the predicted size of APOA1. Figure 36B shows the predicted size of APOA1. [Figure 37] This is a gel profile of UGT1A1 protein production in mammals. Figure 37A shows the predicted size of UGT1A1. Figure 37B shows the predicted size of UGT1A1. [Figure 38]This is a gel profile of THPO protein production in mammals. Figure 38A shows the predicted size of THPO. Figure 38B shows the predicted size of THPO. [Figure 39] This is a gel profile of ASL protein production in mammals. Figure 39A shows the predicted size of ASL. Figure 39B shows the predicted size of ASL. [Figure 40] This is a gel profile of FSHα protein production in mammals. [Figure 41] This is a gel profile of BMP2 protein production in mammals. Figure 41A shows the predicted size of BMP2. Figure 41B shows the predicted size of BMP2. [Figure 42] This is a gel profile of PLG protein production in mammals. Figure 42A shows the predicted size of PLG. Figure 42B shows the predicted size of PLG. [Figure 43] This is a gel profile of FGA protein production in mammals. Figure 43A shows the predicted size of FGA. Figure 43B shows the predicted size of FGA. [Figure 44] This is a gel profile of SERPINC1 protein production in mammals. Figure 44A shows the predicted size of SERPINC1. Figure 44B shows the predicted size of SERPINC1. [Figure 45] This is a gel profile of MTTP protein production in mammals. Figure 45A shows the predicted size of MTTP. Figure 45B shows the predicted size of MTTP. [Figure 46] This is a gel profile of SEPT4 protein production in mammals. Figure 46A shows the predicted size of SEPT4. Figure 46B shows the predicted size of SEPT4. [Figure 47] This is a gel profile of XIAP protein production in mammals. Figure 47A shows the predicted size of XIAP. Figure 47B shows the predicted size of XIAP. [Figure 48]This is a gel profile of SLC16A3 protein production in mammals. Figure 48A shows the predicted size of SLC16A3. Figure 48B shows the predicted size of SLC16A3. [Figure 49] This is a gel profile of ANGPT1 protein production in mammals. Figure 49A shows the predicted size of ANGPT1. Figure 49B shows the predicted size of ANGPT1. [Figure 50] This is a gel profile of IL-10 protein production in mammals. Figure 50A shows the predicted size of IL-10. Figure 50B shows the predicted size of IL-10. [Figure 51] This is a histogram showing insulin protein production in mammals. [Figure 52] This is a histogram showing factor XI protein production in HEK293. [Figure 53] This is a histogram showing factor XI protein production in HeLa. [Figure 54] This is a histogram showing factor XI protein production in HeLa. [Figure 55] This is a histogram showing factor XI protein production in HeLa supernatant. [Figure 56] This is a histogram showing HGH protein production in HeLa cells. [Modes for carrying out the invention]

[0016] In the fields of therapeutics, diagnostics, reagents, and biological assays, the ability to deliver nucleic acids, such as ribonucleic acid (RNA), into cells, whether in vitro, in vivo, insights, or ex vivo, for example, to induce intracellular translation of nucleic acids and production of the encoded target polypeptide, is of great interest. The delivery and function of non-integrated polynucleotides are of particular importance.

[0017] Compositions of polynucleotides encoding one or more target polypeptides (including pharmaceutical compositions), as well as methods for designing, preparing, manufacturing, and / or formulating them, are described herein. Systems, processes, devices, and kits for selecting, designing, and / or utilizing the polynucleotides encoding the target polypeptides described herein are also provided.

[0018] In accordance with the present invention, these polynucleotides are preferably modified to avoid defects in molecules encoding other polypeptides in the Art. Therefore, these polynucleotides are referred to as modified mRNA or mmRNA.

[0019] The inventors have studied the use of modified polynucleotides in various in vivo settings, including, for example, U.S. Provisional Patent Application No. 61 / 470,451 filed March 31, 2011, teaching the in vivo application of mMRNA; U.S. Provisional Patent Application No. 61 / 517,784 filed April 26, 2011, teaching engineered nucleic acids for the production of antibody polypeptides; and teaching the veterinary application of mMRNA technology. Filing No. 61 / 519,158 on May 17, 2011; Filing No. 61 / 533,537 on September 12, 2011, teaching the application of mMRNA technology in antimicrobial agents; Filing No. 61 / 533,554 on September 12, 2011, teaching the application of mMRNA technology to viruses; Filing No. 61 / 542,533 on October 3, 2011, teaching various chemical modifications used in mMRNA technology; Filing December 14, 2011, teaching mobile devices used in the production and use of mMRNA technology. Application No. 61 / 570,690; Application No. 61 / 570,708 of December 14, 2011, instructing the use of mMRNA in emergency medical situations; Application No. 61 / 576,651 of December 16, 2011, instructing the terminal modification structure of mMRNA; Application No. 61 / 576,705 of December 16, 2011, instructing a method of delivery of mMRNA using lipidoids; Application No. 61 / 576,705 of December 21, 2011, instructing a method for increasing organ or tissue viability using mMRNA These are disclosed in patents 1 / 578,271; 61 / 581,322 of December 29, 2011, teaching an mMRNA encoding a cell-permeable peptide; 61 / 581,352 of December 29, 2011, teaching the incorporation of a cytotoxic nucleoside into an mMRNA; and 61 / 631,729 of January 10, 2012, teaching a method using mMRNA to cross the blood-brain barrier, all of which are incorporated herein by reference in their entirety.

[0020] Provided in part herein are polynucleotides, primary constructs, and / or mMRNAs encoding a target polypeptide, designed to improve one or more of the following: stability and / or elimination in tissues, receptor uptake and / or kinetics, cell reach by the composition, involvement with the translation mechanism, mRNA half-life, translation efficiency, immune evasion, protein production capacity, secretion efficiency (where applicable), reachability to the bloodstream, protein half-life, and / or regulation of cellular state, function, and / or activity.

[0021] I. Composition of the present invention (mmRNA) The present invention provides nucleic acid molecules encoding one or more target polypeptides, specifically polynucleotides, primary constructs, and / or mMRNAs. The term “nucleic acid” in its broadest sense includes any compound and / or substance comprising polymers of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the present invention include, but are not limited to, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (including LNA, LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2'-amino-LNA having 2'-amino functionalization, and 2'-amino-α-LNA having 2'-amino functionalization), or hybrids thereof.

[0022] 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 target polypeptide and can be translated to produce the encoded target polypeptide in vitro, in vivo, in sights, or ex vivo.

[0023] Traditionally, the main components of an mRNA molecule include at least a coding region, a 5'UTR, a 3'UTR, a 5' cap, and a poly-A tail. Building on this wild-type modular structure, the present invention expands the functional range of conventional mRNA molecules by providing polynucleotides or primary RNA constructs that include one or more structural and / or chemical modifications or alterations that confer useful properties to the polynucleotide, including, in some embodiments, the absence of substantial induction of the cell's innate immune response to the region where the polynucleotide is introduced. Accordingly, the modified mRNA molecules of the present invention are referred to as "mmRNAs." As used herein, a "structural" feature or modification is a feature or modification in which two or more bound nucleotides are inserted, deleted, replicated, inverted, or randomized in a polynucleotide, primary construct, or mMRNA without significant chemical modification of the nucleotides themselves. Structural modifications are of a chemical nature and are therefore chemical modifications, since chemical bonds are necessarily broken and reformed to result in structural 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". Here, the dinucleotide "CC" is inserted, resulting in a structural modification of the polynucleotide.

[0024] mmRNA mechanism The mRNAs of the present invention are distinguished from wild-type mRNA in their functional and / or structural design features, which help overcome existing problems in effective polypeptide production using nucleic acid-based therapeutics, as demonstrated herein.

[0025] Figure 1 shows a typical polynucleotide primary construct 100 of the present invention. As used herein, the terms “primary construct” or “primary mRNA construct” refer to a polynucleotide transcript that encodes one or more target polypeptides and retains sufficient structural and / or chemical characteristics to enable the encoding of target polypeptides to be translated. A primary construct may be a polynucleotide of the present invention. If structurally or chemically modified, a primary construct may be referred to as an mRNA.

[0026] Referring to Figure 1, the primary construct 100 contains a first region of binding nucleotide 102 adjacent to a first facile region 104 and a second facile region 106. As used herein, the “first region” may be referred to as the “coding region” or “code region” or simply “first region.” This first region may, but is not limited to, the encoded target polypeptide, which may contain one or more signal sequences encoded by the signal sequence region 103 at its 5' end. The facile region 104 may contain a region of binding nucleotide containing one or more complete or incomplete 5'UTR sequences. The facile region 104 may also contain a 5' end cap 108. The second facile region 106 may contain a region of binding nucleotide containing one or more complete or incomplete 3'UTR sequences. The facile region 106 may also contain a 3' tail sequence 110.

[0027] The 5'-terminus bridge between the first region 102 and the first adjacent region 104 is the first manipulation region 105. Conventionally, this manipulation region contains a start codon. Alternatively, this manipulation region may contain any translation start sequence or signal containing a start codon.

[0028] The 3'-terminal bridge between the first region 102 and the second adjacent region 106 is the second manipulation region 107. Conventionally, this manipulation region contains a stop codon. Alternatively, this manipulation region may contain any translation initiation sequence or signal containing a stop codon. Multiple consecutive stop codons may also be used according to the present invention.

[0029] Generally, the shortest length of the first region of the primary construct of the present invention may be a nucleic acid sequence length sufficient to encode a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, or decapeptide. In another embodiment, this length may be sufficient to encode a peptide of 2 to 30 amino acids, e.g., 5 to 30, 10 to 30, 2 to 25, 5 to 25, 10 to 25, or 10 to 20 amino acids. This length may be sufficient to encode 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., a peptide not longer than 35, 30, 25, 20, 17, 15, 14, 13, 12, 11, or 10 amino acids. Examples of dipeptides that a polynucleotide sequence can encode include, but are not limited to, carnosine and anserine.

[0030] Generally, the length of the first region encoding the polypeptide targeted by the present invention is greater than about 30 nucleotides (for example, 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, Up to 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 nucleotide lengths (including 100,000) or exceeding them). As used herein, “first region” may be referred to as “coding region” or “code region” or simply “first region”.

[0031] In some embodiments, the polynucleotide, primary construct, or mMRNA has approximately 30 to approximately 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, Includes 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.

[0032] In accordance with the present invention, the first and second adjacent regions may independently be in the range of 15 to 1,000 nucleotide lengths (e.g., 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, and 900 nucleotide lengths, or at least 30, 40, 45, 50, 55, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, and 1,000 nucleotide lengths).

[0033] According to the present invention, the tail sequence may be in the range of 0 to 500 nucleotide lengths (e.g., at least 60, 70, 80, 90, 120, 140, 160, 180, 200, 250, 300, 350, 400, 450, or 500 nucleotide lengths). If the tail region is a poly(A) tail, the length may be determined in units of poly(A) binding protein binding or as a function thereof. In this embodiment, the poly(A) tail is long enough to bind to at least four monomers of the poly(A) binding protein. A monomer of the poly(A) binding protein binds to a sequence of about 38 nucleotides. Thus, poly(A) tails of about 80 and 160 nucleotides have been observed to be functional.

[0034] According to 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 have a nucleotide length of 1 to 10, for example, 2 to 9, 3 to 8, 4 to 7, 1 to 5, 5 to 10, or at least 2 or 10 or less. In some embodiments, the cap is absent.

[0035] According to the present invention, the first and second operating regions may have nucleotide lengths of 3 to 40, for example, in the range of 5 to 30, 10 to 20, 15, or at least 4, or 30 or less, and may include one or more signal and / or restriction sequences in addition to a start codon and / or stop codon.

[0036] Circular mMRNA According to the present invention, a primary construct or mMRNA can be cyclized or concatemerized to generate a translationally competent molecule that facilitates the interaction between a poly(A) binding protein and a 5' end binding protein. The mechanism of cyclization or concatemerization can occur through at least three different pathways, namely 1) a chemical pathway, 2) an enzymatic pathway, and 3) a ribozyme-catalyzed pathway. The newly formed 5' / 3' linkage can be intramolecular or intermolecular.

[0037] In the first pathway, the 5' and 3' ends of the nucleic acid contain chemically reactive groups that, when in close proximity, form a new covalent bond between the 5' and 3' ends of the molecule. The 5' end may contain an NHS-ester reactive group, and the 3' end may contain a 3'-amino-terminal nucleotide, so that in an 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.

[0038] In a second pathway, a T4 RNA ligase can be used to enzymatically link a 5'-phosphorylated nucleic acid molecule to the 3'-hydroxyl group of the nucleic acid, forming a new phosphorodiester bond. In an example reaction, 1 μg of nucleic acid molecule is incubated with 1 to 10 units of T4 RNA ligase (New England Biolabs, Ipswich, MA) at 37°C for 1 hour, according to the manufacturer's protocol. The ligation reaction may occur in the presence of a fission oligonucleotide that can base-pair alongside both the 5' and 3' regions to assist the enzymatic ligation reaction.

[0039] 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, a delta hepatitis 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 a temperature of 0 to 37°C.

[0040] mmRNA multimer According to the present invention, multiple distinctly different polynucleotides, primary constructs, or mMRNAs can be linked via their 3' ends using nucleotides modified at the 3' ends. The stoichiometry of delivery to cells can be controlled using chemical complex formation. For example, glyoxylate cycle enzymes, isocitrate lyase, and malate synthase can be supplied to HepG2 cells in a 1:1 ratio to alter cellular fatty acid metabolism. This ratio can be controlled by chemically linking the polynucleotide, primary construct, or mMRNA to one polynucleotide, primary construct, or mMRNA species using a 3'-azide-terminal nucleotide and to the other polynucleotide, primary construct, or mMRNA species using a C5-ethynyl or alkynyl-containing nucleotide. These modified nucleotides are added post-transcriptionally using terminal transferase (New England Biolabs, Ipswich, MA) according to the manufacturer's protocol. Following the addition of a 3'-terminal modified nucleotide, these two polynucleotides, primary constructs, or mMRNA species can be combined in aqueous solution, in or out of the presence of copper, to form a new covalent bond via the click chemical mechanism described in the literature.

[0041] In another example, three or more polynucleotides may be linked using a functionalized linker molecule. For instance, a functionalized saccharide molecule may be chemically modified to contain multiple reactive groups (SH-, NH2-, N3, etc.) to react with a congeneral 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 stoichiometrically to directly control the stoichiometric ratio of the complexed polynucleotide, primary construct, or mRNA.

[0042] mmRNA complex and combination To further enhance protein production, the primary construct or mMRNA of the present invention may contain other polynucleotides, dyes, inserts (e.g., acridine), crosslinking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphylline, saffrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), alkylating agents, phosphates, amino acids, mercaptosaccharides, PEGs (e.g., PEG-40K), MPEG, [MPEG]2, polyamino acids, alkyl groups, Substitutive alkyl groups, 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 colligands), antibodies (e.g., antibodies that bind to specific cell types such as cancer cells, endothelial cells, or osteocytes), hormones and hormone receptors, non-peptide species (e.g., lipids, lectins, carbohydrates, vitamins, cofactors), or drugs may be designed to be complexed with these.

[0043] Complexation can result in increased stability and / or half-life, which may be particularly useful when targeting polynucleotides, primary constructs, or mMRNAs to specific sites in cells, tissues, or organisms.

[0044] In accordance with the present invention, an mMRNA or primary construct may be administered together with one or more of the following: RNAi agents, siRNA, shRNA, miRNA, miRNA binding sites, antisense RNA, ribozymes, catalytic DNA, tRNA, triple helix-inducing RNA, aptamers, or vectors, or may further encode these.

[0045] Bifunctional mMRNA One embodiment of the present invention is a bifunctional polynucleotide (e.g., a bifunctional primary construct or bifunctional mMRNA). As the name suggests, a bifunctional polynucleotide is a polynucleotide that has at least two functions or is capable of having at least two functions. These molecules may conventionally also be referred to as multifunctional.

[0046] The multiple functions of a bifunctional polynucleotide may be encoded by RNA (these functions may not manifest until the encoded product is translated) or they may be properties of the polynucleotide itself. These can be structural or chemical. Bifunctional modified polynucleotides may include functions covalently or electrostatically associated with the polynucleotide. Furthermore, these two functions may be provided in relation to a complex of mMRNA and another molecule.

[0047] Bifunctional polynucleotides can encode antiproliferative peptides. These peptides can be linear, cyclic, constrained, or randomly coiled. They can function as aptamers, signaling molecules, ligands, or mimics or mimetics thereof. When translated, antiproliferative peptides can be 3–50 amino acid long. They can be 5–40, 10–30, or about 15 amino acid long. They can be single-stranded, multi-stranded, or branched and, when translated, can form complexes, aggregates, or any multi-unit structures.

[0048] Non-coding polynucleotides and primary constructs As described herein, polynucleotides and primary constructs having partially or substantially non-translatable sequences, e.g., non-coding regions. Such non-coding regions may be the “first region” of a primary construct. Alternatively, the non-coding region may be a region other than the first region. Such molecules, though not normally translated, may influence protein production by binding to and sequestration of one or more translational components, such as ribosomal proteins or transfer RNA (tRNA), thereby effectively reducing protein expression in cells or modulating one or more pathways or cascades in cells, which in turn alter protein levels. Polynucleotides or primary constructs may contain, or encode, one or more long non-coding RNAs (lncRNAs or lincRNAs) or portions thereof, small nuclear RNAs (sno-RNAs), microRNAs (miRNAs), small interfering RNAs (siRNAs), or Piwi interfering RNAs (piRNAs).

[0049] Target polypeptide In accordance with the present invention, a primary construct is designed to encode one or more target polypeptides or fragments thereof. The target polypeptide may include, but is not limited to, a whole polypeptide, multiple polypeptides, or polypeptide fragments, which may independently be encoded by one or more nucleic acids, multiple nucleic acids, nucleic acid fragments, or any of the aforementioned variants. As used herein, the term “target polypeptide” refers to any polypeptide selected and encoded in the primary construct of the present invention. As used herein, “polypeptide” means a polymer of amino acid residues (natural or unnatural) linked most often by peptide bonds. As used herein, the term refers to proteins, polypeptides, and peptides having any size, structure, or function. In some examples, if the encoded polypeptide is smaller than about 50 amino acids, the polypeptide is referred to as a peptide. If a polypeptide is a peptide, it is at least about 2, 3, 4, or at least 5 amino acid residues long. Thus, polypeptides include gene products, naturally occurring polypeptides, synthetic polypeptides, homologs, orthologs, paralogs, the aforementioned fragments and other equivalents, variants, and analogs. Polypeptides can be single molecules or multimolecular complexes such as dimers, trimers, or tetramers. They may also include single-chain or multi-chain polypeptides, such as antibodies or insulin, and may associate or bind with them. Disulfide bonds are most commonly found in multi-chain polypeptides. The term "polypeptide" can also be applied to amino acid polymers, which are artificial chemical analogs of naturally occurring amino acids, with one or more amino acid residues.

[0050] The term "polypeptide variant" refers to a molecule whose amino acid sequence differs from that of the native sequence or reference sequence. Amino acid variants may have substitutions, deletions, and / or insertions at specific positions within the amino acid sequence compared to the native sequence or reference sequence. Typically, variants have at least about 50% identity (homology) with the native sequence or reference sequence, preferably at least about 80%, and more preferably at least about 90% identity (homology) with the native sequence or reference sequence.

[0051] In some embodiments, “muta mimes” are provided. As used herein, the term “muta mime” means a substance containing one or more amino acids that mimic an active sequence. For example, glutamate may act as a mime for phosphorothreonine and / or phosphoroserine. Alternatively, a muta mime may result in the inactivation or deactivation of a product containing the mime; for example, phenylalanine may function as an inactivating substitution for tyrosine, or alanine may function as an inactivating substitution for serine.

[0052] "Homologie," when applied to amino acid sequences, is defined as the percentage of residues in a candidate amino acid sequence that are identical to residues in a second sequence after the sequences have been aligned and gaps introduced as necessary to obtain the maximum homology percentage. Methods and computer programs for alignment are well known in the art. It is understood that homology depends on the calculation of identity percentage, but the value may differ due to gaps and penalties introduced into the calculation.

[0053] When applied to polypeptide sequences, "homologous" refers to a corresponding sequence of another species that shares a significant degree of identity with a second sequence of the second species. "Analogs" are intended to include polypeptide variants that differ by one or more amino acid modifications, e.g., substitution, addition, or deletion of amino acid residues, while still retaining one or more characteristics of the parent or starting polypeptide.

[0054] The present invention aims to provide several types of compositions of polypeptide systems, including mutants and derivatives. These include substitutions, insertions, deletions, and covalent mutants and derivatives. The term “derivative” is used synonymously with the term “mutant,” but generally refers to a molecule that has been modified and / or altered in any way relative to a reference molecule or starting molecule.

[0055] Accordingly, the scope of the invention includes mMRNAs encoding polypeptides that have undergone substitution, insertion and / or addition, deletion, and covalent modification to a reference sequence, specifically, a polypeptide sequence disclosed herein. For example, a sequence tag or amino acids, such as one or more lysines, may be added to the peptide sequence of the invention (e.g., at the N-terminus or C-terminus). Sequence tags may be used for peptide purification or localization. Lysines may be used to increase peptide solubility or enable biotinylation. Alternatively, amino acid residues located in the carboxyl and amino-terminal regions of the amino acid sequence of a peptide or protein may be optionally deleted to provide a cleavage sequence. Alternatively, certain amino acids (e.g., C-terminal or N-terminal residues) may be deleted during sequence expression, for example, as part of a larger soluble sequence, or bound to a solid support, depending on the use of the sequence.

[0056] A "substitutional variant," when referring to a polypeptide, is one in which at least one amino acid residue is removed from the natural or starting sequence and a different amino acid is inserted at the same position. These substitutions may be single substitutions, where only one amino acid in the molecule is replaced, or they may be polysubstituteds, where two or more amino acids in the same molecule are replaced.

[0057] As used herein, the term “conserved amino acid substitution” refers to the substitution of an amino acid normally present in a sequence with a different amino acid having a similar size, charge, or polarity. Examples of conserved substitutions include the substitution of a nonpolar (hydrophobic) residue, such as isoleucine, valine, and leucine, with another nonpolar residue. Similarly, examples of conserved substitutions include the substitution of a polar (hydrophilic) residue, such as arginine with lysine, glutamine with asparagine, and glycine with serine, with another residue. Further examples of conserved substitutions include the substitution of a basic residue, such as lysine, arginine, or histidine, with another basic residue, or the substitution of an acidic residue, such as aspartic acid or glutamic acid, with another acidic residue. Examples of non-conserved substitutions include the substitution of a nonpolar (hydrophobic) amino acid residue, such as isoleucine, valine, leucine, alanine, or methionine, with a polar (hydrophilic) residue, such as cysteine, glutamine, glutamic acid, or lysine, and / or the substitution of a polar residue with a nonpolar residue.

[0058] An "insertion variant" in the context of polypeptides refers to a polypeptide in which one or more amino acids are inserted immediately adjacent to an amino acid at a specific position within the natural or starting sequence. "Immediately adjacent" to an amino acid means that it is linked to either the α-carboxyl or α-amino functional group of the amino acid.

[0059] A "deletion variant" refers to a polypeptide in which one or more amino acids have been removed from the natural or starting amino acid sequence. Typically, a deletion variant involves the deletion of one or more amino acids in a specific region of the molecule.

[0060] When referring to polypeptides, "covalent derivatives" include modifications of native or starting proteins with organic or non-proteinogenic derivatizers, and / or post-translational modifications. Covalent modifications are conventionally introduced by reacting target amino acid residues of a protein with organic derivatizers that can react with selected side-chain or terminal residues, or by utilizing post-translational modification mechanisms that function in selected recombinant host cells. The resulting covalent derivatives are useful in programs aimed at identifying residues important for the biological activity, immunological assays, or preparation of anti-protein antibodies for immunoaffinity purification of recombinant glycoproteins. Such modifications are within the scope of the art and are performed without excessive experimentation.

