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WO2022056117A1 - Compositions d'acide nucléique-polypeptide et leurs utilisations - Google Patents

Compositions d'acide nucléique-polypeptide et leurs utilisations Download PDF

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Publication number
WO2022056117A1
WO2022056117A1 PCT/US2021/049651 US2021049651W WO2022056117A1 WO 2022056117 A1 WO2022056117 A1 WO 2022056117A1 US 2021049651 W US2021049651 W US 2021049651W WO 2022056117 A1 WO2022056117 A1 WO 2022056117A1
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molecule
instances
polynucleotide
antibody
aspects
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Jae B. KIM
Art LEVIN
Rob BURKE
Hanhua Huang
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Avidity Biosciences Inc
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Avidity Biosciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1131—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/31—Chemical structure of the backbone
    • C12N2310/315—Phosphorothioates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/34—Spatial arrangement of the modifications
    • C12N2310/343—Spatial arrangement of the modifications having patterns, e.g. ==--==--==--
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3513—Protein; Peptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/30—Chemical structure
    • C12N2310/35—Nature of the modification
    • C12N2310/351—Conjugate
    • C12N2310/3515—Lipophilic moiety, e.g. cholesterol
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/10—Applications; Uses in screening processes
    • C12N2320/11—Applications; Uses in screening processes for the determination of target sites, i.e. of active nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • RNA interference provides long lasting effect over multiple cell divisions.
  • RNAi represents a viable method useful for anti-infectious agent therapeutics, including interruption of virus replication and modulation of tissue damage resulting from virus infection or the immune response thereto.
  • An example of one such disease is COVID-19 and the impact of infection by SARS-CoV and SARS-CoV-2 on heart muscle cells, which can result in sometimes fatal cardiomyopathy and heart failure.
  • compositions and pharmaceutical formulations that comprise a binding moiety conjugated to a polynucleic acid molecule specific for viral genes and/or their gene products.
  • the composition includes a polymer, and/or a cell membrane penetrating moiety.
  • methods for treating a disease or condition e.g., virus infection
  • a composition or a pharmaceutical formulation comprising a binding moiety conjugated to a polynucleic acid molecule and optionally including a polymer and/or a membrane penetrating moiety.
  • Antibody-oligonucleotide conjugates of the present disclosure targeting SARS-CoV-2 for treatment of COVID-19 myocarditis can be a transformative therapy for COVID-19 patients.
  • Cardiomyocytes express ACE2 receptor and TMPRSS2 receptor (required for SARS-CoV-2 viral entry) as well as transferrin receptor 1 (TfRl) in high abundance. Cardiovascular involvement is common in patients with severe COVID-19 and is associated with worse prognosis and increased death rates. In 68 COVID-19 deaths in Wuhan, 60% had myocardial damage/heart failure. In 39 consecutive autopsy cases in Germany, 24 had cardiac tissue test positive for SARS-CoV-2; viral load above 1,000 copies per pg RNA was documented in 16 cases.
  • COVID-19 patients with preexisting or de novo cardiomyopathy can be treated with the antibody-oligonucleotide conjugates of the present disclosure.
  • siRNA knockdown of SARS-CoV-2 viral genome is achievable with high potency.
  • Antibody-oligonucleotide conjugates of the present disclosure are likely to work well (or better) with emerging therapies (anti-virus mAbs, antiviral drugs, anti-inflammatory drugs).
  • A is a binding moiety
  • B is a polynucleotide
  • n is an integer between 1 and 10.
  • the polynucleotide is capable of binding to a SARS-CoV or SARS-CoV-2 RNA and mediate RNA interference against the SARS-CoV or SARS-CoV-2 RNA.
  • the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.
  • the at least one 2’ modified nucleotide comprises 2’-O-methyl, 2’-O- methoxyethyl (2’-0-M0E), 2’-O-aminopropyl, 2'-deoxy, 2’-deoxy-2'-fluoro, 2'-O-aminopropyl (2'- O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified nucleotide.
  • the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA).
  • the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.
  • the at least one inverted abasic moiety is at at least one terminus.
  • the polynucleotide comprises a single-stranded nucleic acid molecule.
  • the single strand is an antisense oligonucleotide, or a PMO.
  • the polynucleotide comprises two or more strands.
  • the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule.
  • the second polynucleotide comprises at least one modification.
  • the first polynucleotide and the second polynucleotide are RNA molecules. In some aspects, the first polynucleotide and the second polynucleotide are siRNA molecules and/or form a double-stranded siRNA molecules.
  • the first polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the published sequence is found at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NO: 1-20, 21-64, and 109-152.
  • the second polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the published sequence is found at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 65-108, and 153-196.
  • X is a bond or a non-polymeric linker group. In some aspects, X is a bond. In some aspects, X is a Ci-Ce alkyl group. In some aspects, X is a homobifunctional linker or a heterobifunctional linker, optionally conjugated to a Ci-Ce alkyl group.
  • the binding moiety is a cell surface receptor binding moiety or ligand, a cell penetrating moiety such as a peptide, or an antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof comprises a humanized antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof, monovalent Fab’, divalent Fab2, singlechain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or antigen binding fragment thereof.
  • the antibody or binding fragment thereof is an anti-cell surface receptor antibody or binding fragment thereof wherein the receptor is involved in virus attachment to a cell.
  • the antibody or binding fragment thereof is an anti-TfR.1 antibody, an anti-ACE2 antibody, an anti-TMPRSS2 antibody, or antigen binding fragment(s) thereof.
  • A-X is conjugated to the 5’ end or the 3’ end of B. In some aspects, A- X, is conjugated to an intemucleotide linkage group.
  • A is a binding moiety
  • B is a polynucleotide
  • C is a polymer
  • X is a bond or first linker
  • Y is a bond or second linker; n is an integer between 1 and 10; and wherein the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety.
  • the at least one 2’ modified nucleotide comprises 2’-O-methyl, 2’-O- methoxyethyl (2’-0-M0E), 2’-O-aminopropyl, 2'-deoxy, 2’-deoxy-2'-fluoro, 2'-O-aminopropyl (2'- O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified nucleotide.
  • the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA).
  • the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.
  • the at least one inverted abasic moiety is at at least one terminus.
  • the polynucleotide comprises a single-stranded molecule.
  • the single strand is an antisense oligonucleotide, or a PMO.
  • the polynucleotide comprises two or more strands.
  • the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule.
  • the second polynucleotide comprises at least one modification.
  • the first polynucleotide and the second polynucleotide are RNA molecules. In some aspects, the first polynucleotide and the second polynucleotide are siRNA molecules and/or form a double-stranded siRNA molecules.
  • the first polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the published sequence is found at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NO: 1-20, 21-64, and 109-152.
  • the second polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the published sequence is found at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 65-108, and 153-196.
  • X and Y are independently a bond or a non-polymeric linker group.
  • X is a bond.
  • X is a Ci-Ce alkyl group.
  • Y is a Ci- G> alkyl group.
  • X is a homobifunctional linker or a heterobifunctional linker, optionally conjugated to a Ci-Ce alkyl group.
  • Y is a homobifunctional linker or a heterobifunctional linker.
  • the binding moiety is an antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof comprises a humanized antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof, monovalent Fab’, divalent Fab2, singlechain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or binding fragment thereof.
  • the antibody or antigen binding fragment thereof is an anti-TfRl antibody, an anti-ACE2 antibody, an anti-TMPRSS2 antibody, or antigen binding fragment thereof.
  • C is polyethylene glycol. In some aspects, C has a molecular weight of about 5000 Da.
  • A-X is conjugated to the 5’ end of B and Y-C is conjugated to the 3’ end of B. In some aspects, Y-C is conjugated to the 5’ end of B and A-X is conjugated to the 3’ end of B. In some aspects, A-X, Y-C or a combination thereof is conjugated to an internucleotide linkage group.
  • the molecule further comprises D.
  • D is conjugated to C or to A.