[0061] Certain post-translational modifications are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently deamidated post-translation to their corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under weakly acidic conditions. Any form of these residues may be present in polypeptides produced according to the present invention.

[0062] Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, and methylation of α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)).

[0063] When referring to polypeptides, "characteristics" are defined as components based on the distinctly different amino acid sequences of the molecule. The characteristics of polypeptides encoded by the mMRNA of the present invention include surface appearance, local conformation, folding, loops, semiloops, domains, semidomains, sites, terminals, or any combination thereof.

[0064] As used herein and when referring to polypeptides, the term “surface appearance” refers to the appearance of the polypeptide components of a protein on its outermost surface. As used herein and when referring to polypeptides, the term “local conformation” means the appearance of the polypeptide system structure of a protein located within the definable space of the protein.

[0065] As used herein and when referring to polypeptides, the term “folding” refers to the three-dimensional structure resulting from the amino acid sequence at energy minimization. Folding can occur at the secondary or tertiary level of the folding process. Examples of secondary-level folding include β-sheets and α-helices. Examples of tertiary-level folding include domains and regions formed by the aggregation or separation of energy forces. Regions thus formed include hydrophobic and hydrophilic pockets, etc.

[0066] As used herein, the term “rotation” in relation to protein structures means a bend that alters the orientation of the backbone of a peptide or polypeptide, and may involve one, two, or three or more amino acid residues.

[0067] As used herein and when referring to polypeptides, 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 alters the orientation of the backbone, it may 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). Loops can be open or closed. Closed or “cyclic” loops may contain two, three, four, five, six, seven, eight, nine, or ten or more amino acids between the crosslinking segments. Such crosslinking segments may include cysteine-cysteine ​​crosslinks (Cys-Cys), which are typical in polypeptides with disulfide crosslinks, or the crosslinking segments may be non-protein systems, such as dibromodylyl agents used herein.

[0068] As used herein and when referring to polypeptides, the term “half-loop” refers to the portion of a loop having at least half of the amino acid residues identified as the loop from which it originates. It is understood that a loop does not necessarily contain an even number of amino acid residues. Therefore, where a loop contains an odd number of amino acids, or is identified as containing an odd number of amino acids, the half-loop of an odd-numbered loop contains the integer part of the loop or the next integer part (number of amino acids in the loop / 2 + / -0.5 amino acids). For example, a loop identified as a 7-amino acid loop may result in a half-loop of 3 or 4 amino acids (7 / 2 = 3.5 + / -0.5, which is 3 or 4).

[0069] As used herein and when referring to polypeptides, the term “domain” refers to a polypeptide motif having one or more identifiable structural or functional features or properties (e.g., binding ability, acting as a site for protein-protein interactions).

[0070] As used herein and when referring to polypeptides, the term “half-domain” means a portion of a domain having at least half of the amino acid residues identified as the domain from which it originates. It is understood that a domain does not necessarily contain an even number of amino acid residues. Therefore, where a domain contains an odd number of amino acids, or is identified as containing an odd number of amino acids, a half-domain of an odd-numbered domain contains an integer part of the domain or the next integer part (number of amino acids in the domain / 2 + / - 0.5 amino acids). For example, a domain identified as a 7-amino acid domain may result in a half-domain of 3 or 4 amino acids (7 / 2 = 3.5 + / - 0.5, resulting in 3 or 4). It is also understood that subdomains may be identified within a domain or half-domain, and these subdomains may have structural or functional properties that are less than all of the structural or functional properties identified in the domain or half-domain from which they originate. It is also understood that amino acids containing any of the domain types herein do not need to be adjacent along the polypeptide backbone (i.e., non-adjacent amino acids can structurally fold to result in a domain, half-domain, or subdomain).

[0071] As used herein and when referring to polypeptides, the term “site” is used synonymously with “amino acid residue” and “amino acid side chain” when relating to embodiments of amino acid systems. A site represents a location within a peptide or polypeptide that can be modified, manipulated, altered, derivatized, or changed within the polypeptide system molecule of the present invention.

[0072] As used herein and when referring to polypeptides, the terms “terminus” or “terminus” refer to the end of a peptide or polypeptide. Such an end may be limited to the first or final site of a peptide or polypeptide, and may also include additional amino acids in the terminal region. Polypeptide molecules of the present invention may be characterized by having both an N-terminus (terminated with a free amino group (NH2) by an amino acid) and a C-terminus (terminated with a free carboxyl group (COOH) by an amino acid). Proteins of the present invention may, in some cases, consist of multiple polypeptide chains linked by disulfide bonds or non-covalent bonds (multimers, oligomers). These types of proteins may have multiple N-terminuses and C-terminuses. Alternatively, the ends of polypeptides may be modified so that they begin or end with non-polypeptide moieties, such as organic complexes, as they may be.

[0073] Once any of these features is identified or defined as a desired component of the primary construct or polypeptide encoded by the mMRNA of the present invention, any of the operations and / or modifications of these features may be carried out by movement, exchange, inversion, deletion, randomization, or replication. Furthermore, it is understood that operations on features may yield the same results as modifications to the molecules of the present invention. For example, operations involving domain deletion result in alterations of molecular length, such as modifications to nucleic acids encoding less than a full-length molecule.

[0074] Modification and manipulation can be achieved by methods known in the art, including 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.

[0075] In accordance with the present invention, polypeptides may include consensus sequences discovered by a series of experiments. As used herein, a “consensus” sequence is a single sequence representing a collection of sequences that enable variability at one or more sites.

[0076] As will be recognized by those skilled in the art, protein fragments, functional protein domains, and homologous proteins are also considered to be within the scope of polypeptides targeted by the present invention. For example, any protein fragment of a reference protein having an amino acid length of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more than 100 (meaning a polypeptide sequence in which at least one amino acid residue is shorter than the reference polypeptide sequence but otherwise identical) is provided herein. In another example, any protein containing a sequence of about 20, about 30, about 40, about 50, or about 100 amino acids that is about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, or about 100% identical to any of the sequences described herein may be utilized according to the present invention. In a particular embodiment, the polypeptide utilized according to the present invention includes 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations shown in any of the sequences provided or referenced herein.

[0077] Coded polypeptide The primary constructs or mMRNAs of the present invention may be designed to encode a target polypeptide selected from any of several target categories, including, but not limited to, biologics, antibodies, vaccines, therapeutic proteins or peptides, cell-permeable peptides, secreted proteins, plasma membrane proteins, cytoplasmic or cytoskeletal proteins, intracellular membrane-bound proteins, nucleoproteins, proteins associated with human diseases, target regions, or proteins encoded by the human genome that have utility in the field of research and discovery even though no therapeutic indicators have been identified.

[0078] In one embodiment, a primary construct or mMRNA may encode a mutant polypeptide having a certain identity with a reference polypeptide sequence. As used herein, “reference polypeptide sequence” refers to a starting polypeptide sequence. The reference sequence may be a wild-type sequence or any sequence referenced in the design of another sequence. The "reference polypeptide sequence" is, for example, the concurrently pending U.S. Provisional Patent Application No. 61 / 618,862, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Biologics," U.S. Provisional Patent Application No. 61 / 681,645, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Biologics," U.S. Provisional Patent Application No. 61 / 737,130, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Biologics," and U.S. Provisional Patent Application No. 61 / 618,866, filed April 2, 2012, titled "Modified Polynucleotides for the U.S. Provisional Patent Application No. 61 / 681,647, filed August 10, 2012, title "Modified Polynucleotides for the Production of Antibodies", U.S. Provisional Patent Application No. 61 / 737,134, filed December 14, 2012, title "Modified Polynucleotides for the Production of Antibodies", U.S. Provisional Patent Application No. 61 / 618,868, filed April 2, 2012, title "Modified Polynucleotides for the Production of Vaccines", U.S. Provisional Patent Application No. 61 / 681,648, filed August 10, 2012, title "Modified Polynucleotides for the Production of Vaccines", U.S. Provisional Patent Application No. 61 / 737,135, filed December 14, 2012, title "Modified Polynucleotides for the Production of Vaccines, U.S. Provisional Patent Application No. 61 / 618,873, filed April 2, 2012, title: "Modified Polynucleotides for Vaccines" "The Production of Secreted Proteins," U.S. Provisional Patent Application No. 61 / 681,650, filed August 10, 2012, title: "Modified Polynucleotides for the Production of U.S. Provisional Patent Application No. 61 / 737,147, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Secreted Proteins", U.S. Provisional Patent Application No. 61 / 618,878, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Plasma Membrane Proteins", U.S. Provisional Patent Application No. 61 / 681,654, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Plasma U.S. Provisional Patent Application No. 61 / 737,152, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Plasma Membrane Proteins," and U.S. Provisional Patent Application No. 61 / 618,885, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal U.S. Provisional Patent Application No. 61 / 681,658, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins"; U.S. Provisional Patent Application No. 61 / 737,155, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins"; U.S. Provisional Patent Application No. 61 / 618,896, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Proteins" "Bound Proteins," U.S. Provisional Patent Application No. 61 / 668,157, filed July 5, 2012, title "Modified Polynucleotides for U.S. Provisional Patent Application No. 61 / 681,661, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins"; U.S. Provisional Patent Application No. 61 / 737,160, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins"; U.S. Provisional Patent Application No. 61 / 618,911, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Nuclear Proteins"; U.S. Provisional Patent Application No. 61 / 681,667, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Nuclear U.S. Provisional Patent Application No. 61 / 737,168, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Nuclear Proteins", U.S. Provisional Patent Application No. 61 / 618,922, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Proteins", U.S. Provisional Patent Application No. 61 / 681,675, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Proteins", U.S. Provisional Patent Application No. 61 / 737,174, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Proteins", U.S. Provisional Patent Application No. 61 / 618, filed April 2, 2012Patent No. 935, title "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 681,687, filed August 10, 2012, title "Modified Polynucleotides for the Production of Proteins Associated with Human, U.S. Provisional Patent Application No. 61 / 737,184, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 618,945, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with "Human Disease," U.S. Provisional Patent Application No. 61 / 681,696, filed August 10, 2012, title "Modified Polynucleotides for "The Production of Proteins Associated with Human Disease," U.S. Provisional Patent Application No. 61 / 737,191, filed December 14, 2012, title "Modified Polynucleotides" U.S. Provisional Patent Application No. 61 / 618,953, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 681,704, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 737,203, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Proteins International application PCT / US2013 / 030062, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Biologics and Proteins Associated with Human Disease", International application PCT / US2013 / 030064, titled "Modified Polynucleotides for the Production of Secreted Proteins", International application PCT / US2013 / 030059, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Membrane Proteins", International application PCT / US2013 / 030066, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal International application PCT / US2013 / 030067, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Nuclear Proteins", International application PCT / US2013 / 030060, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Proteins", International application PCT / US2013 / 030061, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Proteins Associated" The patents listed in Table 6 are those "with Human Disease," and the concurrently pending U.S. Provisional Patent Application No. 61 / 681,720, filed on August 10, 2012, titled "Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides”, 2012 U.S. Provisional Patent Application No. 61 / 737,213, filed December 14, titled "Modified The following are listed in Tables 6 and 7: Polynucleotides for the Production of Cosmetic Proteins and Peptides, U.S. Provisional Patent Application No. 61 / 681,742 filed August 10, 2012, titled "Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides", International Application No. PCT / US2013 / 030070 filed March 9, 2013, titled "Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides", and concurrently pending International Application No. PCT / US2013 / 030068 filed March 9, 2013, titled "Modified Polynucleotides for the Production of Cosmetic Proteins and The following are listed in Tables 6, 178 and 179, U.S. Provisional Patent Application No. 61 / 618,870, filed on April 2, 2012, entitled "Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides," listed in Tables 6, 28 and 29, U.S. Provisional Patent Application No. 61 / 681,649, filed on August 10, 2012, entitled "Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides," listed in Tables 6, 56 and 57, and U.S. Provisional Patent Application No. 61 / 737,139, filed on December 14, 2012, entitled "Modified Polynucleotides for the Production of Therapeutic Proteins." Any of the protein sequences listed in Tables 6, 186 and 187, as well as those listed in Tables 6, 185 and 186, in concurrently pending international application PCT / US2013 / 030063, filed 9 March 2013, titled "Modified Polynucleotides," the contents of each of these are incorporated herein by reference in their entirety.

[0079] In the art, the term "identity" refers to the relationship between the sequences of two or more peptides determined by comparing their sequences. In the art, identity also means the degree of sequence relevance between peptides, 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 that have gap alignments (if any), which are processed by a specific mathematical model or computer program (i.e., an "algorithm"). The identity of related peptides can be readily calculated by known methods. Such methods include: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part 1, Griffin, AM, and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von This includes, but is 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).

[0080] In some embodiments, polypeptide variants may have the same or similar activity as the reference polypeptide. Alternatively, the variants may have altered (e.g., increased or decreased) activity compared to the reference polypeptide. Generally, the variants of a particular polynucleotide or polypeptide of the present invention have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% but less than 100% sequence identity with a particular reference polynucleotide or polypeptide variant, as determined by the sequence alignment programs and parameters described herein and known to those skilled in the art. Tools for such alignment include a complete BLAST program (Stephen F. Altschul, Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), “Gapped BLAST and PSI-BLAST: a new generation of protein database search programs”, Nucleic Acids Res. 25:3389-3402). Other tools are described herein, specifically in the definition of “identity”.

[0081] Initial parameters for the BLAST algorithm include, for example, a prediction threshold of 10, a word size of 28, a match / mismatch score of 1, -2, and a linear gap cost. Any filters, as well as species-specific repeat sequences, such as human-specific repeat sequences, can be applied.

[0082] Biologics The polynucleotides, primary constructs, or mMRNAs disclosed herein may encode one or more biologics. As used herein, “biologic” is a polypeptide molecule produced by the methods provided herein and which can be used to treat, cure, alleviate, prevent, or diagnose serious or life-threatening diseases or conditions. Biologics include, but are not limited to, allergen extracts (e.g., for allergy injections and tests), blood components, gene therapy products, human tissue or cell products used for transplantation, vaccines, monoclonal antibodies, cytokines, growth factors, enzymes, thrombolytics, and immunomodulators, according to the present invention.

[0083] In accordance with the present invention, one or more biologics currently on the market or under development may be encoded by the polynucleotides, primary constructs, or mMRNAs of the present invention. While we do not wish to be constrained by theory, at least in part, due to their specificity, purity, and / or selectivity of construct design, the incorporation of polynucleotides encoding known biologics into the primary constructs or mMRNAs of the present invention is thought to result in improved therapeutic efficacy.

[0084] antibody The primary constructs or mMRNAs disclosed herein may encode one or more antibodies or fragments thereof. The term “antibody” includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions having polyepitope specificity, polyspecific antibodies (e.g., bispecific antibodies, diabodies, and single-chain molecules), and antibody fragments. The term “immunoglobulin (Ig)” is used herein as synonymous with “antibody.” As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population, i.e., an individual antibody that is identical to that population except for naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific and directed to a single antigen site.

[0085] The monoclonal antibodies described herein explicitly include “chimeric” antibodies (immunoglobulins) and fragments of such antibodies, insofar as they exhibit the desired biological activity, in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence of an antibody originating from a particular species or belonging to a particular antibody class or subclass, while the rest of the chain is identical or homologous to a corresponding sequence of an antibody originating from another species or belonging to another antibody class or subclass. The chimeric antibodies described herein include, but are not limited to, “primatized” antibodies containing variable domain antigen-binding sequences and human constant region sequences derived from non-human primates (e.g., Old World monkeys, apes, etc.).

[0086] An "antibody fragment" is a portion of an intact antibody, preferably comprising 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 polyspecific antibodies formed from antibody fragments.

[0087] The mmRNA of the present invention may encode any of the five classes of immunoglobulins, IgA, IgD, IgE, IgG, and IgM, each containing heavy chains designated as α, δ, ε, γ, and μ. Polynucleotide sequences encoding the subclasses γ and μ are also included. Therefore, any of the antibody subclasses, including the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, may be encoded partially or entirely.

[0088] In accordance with the present invention, one or more antibodies or fragments currently on the market or under development may be encoded by the polynucleotide, primary construct, or mMRNA of the present invention. While we do not wish to be constrained by theory, it is believed, at least in part, that the incorporation of the primary construct of the present invention will result in improved therapeutic efficacy due to its specificity, purity, and the selectivity of its mMRNA design.

[0089] Antibodies encoded in polynucleotides, primary constructs, or mMRNAs of the present invention can be used to treat conditions or diseases in many therapeutic fields, including, but not limited to, hematological, cardiovascular, CNS, toxicology (including antitoxins), dermatology, endocrinology, gastrointestinal, medical imaging, musculoskeletal, oncology, immunology, respiratory, sensory, and anti-infective conditions.

[0090] In one embodiment, the primary construct or mmol RNA disclosed herein may encode a monoclonal antibody and / or a variant thereof. Antibody variants may include, but are not limited to, substitutional variants, conserved amino acid substitutions, insertional variants, deletion variants, and / or covalent derivatives. In one embodiment, the primary construct and / or mmol RNA disclosed herein may encode an immunoglobulin Fc region. In another embodiment, the primary construct and / or mmol RNA may encode a variant immunoglobulin Fc region. As a non-limiting example, the primary construct and / or mmol RNA may encode an antibody having a variant immunoglobulin Fc region as described in U.S. Patent No. 8,217,147, which is incorporated herein in whole by reference.

[0091] vaccine The primary constructs or mMRNAs disclosed herein may encode one or more vaccines. As used herein, “vaccine” is a biological preparation that enhances immunity against a particular disease or infectious agent. According to the present invention, one or more vaccines currently on the market or under development may be encoded by the polynucleotides, primary constructs, or mMRNAs of the present invention. While we do not wish to be bound by theory, at least in part, due to specificity, purity, and selectivity of construct design, it is believed that incorporation into the primary constructs or mMRNAs of the present invention will result in improved therapeutic efficacy.

[0092] The present invention allows for the treatment of conditions or diseases in many therapeutic fields, including but not limited to cardiovascular, CNS, dermatology, endocrinology, oncology, immunology, respiratory, and anti-infective diseases, by utilizing vaccines encoded in polynucleotides, primary constructs, or mMRNA.

[0093] Therapeutic proteins or peptides The primary constructs or mMRNAs disclosed herein may encode one or more effective or "experimental" therapeutic proteins or peptides.

[0094] In accordance with the present invention, one or more therapeutic proteins or peptides currently on the market or under development may be encoded by the polynucleotides, primary constructs, or mMRNAs of the present invention. While we do not wish to be constrained by theory, it is believed, at least in part, that the incorporation of the primary constructs or mMRNAs of the present invention will result in improved therapeutic efficacy due to their specificity, purity, and selectivity in construct design.

[0095] The present invention provides therapeutic proteins and peptides encoded in polynucleotides, primary constructs, or mMRNAs that can be used to treat conditions or diseases in many therapeutic fields, including, but not limited to, hematological, cardiovascular, CNS, poisoning (including antitoxins), dermatology, endocrinology, genetics, urogenital, gastrointestinal, musculoskeletal, oncology, immunology, respiratory, sensory, and anti-infective systems.

[0096] Cell-permeable polypeptide The primary constructs or mMRNAs disclosed herein may encode one or more cell-permeable polypeptides. As used herein, “cell-permeable polypeptide” or CPP refers to a polypeptide that can facilitate the cellular uptake of a molecule. The cell-permeable polypeptides of the present invention may contain one or more detectable labels. Polypeptides may be partially labeled or fully labeled throughout. Polynucleotides, primary constructs, or mMRNAs may fully encode, partially encode, or not encode any detectable labels. Cell-permeable peptides may also contain signal sequences. As used herein, “signal sequence” refers to a sequence of amino acid residues that are bonded to the amino terminus of a nascent protein during protein translation. Signal sequences can be used to signal the secretion of cell-permeable polypeptides.

[0097] In one embodiment, a polynucleotide, primary construct, or mMRNA may also encode a fusion protein. The fusion protein may be created by operably binding a charged protein to the therapeutic protein. As used herein, “operably binding” means that the therapeutic protein and the charged protein are bound in such a way that they enable the expression of a complex when introduced into a cell. As used herein, “charged protein” means a protein having a positive charge, a negative charge, or an overall neutral charge. Preferably, the therapeutic protein may be covalently bound to the charged protein during the formation of the fusion protein. The ratio of surface charge to total amino acids 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.

[0098] Cell-permeable polypeptides encoded by polynucleotides, primary constructs, or mMRNAs may form complexes after translation. These complexes may contain, for example, covalently bonded charged proteins to the cell-permeable polypeptide. A “therapeutic protein” refers to a protein that, when administered to cells, has therapeutic, diagnostic, and / or prophylactic effects and / or induces a desired biological and / or pharmacological effect.

[0099] In one embodiment, the cell-permeable polypeptide may comprise a first domain and a second domain. The first domain may comprise a supercharged polypeptide. The second domain may comprise a protein-binding partner. As used herein, “protein-binding partner” includes, but is not limited to, antibodies and their functional fragments, scaffold proteins, or peptides. The cell-permeable polypeptide may further comprise an intracellular binding partner of the protein-binding partner. The cell-permeable polypeptide may be secreted from a cell into which polynucleotides, primary constructs, or mMRNA may be introduced. The cell-permeable polypeptide may also penetrate the first cell.

[0100] In further embodiments, the cell-permeable polypeptide can penetrate a second cell. The second cell may originate from the same region as the first cell or from a different region. This region may include, but is not limited to, tissues and organs. The second cell may be proximal to or distal to the first cell.

[0101] In one embodiment, a polynucleotide, primary construct, or mMRNA may encode a cell-permeable polypeptide that may contain a protein-binding partner. The protein-binding partner may include, but is not limited to, an antibody, a supercharged antibody, or a functional fragment. The polynucleotide, primary construct, or mMRNA may be introduced into cells into which the cell-permeable polypeptide containing the protein-binding partner is introduced.

[0102] Secretory Protein 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 its function. The cell uses "selection signals," which are amino acid motifs located within proteins, to target proteins to specific organelles.

[0103] A type of sorting signal, called a signal sequence, signal peptide, or leader sequence, directs a protein class to an organelle called the endoplasmic reticulum (ER). Proteins targeted to the ER by a signal sequence can be released into the extracellular space as secreted proteins. Similarly, proteins present on the cell membrane can be secreted into the extracellular space by proteolytic cleavage of the "linker" that holds the protein to the membrane. While we do not wish to be constrained by theory, the molecules of the present invention can be used to utilize the aforementioned cellular transport. Accordingly, in some embodiments of the present invention, polynucleotides, primary constructs, or mMRNAs expressing secreted proteins are provided. The secreted proteins may be selected from those described herein or in U.S. Patent Publication No. 20100255574, the contents of which are incorporated herein by reference in their entirety.

[0104] In one embodiment, these can be used in the production of large quantities of beneficial human gene products. Plasma membrane proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mMRNAs that express plasma membrane proteins are provided.

[0105] Cytoplasmic or cytoskeletal proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mMRNAs expressing cytoplasmic or cytoskeletal proteins are provided.

[0106] Intracellular membrane-bound proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mMRNAs expressing intracellular membrane-bound proteins are provided.

[0107] nucleoprotein In some embodiments of the present invention, polynucleotides, primary constructs, or mMRNAs expressing nucleoproteins are provided.

[0108] Proteins associated with human diseases In some embodiments of the present invention, polynucleotides, primary constructs, or mMRNAs expressing proteins associated with human diseases are provided.

[0109] various proteins In some embodiments of the present invention, polynucleotides, primary constructs, or mMRNAs expressing proteins having currently unknown therapeutic functions are provided.

[0110] target area In some embodiments of the present invention, polynucleotides, primary constructs, or mMRNAs expressing a target region are provided. These include protein-binding partners or receptors on the cell surface that function to target cells to a specific tissue space or interact with a specific region, 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 regions or biomolecules.

[0111] Polypeptide Library In one embodiment, polypeptide libraries can be generated using polynucleotides, primary constructs, or mMRNAs. These libraries may result from the generation of a population of polynucleotides, primary constructs, or mMRNAs having various structural or chemical modification designs. In this embodiment, the polynucleotides, primary constructs, or mMRNA populations may include, but are not limited to, antibodies or antibody fragments, protein-binding partners, scaffold proteins, and other polypeptides taught herein or known in the art, and may include a plurality of encoded polypeptides. In a preferred embodiment, the polynucleotide is a primary construct of the present invention comprising mMRNAs that may be suitable for direct introduction into target cells or cultures from which the subsequently encoded polypeptides can be synthesized.

[0112] In certain embodiments, multiple variants of a protein having 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 level. Such libraries can contain 10, 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 、10 8 、10 9 、or 10 9 or more possible variants (including, but not limited to, substitutions, deletions, and insertions of one or more residues).

[0113] Antibacterial and antiviral polypeptides The polynucleotides, primary constructs, and mMRNAs 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 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 The contents of these publications are described in US No. 6887847, US No. 6906035, US No. 6911524, US No. 6936432, US No. 7001924, US No. 7071293, US No. 7078380, US No. 7091185, US No. 7094759, US No. 7166769, US No. 7244710, US No. 7314858, and US No. 7582301, and the contents of these publications are incorporated in their entirety by reference.

[0114] The antimicrobial polypeptides described herein may block cell fusion and / or viral entry by one or more enveloped viruses (e.g., HIV, HCV). For example, an antimicrobial polypeptide may comprise, or consist of, a synthetic peptide corresponding to a region, e.g., a continuous sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of a transmembrane subunit of a viral envelope protein, e.g., HIV-1 gp120 or gp41. The amino acid and nucleotide sequences of gp120 or gp41 are described, for example, in Kuiken et al., (2008) “HIV Sequence Compendium,” Los Alamos National Laboratory.

[0115] In some embodiments, the antimicrobial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding viral protein sequence.

[0116] In other embodiments, the antimicrobial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, for example, a sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of a capsid-binding protein. In some embodiments, the antimicrobial polypeptide may have at least about 75%, 80%, 85%, 90%, 95%, or 100% sequence homology to the corresponding sequence of a capsid-binding protein.

[0117] The antimicrobial polypeptides described herein can block protease dimerization and inhibit the cleavage of viral proproteins (e.g., HIV Gag-pol treated) into functional proteins, thereby preventing the release of one or more enveloped viruses (e.g., HIV, HCV). 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.

[0118] In other embodiments, the antimicrobial polypeptide may comprise or consist of a synthetic peptide corresponding to a region, for example, a sequence of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 amino acids of the binding domain of a protease-binding protein. In some embodiments, the antimicrobial polypeptide may have sequence homology of at least about 75%, 80%, 85%, 90%, 95%, or 100% to the corresponding sequence of a protease-binding protein.

[0119] The antimicrobial polypeptides described herein may include polypeptides that have evolved in vitro and are targeted against viral pathogens. Antimicrobial polypeptide Antimicrobial polypeptides (AMPs) are small peptides of variable length, variable sequence, and variable structure that exhibit broad activity against a wide range of microorganisms, including but not limited to bacteria, viruses, fungi, protozoa, parasites, prions, and tumor / cancer cells (see, for example, Zaiou, J Mol Med, 2007;85:317, the whole of which is incorporated herein by reference). AMPs have been shown to possess broad, rapidly developing killing activity, low levels of induced resistance, and potentially broad anti-inflammatory effects that occur simultaneously.

[0120] In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may have a molecular weight of less than 10 kDa, e.g., 8 kDa, 6 kDa, 4 kDa, 2 kDa, or less than 1 kDa. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) consists of about 6 to about 100 amino acids, e.g., about 6 to about 75 amino acids, about 6 to about 50 amino acids, about 6 to about 25 amino acids, about 25 to about 100 amino acids, about 50 to about 100 amino acids, or about 75 to about 100 amino acids. In certain embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) may consist of about 15 to about 45 amino acids. In some embodiments, the antimicrobial polypeptide (e.g., antibacterial polypeptide) is substantially cationic.

[0121] In some embodiments, antimicrobial polypeptides (e.g., antibacterial polypeptides) may be substantially amphiphilic. In certain embodiments, antimicrobial polypeptides (e.g., antibacterial polypeptides) may be substantially cationic and amphiphilic. In some embodiments, antimicrobial polypeptides (e.g., antibacterial polypeptides) may be cell growth inhibitory against Gram-positive bacteria. In some embodiments, antimicrobial polypeptides (e.g., antibacterial polypeptides) may be cytotoxic against Gram-positive bacteria. In some embodiments, antimicrobial polypeptides (e.g., antibacterial polypeptides) may be cell growth inhibitory and cytotoxic against Gram-positive bacteria. In some embodiments, antimicrobial polypeptides (e.g., antibacterial polypeptides) may be cell growth inhibitory against Gram-negative bacteria. In some embodiments, antimicrobial polypeptides (e.g., antibacterial polypeptides) may be cytotoxic against Gram-negative bacteria. In some embodiments, antimicrobial polypeptides (e.g., antibacterial polypeptides) may be cell growth inhibitory and cytotoxic against Gram-positive bacteria. In some embodiments, antimicrobial polypeptides may be cell growth inhibitory against viruses, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, antimicrobial polypeptides may be cytotoxic to viruses, fungi, protozoa, parasites, prions, or combinations thereof. In certain embodiments, antimicrobial polypeptides may be cell proliferation inhibitory and cytotoxic to viruses, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, antimicrobial polypeptides may be cytotoxic to tumor or cancer cells (e.g., human tumors and / or cancer cells). In some embodiments, antimicrobial polypeptides may be cell proliferation inhibitory to tumor or cancer cells (e.g., human tumors and / or cancer cells). In certain embodiments, antimicrobial polypeptides may be cytotoxic and cell proliferation inhibitory to tumor or cancer cells (e.g., human tumors or cancer cells).In some embodiments, the antimicrobial polypeptide (e.g., an antibacterial polypeptide) may be a secreted polypeptide.

[0122] In some embodiments, the antimicrobial polypeptide comprises or consists of defensins. 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-defensins. In other embodiments, the antimicrobial polypeptide comprises or consists of cathelicidine (e.g., hCAP18).

[0123] Antiviral polypeptide Antiviral polypeptides (AVPs) are small peptides with variable length, variable sequence, and variable structure that exhibit broad activity against various viruses. For example, Zaiou, J. Mol. See Med, 2007;85:317. AVP has been shown to have broad-spectrum, rapidly occurring killing activity, low levels of induced resistance, and potentially broad-spectrum anti-inflammatory activity occurring simultaneously. In some embodiments, the antiviral polypeptide is less than 10 kDa, e.g., 8 kDa, 6 kDa, 4 kDa, 2 kDa, or less than 1 kDa. In some embodiments, the antiviral polypeptide contains 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 contains 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 amphiphilic. In certain embodiments, the antiviral polypeptide is substantially cationic and amphiphilic. In some embodiments, the antiviral polypeptide is cell growth inhibitory against viruses. In some embodiments, the antiviral polypeptide is cytotoxic against viruses. In some embodiments, the antiviral polypeptide is cell growth inhibitory and cytotoxic against viruses. In some embodiments, the antiviral polypeptide is cell growth inhibitory against bacteria, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, the antiviral polypeptide is cytotoxic against bacteria, fungi, protozoa, parasites, prions, or combinations thereof. In certain embodiments, the antiviral polypeptide is cell growth inhibitory and cytotoxic against bacteria, fungi, protozoa, parasites, prions, or combinations thereof. In some embodiments, the antiviral polypeptide is cytotoxic against tumor or cancer cells (e.g., human cancer cells). In some embodiments, the antiviral polypeptide is cell growth inhibitory against tumor or cancer cells (e.g., human cancer cells).In certain embodiments, the antiviral polypeptide is cytotoxic to tumor or cancer cells (e.g., human cancer cells) and inhibits cell proliferation. In some embodiments, the antiviral polypeptide is a secreted polypeptide.

[0124] Cytotoxic nucleosides In one embodiment, the polynucleotide, primary construct, or mMRNA of the present invention may incorporate one or more cytotoxic nucleosides. For example, a cytotoxic nucleoside may be incorporated into the polynucleotide, primary construct, or mMRNA, such as a bifunctionally modified RNA or mRNA. Cytotoxic nucleoside anticancer agents include, but are not limited to, adenosine arabinoside, cytarabine, cytosine arabinoside, 5-fluorouracil, fludarabine, phloxuridine, FTORAFUR® (a combination of tegafur and uracil), tegafur ((RS)-5-fluoro-1-(tetrahydrofuran-2-yl)pyrimidine-2,4(1H,3H)-dione), and 6-mercaptopurine.

[0125] Several cytotoxic nucleoside analogs are used clinically or are being studied in clinical trials as anticancer agents. Examples of such analogs include, but are not limited to, cytarabine, gemcitabine, troxacitabine, decitabine, tezacitabine, 2'-deoxy-2'-methylidenecytidine (DMDC), cladribine, clofarabine, 5-azacitidine, 4'-thio-aracitidine, cyclopentenylcytosine, and 1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)-cytosine. Another example of such compounds is fludarabine phosphate. These compounds can be administered systemically and may have typical side effects of cytotoxic agents, including, but are not limited to, side effects such as little to no specificity for tumor cells during the proliferation of normal cells.

[0126] Several prodrugs of cytotoxic nucleoside analogs have also been reported in the art. Examples include, but are not limited to, N4-behenoyl-1-β-D-arabinofuranosylcytosine, N4-octadecyl-1-β-D-arabinofuranosylcytosine, N4-palmitoyl-1-(2-C-cyano-2-deoxy-β-D-arabino-pentofuranosyl)cytosine, and P-4055 (cytarabine 5'-elaidic acid ester). Generally, these prodrugs are converted to active drugs primarily in the liver and systemic circulation, exhibiting little to no selective release of the active drug 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" are claimed by Fujiu et al. (U.S. Patent No. 4,966,891) and are incorporated herein by reference. This series of capecitabine analogs, as described by Fujiu, comprises N4 alkyl and aralkylcarbamates of 5'-deoxy-5-fluorocytidine, implying that these compounds are activated by hydrolysis under normal physiological conditions to yield 5'-deoxy-5-fluorocytidine.

[0127] A series of cytarabine N4-carbamates were reported by Fadl et al. (Pharmazie. 1995, 50, 382-7, incorporated herein by reference), in which the compounds were designed to be converted to cytarabine in the liver and plasma. International Publication WO2004 / 041203, incorporated herein by reference, discloses prodrugs of gemcitabine, in which some of the prodrugs are N4-carbamates. These compounds were designed to overcome the gastrointestinal toxicity of gemcitabine and were intended to deliver gemcitabine by hydrolysis release in the liver and plasma after absorption of the intact prodrug from the gastrointestinal tract. Nomura et al. (incorporated herein by reference, Bioorg Med. Chem. 2003, 11, 2453-61) describe an acetal derivative of 1-(3-C-ethynyl-β-D-ribo-pentofaranosyl)cytosine that produced an intermediate requiring further hydrolysis under acidic conditions upon in vivo reduction, thereby producing a cytotoxic nucleoside compound.

[0128] Cytotoxic nucleotides that may be chemotherapeutic agents include, but are not limited to, pyrazolo[3,4-D]-pyrimidine, allopurinol, azathioprine, capecitabine, cytosine arabinoside, fluorouracil, mercaptopurine, 6-thioguanine, acyclovir, ala-adenosine, ribavirin, 7-deaza-adenosine, 7-deaza-guanosine, 6-aza-uracil, 6-aza-cytidine, thymidine ribonucleotide, 5-bromodeoxyuridine, 2-chloropurine, and inosine, or combinations thereof.

[0129] Adjacent regions: Untranslated regions (UTR) The untranslated region (UTR) of a gene is transcribed but not translated. The 5'UTR begins at the transcription start site and follows the start codon, but does not contain the start codon, while the 3'UTR begins immediately after the stop codon and continues to the transcription termination signal. There have been numerous reports on the regulatory role that UTRs play in the stability of nucleic acid molecules and translation. The regulatory features of UTRs can be incorporated into the polynucleotides, primary constructs, and / or mMRNAs of the present invention to enhance molecular stability. Specific features can also be incorporated to ensure controlled downregulation of the transcript in the event that they are misdirected to undesirable organ sites.

[0130] 5'UTR and translation start Natural 5'UTRs possess features involved in translation initiation. They have features similar to Kozak sequences, which are commonly known to be involved in the process by which ribosomes initiate translation of many genes. Kozak sequences have 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.

[0131] By manipulating the characteristics typically found in genes highly expressed in specific target organs, the stability and protein production of the polynucleotides, primary constructs, or mMRNAs of the present invention can be enhanced. For example, by introducing the 5'UTR into mRNA expressed in the liver, such as albumin, serum amyloid A, apolipoproteins A / B / E, transferrin, α-fetoprotein, erythropoietin, or factor VIII, the expression of nucleic acid molecules such as mMRNAs in hepatocyte lines or the liver can be increased. Similarly, enhancing the expression of other tissue-specific mRNAs using their 5'UTRs is possible 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).

[0132] Other non-UTR sequences may be incorporated into the 5'UTR (or 3'UTR). For example, an intron or a portion of an intron sequence may be incorporated into the adjacent region of the polynucleotide, primary construct, or mRNA of the present invention. Incorporation of intron sequences may increase protein production and mRNA levels.

[0133] 3'UTR and AU rich elements 3'UTRs are known to contain a sequence of adenosine and uridine molecules enclosed within them. These AU-rich features are particularly common in genes with high turnover rates. Based on their sequence and functional characteristics, 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 AU-rich region. C-Myc and MyoD contain Class I AREs. Class II AREs have two or more duplicated UUAUUUA(U / A)(U / A) notamers. Molecules containing this type of ARE include GM-CSF and TNF-α. Class III AREs are less clearly defined. These AU-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, particularly HuR, have been demonstrated to improve mRNA stability. HuR binds to three classes of AREs. Manipulating the HuR-specific binding site to insert it into the 3'UTR of nucleic acid molecules leads to HuR binding and, consequently, in vivo message stabilization.

[0134] The stability of the polynucleotides, primary constructs, or mMRNAs of the present invention can be regulated by introducing, removing, or modifying AU-rich elements (AREs) in the 3'UTR. When manipulating specific polynucleotides, primary constructs, or mMRNAs, 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, and consequently increase the translation and production of the resulting protein. Transfection experiments using 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 time points after transfection. For example, cells can be transfected with different ARE-manipulating molecules, and the proteins produced at 6, 12, 24, 48, and 7 days after transfection can be assayed using an ELISA kit for the relevant proteins.

[0135] Integration of microRNA binding sites MicroRNAs (or miRNAs) are 19-25 nucleotide-long non-coding RNAs that bind to the 3'UTR of nucleic acid molecules and downregulate gene expression by either reducing the stability of the nucleic acid molecule or inhibiting translation. The polynucleotides, primary constructs, or mmRNAs of the present invention may comprise one or more microRNA target sequences, microRNA sequences, or microRNA species. Such sequences may correspond to any known microRNAs, such as those taught in U.S. Patent Publications US2005 / 0261218 and U.S. Patent Publications US2005 / 0059005, the contents of which are incorporated herein by reference in their entirety.

[0136] The microRNA sequence includes a “species” region, i.e., the sequence in the region between positions 2 and 8 of the mature microRNA, and this sequence exhibits complete Watson-Crick complementarity with the miRNA target sequence. The microRNA species may include positions 2 to 8 or 2 to 7 of the mature microRNA. In some embodiments, the microRNA species may include seven nucleotides (e.g., nucleotides 2 to 8 of the mature microRNA), and this species-complementary site in the corresponding miRNA target is adjacent to the adenine (A) opposite to microRNA position 1. In some embodiments, the microRNA species may include six nucleotides (e.g., nucleotides 2 to 7 of the mature microRNA), and the species-complementary site in the corresponding miRNA target is adjacent to the adenine (A) opposite to 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, the entirety of which is incorporated herein by reference. The bases of the microRNA species have complete complementarity with the target sequence. The microRNA target sequence can be manipulated to target the molecule for degradation or translation reduction by inserting it into the 3'UTR of the polynucleotide, primary construct, or mMRNA of the present invention, 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 (each of which is incorporated herein by reference: 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). 129:1401-1414).

[0137] For example, if a nucleic acid molecule is mRNA and is not intended to be delivered to the liver but settles there, then miR-122, an abundant microRNA in the liver, can inhibit the expression of a target gene if one or more target sites of miR-122 are manipulated and inserted into the 3'UTR of a polynucleotide, primary construct, or mMRNA. By manipulating the introduction of one or more binding sites of different microRNAs, the longevity, stability, and protein translation of the polynucleotide, primary construct, or mMRNA can be further reduced.

[0138] As used herein, the term “microRNA site” refers to a microRNA target site, a microRNA recognition site, or any nucleotide sequence to which a microRNA binds or associates. “Binding” should be understood to mean either following conventional Watson-Crick hybridization rules or representing any stable association of a microRNA with a target sequence at or adjacent to a microRNA site.

[0139] Conversely, for the purposes of the polynucleotides, primary constructs, or mMRNAs of the present invention, naturally occurring microRNA binding sites can be manipulated to remove them from the sequence (i.e., removed from the sequence) in order to increase protein expression in specific tissues. For example, the removal of the miR-122 binding site can improve protein expression in the liver. Control of expression in multiple tissues can be achieved by introducing or removing one or more microRNA binding sites.

[0140] Tissues in which microRNAs are known to regulate mRNA and consequently control protein expression include, but are not limited to, the 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 can also control complex biological processes such as angiogenesis (miR-132) (Anand and Cheresh Curr Opin Hematol 2011 18:171-176, the whole is incorporated herein by reference). In the polynucleotides, primary constructs, or mMRNAs of the present invention, microRNA binding sites involved in such processes may be removed or introduced to modulate the expression of the polynucleotides, primary constructs, or mMRNAs for biologically relevant cell types or in relation to relevant biological processes. Lists of microRNAs, miR sequences, and miR binding sites are provided 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, the whole of which is incorporated herein by reference.

[0141] Finally, through understanding microRNA expression patterns in different cell types, polynucleotides, primary constructs, or mMRNAs can be manipulated for more targeted expression in specific cell types or under specific biological conditions only. By introducing tissue-specific microRNA binding sites, polynucleotides, primary constructs, or mMRNAs optimized for protein expression in tissues or for protein expression related to biological conditions can be designed. Examples of microRNA use to drive tissue or disease-specific gene expression are listed (the entire text is incorporated herein by reference, Getner). (and Naldini, Tissue Antigens. 2012, 80:393-403). In addition, microRNA species sites can be incorporated into mRNA and reduce its expression in certain cells, which can lead to biological improvements. An example of this is the incorporation of the miR-142 site into a UGT1A1 expression lentiviral vector. The presence of the miR-142 species site reduced expression in hematopoietic cells, resulting in reduced expression in antigen-presenting cells and the absence of an immune response to UGT1A1 expressed by the virus (both are incorporated herein by reference in their entirety: Schmitt et al., Gastroenterology 2010;139:999-1007, Gonzalez-Asequinolaza et al. Gastroenterology 2010, 139:726-729). Incorporation of the miR-142 site into modified mRNA not only reduced the expression of the encoded protein in hematopoietic cells, but also reduced or neutralized the immune response to the mRNA-encoded protein. Incorporation of one or more miR-142 sites into mRNA is important in the treatment of patients with complete protein deficiency (e.g., type I UGT1A1, LDLR deficiency patients, CRIM-negative Pompe patients).

[0142] Transfection experiments using manipulated polynucleotides, primary constructs, or mMRNAs can be performed in relevant cell lines, and protein production can be assayed at various time points after transfection. For example, cells can be transfected with different microRNA-binding site-manipulated polynucleotides, primary constructs, or mMRNAs, and the proteins produced can be assayed using ELISA kits at 6, 12, 24, 48, 72, and 7 days after transfection. In vivo experiments using microRNA-binding site-manipulated molecules can also be performed to test changes in tissue-specific expression of formulated polynucleotides, primary constructs, or mMRNAs.

[0143] 5' capping The 5' cap structure of mRNA is involved in nuclear export, enhances mRNA stability, and binds to mRNA cap-binding proteins (CBPs), which, through association with poly(A)-binding proteins of CBPs, contribute to mRNA stability and translational readiness in cells, forming mature circular mRNA species. This cap also assists in the removal of 5' proximal introns during mRNA splicing.

[0144] Endogenous mRNA molecules can be 5'-capped, resulting in a 5'-ppp-5'-triphosphate bond between the terminal guanosine cap residue and the 5'-terminal transcription sense nucleotide of the mRNA molecule. Subsequently, this 5'-guanylate cap can be methylated to produce an N7-methyl-guanylate residue. The ribose sugars of the terminal and / or pre-terminal transcription nucleotides at the 5' end of the mRNA can also be optionally 2'-O-methylated. 5' decapping via hydrolysis and cleavage of the guanylate cap structure can target nucleic acid molecules such as mRNA molecules for degradation.

[0145] The polynucleotides, primary constructs, and mRNA modifications of the present invention can generate non-hydrolyzable cap structures that inhibit decapping, thereby increasing the mRNA half-life. 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, the vaccinia capping enzyme from New England Biolabs (Ipswich, MA) can be used with α-thio-guanosine nucleotides according to the manufacturer's instructions to induce phosphorothioate bonding at the 5'-ppp-5' cap. Further modified guanosine nucleotides, such as α-methylphosphonate and selenophosphate nucleotides, can be used.

[0146] Further modifications include, but are not limited to, 2'-O-methylation of the ribose sugar at the 5' end and / or pre-5' end nucleotide of mRNA at the 2'-hydroxyl group of the sugar ring (as described above). Multiple distinctly different 5'-cap structures can be used to generate the 5' cap of nucleic acid molecules such as mRNA molecules.

[0147] Cap analogs, also referred to herein as synthetic cap analogs, chemical caps, chemical cap analogs, or structural or functional cap analogs, differ in their chemical structure from natural (i.e., endogenous, wild-type, or physiological) 5' caps, but retain their cap function. Cap analogs can be synthesized chemically (i.e., non-enzymatically) or enzymatically and / or bound to nucleic acid molecules.

[0148] For example, the Reverse Direction Cap Analogue (ARCA) cap contains two guanines linked by a 5'-5'-triphosphate group, one of which is linked to an N7 methyl group and the other to a 3'-O-methyl group (i.e., N7,3'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine(m)). 7It contains G-3'mppp-G (which can be equivalently designated as 3'O-Me-m7G(5')ppp(5')G). The 3'-O atom of the other unmodified guanine is bound to the 5' terminal nucleotide of the capped nucleic acid molecule (e.g., mRNA or mmRNA). N7- and 3'-O-methylated guanines provide terminal portions of the capped nucleic acid molecule (e.g., mRNA or mmRNA).

[0149] Another example of a cap is mCAP, which is similar to ARCA but has a 2'-O-methyl group on guanosine (i.e., N7,2'-O-dimethyl-guanosine-5'-triphosphate-5'-guanosine, m 7 It has Gm-ppp-G.

[0150] While cap analogs enable simultaneous capping of nucleic acid molecules in in vitro transcription reactions, up to 20% of the transcript may remain uncapped. This, along with structural differences between the cap analogs and the endogenous 5'-cap structure of nucleic acids produced by endogenous cellular transcription mechanisms, can lead to reduced translational qualification and decreased cellular stability.

[0151] The polynucleotides, primary constructs, and mMRNAs of the present invention may also be post-transcriptionally capped using enzymes to produce more authentic 5'-cap structures. As used herein, the expression “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 compared to a synthetic feature or analogue of the prior art, or it surpasses the corresponding endogenous, wild-type, natural, or physiological feature in one or more respects. Non-limiting examples of more authentic 5'-cap structures of the present invention include, among others, enhanced cap-binding protein binding, increased half-life, reduced sensitivity to 5' endonucleases, and / or reduced 5' decapping compared to synthetic 5'-cap structures (or wild-type, natural, or physiological 5'-cap structures) known in the art. For example, recombinant vaccinia virus capping enzymes and recombinant 2'-O-methyltransferase enzymes can produce a standard 5'-5'-triphosphate bond between the 5' terminal nucleotide of mRNA and the guanine cap nucleotide, where the cap guanine contains N7 methylation and the 5' terminal nucleotide of mRNA contains 2'-O-methylation. Such a structure is referred to as a cap 1 structure. This cap results in higher translational qualification and cellular stability, as well as reduced activation of pro-inflammatory cytokines, compared, for example, with 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).