  • D is conjugated to the molecule of Formula (I) or Formula (II) according to Formula (III):
  • A is a binding moiety
  • B is a polynucleotide
  • C is a polymer
  • X is a bond or linker
  • Y is a bond or linker
  • L is a bond or linker
  • D is a cell-penetrating or endosomolytic moiety; and z is an integer between 1 and 10; and n is an integer between 0 and 10; and wherein the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety; and D is conjugated anywhere on A, B, or C.
  • D is INF7 or melittin.
  • D is a cell-penetrating or endosomolytic polymer or peptide.
  • L is a Ci-Ce alkyl group.
  • L is a homobifunctional linker or a heterobifunctional linker.
  • the molecule further comprises at least a second binding moiety A.
  • the at least second binding moiety A is conjugated to A, to B, or to C.
  • the at least second binding moiety A is cholesterol.
  • binding moiety A is an antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof is an anti-TfRl antibody, an anti-ACE2 antibody, an anti-TMPRSS2 antibody, or antigen binding fragment thereof.
  • the molecule further comprises at least an additional polynucleotide B.
  • the at least an additional polynucleotide B is conjugated to A, to B, or to C.
  • the molecule further comprises at least an additional polymer C.
  • the at least an additional polymer C is conjugated to A, to B, or to C.
  • a pharmaceutical composition comprising a molecule described above, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is formulated as a nanoparticle formulation, including lipid nanoparticle formulation.
  • the pharmaceutical composition is formulated for parenteral, oral, intranasal, pulmonary, buccal, rectal, or transdermal administration.
  • the disease or disorder is a virus infection.
  • the disease is caused or exacerbated by the virus infection.
  • the disease is cardiomyopathy.
  • the cardiomyopathy is caused by SARS-CoV-2 infection.
  • the cardiomyopathy is associated with COVID-19 or infection with SARS-CoV-2 resulting in de novo or exacerbated pre-existing cardiomyopathy.
  • a method of inhibiting the expression of a target nucleotide in a cell of a patient comprising administering a molecule described herein.
  • the method is an in vivo method.
  • the patient is a human with or recovering from COVID-19, with or without cardiomyopathy.
  • the polynucleic acid molecule for inhibiting expression of the replication, transcription, or expression of a target SARS-COV or SARS-COV-2 gene or gene product.
  • the polynucleic acid molecule comprises a sense strand and an antisense strand, wherein the sense strand comprises a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 21-64, and 109-152.
  • the antisense strand comprises a nucleic acid sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence of GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 65-108, and 153-196.
  • the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleic acid sequences of GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 21-64, and 109-152.
  • the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleic acid sequences of GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 65-108, and 153-196.
  • the sense strand and antisense strand form a double-stranded siRNA molecule.
  • the double-stranded region is 15-30 nucleotide pairs in length
  • FIG. 1 shows the graphs of in vitro dose-response antiviral activity of the modified siRNA COV2-17 and COV2-37 against SARS-CoV-2 with the final concentration of 500 nM.
  • FIG. 2 shows the graphs of in vitro dose-response antiviral activity of the modified siRNA COV2-17 and COV2-37 against SARS-CoV-2 with the final concentration of 50 nM.
  • FIG. 3 shows the graph of in vitro SARS-Cov-2 viral yield of the modified siRNA COV2-17 and COV2-37 against SARS-CoV-2.
  • FIGs. 4-8 illustrate conjugation schemes described herein.
  • Nucleic acid (e.g., RNAi) therapy is a targeted therapy with high selectivity and specificity.
  • nucleic acid therapy is also hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation.
  • various modifications of the nucleic acid composition are explored, such as for example, novel linkers for better stabilizing and/or lower toxicity, optimization of binding moiety for increased target specificity and/or target delivery, and nucleic acid polymer modifications for increased stability and/or reduced off-target effect.
  • the arrangement or order of the different components that make-up the nucleic acid composition further effects intracellular uptake, stability, toxicity, efficacy, and/or nonspecific immune stimulation.
  • the nucleic acid component includes a binding moiety, a polymer, and a polynucleic acid molecule (or polynucleotide)
  • the order or arrangement of the binding moiety, the polymer, and/or the polynucleic acid molecule (or polynucleotide) e.g., binding moiety-polynucleic acid molecule-polymer, binding moiety-polymer-polynucleic acid molecule, or polymer-binding moiety-polynucleic acid molecule
  • the molecule comprises a binding moiety conjugated to a polynucleic acid molecule and optionally to a polymer.
  • the molecule comprises a molecule according to Formula (I): A-X-B; in which A is a binding moiety, B is a polynucleotide, and X is a bond or first linker.
  • the molecule comprises a molecule of Formula (II): A-X-B-Y-C; in which A is a binding moiety, B is a polynucleotide, C is a polymer, X is a bond or first linker, and Y is a bond or second linker.
  • the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety.
  • the molecule of Formula (I) or Formula (II) further comprises D, a cell-penetrating or endosomolytic moiety.
  • a molecule described herein is further used to treat a disease or disorder.
  • a molecule for the treatment of a disease or disorder is a molecule according to Formula (I): A-X-B-Y-C; in which A is a binding moiety, B is a polynucleotide, C is a polymer, X is a bond or first linker, and Y is a bond or second linker.
  • the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety.
  • the molecule of Formula (I) further comprises D, a cell-penetrating or endosomolytic moiety.
  • a molecule described herein is also used for inhibiting the expression of a target gene or gene product in a primary cell of a COVID-19 infected patient or a patient suffering from COVID-19 related symptoms or diseases in need thereof.
  • a molecule for such use is a molecule according to Formula (I): A-X-B; in which A is a binding moiety, B is a polynucleotide, and X is a bond or linker.
  • the molecule comprises a molecule according to Formula (II): A-X-B-Y-C; in which A is a binding moiety, B is a polynucleotide, C is a polymer, X is a bond or first linker, and Y is a bond or second linker.
  • the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.
  • the molecule of Formula (I) or Formula (II) further comprises D, a cell-penetrating or endosomolytic moiety.
  • a molecule described herein is additionally used as COVID-19 therapy, or for the treatment of a symptom, a disease or disorder associated with COVID-19 infection including cardiomyopathy or heart failure.
  • the molecule is a molecule according to Formula (I): A-X-B; in which A is a binding moiety, B is a polynucleotide, and X is a bond or linker.
  • the molecule is a molecule according to Formula (II): A-X-B-Y-C; in which A is a binding moiety, B is a polynucleotide, C is a polymer, X is a bond or first linker, and Y is a bond or second linker.
  • the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety.
  • the molecule of Formula (I) further comprises D, a cell-penetrating or endosomolytic moiety.
  • kits which comprises one or more of the molecules described herein.
  • a molecule e.g., a therapeutic molecule described herein comprises a binding moiety conjugated to a polynucleic acid molecule and a polymer.
  • A is a binding moiety
  • B is a polynucleotide
  • n is an integer between 1 and 10;
  • the polynucleotide is capable of binding to a SARS-CoV or SARS-CoV-2 RNA and mediate RNA interference against the SARS-CoV or SARS-CoV-2 RNA.
  • the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.
  • the at least one 2’ modified nucleotide comprises 2’-O-methyl, 2’-O- methoxyethyl (2’-O-MOE), 2’-O-aminopropyl, 2'-deoxy, 2’-deoxy-2'-fluoro, 2'-O-aminopropyl (2'- O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified nucleotide.
  • the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA).
  • the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.
  • the at least one inverted abasic moiety is at at least one terminus.
  • the polynucleotide comprises a single-stranded molecule.
  • the single strand is an antisense oligonucleotide, or a PMO.
  • the polynucleotide comprises two or more strands.
  • the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule.
  • the second polynucleotide comprises at least one modification.
  • the first polynucleotide and the second polynucleotide are RNA molecules. In some aspects, the first polynucleotide and the second polynucleotide are siRNA molecules or form a double-stranded siRNA molecule.