[0152] Polynucleotides, primary constructs, or mMRNAs can be capped post-transcriptionally, and because this process is more efficient, nearly 100% of polynucleotides, primary constructs, or mMRNAs can be capped. This is a significant difference from the approximately 80% that occur when capping analogs bind to mRNA during the in vitro transcription reaction.

[0153] According to the present invention, the 5'-terminated cap may include an endogenous cap or a cap analog. According to the present invention, the 5'-terminated cap may 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.

[0154] Virus sequence Further viral sequences, including but not limited to translational enhancer sequences of barley stripe molar virus (BYDV-PAV), Jagziktehitsuji retrovirus (JSRV), and / or endemic rhinotumor virus (see, for example, International Publication WO2012129648, which is incorporated herein in whole by reference), may be manipulated and inserted into the 3'UTR of the polynucleotides, primary constructs, or mMRNAs of the present 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, 24, 48, 72, and 7 days post-transfection.

[0155] IRES array Furthermore, polynucleotides, primary constructs, or mMRNAs that may contain an internal ribosome entry site (IRES) are provided. The IRES, initially identified as a characteristic picornavirus RNA, plays a crucial role in initiating protein synthesis in the absence of a 5' cap structure. The IRES may function as the sole ribosome binding site of the mRNA, or as one of several ribosome binding sites. Polynucleotides, primary constructs, or mMRNAs containing two or more functional ribosome binding sites may encode several peptides or polypeptides that are independently translated by ribosomes ("polycistronic nucleic acid molecules"). When an IRES is provided to a polynucleotide, primary construct, or mMRNA, a second translatable region may be further optionally provided. Examples of IRES sequences that can be used in accordance with the present invention include, without limitation, those derived from picornaviruses (e.g., FMDV), plague virus (CFFV), poliovirus (PV), encephalomyocarditis virus (ECMV), foot-and-mouth disease virus (FMDV), hepatitis C virus (HCV), classical swine fever virus (CSFV), mouse leukemia virus (MLV), simian immunodeficiency virus (SIV), or cricket paralysis virus (CrPV).

[0156] Poly A tail During RNA processing, a long adenine nucleotide chain (poly-A tail) can be added to polynucleotides such as mRNA molecules to improve stability. Immediately after transcription, the 3' end of the transcript may be cleaved, releasing a 3' hydroxyl group. Subsequently, poly-A polymerase adds the adenine nucleotide chain to the RNA. This process, called polyadenylation, adds a poly-A tail that can be approximately 100-250 residues long, for example.

[0157] The unique length of the poly(A) tail has been found to provide specific advantages to the polynucleotides, primary constructs, or mMRNAs of the present invention. Generally, the length of the poly-A tail of the present invention is greater than 30 nucleotides. In another embodiment, the poly-A tail is greater than 35 nucleotides (for example, 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, 1,600, 1,700, 1,800, 1,900, 2,000, 2,500, and 3,000 nucleotides, or greater). In some embodiments, the polynucleotide, primary construct, or mMRNA has approximately 30 to approximately 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 Includes 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.

[0158] In one embodiment, the polyA tail is designed relative to the length of the entire polynucleotide, primary construct, or mMRNA. This design may be based on the length of the coding region, the length of a specific feature or region (such as a first or adjacent region), or the length of the final product expressed from the polynucleotide, primary construct, or mMRNA.

[0159] In this context, the polyA tail may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% longer than the polynucleotide, primary construct, or mMRNA, or its features. The polyA tail may also be designed as a fraction of the polynucleotide, primary construct, or mMRNA to which it belongs. In this context, the polyA tail may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more of the total length of the construct or the total length of the construct minus the polyA tail. Furthermore, manipulation of the binding site and complexation of the polynucleotide, primary construct, or mMRNA with the polyA-binding protein may enhance expression.

[0160] Furthermore, multiple distinctly different polynucleotides, primary constructs, or mMRNAs can be bound to PABP (poly-A binding protein) via the 3' end using nucleotides modified at the 3' end of the poly-A tail. Transfection experiments can be performed in relevant cell lines, and protein production can be assayed by ELISA at 12, 24, 48, 72, and 7 days post-transfection.

[0161] In one embodiment, the polynucleotide primary construct of the present invention is designed to include a poly-A-G quadruplet. The G quadruplet is a cyclic hydrogen-bonded array of four guanine nucleotides that can be formed by G-rich sequences in both DNA and RNA. In this embodiment, the G quadruplet is incorporated at the end of the poly-A tail. The resulting mMRNA construct is assayed for stability, protein production, and half-life at various time points and other parameters. It has been found that the poly-A-G quadruplet yields protein production equivalent to at least 75% of the protein production obtained using the 120-nucleotide poly-A tail alone.

[0162] Quantification In one embodiment, the polynucleotides, primary constructs, or mMRNAs of the present invention can be quantified in exosomes derived from one or more bodily fluids. As used herein, “bodily fluids” include peripheral blood, serum, plasma, ascites, urine, cerebrospinal fluid (CSF), sputum, saliva, bone marrow, synovial fluid, aqueous humor, amniotic fluid, earwax, breast milk, bronchoalveolar lavage fluid, semen, prostatic fluid, Cooper’s gland fluid or preejaculatory fluid, sweat, feces, hair, tears, cystic fluid, pleura and ascites, pericardial fluid, lymph, atherosclerotic fluid, chyle, bile, interstitial fluid, vaginal discharge, pus, sebum, vomit, vaginal secretions, mucosal secretions, watery stool, pancreatic juice, nasal lavage fluid, bronchopulmonary aspirate, blastocyst cavity fluid, and umbilical cord blood. Alternatively, exosomes may be recovered from organs selected from the group consisting of the lungs, heart, pancreas, stomach, intestines, bladder, kidneys, ovaries, testes, skin, colon, breasts, prostate, brain, esophagus, liver, and placenta.

[0163] In the quantification method, less than 2 mL of sample is obtained from the subject, and exosomes are isolated by size exclusion chromatography, density gradient centrifugation, fractional centrifugation, nanomembrane ultrafiltration, immunoabsorption capture, affinity purification, microfluidic separation, or a combination thereof. In the analysis, the levels or concentrations of polynucleotides, primary constructs, or mMRNA may be the expression level, presence, absence, cleavage, or modification of the administered construct. Correlating these levels with an assay for one or more clinical phenotypes or human disease biomarkers is advantageous. While this assay may be performed using construct-specific probes, cytometry, qRT-PCR, real-time PCR, PCR, flow cytometry, electrophoresis, mass spectrometry, or a combination thereof, exosomes can be isolated using immunohistochemistry methods such as enzyme-linked immunosorbent assay (ELISA). Exosomes can also be isolated by size exclusion chromatography, density gradient centrifugation, fractional centrifugation, nanomembrane ultrafiltration, immunoabsorption capture, affinity purification, microfluidic separation, or a combination thereof.

[0164] These methods provide researchers with the ability to monitor in real time the levels of residual or delivered polynucleotides, primary constructs, or mMRNAs. This is possible because the polynucleotides, primary constructs, or mMRNAs of the present invention differ from their endogenous forms due to structural or chemical modifications.

[0165] II. Design and Synthesis of mMRNA The polynucleotides, primary constructs, or mMRNAs used in accordance with the present invention may be prepared by 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 incorporated herein by reference).

[0166] The process for designing and synthesizing the primary construct of the present invention generally comprises 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 the 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 may be codon-optimized. mRNA is then produced using the cDNA template by in vitro transcription (IVT). After production, the mRNA may undergo purification and cleansing processes. These steps are provided in more detail below.

[0167] Genetic construction The gene construction steps may include, but are not limited to, gene synthesis, vector amplification, plasmid purification, plasmid linearization and cleansing, and cDNA template synthesis and cleansing.

[0168] gene synthesis A primary construct is designed once the target polypeptide or target has been selected for production. Within the primary construct, the first region of the binding nucleoside encoding the target polypeptide may be constructed using an open reading frame (ORF) of the selected nucleic acid (DNA or RNA) transcript. The ORF may include a wild-type ORF, isoform, variant, or fragment thereof. As used herein, “open reading frame” or “ORF” is intended to refer to a nucleic acid sequence (DNA or RNA) capable of encoding the target polypeptide. ORFs often begin with a start codon ATG and end with a nonsense or stop codon or signal.

[0169] Furthermore, the nucleotide sequence of the first region can be codon-optimized. Codon optimization methods are known in the art and may be useful in efforts to achieve one or more of the following goals: matching codon frequencies in the target and host organisms to ensure proper folding; boosting GC content to increase mRNA stability or reducing secondary structures; minimizing tandem repeat codons or nucleotide electrophoresis that may impair gene construction or expression; customizing transcriptional and translational regulatory regions; inserting or removing protein transport sequences; removing / adding post-translational modification sites (e.g., glycosylation sites) in encoded proteins; adding, removing, or rearranging protein domains; inserting or deleting restriction sites; modifying ribosome binding sites and mRNA degradation sites; regulating translation rates to enable proper folding of various domains of the protein; or reducing or eliminating problematic secondary structures within the mRNA. Codon optimization tools, algorithms, and services are known in the art, and non-limiting examples include the GeneArt service (Life Technologies), DNA2.0 (Menlo Park CA), and / or proprietary methods. In one embodiment, an ORF sequence is optimized using an optimization algorithm. Codon options for each amino acid are provided in Table 1.

[0170] Table 1. Codon options

[0171] [Table 0001] Features that may be beneficial in some embodiments of the present invention may be encoded by the primary constructor and may be adjacent to the ORF as a first or second adjacent region. This adjacent region may be incorporated into the primary constructor before and / or after optimization of the ORF. The primary constructor does not need to contain both the 5' and 3' adjacent regions. Examples of such features include, but are not limited to, untranslated regions (UTRs), Kozak sequences, oligo (dT) sequences, and detectable tags, as well as multiple cloning sites that may have XbaI recognition.

[0172] In some embodiments, a 5'UTR and / or a 3'UTR may be provided as an adjacent region. Multiple 5' or 3'UTRs may be contained within the adjacent region and may be identical or different sequences. Any portion of the adjacent region (including cases where no such portion exists) may be codon-optimized, and any of them may independently contain one or more different structural or chemical modifications before and / or after codon optimization. Combinations of features may be contained within the first and second adjacent regions and within other features. For example, an ORF may be adjacent to a 5'UTR that may contain a potent Kosack translation initiation signal for poly-A tail template addition and a 3'UTR that may contain an oligo(dT) sequence. A 5'UTR, such as the one described in U.S. Patent Application Publication No. 20100293625, which is incorporated herein by reference in whole, may contain a first polynucleotide fragment and a second polynucleotide fragment from the same and / or different genes.

[0173] Tables 2 and 3 provide a list of exemplary UTRs that may be used as adjacent regions in the primary construct of the present invention. A list of 5' untranslated regions of the present invention is shown in Table 2. Mutations of the 5' UTR may be used in which one or more nucleotides containing A, T, C, or G are added to or removed from the terminus.

[0174] Table 2. 5' untranslated region

[0175] [Table 0002] Table 3 shows a representative list of the 3' untranslated regions of the present invention. Mutations of the 3'UTR in which one or more nucleotides containing A, T, C, or G are added to or removed from the terminal may be used.

[0176] Table 3. 3' untranslated region

[0177] [Table 0003-1]

[0178] [Table 0003-2]

[0179] [Table 0003-3]

[0180] [Table 0003-4]

[0181] [Table 0003-5]

[0182] [Table 0003-6]

[0183] [Table 0003-7] The examples listed in the table above are for illustrative purposes only, and it should be understood that any UTR derived from any gene can be incorporated into the first or second adjacent region of each primary construct. Furthermore, multiple wild-type UTRs of any known gene can be utilized. Providing artificial UTRs that are not variants of wild-type genes is also within the scope of the invention. These UTRs or portions thereof can be positioned in the same orientation as the UTR or portion thereof of the transcript from which they may be selected, or their orientation or position can be altered. Thus, a 5' or 3' UTR can be inverted, shortened, lengthened, and chimerized with one or more other 5' UTRs or 3' UTRs. As used herein, the term “modified” means that, when relating to a UTR sequence, the UTR has been altered in relation to the reference sequence. For example, a 3' or 5' UTR can be modified from a wild-type or native UTR by the orientation or positional changes taught above, or by the inclusion of further nucleotides, deletion of nucleotides, exchange or transposition of nucleotides. Any of these changes that result in a “modified” UTR (whether 3' or 5') includes a variant UTR.

[0184] In one embodiment, a double, triple, or quadruple UTR, such as a 5' or 3' UTR, may be used. As used herein, a “double” UTR is one in which two copies of the same UTR are coded either consecutively or substantially consecutively. For example, a double β-globin 3' UTR described in U.S. Patent Publication No. 20100129877, whose contents are incorporated herein by reference in their entirety, may be used.

[0185] Having patterned UTRs is also within the scope of the present invention. As used herein, “patterned UTR” refers to a UTR that exhibits a repeating or alternating pattern that is repeated once, twice, or three or more times, for example, ABABAB, AABBABBAABB, or ABCABCABC, or a variant thereof. In these patterns, each letter, A, B, or C, represents a different UTR at the nucleotide level.

[0186] In one embodiment, the adjacent region is selected from a family of transcripts from which the protein shares common function, structure, or characteristic features. For example, the target polypeptide may belong to a family of proteins that are expressed in specific cells or tissues, or at some point during development. Any UTR of any of these genes can be replaced with any other UTR of the same or different protein families to create a new chimeric primary transcript. As used herein, “family of proteins” is used in its broadest sense to refer to a group of two or more target polypeptides that share at least one function, structure, characteristic, localization, origin, or expression pattern.

[0187] After optimization (if desired), the primary construct components can be reconstructed and converted into vectors, including but not limited to plasmids, viruses, cosmids, and artificial chromosomes. For example, the optimized construct can be reconstructed and converted into chemically competent E. coli, yeast, Neurospora crassa, maize, Drosophila melanogaster, etc., and high copy number plasmid-like or chromosomal structures can be produced by the methods described herein.

[0188] The untranslated region may also include translation enhancer elements (TEEs). In non-limiting examples, TEEs may include the TEE described in U.S. Patent Application No. 20090226470, which is incorporated herein in whole 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 is isolated and purified using methods known in the art, such as, but not limited to, maxiprep using the Invitrogen PURELINK® HiPure Maxiprep kit (Carlsbad, CA).

[0191] Plasmid linearization The plasmid can then be linearized using methods known in the art, including but not limited to the use of restriction enzymes and buffers. The linearized reaction product can be purified using, for example, Invitrogen's PURELINK® PCR Micro kit (Carlsbad, CA), as well as HPLC-based purification methods, including but not limited to strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), and methods including Invitrogen's standard PURELINK® PCR kit (Carlsbad, CA). The purification method may be modified depending on the size of the resulting linearized reaction product. The linearized plasmid is then used to generate cDNA for in vitro transcription (IVT) reactions.

[0192] cDNA template synthesis cDNA templates can be synthesized by polymerase chain reaction (PCR) on a linearized plasmid. Table 4 lists primers and probes that may be useful in the PCR reaction of the present invention. It should be understood that this list is not exhaustive and that primer-probe designs for any amplification are within the scope of the art. Probes may also contain chemically modified bases to enhance base pairing fidelity and base pairing strength to the target molecule. Such modifications may include 5-methylcytidine, 2,6-diaminopurine, 2'-fluoro, phosphorothioate, or locked nucleic acids.

[0193] Table 4. Primers and probes

[0194] [Table 0004] *UFP stands for Universal Forward Primer, and URP stands for Universal Reverse Primer.

[0195] In one embodiment, the cDNA may be submitted for sequencing analysis before transcription. mRNA production The mRNA or mMRNA production process may include, but is not limited to, in vitro transcription, cDNA template removal and RNA purification, as well as mRNA capping and / or tailing reactions.

[0196] In vitro transfer The cDNA produced in the previous step can be transcribed using an in vitro transcription (IVT) system. This system typically comprises a transcription buffer, a nucleotide triphot (NTP), an RNase inhibitor, and a polymerase. The NTP may be manufactured in-house, selected from a supplier, or synthesized as described herein. The NTP may be selected from, but is not limited to, natural and non-natural (modified) NTPs as described herein. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase, and polymerases capable of incorporating modified nucleic acids, but is not limited to, mutant polymerases.

[0197] RNA polymerase Any number of RNA polymerases or variants may be used in the design of the primary construct of the present invention.

[0198] RNA polymerase can be modified by inserting or deleting amino acids from the RNA polymerase sequence. As a non-limiting example, RNA polymerase can be modified to exhibit an improved ability to incorporate 2'-modified nucleotide triphosphates compared to unmodified RNA polymerase (see International Publication WO2008078180 and U.S. Patent No. 8,101,385, these in whole are incorporated herein by reference).

[0199] Mutants can be obtained by evolving RNA polymerase, optimizing the RNA polymerase amino acid and / or nucleic acid sequence, and / or by using other methods known in the art. As a non-limiting example, T7 RNA polymerase mutants can be evolved using a sequentially oriented evolutionary system designed by Esvelt et al. (Nature (2011) 472(7344):499-503, which is incorporated herein by reference in its entirety), and T7 RNA polymerase clones include mutations such as K93T (lysine substituted with threonine at position 93), I4M, A7T, E63V, V64D, A65E, D66Y, T76N, C125R, S128R, A136T, N165S, G175R, H176L, Y178H, F182L, L196F, G198V, D208Y, E222K, S228A, Q239R, T243N, G259D, M267I, G280C, H300R, D351A, A354S, E356D, L360P, A383V, Y385C, D388Y, S397R, M401T, N410S, K450R, P451T, G452V, E484A, H523L, H524N, G542V, E565K, K577E, K577M, N601S, S684Y, L699I, K713E, N748D, Q754R, E775K, A827V, D851N, or L864F, etc., may encode at least one mutation, but is not limited to these. As another non-limiting example, the T7 RNA polymerase mutant may encode at least one mutation described in U.S. Patent Publications 20100120024 and 20070117112, which are incorporated herein by reference in their entirety. Mutations of RNA polymerase may include, but are not limited to, substitutional mutations, conserved amino acid substitutions, insertional mutations, deletion mutations, and / or covalent derivatives.

[0200] In one embodiment, the primary construct may be designed to be recognized by wild-type or mutant RNA polymerase. In this way, the primary construct may be modified to contain sequence alteration sites or regions derived from the wild-type or parent primary construct.

[0201] In one embodiment, the primary construct may be designed to include 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.

[0202] 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 nucleotide region and / or string may, but is not limited to, contain at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 nucleotides, and these nucleotides may be natural and / or non-natural. As a non-limiting example, this nucleotide group may contain a string of 5 to 8 adenines, cytosines, thymines, any of the other nucleotides disclosed herein, and / or combinations thereof.

[0203] 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, including 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 to 8 adenine bases, followed by 5 to 8 cytosine bases. In another example, the 5'UTR may be replaced by inserting 5 to 8 cytosine bases, followed by 5 to 8 adenine bases.

[0204] In one embodiment, the primary construct may include at least one substitution and / or insertion downstream of a transcription start site that can be recognized by RNA polymerase. As a non-limiting example, at least one substitution and / or insertion may occur downstream of the transcription start site by substituting at least one nucleic acid in a region immediately downstream of the transcription start site (e.g., +1 to +6, but not limited to these regions). By altering the nucleotide region immediately downstream of the transcription start site, it may be possible to affect the initiation rate, increase the apparent constant value of the nucleotide triphot (NTP) reaction, and increase the dissociation of short transcripts from the transcription complex by curing the initial transcript (the whole is incorporated herein by reference, Brieba et al, Biochemistry (2002) 41:5144-5149). Modification, substitution, and / or insertion of at least one nucleic acid may result in silent mutations of the nucleic acid sequence or mutations in the amino acid sequence.

[0205] In one embodiment, the primary construct may include 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 base substitutions downstream of the transcription start site.

[0206] 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, or at least 6 guanine bases in the region immediately downstream of the transcription start site. In an unrestricted example, when the nucleotide in that region is GGGAGA, the guanine bases may be substituted with at least 1, at least 2, at least 3, or at least 4 adenine nucleotides. In another unrestricted example, when the nucleotide in that region is GGGAGA, the guanine bases may be substituted with at least 1, at least 2, at least 3, or at least 4 cytosine bases. In yet another unrestricted example, when the nucleotide in that region is GGGAGA, the guanine bases may be substituted with at least 1, at least 2, at least 3, or at least 4 thymine and / or any of the nucleotides described herein.

[0207] In one embodiment, the primary construct may include at least one substitution and / or insertion upstream of the start codon. For clarification, those skilled in the art will understand that the start codon is the first codon of the protein coding region, while the transcription start site is the site where transcription begins. The primary construct may include, 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 identical bases (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, a guanine base upstream of the coding region in the primary construct may be substituted with adenine, cytosine, thymine, or any of the nucleotides described herein. In another non-limiting example, a guanine base substitution in the primary construct may be designed to leave one guanine base downstream of the transcription start site and prior to 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 may be inserted at one position downstream of the transcription start site and upstream of the start codon, and at least five nucleotides may be of the same base type.

[0208] cDNA template removal and purification cDNA templates can be removed using methods known in the art, such as treatment with deoxyribonuclease I (DNase I), but are not limited to these. RNA purification can be performed using Beckman Coulter (Danvers, MA)'s AGENCOURT® CLEANSEQ® system; strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC), but are not limited to these HPLC-based purification methods, but may also include purification methods not limited to these.

[0209] Capping and / or tailing reactions The primary construct or mMRNA may also undergo capping and / or tailing reactions. 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 enzymes (New England Biolabs, Ipswich, MA).

[0210] The poly-A tailing reaction can be carried out 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 poly-T, it may be beneficial to perform the poly-A tailing reaction before the primary construct is purified.

[0211] mRNA purification Primary construct or mMRNA purification may include, but is not limited to, mRNA or mMRNA cleansing, quality assurance, and quality control. mRNA or mMRNA cleansing may be performed using methods known in the art, such as AGENCOURT® beads (Beckman Coulter Genomics, Danvers, MA); Poly-T beads; LNA® oligo-T capture probe (EXIQON® Inc, Vedbaek, Denmark); or HPLC-based purification methods, such as, but is not limited to, strong anion exchange HPLC, weak anion exchange HPLC, reverse-phase HPLC (RP-HPLC), and hydrophobic interaction HPLC (HIC-HPLC). The term "purified," as in "purified mRNA or mMRNA," when used in relation to polynucleotides, means separated from at least one contaminant. As used herein, "contaminant" is any substance that makes another substance unsuitable, impure, or inferior. Therefore, purified polynucleotides (e.g., DNA and RNA) exist in a form or environment different from that which they exist in naturally occurring forms or environments, or in a form or environment different from that which they existed in before being subjected to processing or purification methods.

[0212] Quality assurance and / or quality control inspections may be performed using methods such as, but are not limited to, gel electrophoresis, ultraviolet absorption, or analytical HPLC. In another embodiment, mRNA or mmRNA may be sequenced using methods including, but not limited to, reverse transcription PCR.

[0213] In one embodiment, 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 is of appropriate size and to confirm that no mRNA or mmRNA degradation has occurred. mRNA and / or mmRNA degradation 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, reverse-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 Primary constructs or mMRNAs may also encode further features that facilitate the transport of polypeptides to therapeutically relevant sites. One such feature that assists protein transport is a signal sequence. As used herein, “signal sequence” or “signal peptide” are, respectively, polynucleotides or polypeptides approximately 9–200 nucleotides (3–60 amino acids) long, which are incorporated into the coding region or the 5' (or N-terminus) of the encoded polypeptide. The addition of these sequences results in the transport of the encoded polypeptide into the endoplasmic reticulum via one or more secretory pathways. Some signal peptides are cleaved from the protein by signal peptidases after the protein has been transported.