  • the first polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the published sequence is found at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NO: 1-20, 21-64, and 109-152.
  • the second polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the published sequence is found at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 65-108, and 153-196.
  • X is a bond or a non-polymeric linker group. In some aspects, X is a bond. In some aspects, X is a Ci-Ce alkyl group. In some aspects, X is a homobifunctional linker or a heterobifunctional linker, optionally conjugated to a Ci-Ce alkyl group.
  • the binding moiety is a cell surface receptor binding moiety or ligand, a cell penetrating moiety such as a peptide, or an antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof comprises a humanized antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof, monovalent Fab’, divalent Fab2, singlechain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof is an anti-cell surface receptor antibody or b antigen binding fragment thereof wherein the receptor is involved in virus attachment to a cell. In some aspects, the antibody or binding fragment thereof is an anti-TfRl antibody, an anti-ACE2 antibody, an anti-TMPRSS2 antibody, or antigen binding fragment thereof.
  • A-X is conjugated to the 5’ end of B. In some aspects, A-X, is conjugated to an intemucleotide linkage group.
  • A is a binding moiety
  • B is a polynucleotide
  • C is a polymer
  • X is a bond or first linker
  • Y is a bond or second linker
  • n is an integer between 1 and 10;
  • the polynucleotide is capable of binding to a SARS-CoV or SARS-CoV-2 RNA and mediate RNA interference against the SARS-CoV or SARS-CoV-2 RNA.
  • the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety.
  • the at least one 2’ modified nucleotide comprises 2’-O-methyl, 2’-O- methoxyethyl (2’-0-M0E), 2’-O-aminopropyl, 2'-deoxy, 2’-deoxy-2'-fluoro, 2'-O-aminopropyl (2'- O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified nucleotide.
  • the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA).
  • the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.
  • the at least one inverted abasic moiety is at at least one terminus.
  • the polynucleotide comprises a single-stranded molecule.
  • the single strand is an antisense oligonucleotide, or a PMO.
  • the polynucleotide comprises two or more strands.
  • the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule.
  • the second polynucleotide comprises at least one modification.
  • the first polynucleotide and the second polynucleotide are RNA molecules. In some aspects, the first polynucleotide and the second polynucleotide are siRNA molecules or form a double-stranded siRNA molecule.
  • the first polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the published sequence is found at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NO: 1-20, 21-64, and 109-152.
  • the second polynucleotide comprises a sequence having at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the published sequence is found at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 65-108, and 153-196.
  • X and Y are independently a bond or a non-polymeric linker group.
  • X is a bond.
  • X is a Ci-Ce alkyl group.
  • Y is a Ci- G> alkyl group.
  • X is a homobifunctional linker or a heterobifunctional linker, optionally conjugated to a Ci-Ce alkyl group.
  • Y is a homobifunctional linker or a heterobifunctional linker.
  • the binding moiety is an antibody or binding fragment thereof.
  • the antibody or antigen binding fragment thereof comprises a humanized antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof, monovalent Fab’, divalent Fab2, singlechain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or antigen binding fragment thereof.
  • the antibody or antigen binding fragment thereof is an anti-TfRl antibody, an anti-ACE2 antibody, an anti-TMPRSS2 antibody, or antigen binding fragment thereof.
  • C is polyethylene glycol. In some aspects, C has a molecular weight of about 5000 Da.
  • A-X is conjugated to the 5’ end of B and Y-C is conjugated to the 3’ end of B. In some aspects, Y-C is conjugated to the 5’ end of B and A-X is conjugated to the 3’ end of B. In some aspects, A-X, Y-C or a combination thereof is conjugated to an internucleotide linkage group.
  • the molecule further comprises D.
  • D is conjugated to C or to A.
  • at least one A and/or at least one C are conjugated to the 5’ terminus of B, the 3’ terminus of B, an internal site on B, or in any combinations thereof.
  • at least one A is conjugated at one terminus of B while at least one C is conjugated at the opposite terminus of B.