[0215] Table 5 is a representative list of protein signal sequences that may be incorporated to encode polynucleotides, primary constructs, or mMRNAs according to the present invention. Table 5. Signal Sequences

[0216] [Table 0005-1]

[0217] [Table 0005-2]

[0218] [Table 0005-3]

[0219] [Table 0005-4]

[0220] [Table 0005-5]

[0221] [Table 0005-6] In this table, SS is a secretion signal and MLS is a mitochondrial leader signal. The primary constructs or mMRNAs of the present invention may be designed to encode any of the signal sequences of SEQ ID NOs. 94-155, or fragments or variants thereof. These sequences may be located at the beginning, middle, or end of the polypeptide coding region, or in an adjacent region. Furthermore, any of the polynucleotide primary constructs of the present invention may also include one or more sequences defined by SEQ ID NOs. 32-93. These may be located in the first region or in any of the adjacent regions.

[0222] Further signal sequences that may be used in the present invention include, for example, signal sequences taught in databases such as the database found at http: / / www.signalpeptide.de / or http: / / proline.bic.nus.edu.sg / spdb / . The provisions of U.S. Patents 8,124,379, 7,413,875, and 7,385,034 are also within the scope of the present invention, and the contents of each of these are incorporated herein by reference in their entirety.

[0223] Target selection According to the present invention, the primary construct comprises at least one first region of a bound nucleoside encoding at least one target polypeptide. The target polypeptide, i.e., the "target," or the protein and peptide of the present invention, is concurrently pending in U.S. Provisional Patent Application No. 61 / 618,862, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Biologics," U.S. Provisional Patent Application No. 61 / 681,645, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Biologics," U.S. Provisional Patent Application No. 61 / 737,130, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Biologics," U.S. Provisional Patent Application No. 61 / 618,866, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Antibodies," and U.S. Provisional Patent Application No. 61 / 681,647, filed August 10, 2012, titled "Modified Polynucleotides for "The Production of Antibodies," U.S. Provisional Patent Application No. 61 / 737,134, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Antibodies," U.S. Provisional Patent Application No. 61 / 618,868, filed April 2, 2012, titled "Modified U.S. Provisional Patent Application No. 61 / 681,648, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Vaccines", U.S. Provisional Patent Application No. 61 / 737,135, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Vaccines", U.S. Provisional Patent Application No. 61 / 618,873, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Secreted Proteins", U.S. Provisional Patent Application No. 61 / 681,650, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Secreted Proteins", U.S. Provisional Patent Application No. 61 / 737,147, filed December 14, 2012, titled "Modified Polynucleotides for the Production of "Secreted Proteins", U.S. Provisional Patent Application No. 61 / 618,878 filed April 2, 2012, title "Modified Polynucleotides for the Production of Plasma Membrane Proteins", U.S. Provisional Patent Application No. 61 / 681,654 filed August 10, 2012, title "Modified Polynucleotides for the Production of Plasma Membrane Proteins", U.S. Provisional Patent Application No. 61 / 737,152 filed December 14, 2012, title "Modified Polynucleotides for the Production of Plasma Membrane Proteins", U.S. Provisional Patent Application No. 61 / 618 filed April 2, 2012,U.S. Provisional Patent Application No. 61 / 681,658, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins", filed December 14, 2012, titled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins", U.S. Provisional Patent Application No. 61 / 737,155, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal Proteins", U.S. Provisional Patent Application No. 61 / 618,896, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins", U.S. Provisional Patent Application No. 61 / 668,157, filed July 5, 2012, titled "Modified Polynucleotides for the Production of Intracellular U.S. Provisional Patent Application No. 61 / 681,661, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins", U.S. Provisional Patent Application No. 61 / 737,160, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Intracellular Membrane Bound Proteins", U.S. Provisional Patent Application No. 61 / 618,911, filed April 2, 2012, titled "Modified Polynucleotides for the Production, U.S. Provisional Patent Application No. 61 / 681,667, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Nuclear Proteins", U.S. Provisional Patent Application No. 61 / 737,168, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Nuclear Proteins", U.S. Provisional Patent Application No. 61 / 618,922, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Proteins", U.S. Provisional Patent Application No. 61 / 681,675, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Proteins", U.S. Provisional Patent Application No. 61 / 737,174, filed December 14, 2012, titled "Modified Polynucleotides for the Production of U.S. Provisional Patent Application No. 61 / 618,935, filed April 2, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 681,687, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 737,184, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 618, filed April 2, 2012U.S. Provisional Patent Application No. 61 / 681,696, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 737,191, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 618,953, filed April 2, 2012, titled "Modified Polynucleotides for the Production, U.S. Provisional Patent Application No. 61 / 681,704, filed August 10, 2012, titled "of Proteins Associated with Human Disease", U.S. Provisional Patent Application No. 61 / 737, filed December 14, 2012, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease",International application PCT / US2013 / 030062, filed March 9, 2013, title: Modified Polynucleotides for the Production of Proteins Associated with Human Disease; International application PCT / US2013 / 030064, title: Modified Polynucleotides for the Production of Secreted Proteins; International application PCT / US2013 / 030059, filed March 9, 2013, title: Modified Polynucleotides for the Production of Membrane Proteins; International application PCT / US2013 / 030066, filed March 9, 2013, title: Modified Polynucleotides for the Production of Cytoplasmic and Cytoskeletal The following are listed in Table 6: International application PCT / US2013 / 030067, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Nuclear Proteins"; International application PCT / US2013 / 030060, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Proteins"; and the concurrently pending US provisional patent application 61 / 681,720, filed August 10, 2012, titled "Modified Polynucleotides, for the Production of Cosmetic Proteins "Modified Polynucleotides for Peptides", U.S. Provisional Patent Application No. 61 / 737,213, filed December 14, 2012. "The Production of Cosmetic Proteins and Peptides," U.S. Provisional Patent Application No. 61 / 681,742, filed August 10, 2012, titled "Modified Polynucleotides for the Production of Oncology-Related Proteins and Peptides," and International Patent Application No. PCT / US2013 / 030070, filed March 9, 2013, titled "Modified Polynucleotides for The following are listed in Tables 6 and 7, concurrently pending international application PCT / US2013 / 030068 filed on March 9, 2013, titled "Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides," listed in Tables 6, 178 and 179, concurrently pending US provisional patent application 61 / 618,870 filed on April 2, 2012, titled "Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides," listed in Tables 6, 28 and 29, concurrently pending US provisional patent application 61 / 681,649 filed on August 10, 2012, titled "Modified Polynucleotides for the Production of Therapeutic Proteins and Peptides listed in Tables 6, 56, and 57, and U.S. Provisional Patent Application No. 61 / 737,139 "Modified" filed on December 14, 2012, which is concurrently pending. The polynucleotides listed in Tables 6, 186 and 187, as well as the concurrently pending international application PCT / US2013 / 030063, filed 9 March 2013, titled "Modified Polynucleotides," listed in Tables 6, 185 and 186, the contents of which are incorporated herein by reference in their entirety.

[0224] As a non-limiting example, the targets of the present invention are shown in Table 6, and in addition to the name and description of the gene encoding the target polypeptide, the ENSEMBL transcript sequence number (ENST), the reference application number, and the target number in the reference application in which the target is described are also shown. The sequences related to the targets listed in Table 6 are concurrently pending in the following applications: International PCT / US2013 / 030062, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Biologics and Proteins Associated with Human Disease"; International PCT / US2013 / 030061, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Proteins Associated with Human Disease"; International PCT / US2013 / 030068, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Cosmetic Proteins and Peptides"; and International PCT / US2013 / 030070, filed March 9, 2013, titled "Modified Polynucleotides for the Production of Oncology-Related Proteins and Those concerning "Peptides" are incorporated herein by reference in their entirety. For any given 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 faciest regions. These are encoded in either the 5' (upstream) or 3' (downstream) of the ORF or coding region in each ENST transcript. The coding region is disclosed definitively and specifically by teaching the ENSP sequence. As a result, sequences of faciest teachings that code for a protein are considered faciest regions. It is also possible to further characterize the 5' and 3' faciest regions by utilizing one or more available databases or algorithms. Databases annotating features contained in faciest regions of ENST transcripts are available in the art.

[0225] Table 6. Target

[0226] [Table 0006-1]

[0227] [Table 0006-2]

[0228] [Table 0006-3]

[0229] [Table 0006-4]

[0230] [Table 0006-5]

[0231] [Table 0006-6]

[0232] [Table 0006-7]

[0233] [Table 0006-8] Protein cleavage signals and sites In one embodiment, the polypeptide of the present invention may contain at least one protein cleavage signal containing at least one protein cleavage site. The protein cleavage site may be located at the N-terminus or C-terminus in any space between the N-terminus and C-terminus, such as midway between the N-terminus and the C-terminus, between the N-terminus and the midpoint, between the midpoint and the C-terminus, or a combination thereof, but is not limited to these locations.

[0234] The polypeptides of the present invention may, but are not limited to, proprotein convertases (or prohormone convertases), 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 associated with yeast kexins known as prohormone convertase 1 / 3 (PC1 / 3), PC2, furin, PC4, PC5 / 6, paired basic amino acid cleavage 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, and "n" can be 0, 2, 4, or 6 amino acids. *" refers to the protein cleavage site. In Table 7, sequence number 158 refers to the case when n is 4, and sequence number 159 refers to the case when n is 6.

[0235] Table 7. Sequence of protein cleavage sites

[0236] [Table 0007] In one embodiment, the primary construct and mMRNA of the present invention may be manipulated so that the primary construct or mMRNA contains at least one encoded protein cleavage signal. The encoded protein cleavage signal may be located before the start codon, after the start codon, before the coding region, within the coding region, for example, 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, etc.

[0237] In one embodiment, the primary construct or mMRNA of the present invention may contain at least one encoded protein cleavage signal containing at least one protein cleavage site. The encoded protein cleavage signal may include, but is not limited to, a proprotein convertase (or prohormone convertase), thrombin, and / or a factor Xa protein cleavage signal. Those skilled in the art can determine a suitable encoded protein cleavage signal to include in the primary construct or mMRNA of the present invention using Table 1 above or other known methods. For example, starting with the signals in Table 7 and considering the codons in Table 1, one can design a signal for a primary construct that can generate a protein signal in the resulting polypeptide.

[0238] In one embodiment, the polypeptide of the present invention comprises at least one protein cleavage signal and / or site. As non-limiting examples, U.S. Patent No. 7,374,930 and U.S. Patent Publication No. 20090227660, which are incorporated herein by reference in their entirety, describe using a furin cleavage site to cleave the N-terminal methionine of GLP-1 in an expression product from the Golgi apparatus of that cell. In one embodiment, the polypeptide of the present invention comprises at least one protein cleavage signal and / or site, provided that the polypeptide is not GLP-1.

[0239] In one embodiment, the primary construct or mMRNA of the present invention comprises at least one encoded protein cleavage signal and / or site. 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.

[0240] In one embodiment, the primary construct or mMRNA of the present invention may include two or more coding regions. If 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 form, where A and B 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. A second such pattern follows the AXYBZ form, where A and B are coding regions that may be the same or different coding regions and / or may encode the same or different polypeptides, and X, Y, and Z are encoded protein cleavage signals that may encode the same or different protein cleavage signals. The third pattern follows the ABXCY morphology, where A, B, and C are coding regions that may be identical or different and / or capable of encoding identical or different polypeptides, and X and Y are encoded protein cleavage signals that may encode identical or different protein cleavage signals.

[0241] In one embodiment, the polypeptide, primary construct, and mMRNA may also contain sequences encoding the aforementioned protein cleavage site, such that the polypeptide, primary construct, and mMRNA can be released from the carrier region or fusion partner by treatment with a protein cleavage site-specific protease.

[0242] In one embodiment, the polypeptide, primary construct, and mMRNA of the present invention may include a sequence encoding the 2A peptide. In one embodiment, this sequence can be used to isolate the coding regions of two or more target polypeptides. As a non-limiting example, the sequence encoding the 2A peptide may be located between coding region A and coding region B (A-2Apep-B). The presence of the 2A peptide results in the cleavage of a single long protein into protein A, protein B, and the 2A peptide. Protein A and protein B may be the same or different target polypeptides. In another embodiment, the 2A peptide can be used in the polynucleotide, primary construct, and / or mMRNA of the present invention to produce two, three, four, five, six, seven, eight, nine, ten or more proteins.

[0243] Incorporation of post-transcriptional regulatory factors In one embodiment, the polynucleotides, primary constructs, and / or mMRNAs of the present invention may comprise at least one post-transcriptional regulatory factor. These post-transcriptional regulatory factors may be, but are not limited to, small molecules, 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 the GEMS® (Gene Expression Modulation by Small-Moleclues) screening technology.

[0244] Post-transcriptional regulatory factors may be gene expression regulators screened by the method described in International Publication WO2006022712, which is incorporated entirely herein by reference, or gene expression regulators described therein. A method for identifying RNA regulatory sequences involved in translational control is described in International Publication WO2004067728, which is incorporated entirely herein by reference, and a method for identifying compounds that regulate gene expression in a non-translation region-dependent manner is described in International Publication WO2004065561, which is incorporated entirely herein by reference.

[0245] In one embodiment, the polynucleotide, primary construct, and / or mMRNA of the present invention may comprise at least one post-transcriptional regulatory factor located in the 5' and / or 3' untranslated regions of the polynucleotide, primary construct, and / or mMRNA of the present invention.

[0246] In another embodiment, the polynucleotides, primary constructs, and / or mMRNAs of the present invention may comprise at least one posttranscriptional regulatory factor to modulate immature translation termination. The posttranscriptional regulatory factors may be compounds described in International Publications WO2004010106, WO2006044456, WO2006044682, WO2006044503, and WO2006044505, which are incorporated herein in their entirety by reference, or compounds found by the methods outlined therein. As a non-limiting example, such compounds may bind to a region of 28S ribosomal RNA to modulate immature translation termination (see, for example, International Publication WO2004010106, which is incorporated herein in its entirety by reference).

[0247] In one embodiment, the polynucleotides, primary constructs, and / or mMRNAs of the present invention may comprise at least one post-transcriptional regulatory factor to alter protein expression. As a non-limiting example, VEGF expression may be controlled using compounds described in International Publications WO2005118857, WO2006065480, WO2006065479, and WO2006058088, respectively, which are incorporated herein in whole by reference, or compounds that may be found by methods thereof.

[0248] The polynucleotides, primary constructs, and / or mMRNAs of the present invention may comprise at least one post-transcriptional regulatory factor to control translation. In one embodiment, the post-transcriptional regulatory factor may be an RNA regulatory sequence. As a non-limiting example, the RNA regulatory sequence may be identified by the method described in International Publication No. WO2006071903, which is incorporated in whole by reference herein.

[0249] III. Qualification In this specification, the terms “modified” or, as appropriate, “modified” refer to modifications to A, G, U, or C ribonucleotides in polynucleotides (primary constructs or mRNA molecules, etc.). In general, these terms are not intended to refer to ribonucleotide modifications in naturally occurring 5' mRNA cap portions. In polypeptides, the term “modified” refers to modifications compared to the 20 amino acids (or portion) of a reference set.

[0250] This modification can be a variety of distinctly different modifications. In some embodiments, the coding region, adjacent regions, and / or terminal regions may contain one, two, or more (arbitrarily different) nucleoside or nucleotide modifications. In some embodiments, modified polynucleotides, primary constructs, or mMRNAs introduced into cells may exhibit reduced degradation in cells compared to unmodified polynucleotides, primary constructs, or mMRNAs.

[0251] Polynucleotides, primary constructs, and mMRNAs may include any useful modifications to sugars, nucleic acid bases, or nucleoside bonds (e.g., to bound phosphate / phosphodiester bonds / phosphodiester backbones). One or more atoms of pyrimidine nucleic acid bases may be replaced with or substituted with optionally substituted aminos, optionally substituted thiols, optionally substituted alkyls (e.g., methyl or ethyl), or halos (e.g., chloro or fluoro). In certain embodiments, the modification (e.g., one or more modifications) is present in each of the sugar and nucleoside bonds. Modifications according to the present invention may be modifications 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 hybrids thereof. Further modifications are described herein.

[0252] As described herein, the polynucleotides, primary constructs, and mRNAs of the present invention do not substantially induce an innate immune response in cells into which mRNA is introduced. Characteristic features of an induced innate immune response include 1) increased expression of pro-inflammatory cytokines, 2) activation of intracellular PRRs (RIG-I, MDA5, etc.), and / or 3) termination or reduction of protein translation.

[0253] In certain embodiments, it may be desirable to degrade modified nucleic acid molecules introduced into cells within the cell. For example, degradation of modified nucleic acid molecules may be desirable when precise timing of protein production is desired. Therefore, in some embodiments, the present invention provides modified nucleic acid molecules containing degradation domains that can act within cells in a directed manner. In another embodiment, the disclosure provides polynucleotides comprising nucleosides or nucleotides that can disrupt the binding of major groove interaction (e.g., binding) partners to the polynucleotide (e.g., modified nucleotides have reduced binding affinity to major groove interaction partners compared to unmodified nucleotides).

[0254] Polynucleotides, primary constructs, and mMRNAs may optionally contain other active ingredients (e.g., RNAi inducers, RNAi agents, siRNA, shRNA, miRNA, antisense RNA, ribozymes, catalytic DNA, tRNA, RNA that induces triple helix formation, aptamers, vectors, etc.). In some embodiments, polynucleotides, primary constructs, or mMRNAs may contain one or more messenger RNAs (mRNAs) and one or more modified nucleosides or nucleotides (e.g., mMRNA molecules). Further details regarding these polynucleotides, primary constructs, and mMRNAs follow below.

[0255] Polynucleotides and primary constructs The polynucleotide, primary construct, and mMRNA of the present invention include a first region of a binding nucleoside encoding the target polypeptide, a first adjacent region located at the 5' end of the first region, and a second adjacent region located at the 3' end of the first region.

[0256] In some embodiments, a polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is given by formula (Ia) or formula (Ia-1):

[0257] [ka] It comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, During the ceremony, U is O, S, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer between 0 and 2, and each R U However, independently, they are H, halo, or optionally substituted alkyl groups. --- is either a single bond or absent, R 1’ , R 2’ , R1” , R 2” , R 1 , R 2 , R 3 , R 4 , and R 5 Each of these can be, independently, if present, H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide, 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 (for example, the combination of R1' and R3, the combination of R1'' and R3, the combination of R2' and R3, the combination of R2'' and R3, or A combination of R5 and R3 may together form an optionally substituted alkylene or optionally substituted heteroalkylene, and together with the carbon to which they are bound, may provide an optionally substituted heterocycline (e.g., a bicyclic, tricyclic, or tetracyclic heterocycline), and a combination of R5 and one or more of R1', R1'', R2', or R2'' (e.g., a combination of R1' and R5, a combination of R1'' and R5, a combination of R2' and R5, or a combination of R2'' and R5) may together form an optionally substituted alkylene or optionally substituted heteroalkylene, and together with the carbon to which they are bound, may provide an optionally substituted heterocycline (e.g., a bicyclic, tricyclic, or tetracyclic heterocycline), R 4 And, R 1’ , R 1” , R 2’ , R 2” , R 3 , or R 5A combination of one or more of these may form an arbitrarily substituted alkylene or an arbitrarily substituted heteroalkylene, and together with the carbons to which they are bonded may provide an arbitrarily substituted heterocycline (e.g., a bicyclic, tricyclic, or tetracyclic heterocycline), where each of m' and m'' is independently an integer between 0 and 3 (e.g., 0 to 2, 0 to 1, 1 to 3, or 1 to 2). Y 1 , Y 2 , and Y 3 Each of these independently corresponds to O, S, Se, -NR N1 - is an optionally substituted alkylene or an optionally substituted heteroalkylene, where R N1 However, H is either optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent. Each Y 4 However, independently, these are 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. Each Y 5 However, independently, these are O, S, Se, an optionally substituted alkylene (e.g., methylene), or an optionally substituted heteroalkylene. n is an integer between 1 and 100,000. B is a nucleic acid base (e.g., purine, pyrimidine, or a derivative thereof), and B and R 1’ The combination of B and R 2’ The combination of B and R 1” A combination of B and R 2” The combination of these elements, together with the carbon atoms to which they are bonded, can arbitrarily form a bicyclic group (e.g., a bicyclic heterocycline), or B, R 1” , and R 3 A combination of B and R 2” , and R 3The combination may optionally form a tricyclic or tetracyclic group (e.g., a tricyclic or tetracyclic heterocyclil 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., a first region, a first adjacent region, or a second adjacent region) comprises n bound nucleosides having formula (Ia-2)-(Ia-5), or pharmaceutically acceptable salts or stereoisomers thereof.

[0258] [ka] In some embodiments, a polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is expressed by formula (Ib) or formula (Ib-1):

[0259] [ka] It comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, During the ceremony, U is O, S, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer between 0 and 2, and each R U However, independently, they are H, halo, or optionally substituted alkyl groups. --- is either a single bond or absent, R 1 , R 3’ , R 3” , and R 4Each of them 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, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent, R 1 and R 3’ A combination or R 1 and R 3” These combinations can form arbitrarily substituted alkylenes or arbitrarily substituted heteroalkylenes (for example, to produce locked nucleic acids), Each R 5 However, independently, H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, or absent. Y 1 , Y 2 , and Y 3 Each of these independently corresponds to O, S, Se, -NR N1 - is an optionally substituted alkylene or an optionally substituted heteroalkylene, where R N1 However, H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl, Each Y 4 However, independently, these are 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. n is an integer between 1 and 100,000. B is a nucleic acid base.

[0260] In some embodiments, a polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is expressed by formula (Ic):

[0261] [ka] It comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, During the ceremony, U is O, S, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer between 0 and 2, and each R U However, independently, they are H, halo, or optionally substituted alkyl groups. --- is either a single bond or absent, B 1 B 2 , and B 3 Each of these is independently a nucleic acid base (e.g., purines, pyrimidines, or derivatives thereof as described herein), H, halo, hydroxy, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, optionally substituted hydroxyalkoxy, optionally substituted amino, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl, and B 1 B 2 , and B 3 Only one of them is a nucleic acid base. R b1 , R b2 , R b3 , R 3 , and R 5Each of them 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, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl, Y 1 Y, 2 and Y 3 each of which is independently O, S, Se, -NR N1 -, optionally substituted alkylene, or optionally substituted heteroalkylene, wherein R N1 is H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, or optionally substituted aryl, Each Y 4 is independently H, hydroxy, thiol, boranyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino, Each Y 5 is independently O, S, Se, optionally substituted alkylene (e.g., methylene), or optionally substituted heteroalkylene, n is an integer from 1 to 100,000, The ring containing U may contain one or more double bonds.

[0262] In certain embodiments, the ring containing U does not have a double bond between U-CB 3 R b3 or between CB 3 R b3 -C B2 R b2 . In some embodiments, a polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is expressed by formula (Id):

[0263] [ka] It comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, During the ceremony, U is O, S, N(R U ) nu , or C(R U ) nu In the formula, nu is an integer between 0 and 2, and each R U However, independently, they are H, halo, or optionally substituted alkyl groups. Each R 3 However, independently, these are 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, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl. Y 1 , Y 2 , and Y 3 Each of these independently corresponds to O, S, Se, -NR N1 - is an optionally substituted alkylene or an optionally substituted heteroalkylene, where R N1 However, H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl, Each Y 4However, independently, these are 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. Each Y 5 However, independently, they are O, S, an arbitrarily substituted alkylene (e.g., methylene), or an arbitrarily substituted heteroalkylene. n is an integer between 1 and 100,000. B is a nucleic acid base (for example, a purine, pyrimidine, or a derivative thereof).

[0264] In some embodiments, a polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is expressed by formula (Ie):

[0265] [ka] It comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, During the ceremony, U' and U'' are independently O, S, N(R) U ) nu , or C(R U ) nu In the formula, nu is an integer between 0 and 2, and each R U However, independently, they are H, halo, or optionally substituted alkyl groups. Each R 6However, independently, these are 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, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl. Each Y 5’ However, independently, these are O, S, optionally substituted alkylenes (e.g., methylene or ethylene), or optionally substituted heteroalkylenes. n is an integer between 1 and 100,000. B is a nucleic acid base (for example, a purine, pyrimidine, or a derivative thereof).

[0266] In some embodiments, a polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is expressed by formula (If) or (If-1):

[0267] [ka] It comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, During the ceremony, U' and U'' are independently O, S, N, N(R) U ) nu , or C(R U ) nu In the formula, nu is an integer between 0 and 2, and each R U However, independently, they are H, halo, or optionally substituted alkyl (for example, U' is O and U'' is N), --- is either a single bond or absent, R 1’ , R 2’ , R 1” , R 2” , R 3, and R 4 Each of them 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, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent, R 1’ and R 3 The combination, R 1” and R 3 The combination, R 2’ and R 3 A combination of R 2” and R 3 The combination of these elements can form an arbitrarily substituted alkylene or an arbitrarily substituted heteroalkylene (for example, to produce locked nucleic acids), where each of m' and m'' is independently an integer between 0 and 3 (e.g., 0 to 2, 0 to 1, 1 to 3, or 1 to 2). Y 1 , Y 2 , and Y 3 Each of these independently corresponds to O, S, Se, -NR N1 - is an optionally substituted alkylene or an optionally substituted heteroalkylene, where R N1 However, H is either optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aryl, or absent. Each Y 4 However, independently, these are 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. Each Y 5However, independently, these are O, S, Se, an optionally substituted alkylene (e.g., methylene), or an optionally substituted heteroalkylene. n is an integer between 1 and 100,000. B is a nucleic acid base (for example, a purine, pyrimidine, or a derivative thereof).

[0268] In some embodiments of polynucleotides, primary constructs, or mMRNAs (e.g., formula (Ia), (Ia-1) to (Ia-3), (Ib) to (If), and (IIa) to (IIp)), the U-containing ring has one or two double bonds.

[0269] Polynucleotides, primary constructs, or some embodiments of 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 of them is H if present. In a further embodiment, R 2 , R 2’ , and R 2” Each of these, if present, is independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In certain embodiments, the alkoxyalkoxy is -(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 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 It is alkyl. In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R' is C 1~6 It is alkyl.

[0270] Polynucleotides, primary constructs, or some embodiments of 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 of them is H if present. In a further embodiment, R 1 , R 1’ , and R 1” Each of these, if present, is independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy. In certain embodiments, the alkoxyalkoxy is -(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 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 It is alkyl. In some embodiments, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R' is C 1~6 It is alkyl.

[0271] Polynucleotides, primary constructs, or some embodiments of 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 these 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 H is R 4 H is R 5 Is H or R 3 , R 4 , and R 5 All of them are H. In a particular embodiment, R 3 C 1~6 It is alkyl, R 4 C 1~6 It is alkyl, R 5 C 1~6 Alkyl or R 3 , R 4 , and R 5 All of C 1~6 It is alkyl. In certain embodiments, R 3 R 4 Both are H, and R 5 is C 1~6 It is alkyl.

[0272] Polynucleotides, primary constructs, or some embodiments of 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 These combine to form optionally substituted alkylenes or optionally substituted heteroalkylenes, and together with the carbons to which they are bonded, provide optionally substituted heterocyclines (e.g., bicyclic, tricyclic, or tetracyclic heterocyclines) such as trans-3',4' analogs, and R 3 and R 5 These combine to form heteroalkylenes (for example, -(CH2) b1 O(CH2) b2 O(CH2) b3-where b1, b2, and b3 are independently integers between 0 and 3).

[0273] Polynucleotides, primary constructs, or some embodiments of 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 these combine to form an optionally substituted alkylene or optionally substituted heteroalkylene, and together with the carbons they bond to, provide an optionally substituted heterocycline (e.g., a bicyclic, tricyclic, or tetracyclic heterocycline), R 3 And, R 1’ , R 1” , R 2’ , R 2” , or R 5 One or more of these combine to form a heteroalkylene (e.g., -(CH2) b1 O(CH2) b2 O(CH2) b3 -where b1, b2, and b3 are independently integers between 0 and 3).

[0274] Polynucleotides, primary constructs, or some embodiments of 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 R 2”One or more of these combine to form an optionally substituted alkylene or optionally substituted heteroalkylene, and together with the carbons they bond to, provide an optionally substituted heterocycline (e.g., a bicyclic, tricyclic, or tetracyclic heterocycline), R 5 And, R 1’ , R 1” , R 2’ , or R 2” One or more of these combine to form a heteroalkylene (e.g., -(CH2) b1 O(CH2) b2 O(CH2) b3 -where b1, b2, and b3 are independently integers between 0 and 3).

[0275] In several embodiments of 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)), each Y 2 These are independently O, S, or -NR N1 - and in the formula, R N1 However, H is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl. In certain embodiments, Y 2 , NR N1 - and in the formula, R N1 However, H or optionally substituted alkyl (e.g., C 1~6 Alkyl compounds (e.g., methyl, ethyl, isopropyl, or n-propyl).

[0276] In several embodiments of 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)), each Y 3These are independently either O or S.

[0277] Polynucleotides, primary constructs, or some embodiments of 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 H is H, and each R 2 These are independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g., -(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 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 It is an alkyl group, for example, in the formula s2 is 0, s1 is 1 or 2, and s3 is 0 or 1 (for example, R' is C 1~6 (It is alkyl), and each Y 2 These are, independently, O or -NR N1 - and in the formula, R N1 However, H is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl (for example, in the formula, R N1 However, H or optionally substituted alkyl (e.g., C 1~6 Alkyl (e.g., methyl, ethyl, isopropyl, or n-propyl), each Y 3 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 further embodiments, each Y1 These are, independently, O or -NR N1 - and in the formula, R N1 However, H is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl (for example, in the formula, R N1 However, H or optionally substituted alkyl (e.g., C 1~6 Alkyl (e.g., methyl, ethyl, isopropyl, or n-propyl), each Y 4 These are independently H, hydroxyl, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.

[0278] In several embodiments of 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)), each R 1 These are independently H, halo (e.g., fluoro), hydroxy, optionally substituted alkoxy (e.g., methoxy or ethoxy), or optionally substituted alkoxyalkoxy (e.g., -(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR', where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 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 It is an alkyl group (for example, in the formula, s2 is 0, s1 is 1 or 2, s3 is 0 or 1, and R' is C 1~6 (It is alkyl), R 2 H is and each Y 2 These are, independently, O or -NR N1 - and in the formula, R N1However, H is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl (for example, in the formula, R N1 However, H or optionally substituted alkyl (e.g., C 1~6 Alkyl (e.g., methyl, ethyl, isopropyl, or n-propyl), each Y 3 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 further embodiments, each Y 1 These are, independently, O or -NR N1 - and in the formula, R N1 However, H is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl (for example, in the formula, R N1 However, H or optionally substituted alkyl (e.g., C 1~6 Alkyl (e.g., methyl, ethyl, isopropyl, or n-propyl), each Y 4 These are independently H, hydroxyl, thiol, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted alkoxyalkoxy, or optionally substituted amino.

[0279] In some embodiments of polynucleotides, primary constructs, or mMRNAs (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 U is in a β-D (e.g., β-D-ribo) configuration.

[0280] In some embodiments of polynucleotides, primary constructs, or mMRNAs (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 U is in an α-L (e.g., α-L-ribo) configuration.

[0281] In some embodiments of polynucleotides, primary constructs, or mMRNAs (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 are pseudouridine (ψ) or 5-methylcytidine (m 5 C) is not the case. In some embodiments, about 10% to about 100% of the n B nucleic acid bases are ψ or m 5 Not C either (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%) %, 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 expressed as ψ or m 5 (Not C either). In some embodiments, B is either ψ or m 5 It's not C either.

[0282] In some embodiments of polynucleotides, primary constructs, or mMRNAs (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 nucleic acid base selected from cytosine, guanine, uracil, and adenine, Y 1 , Y 2 , or Y 3 At least one of them is not O.

[0283] In some embodiments, the polynucleotide, primary construct, or mMRNA contains modified ribose. In some embodiments, the polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is given by formulas (IIa) to (IIc):

[0284] [ka] It comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof. In certain embodiments, U is O or C(R U ) nu In the formula, nu is an integer between 0 and 2, and each R U However, independently, these are H, halo, or optionally substituted alkyl (for example, U is -CH2- or -CH-). In other embodiments, R 1 , R 2 , R 3 , R 4 , and R 5Each of them 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, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent (for example, each R 1 and R 2 R is independently H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, and each R 3 and R 4 R is independently H or an optionally substituted alkyl, 5 ( is H or hydroxyl), and --- is a single bond or a double bond.

[0285] In certain embodiments, polynucleotides or mMRNAs are expressed by formulas (IIb-1)~(IIb-2):

[0286] [ka] It comprises n bonded nucleosides having , or pharmaceutically acceptable salts or stereoisomers thereof. In some embodiments, U is O or C(R U ) nu In the formula, nu is an integer between 0 and 2, and each R U However, independently, these are H, halo, or optionally substituted alkyl (for example, U is -CH2- or -CH-). In other embodiments, R 1 and R 2Each of them 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, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent (for example, each R 1 and R 2 R 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 a hydroxyl or optionally substituted alkoxy (e.g., methoxy, ethoxy, or any of those described herein).

[0287] In certain embodiments, polynucleotides, primary constructs, or mMRNAs are expressed using formulas (IIc-1) to (IIc-4):

[0288] [ka] It comprises n bonded nucleosides having , or pharmaceutically acceptable salts or stereoisomers thereof. In some embodiments, U is O or C(R U ) nu In the formula, nu is an integer between 0 and 2, and each R U However, independently, it is H, halo, or an optionally substituted alkyl (for example, U is -CH2- or -CH-). In some embodiments, R 1 , R 2 , and R 3Each of them 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, azide, optionally substituted aryl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, or absent (for example, each R 1 and R 2 R is independently H, halo, hydroxy, optionally substituted alkyl, or optionally substituted alkoxy, e.g., H, halo, hydroxy, alkyl, or alkoxy, and each R 3 R is independently H or an optionally substituted alkyl group). In certain embodiments, R 2 R is an optionally substituted alkoxy (e.g., methoxy or ethoxy, or any of those described herein). In certain embodiments, R 1 is an optionally substituted alkyl, and R 2 is hydroxyl. In other embodiments, R 1 is hydroxyl, and R 2 is an optionally substituted alkyl group. In further embodiments, R 3 This is an optionally substituted alkyl group.

[0289] In some embodiments, the polynucleotide, primary construct, or mMRNA contains an acyclic modified ribose. In some embodiments, the polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is of formula (IId)~(IIf):

[0290] [ka] It comprises n bonded nucleosides having a specific property, or pharmaceutically acceptable salts or stereoisomers thereof.

[0291] In some embodiments, the polynucleotide, primary construct, or mMRNA contains an acyclic modified hexitol. In some embodiments, the polynucleotide, primary construct, or mMRNA (e.g., the first region, the first adjacent region, or the second adjacent region) is of formula (IIg) to (IIj):

[0292] [ka] It comprises n bonded nucleosides, or pharmaceutically acceptable salts or stereoisomers thereof.

[0293] 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., a first region, a first adjacent region, or a second adjacent region) is given by formula (IIk) to (IIm):

[0294] [ka] The formula comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, where R 1’ , R 1” , R 2’ , and R 2” Each of them independently is either H, halo, hydroxy, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted alkynyloxy, optionally substituted aminoalkoxy, optionally substituted alkoxyalkoxy, or absent, R 2’ and R 3 A combination or R 2” and R 3 These combinations can form arbitrarily substituted alkylenes or arbitrarily substituted heteroalkylenes.

[0295] In some embodiments, the polynucleotide, primary construct, or mMRNA contains locked modified ribose. In some embodiments, the polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is of formula (IIn):

[0296] [ka] The formula comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, where R 3’ However, O, S, or -NR N1 - and in the formula, R N1 However, H is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl, and R 3” However, it is an arbitrarily substituted alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-) or an arbitrarily substituted heteroalkylene (e.g., -CH2NH-, -CH2CH2NH-, -CH2OCH2-, or -CH2CH2OCH2-) (e.g., R 3’ O is R 3” (These are alkylenes in which the parentheses are arbitrarily substituted (for example, -CH2-, -CH2CH2-, or -CH2CH2CH2-)).

[0297] In some embodiments, polynucleotides, primary constructs, or mMRNAs are expressed using the formula (I-n-1)~(II-n2):

[0298] [ka] The formula comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, where R 3’ However, O, S, or -NR N1 - and in the formula, R N1However, H is an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted aryl, and R 3” However, it is an arbitrarily substituted alkylene (e.g., -CH2-, -CH2CH2-, or -CH2CH2CH2-) or an arbitrarily substituted heteroalkylene (e.g., -CH2NH-, -CH2CH2NH-, -CH2OCH2-, or -CH2CH2OCH2-) (e.g., R 3’ O is R 3” (These are alkylenes in which the parentheses are arbitrarily substituted (for example, -CH2-, -CH2CH2-, or -CH2CH2CH2-)).

[0299] In some embodiments, the polynucleotide, primary construct, or mMRNA comprises locked-modified ribose forming a tetracyclic heterocycline. In some embodiments, the polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) is of formula (IIo):

[0300] [ka] The formula comprises n bonded nucleosides having, or pharmaceutically acceptable salts or stereoisomers thereof, where R 12a , R 12c , T 1’ , T 1” , T 2’ , T 2” , V 1 , and V 3 This is as described in this specification.

[0301] A polynucleotide, primary construct, or mMRNA formula may contain one or more nucleic acid bases as described herein (e.g., formulas (b1) to (b43)). In one embodiment, the present invention provides a method for preparing a polynucleotide, a primary construct, or mMRNA, wherein the polynucleotide is defined by formula (Ia) as defined herein:

[0302] [ka] This method comprises n nucleosides having the formula (IIIa) defined herein:

[0303] [ka] This involves reacting the compound with RNA polymerase and a cDNA template.

[0304] In further embodiments, the present invention provides a polynucleotide, a primary construct, or a method for amplifying an 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.

[0305] In one embodiment, the present invention provides a method for preparing a polynucleotide, a primary construct, or an mMRNA comprising at least one nucleotide (e.g., an mMRNA molecule), wherein the polynucleotide is defined by formula (Ia) as defined herein:

[0306] [ka] This method comprises n nucleosides having the formula (IIIa-1) defined herein:

[0307] [ka] This involves reacting the compound with RNA polymerase and a cDNA template.

[0308] In a further embodiment, the present invention provides a method for amplifying a polynucleotide, a primary construct, or an mMRNA comprising at least one nucleotide (e.g., an mMRNA molecule), the method comprising: This method involves reacting a compound of formula (IIIa-1) as defined herein with a primer, a cDNA template, and an RNA polymerase.

[0309] In one embodiment, the present invention provides a method for preparing modified mRNA comprising at least one nucleotide (e.g., an mMRNA molecule), wherein the polynucleotide is defined by formula (Ia-2) as defined herein:

[0310] [ka] This method comprises n nucleosides having the formula (IIIa-2) defined herein:

[0311] [ka] This involves reacting the compound with RNA polymerase and a cDNA template.

[0312] In a further embodiment, the present invention provides a method for amplifying modified mRNA containing at least one nucleotide (e.g., an mMRNA molecule), the method being: This method involves reacting a compound of formula (IIIa-2) as defined herein with a primer, a cDNA template, and an RNA polymerase.

[0313] In some embodiments, the reaction may be repeated 1 to about 7,000 times. In any of the embodiments herein, B may be a nucleic acid base of formula (b1) to (b43).

[0314] Polynucleotides, primary constructs, and mMRNAs may optionally include the 5' and / or 3' facile regions described herein. Modified RNA (mRNA) molecules The present invention also includes building blocks for modified RNA (mmRNA) molecules, such as modified ribonucleosides and modified ribonucleotides. For example, these building blocks may be useful in the preparation of polynucleotides, primary constructs, or mmRNAs of the present invention.

[0315] In some embodiments, the building block molecule is of formula (IIIa) or (IIIa-1):

[0316] [ka] or having a pharmaceutically acceptable salt or stereoisomer thereof, wherein the substituents are those described herein (e.g., formulas (Ia) and (Ia-1)), and when B is an unmodified nucleic acid base selected from cytosine, guanine, uracil, and adenine, Y 1 , Y 2 , or Y 3 At least one of them is not O.

[0317] In some embodiments, the building block molecules that can be incorporated into polynucleotides, primary constructs, or mMRNAs are given by formulas (IVa) to (IVb):

[0318] [ka] or having a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is one of those described herein (e.g., any one of (b1) to (b43)). In certain embodiments, formula (IVa) or (IVb) is combined with modified uracil (e.g., any one of formulas (b1) to (b9), (b21) to (b23), and (b28) to (b31), e.g., formulas (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, formula (IVa) or (IVb) is combined with modified cytosine (e.g., any one of formulas (b10) to (b14), (b24), (b25), and (b32) to (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) to (b17) and (b37) to (b40)). In certain embodiments, formula (IVa) or (IVb) is combined with a modified adenine (e.g., any one of formulas (b18) to (b20) and (b41) to (b43)).

[0319] In some embodiments, the building block molecules that can be incorporated into polynucleotides, primary constructs, or mMRNAs are given by formulas (IVc) to (IVk):

[0320] [ka] or having a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is one of those described herein (e.g., any one of (b1) to (b43)). In certain embodiments, one of formulas (IVc) to (IVk) is combined with modified uracil (e.g., any one of formulas (b1) to (b9), (b21) to (b23), and (b28) to (b31), e.g., formulas (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of formulas (IVc) to (IVk) is combined with modified cytosine (e.g., any one of formulas (b10) to (b14), (b24), (b25), and (b32) to (b36), e.g., formula (b10) or (b32)). In certain embodiments, one of formulas (IVc) to (IVk) is combined with a modified guanine (e.g., any one of formulas (b15) to (b17) and (b37) to (b40)). In certain embodiments, one of formulas (IVc) to (IVk) is combined with a modified adenine (e.g., any one of formulas (b18) to (b20) and (b41) to (b43)).

[0321] In other embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mMRNA is given by formula (Va) or (Vb):

[0322] [ka] or having a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is one of those described herein (e.g., any one of (b1) to (b43)).

[0323] In other embodiments, building block molecules that can be incorporated into polynucleotides, primary constructs, or mMRNAs are given by formulas (IXa) to (IXd):

[0324] [ka] or having a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is one of those described herein (e.g., any one of (b1) to (b43)). In certain embodiments, one of formulas (IXa) to (IXd) is combined with modified uracil (e.g., any one of formulas (b1) to (b9), (b21) to (b23), and (b28) to (b31), e.g., formulas (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of formulas (IXa) to (IXd) is combined with modified cytosine (e.g., any one of formulas (b10) to (b14), (b24), (b25), and (b32) to (b36), e.g., formula (b10) or (b32)). In certain embodiments, one of formulas (IXa) to (IXd) is combined with a modified guanine (e.g., any one of formulas (b15) to (b17) and (b37) to (b40)). In certain embodiments, one of formulas (IXa) to (IXd) is combined with a modified adenine (e.g., any one of formulas (b18) to (b20) and (b41) to (b43)).

[0325] In other embodiments, building block molecules that can be incorporated into polynucleotides, primary constructs, or mMRNAs are given by formulas (IXe) to (IXg):

[0326] [ka] or having a pharmaceutically acceptable salt or stereoisomer thereof, wherein B is one of those described herein (e.g., any one of (b1) to (b43)). In certain embodiments, one of formulas (IXe) to (IXg) is combined with modified uracil (e.g., any one of formulas (b1) to (b9), (b21) to (b23), and (b28) to (b31), e.g., formulas (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of formulas (IXe) to (IXg) is combined with modified cytosine (e.g., any one of formulas (b10) to (b14), (b24), (b25), and (b32) to (b36), e.g., formula (b10) or (b32)). In certain embodiments, one of formulas (IXe) to (IXg) is combined with a modified guanine (e.g., any one of formulas (b15) to (b17) and (b37) to (b40)). In certain embodiments, one of formulas (IXe) to (IXg) is combined with a modified adenine (e.g., any one of formulas (b18) to (b20) and (b41) to (b43)).

[0327] In other embodiments, building block molecules that can be incorporated into polynucleotides, primary constructs, or mMRNAs are given by formulas (IXh)~(IXk):

[0328] [ka] or having pharmaceutically acceptable salts or stereoisomers thereof, wherein B is one of those described herein (e.g., any one of (b1) to (b43)). In certain embodiments, one of formulas (IXh) to (IXk) is combined with modified uracil (e.g., any one of formulas (b1) to (b9), (b21) to (b23), and (b28) to (b31), e.g., formulas (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of formulas (IXh) to (IXk) is combined with modified cytosine (e.g., any one of formulas (b10) to (b14), (b24), (b25), and (b32) to (b36), e.g., formula (b10) or (b32)). In certain embodiments, one of the formulas (IXh) to (IXk) is combined with a modified guanine (e.g., any one of the formulas (b15) to (b17) and (b37) to (b40)). In certain embodiments, one of the formulas (IXh) to (IXk) is combined with a modified adenine (e.g., any one of the formulas (b18) to (b20) and (b41) to (b43)).

[0329] In other embodiments, building block molecules that can be incorporated into polynucleotides, primary constructs, or mMRNAs are given by formulas (IXl) to (IXr):

[0330] [ka] or having pharmaceutically acceptable salts or stereoisomers thereof, where each r1 and r2 is independently an integer between 0 and 5 (e.g., 0 to 3, 1 to 3, or 1 to 5), and B is one of those described herein (e.g., any one of (b1) to (b43)). In certain embodiments, one of the formulas (IXl) to (IXr) is combined with a modified uracil (e.g., any one of the formulas (b1) to (b9), (b21) to (b23), and (b28) to (b31), e.g., formulas (b1), (b8), (b28), (b29), or (b30)). In certain embodiments, one of formulas (IXl) to (IXr) is combined with a modified cytosine (e.g., any one of formulas (b10) to (b14), (b24), (b25), and (b32) to (b36), e.g., formula (b10) or (b32)). In certain embodiments, one of formulas (IXl) to (IXr) is combined with a modified guanine (e.g., any one of formulas (b15) to (b17) and (b37) to (b40)). In certain embodiments, one of formulas (IXl) to (IXr) is combined with a modified adenine (e.g., any one of formulas (b18) to (b20) and (b41) to (b43)).

[0331] In some embodiments, the building block molecules that can be incorporated into polynucleotides, primary constructs, or mMRNAs are:

[0332] [ka]

[0333] [ka] Alternatively, it may be selected from the group consisting of pharmaceutically acceptable salts or stereoisomers thereof, where each r is an independent integer between 0 and 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0334] In some embodiments, the building block molecules that can be incorporated into polynucleotides, primary constructs, or mMRNAs are:

[0335] [ka] or selected from the group consisting of pharmaceutically acceptable salts or stereoisomers thereof, where each r is an integer between 0 and 5 (e.g., 0 to 3, 1 to 3, or 1 to 5), and s1 is as described herein.