  • at least one of A is conjugated at one terminus of B while at least one of C is conjugated at an internal site on B.
  • At least one B and/or at least one C, and optionally at least one D are conjugated to a first A.
  • the at least one B is conjugated at a terminus (e.g., a 5’ terminus or a 3’ terminus) to the first A or are conjugated via an internal site to the first A.
  • the at least one C is conjugated either directly to the first A or indirectly via the two or more Bs. If indirectly via the two or more Bs, the two or more Cs are conjugated either at the same terminus as the first A on B, at opposing terminus from the first A, or independently at an internal site.
  • at least one additional A is further conjugated to the first A, to B, or to C.
  • the at least one D is optionally conjugated either directly or indirectly to the first A, to the at least one B, or to the at least one C. If directly to the first A, the at least one D is also optionally conjugated to the at least one B to form a A-D-B conjugate or is optionally conjugated to the at least one B and the at least one C to form a A-D-B-C conjugate. In some cases, the at least one additional A is different than the first A.
  • two or more Bs and/or two or more Cs are conjugated to a first A.
  • the two or more Bs are conjugated at a terminus (e.g., a 5’ terminus or a 3’ terminus) to the first A or are conjugated via an internal site to the first A.
  • the two or more Cs are conjugated either directly to the first A or indirectly via the two or more Bs. If indirectly via the two or more Bs, the two or more Cs are conjugated either at the same terminus as the first A on B, at opposing terminus from the first A, or independently at an internal site.
  • At least one additional A is further conjugated to the first A, to two or more Bs, or to two or more Cs.
  • at least one D is optionally conjugated either directly or indirectly to the first A, to the two or more Bs, or to the two or more Cs. If indirectly to the first A, the at least one D is conjugated to the first A through the two or more Bs, through the two or more Cs, through a B- C orientation to form a A-B-C-D type conjugate, or through a C-B orientation to form a A-C-B-D type conjugate.
  • the at least one additional A is different than the first A.
  • the two or more Bs are different.
  • the two or more Bs are the same. In some instances, the two or more Cs are different. In other instances, the two or more Cs are the same. In additional instances, the two or more Ds are different. In additional instances, the two or more Ds are the same. [0075] In other cases, two or more Bs and/or two or more Ds, optionally two or more Cs are conjugated to a first A. In some instances, the two or more Bs are conjugated at a terminus (e.g., a 5’ terminus or a 3’ terminus) to the first A or are conjugated via an internal site to the first A. In some instances, the two or more Ds are conjugated either directly to the first A or indirectly via the two or more Bs.
  • a terminus e.g., a 5’ terminus or a 3’ terminus
  • the two or more Ds are conjugated either at the same terminus as the first A on B, at opposing terminus from the first A, or independently at an internal site.
  • at least one additional A is further conjugated to the first A, to the two or more Bs, or to the two or more Ds.
  • the two or more Cs are optionally conjugated either directly or indirectly to the first A, to the two or more Bs, or to the two or more Ds.
  • the at least one additional A is different than the first A.
  • the two or more Bs are different.
  • the two or more Bs are the same.
  • the two or more Cs are different.
  • the two or more Cs are the same.
  • the two or more Ds are different. In additional instances, the two or more Ds are the same.
  • D is conjugated to the molecule of Formula (I) or Formula (II) according to Formula (III):
  • A is a binding moiety
  • B is a polynucleotide
  • C is a polymer
  • X is a bond or linker
  • D is INF7 or melittin.
  • D is a cell-penetrating or endosomolytic polymer or peptide.
  • L is a Ci-Ce alkyl group. In some aspects, L is a homobifunctional linker or a heterobifunctional linker.
  • the molecule further comprises at least a second binding moiety A.
  • the at least second binding moiety A is conjugated to A, to B, or to C.
  • the at least second binding moiety A is cholesterol.
  • the molecule further comprises at least an additional polynucleotide B.
  • the at least an additional polynucleotide B is conjugated to A, to B, or to C.
  • the molecule further comprises at least an additional polymer C.