[0336] In some embodiments, the building block molecule that can be incorporated into nucleic acids (e.g., RNA, mRNA, polynucleotides, primary constructs, or mMRNA) is a modified uridine (e.g., selected from the group consisting of the following, or a pharmaceutically acceptable salt or stereoisomer thereof, where Y 1 , Y 3 , Y 4 , Y 6 , and r are as described herein (for example, each r is an integer between 0 and 5, e.g., 0 to 3, 1 to 3, or 1 to 5):

[0337] [ka]

[0338] [ka]

[0339] [ka]

[0340] [ka]

[0341] [ka]

[0342]

change

[0343]

change

[0344]

change

[0345]

change

[0346]

change

[0347]

change

[0348]

change

[0349]

change

[0350]

change

[0351]

change

[0352] [ka] In some embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mMRNA is a modified cytidine (for example, selected from the group consisting of the following, or a pharmaceutically acceptable salt or stereoisomer thereof, where Y 1 , Y 3 , Y 4 , Y 6 , and r are as described herein (for example, each r is an integer between 0 and 5, e.g., 0 to 3, 1 to 3, or 1 to 5):

[0353] [ka]

[0354] [ka]

[0355] [ka]

[0356] [ka]

[0357] [ka] For example, polynucleotides, primary constructs, or building block molecules that can be incorporated into mMRNA are,

[0358] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, where each r is an integer between 0 and 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0359] In some embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mMRNA is modified adenosine (for example, selected from the group consisting of the following, or a pharmaceutically acceptable salt or stereoisomer thereof, where Y 1 , Y 3 , Y 4 , Y 6 , and r are as described herein (for example, each r is an integer between 0 and 5, e.g., 0 to 3, 1 to 3, or 1 to 5):

[0360] [ka]

[0361] [ka]

[0362] [ka]

[0363] [ka]

[0364] [ka] In some embodiments, the building block molecule that can be incorporated into a polynucleotide, primary construct, or mMRNA is modified guanosine (for example, selected from the group consisting of the following, or a pharmaceutically acceptable salt or stereoisomer thereof, where Y 1 , Y 3 , Y4 , Y 6 , and r are as described herein (for example, each r is an integer between 0 and 5, e.g., 0 to 3, 1 to 3, or 1 to 5):

[0365] [ka]

[0366] [ka]

[0367] [ka]

[0368] [ka] In some embodiments, the chemical modification involves substitution of the C-5 C group with N in the ring (e.g., >CH group of C-5 >NR) (e.g., >NR of >CH group of C-5) (e.g., >NR of >CH group of C-5) N1 Substitution with a group (in the formula, R N 1 may contain H or an optionally substituted alkyl. For example, polynucleotides, primary constructs, or building block molecules that can be incorporated into mMRNA are

[0369] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, where each r is an integer between 0 and 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0370] In another embodiment, the chemical modification may include substitution of the C-5 hydrogen of cytosine with a halo (e.g., Br, Cl, F, or I) or an optionally substituted alkyl (e.g., methyl). For example, a building block molecule that can be incorporated into a polynucleotide, primary construct, or mMRNA may be:

[0371] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, where each r is an integer between 0 and 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0372] In further embodiments, the chemical modification may include a fused ring formed by NH2 at the C-4 position and by a carbon atom at the C-5 position. For example, a building block molecule that can be incorporated into a polynucleotide, primary construct, or mMRNA is:

[0373] [ka] or a pharmaceutically acceptable salt or stereoisomer thereof, where each r is an integer between 0 and 5 (e.g., 0 to 3, 1 to 3, or 1 to 5).

[0374] Modifications in sugars Modified nucleosides and nucleotides (e.g., building block molecules) that can be incorporated into polynucleotides, primary constructs, or mMRNA (e.g., RNA or mRNA as described herein) may be modified at the sugars of ribonucleic acid. For example, the 2' hydroxyl group (OH) may be modified or substituted with several different substituents. Exemplary substitutions at the 2' position include H, halo, and optionally substituted C. 1~6 Alkyl, optionally substituted C 1~6 alkoxy, optionally substituted C 6~10 Aryloxy, optionally substituted C 3~8 Cycloalkyl, optionally substituted C3~8 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 the ingredients specified herein), polyethylene glycol (PEG), -O(CH2CH2O) n CH2CH2OR (wherein R is H or an optionally substituted alkyl group, n is an integer between 0 and 20 (e.g., 0-4, 0-8, 0-10, 0-16, 1-4, 1-8, 1-10, 1-16, 1-20, 2-4, 2-8, 2-10, 2-16, 2-20, 4-8, 4-10, 4-16, and 4-20)), and 2'-hydroxyl is C 1~6 Alkylene or C 1~6This includes, but is not limited to, “locked” nucleic acids (LNAs) bonded to the 4'-carbon of the same ribose sugar by heteroalkylene crosslinks (examples of crosslinks include methylene, propylene, ether, or amino crosslinks), aminoalkyls as defined herein, aminoalkoxys as defined herein, aminos as defined herein, and amino acids as defined herein. Generally, RNA contains a five-membered ring of ribose sugar with oxygen. Non-limiting examples of modified nucleotides include substitution of ribose oxygen (e.g., with S, Se, or alkylene, e.g., methylene or ethylene), addition of double bonds (e.g., to substitute ribose with cyclopentenyl or cyclohexenyl), ring contraction of ribose (e.g., to form a four-membered ring of cyclobutane or oxetane), ring expansion of ribose (e.g., to form a six or seven-membered ring with additional carbon or heteroatoms, such as anhydrous hexitol, althritol, mannitol, cyclohexanyl, cyclohexenyl, and morpholino, which also have a phosphoramide skeleton), polycyclic forms (e.g., tricyclo, and "unlocked" forms, e.g.) Examples include glycol nucleic acids (GNAs) (e.g., R-GNA or S-GNA, the portion where ribose is replaced by a glycol unit attached to a phosphodiester bond), threose nucleic acids (TNAs, the portion where ribose is replaced by an α-L-treophranosyl-(3'→2') bond), and peptide nucleic acids (PNAs, the portion where a 2-amino-ethyl-glycine bond replaces ribose and the phosphodiester skeleton). The sugar group may also contain one or more carbon atoms having a stereochemical configuration opposite to that of the corresponding carbon atoms of ribose. Therefore, polynucleotides, primary constructs, or mMRNA molecules may contain nucleotides containing, for example, arabinose as a sugar.

[0375] Modifications in nucleic acid bases This disclosure provides modified nucleosides and nucleotides. As used herein, “nucleoside” is defined as a compound containing a sugar molecule (e.g., pentose or ribose) or a derivative thereof in combination with an organic base (e.g., purine or pyrimidine) or a derivative thereof (also referred herein as “nucleic acid base”). As used herein, “nucleotide” is defined as a nucleoside containing a phosphate group. Modified nucleotides can be synthesized by any useful method described herein (e.g., chemically, enzymatically, or recombinantly, to include one or more modifications or non-natural nucleosides).

[0376] Modified nucleotide base pairings include not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between and / or between modified nucleotides that include non-standard or modified bases, where the arrangement of hydrogen bond donors and acceptors enables hydrogen bonding between non-standard and standard bases or between two complementary non-standard base structures. An example of such a non-standard base pairing is the base pairing of the modified nucleotide inosine with adenine, cytosine, or uracil.

[0377] Modified nucleosides and nucleotides may contain modified nucleic acid bases. Examples of nucleic acid bases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleic acid bases found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleic acid bases can be modified or completely substituted to provide enhanced properties, such as resistance to nucleases by disruption of the binding of the major groove junction partner, in polynucleotides, primary constructs, or mMRNA molecules. Table 8 below identifies the chemical appearance of nucleotides for each criterion. Circles identify the atoms containing the respective chemical regions.

[0378] Table 8

[0379] [Table 0008] In some embodiments, B is modified uracil. Exemplary modified uracils are given by formulas (b1) to (b5):

[0380] [ka] Having, or containing a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony,

[0381] [ka] However, it is a single or double bond, T 1’ , T 1” , T 2’ , and T 2” Each of these is independently H, an optionally substituted alkyl, an optionally substituted alkoxy, or an optionally substituted thioalkoxy, or T 1’ and T 1” A combination or T 2’ and T 2” When a combination comes together (for example, T 2 As shown in the example, it forms O (oxo), S (thio), or Se (seleno), V 1 and V 2 Each of these independently represents O, S, N(R) Vb ) nv , or C(R Vb ) nv In the formula, nv is an integer between 0 and 2, and each R VbHowever, independently, H, halo, optionally substituted amino acids, 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., N-protecting group, e.g., any of those described herein, e.g., substituted with trifluoroacetyl), optionally A 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), optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, or optionally substituted alkynyloxy (e.g., optionally substituted with any of those substituents described herein, e.g., substituents selected from (1) to (21) of alkyl), R 10 However, H, halo, optionally substituted amino acid, hydroxy, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted aminoalkyl, optionally substituted hydroxyalkyl, optionally substituted hydroxyalkenyl, optionally substituted hydroxyalkynyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkoxy, optionally substituted alkoxycarbonylalkyl, optionally substituted alkoxycarbonylalkenyl, optionally substituted alkoxycarbonylalkynyl, optionally substituted alkoxycarbonylalkoxy, optionally substituted carboxyalkoxy, optionally substituted carboxyalkyl, or optionally substituted carbamoylalkyl. R 11 However, it is H or an optionally substituted alkyl, R 12aHowever, 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 hydroxyl), optionally substituted carboxyalkoxy, optionally substituted carboxyaminoalkyl, or optionally substituted carbamoylalkyl, R 12c However, it 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.

[0382] For other examples of modified uracil, see formulas (b6)~(b9):

[0383] [ka] Having, or containing a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony,

[0384] [ka] However, it is a single or double bond, T 1’ , T 1” , T 2’ , and T 2” Each of these is independently H, an optionally substituted alkyl, an optionally substituted alkoxy, or an optionally substituted thioalkoxy, or T 1’ and T 1” When a combination comes together (for example, T 1 (Like those found in T) or T 2’and T 2” When a combination comes together (for example, T 2 (As shown in the example), it forms O (oxo), S (thio), or Se (seleno), or each T 1 and T 2 However, independently, they are O (oxo), S (thio), or Se (seleno), W 1 and W 2 Each of these independently, N(R Wa ) nw or C(R Wa ) nw In the formula, nw is an integer between 0 and 2, and each R Wa However, independently, H, an optionally substituted alkyl, or an optionally substituted alkoxy, Each V 3 However, independently, O, S, N(R) Va ) nv , or C(R Va ) nv In the formula, nv is an integer between 0 and 2, and each R VaHowever, independently, H, halo, optionally substituted amino acids, 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., 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.) The N-protecting group is, for example, 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 substituent described herein, e.g., substituents selected from (1) to (21) of alkyl), and R Va and R 12c However, together with the carbon atoms to which they are bonded, they can form optionally substituted cycloalkyl, optionally substituted aryl, or optionally substituted heterocyclyl (e.g., a 5- or 6-membered ring). R 12aHowever, H is either 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. 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. R 12b and T 1’ A combination or R 12b and R 12c These combinations can form arbitrarily substituted heterocyclines. R 12cThis is H, halo, optionally substituted alkyl, optionally substituted alkoxy, optionally substituted thioalkoxy, optionally substituted amino, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl.

[0385] Further examples of modified uracil are given in formulas (b28)~(b31):

[0386] [ka] Having, or containing a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony, T 1 and T 2 Each of these is independently O (oxo), S (thio), or Se (seleno), Each R Vb’ and R Vb”However, independently, H, halo, optionally substituted amino acids, 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., an N-protecting group, e.g., as described herein). Any of the compounds listed in the subscript (e.g., those substituted with trifluoroacetyl), optionally substituted alkoxycarbonylalkyls, optionally substituted alkoxycarbonylalkenyls, optionally substituted alkoxycarbonylalkynyls, optionally substituted alkoxycarbonylacyls, optionally substituted alkoxycarbonylalkoxys, optionally substituted carboxyalkyls (e.g., optionally substituted with hydroxy and / or O-protecting groups), optionally substituted carboxyalkoxys, optionally substituted carboxyaminoalkyls, or optionally substituted carbamoylalkyls (e.g., optionally substituted with any substituents listed herein, e.g., substituents selected from (1) to (21) of alkyl) (e.g., R Vb’ However, optionally substituted alkyl, optionally substituted alkenyl, or optionally substituted aminoalkyl, for example, an N-protecting group (for example, any of those described herein, for example, trifluoroacetyl or sulfoalkyl), R 12a However, the aminoalkyl group is H, optionally substituted alkyl, optionally substituted carboxyaminoalkyl, 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, or optionally substituted aminoalkynyl. R12b However, 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 (for example, substituted with an N-protecting group, for example, any of those described herein, for example, trifluoroacetyl or sulfoalkyl), These are optionally substituted alkoxycarbonylacyls, optionally substituted alkoxycarbonylalkoxys, optionally substituted alkoxycarbonylalkyls, optionally substituted alkoxycarbonylalkenyls, optionally substituted alkoxycarbonylalkynyls, optionally substituted alkoxycarbonylalkoxys, optionally substituted carboxyalkoxys, optionally substituted carboxyalkyls, or optionally substituted carbamoylalkyls.

[0387] In a particular embodiment, T 1 O (oxo) and T 2 is S (thio) or Se (seleno). In other embodiments, T 1 It is S (thio) and T 2 is O (oxo) or Se (seleno). In some embodiments, R Vb’ is H, an optionally substituted alkyl, or an optionally substituted alkoxy.

[0388] In other embodiments, each R 12a and R 12b R is independently H, an optionally substituted alkyl, an optionally substituted alkenyl, an optionally substituted alkynyl, or an optionally substituted hydroxyalkyl. In certain embodiments, R 12a In other embodiments, R 12a R 12b All of these are H.

[0389] In some embodiments, R 12b Each RVb’ These 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 is substituted with one or more of the following: 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 a particular embodiment, R 12a R Vb” All of these are H. In a particular embodiment, T 1 O (oxo) and T 2 It is either S (thio) or Se (seleno).

[0390] In some embodiments, R Vb’ This is an optionally substituted alkoxycarbonylalkyl or an optionally substituted carbamoylalkyl. In a particular embodiment, R 12a , R 12b , R 12c , or R Va Any substituent of the polyethylene glycol group (e.g., -(CH2) s2 (OCH2CH2) s1 (CH2) s3OR', where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and R' is either H or C 1~20 (It is alkyl), or an amino-polyethylene glycol group (e.g., -NR) N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 In the formula, s1 is an integer between 1 and 10 (for example, 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (for example, 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 However, independently, hydrogen or optionally substituted C 1~6 It is alkyl.

[0391] In some embodiments, B is modified cytosine. Exemplary modified cytosines are given by formulas (b10) to (b14):

[0392] [ka] The compound, or a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony, T 3’ and T 3” Each of these is independently H, an optionally substituted alkyl, an optionally substituted alkoxy, or an optionally substituted thioalkoxy, or T 3’ and T 3” When a combination comes together (for example, T 3 As shown in the example, it forms O (oxo), S (thio), or Se (seleno), Each V 4 However, independently, O, S, N(R) Vc ) nv , or C(R Vc ) nv In the formula, nv is an integer between 0 and 2, and each R VcHowever, independently, H, halo, optionally substituted amino acids, optionally substituted alkyls, optionally substituted alkenyls, optionally substituted alkynyls, optionally substituted alkoxys, optionally substituted alkenyloxys, optionally substituted heterocyclyls, optionally substituted alkylheterocyclyls, or optionally substituted alkynyloxys (for example, optionally substituted with any substituents described herein, e.g., substituents selected from (1) to (21) of alkyls), and R 13b and R Vc These combinations can form arbitrarily substituted heterocyclines. Each V 5 However, independently, N(R Vd ) nv , or C(R Vd ) nv In the formula, nv is an integer between 0 and 2, and each R Vd However, independently, H, halo, optionally substituted amino acids, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, or optionally substituted alkynyloxy (for example, optionally substituted with any substituents described herein, e.g., alkyls selected from (1) to (21)) (e.g., V 5 However, it is -CH or N), R 13a and R 13b Each of these is independently H, an optionally substituted acyl, an optionally substituted acyloxyalkyl, an optionally substituted alkyl, or an optionally substituted alkoxy, and R 13b and R 14 These combinations can form arbitrarily substituted heterocyclines. Each R 14However, independently, these are 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 (wherein R is H, alkyl, aryl, or phosphoryl)), azide, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkyl. R 15 and R 16 Each of these is independently H, an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted alkynyl.

[0393] Further examples of modified cytosine are given in equations (b32)~(b35):

[0394] [ka] Having, or containing a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony, T 1 and T 3 Each of these is independently O (oxo), S (thio), or Se (seleno), R 13a and R 13b Each of these is independently H, an optionally substituted acyl, an optionally substituted acyloxyalkyl, an optionally substituted alkyl, or an optionally substituted alkoxy, and R 13b and R 14These combinations can form arbitrarily substituted heterocyclines. Each R 14 However, independently, these are 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)), azide, 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. R 15 and R 16 Each of these is independently H, an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted alkynyl (for example, R 15 However, H is R 16 (However, it is H or an optionally substituted alkyl group).

[0395] In some embodiments, R 15 H is R 16 is H or optionally substituted alkyl. In certain embodiments, R 14 is H, acyl, or hydroxyalkyl. In some embodiments, R 14 is a halo. In some embodiments, R 14 R 15 In all of these, H. In some embodiments, R 15 R 16In all of these, H. In some embodiments, R 14 and R 15 and R 16 Each of these is H. In a further embodiment, R 13a and R 13b Each of them is independently H or an optionally substituted alkyl group.

[0396] A further non-restrictive example of modified cytosine is given by equation (b36):

[0397] [ka] Examples include compounds thereof, or pharmaceutically acceptable salts or stereoisomers thereof. During the ceremony, Each R 13b However, independently, H, an optionally substituted acyl, an optionally substituted acyloxyalkyl, an optionally substituted alkyl, or an optionally substituted alkoxy, and R 13b and R 14b These combinations can form arbitrarily substituted heterocyclines. Each R 14a and R 14bHowever, independently, these are 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 (wherein R is H, alkyl, aryl, phosphoryl, optionally substituted aminoalkyl, or optionally substituted carboxyaminoalkyl)), azide, optionally substituted aryl, optionally substituted heterocyclyl, optionally substituted alkylheterocyclyl, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, or optionally substituted aminoalkynyl. R 15 Each of these is independently H, an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted alkynyl.

[0398] In a particular embodiment, R 14b R is an optionally substituted amino acid (for example, an optionally substituted lysine). In some embodiments, R 14a H is H. In some embodiments, B is modified guanine. Exemplary modified guanines are given by formulas (b15)~(b17):

[0399] [ka] The compound, or a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony, T 4’ , T 4” , T 5’ , T 5” , T 6’ , and T6” Each of them is independently H, an optionally substituted alkyl, or an optionally substituted alkoxy, and T 4’ and T 4” combinations (for example, T 4 (Like the one in T) or T 5’ and T 5” combinations (for example, T 5 (Like the one in T) or T 6’ and T 6” combinations (for example, T 6 These (as shown in the image) come together to form O (oxo), S (thio), or Se (seleno), V 5 and V 6 Each of these independently represents O, S, N(R) Vd ) nv , or C(R Vd ) nv In the formula, nv is an integer between 0 and 2, and each R Vd However, independently, these are H, halo, thiol, optionally substituted amino acid, cyano, amidine, optionally substituted aminoalkyl, optionally substituted aminoalkenyl, optionally substituted aminoalkynyl, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted alkenyloxy, or optionally substituted alkynyloxy (for example, optionally substituted with any substituents described herein, e.g., substituents selected from (1) to (21) of alkyl), optionally substituted thioalkoxy, or optionally substituted amino. R 17 , R 18 , R 19a , R 19b , R 21 , R 22 , R 23 , and R 24 Each of these is independently H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted amino acid.

[0400] The modified guanosine shown in the example is given by formulas (b37)~(b40):

[0401] [ka] The compound, or a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony, T 4’ Each of them is independently H, an optionally substituted alkyl, or an optionally substituted alkoxy, and each T 4 However, independently, they are O (oxo), S (thio), or Se (seleno), R 18 , R 19a , R 19b , and R 21 Each of these is independently H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, optionally substituted amino, or optionally substituted amino acid.

[0402] In some embodiments, R 18 is H or an optionally substituted alkyl group. In further embodiments, T 4 is an oxo. In some embodiments, R 19a and R 19b Each of them is independently H or an optionally substituted alkyl group.

[0403] In some embodiments, B is modified adenine. Exemplary modified adenine is given by formulas (b18)~(b20):

[0404] [ka] The compound, or a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony, Each V 7 However, independently, O, S, N(R) Ve ) nv, or C(R Ve ) nv In the formula, nv is an integer between 0 and 2, and each R Ve However, independently, these are H, halo, optionally substituted amino acids, optionally substituted alkyls, optionally substituted alkenyls, optionally substituted alkynyls, optionally substituted alkoxys, optionally substituted alkenyloxys, or optionally substituted alkynyloxys (for example, optionally substituted with any substituents described herein, e.g., alkyls selected from (1) to (21)), Each R 25 However, independently, these are H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino. R 26a and R 26b Each of these can independently be H, an 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 hydroxyalkynyl, an optionally substituted alkoxy, or a polyethylene glycol group (e.g., -(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR'(where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 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 group) or aminopolyethylene glycol group (e.g., -NR) N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (In the formula, s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each RN1 However, independently, hydrogen or optionally substituted C 1~6 It is alkyl)) Each R 27 However, independently, these are H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy, or optionally substituted amino. Each R 28 However, independently, these are H, an optionally substituted alkyl, an optionally substituted alkenyl, or an optionally substituted alkynyl. Each R 29 However, independently, these are 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.

[0405] The example modified adenine is given by formulas (b41)~(b43):

[0406] [ka] The compound, or a pharmaceutically acceptable salt or stereoisomer thereof, During the ceremony, Each R 25 However, independently, these are H, halo, thiol, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted thioalkoxy, or optionally substituted amino. R 26a and R 26bEach of these can independently be H, an 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 hydroxyalkynyl, an optionally substituted alkoxy, or a polyethylene glycol group (e.g., -(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR'(where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 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 aminopolyethylene glycol group (e.g., -NR) N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (In the formula, s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 However, independently, hydrogen or optionally substituted C 1~6 It is alkyl)) Each R 27 However, independently, these are H, optionally substituted alkyl, optionally substituted alkenyl, optionally substituted alkynyl, optionally substituted alkoxy, optionally substituted thioalkoxy, or optionally substituted amino.

[0407] In some embodiments, R 26a H is R 26b is an optionally substituted alkyl group. In some embodiments, R 26a and R 26b Each of them is independently an optionally substituted alkyl. In certain embodiments, R 27R is an optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy. In other embodiments, R 25 This is an optionally substituted alkyl, optionally substituted alkoxy, or optionally substituted thioalkoxy.

[0408] In a particular embodiment, R 26a , R 26b , or R 29 Any substituent of the polyethylene glycol group (e.g., -(CH2) s2 (OCH2CH2) s1 (CH2) s3 OR'(where s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), s2 and s3 are independently integers between 0 and 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 aminopolyethylene glycol group (e.g., -NR) N1 (CH2) s2 (CH2CH2O) s1 (CH2) s3 NR N1 (In the formula, s1 is an integer between 1 and 10 (e.g., 1 to 6 or 1 to 4), and each of s2 and s3 is an integer between 0 and 10 (e.g., 0 to 4, 0 to 6, 1 to 4, 1 to 6, or 1 to 10), and each R N1 However, independently, hydrogen or optionally substituted C 1~6 It is alkyl.

[0409] In some embodiments, B is formula (b21):

[0410] [ka] It may have, in the formula, X 12 xa is an integer between 0 and 3, and R is an integer between 0 and 3. 12a and T 2This is as described in this specification.

[0411] In some embodiments, B is formula (b22):

[0412] [ka] It may have, in the formula, R 10’ R is independently an 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. 11 , R 12a , T 1 , and T 2 This is as described in this specification.

[0413] In some embodiments, B is formula (b23):

[0414] [ka] It may have, in the formula, R 10 This includes optionally substituted heterocyclyls (e.g., optionally substituted furyl, optionally substituted thienyl, or optionally substituted pyrrolyl), optionally substituted aryls (e.g., optionally substituted phenyl or optionally substituted naphthyl), or any substituent as specified herein (e.g., R 10 It is (of R) 11 (e.g., H or any substituent as described herein), R 12a(e.g., H or any substituent as described herein), T 1 (e.g., oxo or any substituent as described herein), and T 2 (For example, oxo or any substituent as described herein) is as described herein.

[0415] In some embodiments, B is formula (b24):

[0416] [ka] It may have, in the formula, R 14’ R is independently an 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. 13a , R 13b , R 15 , and T 3 This is as described in this specification.

[0417] In some embodiments, B is formula (b25):

[0418] [ka] It may have, in the formula, R 14’This includes optionally substituted heterocyclyls (e.g., optionally substituted furyl, optionally substituted thienyl, or optionally substituted pyrrolyl), optionally substituted aryls (e.g., optionally substituted phenyl or optionally substituted naphthyl), or any substituent as specified herein (e.g., R 14 or R 14’ It is (of R) 13a (e.g., H or any substituent as described herein), R 13b (e.g., H or any substituent as described herein), R 15 (e.g., H or any substituent as specified herein), and T 3 (For example, oxo or any substituent as described herein) is as described herein.

[0419] In some embodiments, B is a nucleic acid base selected from the group consisting of cytosine, guanine, adenine, and uracil. In some embodiments, B is

[0420] [ka] It is possible.

[0421] In some embodiments, the modified nucleic acid base is modified uracil. Exemplary nucleic acid bases and nucleosides having modified uracil include pseudouridine (ψ), pyridine-4-onribonucleoside, 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, and 2-thiouridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudridine, 2-thio-pseudridine, 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-methoxyuridine (mo 5 U), Uridine 5-oxyacetic acid (cmo 5U), Uridine 5-oxyacetate methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1-carboxymethyl-pseudridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridinemethyl ester (mchm 5 U), 5-methoxycarbonylmethyluridine (mcm 5 U), 5-methoxycarbonylmethyl-2-thiouridine (mcm 5 s 2 U), 5-aminomethyl-2-thiouridine (nm 5 s 2 U), 5-methylaminomethyluridine (mnm 5 U), 5-methylaminomethyl-2-thiouridine (mnm 5 s 2 U), 5-methylaminomethyl-2-seleno-uridine (mnm 5 se 2 U), 5-Carbamoylmethyluridine (ncm 5 U), 5-carboxymethylaminomethyluridine (cmnm 5 U), 5-carboxymethylaminomethyl-2-thiouridine (cmnm 5 s 2 U), 5-propynyl-uridine, 1-propynyl-pseudolidine, 5-taurinomethyl-uridine (τm 5 U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine (τm 5 s 2 U), 1-taurinomethyl-4-thio-pseuduridine, 5-methyl-uridine (m 5 U, i.e., those having the nucleic acid base deoxythymine), 1-methylpseudridine (m 1 ψ), 5-methyl-2-thiouridine (m 5 s 2 U), 1-methyl-4-thio-pseuduridine (m 1 s 4 ψ), 4-thio-1-methyl-pseuduridine, 3-methyl-pseuduridine (m3 ψ), 2-thio-1-methyl-pseuduridine, 1-methyl-1-deaza-pseuduridine, 2-thio-1-methyl-1-deaza-pseuduridine, dihydrouridine(D), dihydropseuduridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine(m 5 D) 2-thio-dihydrouridine, 2-thio-dihydropsuduridine, 2-methoxy-uridine, 2-methoxy-4-thiouridine, 4-methoxy-psuduridine, 4-methoxy-2-thio-psuduridine, N1-methylpsuduridine (1-methylpsuduridine (m 1 ψ) (also known as 3-(3-amino-3-carboxypropyl)uridine (acp 3 U), 1-methyl-3-(3-amino-3-carboxypropyl)pseudridine (acp 3 ψ), 5-(isopentenylaminomethyl)uridine (inm 5 U), 5-(isopentenylaminomethyl)-2-thiouridine(inm 5 s 2 U), α-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m 5 Um), 2'-O-methyl-pseudridine(ψm), 2-thio-2'-O-methyl-uridine(s 2 Um), 5-methoxycarbonylmethyl-2'-O-methyluridine (mcm 5 Um), 5-Carbamoylmethyl-2'-O-methyluridine (ncm 5 Um), 5-carboxymethylaminomethyl-2'-O-methyluridine (cmnm 5 Um), 3,2'-O-dimethyluridine (m 3 Um), 5-(isopentenylaminomethyl)-2'-O-methyluridine(inm 5 Examples include Um), 1-thiouridine, deoxythymidine, 2'-F-ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl)uridine, and 5-[3-(1-E-propenylamino)uridine.

[0422] In some embodiments, the modified nucleic acid base is modified cytosine. Exemplary nucleic acid bases and nucleosides having modified cytosine include 5-azacytidine, 6-azacytidine, pseudoisocytidine, and 3-methylcytidine (m 3 C), N4-acetylcytidine (ac 4 C), 5-formyl-cytidine (f 5 C), N4-methylcytidine (m 4 C), 5-methylcytidine (m 5 C), 5-halo-cytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine (hm 5 C) 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thiocytidine (s 2 C), 2-thio-5-methylcytidine, 4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine, 4-thio-1-methyl-1-deaza-pseudoisocytidine, 1-methyl-1-deaza-pseudoisocytidine, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, lysidine (k2C), α-thiocytidine, 2'-O-methylcytidine (Cm), 5,2'-O-dimethylcytidine (m 5 Cm), N4-acetyl-2'-O-methylcytidine (ac 4 Cm), N4,2'-O-dimethylcytidine (m 4 Cm), 5-formyl-2'-O-methylcytidine (f 5 Cm), N4,N4,2'-O-trimethylcytidine (m 4 Examples include 2Cm), 1-thiocytidine, 2'-F-alacytidine, 2'-F-cytidine, and 2'-OH-alacytidine.

[0423] In some embodiments, the modified nucleic acid base is modified adenine. Exemplary nucleic acid bases and nucleosides having modified adenine include 2-amino-purine, 2,6-diaminopurine, 2-amino-6-halo-purine (e.g., 2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine), 2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-amino-purine, 7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-diaminopurine, and 1-methyl-adenosine (m 1 A) 2-methyl-adenine (m 2 A) N6-methyl-adenosine (m 6 A) 2-methylthio-N6-methyladenosine (ms 2 m 6 A) N6-isopentenyl-adenosine (i 6 A) 2-methylthio-N6-isopentenyl-adenosine (ms 2 i 6 A) N6-(cis-hydroxyisopentenyl)adenosine (io 6 A) 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms 2 io 6 A) N6-Glycinylcarbamoyl-adenosine (g 6 A) N6-Threonylcarbamoyl-adenosine (t 6 A) N6-methyl-N6-threonylcarbamoyl-adenosine (m 6 t 6 A) 2-methylthio-N6-threonylcarbamoyl-adenosine (ms 2 g 6 A) N6,N6-dimethyl-adenosine (m 6 2A), N6-hydroxynorvalylcarbamoyl-adenosine (hn 6 A) 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms 2 hn 6 A) N6-acetyl-adenosine (ac 6A) 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, α-thio-adenosine, 2'-O-methyl-adenosine (Am), N6,2'-O-dimethyl-adenosine (m 6 Am), N6,N6,2'-O-trimethyl-adenosine (m 6 2Am), 1,2'-O-dimethyl-adenosine (m 1 Examples include Am), 2'-O-ribosyladenosine (phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine, 8-azido-adenosine, 2'-F-ala-adenosine, 2'-F-adenosine, 2'-OH-ala-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

[0424] In some embodiments, the modified nucleic acid base is modified guanine. Exemplary nucleic acid bases and nucleosides having modified guanine include inosine(I), 1-methyl-inosine(m) 1 I) Wyosin (imG), Methyl Wyosin (mimG), 4-Demethyl Wyosin (imG-14), Iso Wyosin (imG2), Wybutosin (yW), Peroxywybutosin (o2yW), Hydroxywybutosin (OHyW), Unmodified Hydroxywybutosin (OHyW*), 7-Deaza-Guanosine, Queosin (Q), Epoxy Queosin (oQ), Galactosyl Queosin (galQ), Mannosyl Queosin (manQ), 7-Cyano-7-Deaza-Guanosine (preQ0), 7-Aminomethyl-7-Deaza-Guanosine (preQ1), Alkaeosin (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 2G), N2,7-dimethyl-guanosine (m2,7 G), N2, N2,7-dimethyl-guanosine (m 2,2,7 G), 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine, α-thio-guanosine, 2'-O-methyl-guanosine (Gm), N2-methyl-2'-O-methyl-guanosine (m 2 Gm), N2,N2-dimethyl-2'-O-methyl-guanosine (m 2 2Gm), 1-methyl-2'-O-methyl-guanosine (m 1 Gm), N2,7-dimethyl-2'-O-methyl-guanosine (m 2,7 Gm), 2'-O-methyl-inosine (Im), 1,2'-O-dimethyl-inosine (m 1 Examples include Im) and 2'-O-ribosylguanosine (phosphate) (Gr(p)).

[0425] The nucleic acid bases of a nucleotide can be independently selected from purines, pyrimidines, purines, or pyrimidine analogs. For example, the nucleic acid bases can be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine, respectively. In another embodiment, nucleic acid bases 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 (pseudolacil), 4-thiouracil, 8-halo (e.g., 8-bromo), 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-thiol derivatives. This may also include naturally occurring and synthetic derivatives of bases, particularly substituted adenines and guanines, 5-halos, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deazaguanine, 7-deazaguanine, 3-deazaguanine, deazaadenine, 7-deazaadenine, 3-deazaadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinon, 9-deazapurine, imidazo[4,5-d]pyrazine, thiazolo[4,5-d]pyrimidine, pyrazine-2-one, 1,2,4-triazine, pyridazine, and 1,3,5-triazine. When nucleotides are represented using the abbreviations A, G, C, T, or U, each letter refers to a representative base and / or its derivatives, for example, A includes adenine or adenine analogs such as 7-deazaadenine.

[0426] Modification of nucleoside bonds Modified nucleotides that can be incorporated into polynucleotides, primary constructs, or mMRNA molecules may be modified at the nucleoside bond (e.g., the phosphate backbone). In this specification, the terms “phosphate” and “phosphodiester” are used synonymously in relation to the polynucleotide backbone. The phosphate group of the backbone may be modified by substituting one or more of the oxygen atoms with different substituents. Furthermore, modified nucleosides and nucleotides may involve extensive substitution of the unmodified phosphate moiety with other nucleoside bonds described herein. Examples of modified phosphate groups include, but are not limited to, phosphorothioates, phosphoroselenates, boranophosphates, boranophosphate esters, hydrogen phosphonates, phosphorolamides, phosphorodiamidates, alkyl or aryl phosphonates, and phosphotryesters. Phosphorodithioates have both unbound oxygen atoms substituted with sulfur. The phosphate linker can also be modified by substitution of the bound oxygen with nitrogen (bridged phosphoramide), sulfur (bridged phosphorothioate), and carbon (bridged methylene phosphonate).

[0427] The α-thio-substituted phosphate moiety is provided to confer stability to RNA and DNA polymers via non-natural phosphorothioate backbone binding. Phosphothioate-bound DNA and RNA exhibit increased nuclease resistance, followed by a longer half-life in the cellular environment. Phosphothioate-bound polynucleotides, primary constructs, or mMRNA molecules are also expected to reduce the innate immune response by weaker binding / activation of innate immune molecules in cells.

[0428] In certain embodiments, the modified nucleoside includes α-thio-nucleosides (e.g., 5'-O-(1-thiophosphate)-adenosine, 5'-O-(1-thiophosphate)-cytidine (α-thiocytidine), 5'-O-(1-thiophosphate)-guanosine, 5'-O-(1-thiophosphate)-uridine, or 5'-O-(1-thiophosphate)-pseudolidine).

[0429] Other nucleoside bonds that may be used in accordance with the present invention, including nucleoside bonds that do not contain a phosphorus atom, are described below in this specification. A combination of modified sugars, nucleic acid bases, and nucleoside bonds. The polynucleotides, primary constructs, and mMRNAs of the present invention may include combinations of modifications to sugars, nucleic acid bases, and / or nucleoside-to-nucleoside bonds. These combinations may include one or more of the modifications described herein. For example, any of the nucleotides described herein in formulas (Ia), (Ia-1) to (Ia-3), (Ib) to (If), (IIa) to (IIp), (IIb-1), (IIb-2), (IIc-1) to (IIc-2), (IIn-1), (IIn-2), (IVa) to (IVl), and (IXa) to (IXr) may be combined with any of the nucleic acid bases described herein (e.g., formulas (b1) to (b43) or any other described herein).

[0430] Synthesis of polypeptides, primary constructs, and mMRNA molecules Polypeptides, primary constructs, and mMRNA molecules used in accordance with the present invention can be prepared according to any useful techniques described herein. 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. Where 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. Optimal reaction conditions may vary depending on the specific reactants or solvents used, but such conditions can be determined by those skilled in the art using routine optimization procedures.

[0431] The processes described herein may be monitored according to any preferred method known in the art. For example, product formation may be monitored by nuclear magnetic resonance spectroscopy (e.g., 1 H or 13C) It can be monitored by spectroscopic means such as infrared spectroscopy, spectrophotometry (e.g., ultraviolet-visible), or mass spectrometry, or by chromatography such as high-performance liquid chromatography (HPLC) or thin-layer chromatography.

[0432] The preparation of polypeptides, primary constructs, and mMRNA molecules of the present invention may involve the protection and deprotection of various chemical groups. The need for protection and deprotection, as well as the selection of appropriate protecting groups, can be readily 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, 2nd Ed., Wiley & Sons, 1991, which is incorporated herein by reference in its entirety.

[0433] The reactions of the processes described herein may be carried out in suitable solvents that can be readily selected by technicians in the field of organic synthesis. Suitable solvents may be substantially inactive with the starting materials (reactants), intermediates, or products at the temperature in which the reaction takes place, i.e., a temperature that can range from the freezing point to the boiling point of the solvent. A given reaction may be carried out in one solvent or a mixture of two or more solvents. Depending on the specific reaction step, a solvent suitable for that particular reaction step may be selected.

[0434] The decomposition of racemic mixtures of modified nucleosides and nucleotides can be carried out by any of the many methods known in the art. An example of such a method is fractional recrystallization using "chiral decomposition acids," which are optically active salt-forming organic acids. Suitable decomposition acids for fractional recrystallization include, for example, optically active acids such as tartaric acid, diacetyltartaric acid, dibenzoyltartaric acid, mandelic acid, malic acid, lactic acid, or the D and L forms of various optically active camphor sulfonic acids. The decomposition of racemic mixtures can also be carried out by elution on a column packed with an optically active decomposition agent (e.g., dinitrobenzoylphenylglycine). Suitable elution solvent compositions can be determined by those skilled in the art.

[0435] 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), in which each is incorporated as a whole by reference.

[0436] The polypeptides, primary constructs, and mMRNAs 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 one or more or all of A, G, U, and C) may or may not be uniformly modified in the polynucleotide of the present invention or in a given predetermined sequence region (e.g., one or more of the sequence regions shown in Figure 1). In some embodiments, all nucleotides X in the polynucleotide of the present invention (or a given sequence region thereof) are modified, where X may be one of the nucleotides A, G, U, and C, or 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.

[0437] Different sugar modifications, nucleotide modifications, and / or nucleoside-to-nucleoside bonds (e.g., skeletal structures) can be present at various positions on polynucleotides, primary constructs, or mMRNAs. Those skilled in the art will recognize that nucleotide analogs or other modifications may be located at any position on a polynucleotide, primary construct, or mMRNA such that their function is substantially diminished. The modifications may also be 5' or 3' terminal modifications. Polynucleotides, primary constructs, or mMRNAs contain approximately 1% to 100% modified nucleotides (total nucleotide content or related to one or more nucleotides, i.e., one or more of A, G, U, or C) or any intermediate proportion (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%). It may contain 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%.

[0438] In some embodiments, the polynucleotide, primary construct, or mMRNA contains 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 approximately 1% to approximately 100% modified uracil or modified 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%) It can be substituted with modified uracil or modified uridine in the following percentages: 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%. Modified uracil or uridine may be substituted with a single unique compound or with multiple compounds having different structures (e.g., two, three, four or more unique structures as described herein).In some embodiments, cytosine or cytidine (generally, C) in polynucleotides, primary constructs, or mMRNA molecules is approximately 1% to approximately 100% modified cytosine or modified cytidine (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%). It can be substituted with modified cytosine or modified cytidine in the following percentages: 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%. Modified cytosine or cytidine may be substituted with a single unique compound or with multiple compounds having different structures (e.g., two, three, four or more unique structures as described herein).

[0439] In some embodiments, this disclosure relates to formula (Ia-1):

[0440] [ka] The present invention provides a method for synthesizing a polynucleotide, primary construct, or mMRNA (e.g., a first region, a first adjacent region, or a second adjacent region) containing n bound nucleosides, wherein the method is a) Equation (IV-1):

[0441] [ka] The nucleotides of formula (V-1):

[0442] [ka] It is reacted with a phosphoramidite compound (wherein Y in the formula). 9 However, each P is H, hydroxy, phosphoryl, pyrophosphate, sulfate, amino, thiol, optionally substituted amino acid, or peptide (e.g., containing 2 to 12 amino acids), and each P 1 , P 2 , and P 3 However, independently, it is a suitable protecting group.

[0443] [ka] However, it indicates a solid support. To provide a polynucleotide, primary construct, or mMRNA of formula (VI-1),

[0444] [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),

[0445] [ka] and c) comprising removing a protecting group to produce a polynucleotide, primary construct, or mMRNA of formula (Ia).

[0446] 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 embodiment...

Claims

1. A method for producing a target polypeptide in vivo, comprising contacting a mammalian cell, tissue, or organism with at least one isolated mRNA encoding the target polypeptide, wherein the target polypeptide is fructose diphosphate aldolase A (ALDOA), α-methylacyl-CoA racemase (AMACR), serum amyloid-P component (APCS), angiopoietin 1 (ANGPT1), apolipoprotein A-I (APOA1) Milano, apolipoprotein A-I (APOA1) Paris, apolipoprotein A-I (APOA1), argininosuccinate triase (ASL), artemin (ARTN), arylsulfatase B (ARSB), bactericidal / permeability-enhancing protein (rBPI-21), bone formation protein Protein 2 (BMP2), bone morphogenetic protein 7 (BMP7), branched chain keto acid dehydrogenase E1 α-polypeptide (BCKDHA), colony-stimulating factor 2 (granulocyte-macrophage) (GM-CSF), colony-stimulating factor 3 (granulocyte) (GCSF), deoxyribonuclease I (DNAse1), erythropoietin (EPO), factor IX, factor VII, factor XI, fibrinogen A (FGA), fibroblast growth factor 18 (FGF18), fibroblast growth factor 23 (FGF23), fibroblast growth factor 7 (FGF7 or KGF), follistatin (FST), fumarylacetoacetate hydrolase (FAH), galactokinase 1 (GALK1), α-galactosidase (GLA), glucan (1,4-α-)Branched branch enzyme 1 (GBE1), glycoprotein hormone α-polypeptide (CGA or FSH-α), β-hemoglobin (HBB), hepatocyte growth factor (HGF), human growth hormone (hGH), insulin aspart, insulin glargine, insulin glulisin, insulin lispro, interferon β (IFNB), interferon α2 (IFNA2), interleukin 10 (IL-10), interleukin 15 (IL-15), interleukin 7 (IL-7), clozole (KL), lecithin / cholesterol Sterol acyltransferase (LCAT), lipase A / lysosomal acid cholesterol esterase (LIPA), lipoprotein lipase (LPL), low-density lipoprotein receptor (LDLR), α-mannosidase class 2B member 1 (MAN2B1), microsomal triglyceride transfer protein (MTTP), N-acetylglutamate synthase (NAGS), neuregulin 1 (NRG1), ornithine carbamoyltransferase (OTC), phosphorylase kinase α2 (Liver) (PHKA2), Plasminogen (PLAT), Septin 4 (ARTS or SEPT4), Serpine peptidase inhibitor, Klade C (antithrombin), Member 1 (SERPINC1), Serpine peptidase inhibitor, Klade F (α-2 antiplasmin, pigment epithelial-derived factor), Member 2 (SERPINF2), Sirtuin 1 (SIRT1), Sirtuin 6 (SIRT6), Solute transporter family 16, Member 3 (monocarboxylic acid transporter 4) (SLC16A3), Solute transporter family The method is selected from the group consisting of 2 (glucose-promoting transporter) member 1 (SLC2A1 or GLUT1), soltirin 1 (SORT1), thrombopoietin (THPO), transforming growth factor β (TGFB1), tuffterin 1 (TUFT1), tumor protein p53 (TP53), tyrosinase (TYR), UDP-glucuronosyltransferase 1 family polypeptide A1 (UGT1A1), vascular endothelial growth factor (VEGF), and X-linked apoptosis inhibitor (XIAP).

2. The method according to claim 1, wherein the isolated mRNA encoding the target polypeptide has a sequence selected from the group consisting of SEQ ID NOs: 168-234, 263-272, and 312-319.

3. The method according to claim 1, wherein the isolated mRNA contains a 3' tailing sequence of a nucleotide-binding nucleoside consisting of approximately 140 nucleotides.

4. The method according to claim 1, wherein the isolated mRNA contains a 3' tailing sequence of a nucleotide-binding nucleoside consisting of approximately 160 nucleotides.

5. The method according to claim 1, wherein the isolated mRNA includes the 5' terminal cap of cap 1.

6. The method according to claim 1, wherein the isolated mRNA comprises at least one chemically modified nucleoside.

7. The aforementioned at least one chemically modified nucleoside is pyridine-4-one ribonucleoside, 5-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thio-pseudridine, 2-thio-pseudridine, 5-hydroxyuridine, 3-methyluridine, 5-carboxymethyluridine, 1-carboxymethyl-pseudridine, 5-propynyluridine, 1-propynyl-pseudridine, 5-taurinomethyluridine, 1-taurinomethyl-pseudridine, 5-taurinomethyl-2-thio Uridine, 1-taurinomethyl-4-thiouridine, 5-methyluridine, 1-methylpsuduridine, 4-thio-1-methylpsuduridine, 2-thio-1-methylpsuduridine, 1-methyl-1-deazapsuduridine, 2-thio-1-methyl-1-deazapsuduridine, dihydrouridine, dihydropsuduridine, 2-thio-dihydrouridine, 2-thio-dihydropsuduridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4-methoxypsuduridine, 4-methoxy-2-thiouridine Eu-pseudolidine, 5-azacytidine, pseudoisocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudolidine, pyrrolocytidine, pyrrolocytidine, 2-thiocytidine, 2-thio-5-methylcytidine, 4-thio-pseudolidine, 4-thio-1-methyl-pseudolidine, 4-thio-1-methyl-1-deazapseudolidine, 1-methyl-1-deazapseudolidine Zin, zebralin, 5-aza-zebralin, 5-methyl-zebralin, 5-aza-2-thio-zebralin, 2-thio-zebralin, 2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine, 2-aminopurine, 2,6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2,6-diaminopurine, 7-deaza-8-aza-2,6-Diaminopurine, 1-Methyladenosine, N6-Methyladenosine, N6-Isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-Methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-Glycinylcarbamoyladenosine, N6-Threonylcarbamoyladenosine, 2-Methylthio-N6-Threonylcarbamoyladenosine, N6,N6-Dimethyladenosine, 7-Methyladenine, 2-Methylthio-Adenine, 2-Methoxy-Adenine, Inosine, 1-Methyl-Inosine, Wyosin, Wyobutosin, 7-Deaza-Guanosine, The method according to claim 6, selected from the group consisting of 7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6-thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, and N2,N2-dimethyl-6-thio-guanosine.

8. The method according to claim 1, wherein the isolated mRNA is formulated into a pharmaceutical product.

9. The method according to claim 8, wherein the preparation is a lipoplex preparation.

10. The method according to claim 8, wherein the formulation comprises a lipid, the lipid being selected from DLin-DMA, DLin-K-DMA, DLin-KC2-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DODMA, DSDMA, DLenDMA, reLNPs, PLGA, and pegylated lipids, and mixtures thereof.

11. The method according to claim 8, wherein the isolated mRNA is administered in a total dose of 1 ug to 150 ug per day.

12. The method according to claim 1, wherein the isolated mRNA is administered in two or more equally or unequally divided doses.

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