  • the at least an additional polymer C is conjugated to A, to B, or to C.
  • a pharmaceutical composition comprising a molecule described above, and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition is formulated as a nanoparticle formulation, including lipid nanoparticle formulation.
  • the pharmaceutical composition is formulated for parenteral, oral, intranasal, pulmonary, buccal, rectal, or transdermal administration.
  • the disease or disorder is a virus infection.
  • the disease is caused or exacerbated by the virus infection.
  • the disease is cardiomyopathy.
  • the cardiomyopathy is caused by SARS-CoV-2 infection.
  • the cardiomyopathy is associated with COVID-19 or infection with SARS-CoV-2 resulting in de novo or exacerbated pre-existing cardiomyopathy.
  • a method of inhibiting the expression of a target nucleotide in a cell of a patient comprising administering a molecule as disclosed herein to the patient.
  • the method is an in vivo method.
  • the patient is a human with or recovering from COVID-19, with or without cardiomyopathy.
  • kits comprising a molecule described herein.
  • the polynucleic acid molecule B is a polynucleic acid molecule (or polynucleotide) that hybridizes to a target region on a virus genome or its gene product(s).
  • the virus is SARS-CoV or SARS-CoV variant, and in some instances the virus is SARS- CoV-2 or SARS-CoV-2 variant.
  • the polynucleic acid molecule comprises a sequence that hybridizes to a target sequence selected from SEQ ID NO: 1-20, 21-64, and 109-152.
  • the polynucleic acid molecule B comprises a single antisense strand.
  • the polynucleic acid molecule B comprises a single antisense strand sequence that hybridizes to a target sequence selected from SEQ ID NO: 1-20, 21-64, and 109-152.
  • the single antisense strand polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a virus genome or its gene product(s).
  • the virus is SARS-CoV or SARS-CoV variant, and in some instances the virus is SARS-CoV-2 or SARS-CoV-2 variant.
  • the polynucleic acid molecule B comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1-20, 65-108, and 153-196.
  • the polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide.
  • the first polynucleotide comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a virus genome or its gene product(s).
  • the virus is SARS- CoV or SARS-CoV variant, and in some instances the virus is SARS-CoV-2 or SARS-CoV-2 variant.
  • the polynucleic acid molecule described herein comprises RNA or DNA.
  • the polynucleic acid molecule comprises RNA.
  • RNA comprises short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), doublestranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), antisense RNA, PMO, or heterogeneous nuclear RNA (hnRNA).
  • RNA comprises shRNA.
  • RNA comprises miRNA.
  • RNA comprises dsRNA.
  • RNA comprises tRNA.
  • RNA comprises rRNA.
  • RNA comprises hnRNA.
  • the RNA comprises siRNA.
  • the polynucleic acid molecule comprises siRNA.
  • B comprises siRNA, antisense RNA, or PMO.
  • the polynucleic acid molecule is from about 10 to about 50 nucleotides in length. In some instances, the polynucleic acid molecule is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, form about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length. [0093] In some aspects, the polynucleic acid molecule is about 50 nucleotides in length. In some instances, the polynucleic acid molecule is about 45 nucleotides in length. In some instances, the polynucleic acid molecule is about 40 nucleotides in length. In some instances, the polynucleic acid molecule is about 35 nucleotides in length.
  • the polynucleic acid molecule is about 30 nucleotides in length. In some instances, the polynucleic acid molecule is about 25 nucleotides in length. In some instances, the polynucleic acid molecule is about 20 nucleotides in length. In some instances, the polynucleic acid molecule is about 19 nucleotides in length. In some instances, the polynucleic acid molecule is about 18 nucleotides in length. In some instances, the polynucleic acid molecule is about 17 nucleotides in length. In some instances, the polynucleic acid molecule is about 16 nucleotides in length. In some instances, the polynucleic acid molecule is about 15 nucleotides in length.
  • the polynucleic acid molecule is about 14 nucleotides in length. In some instances, the polynucleic acid molecule is about 13 nucleotides in length. In some instances, the polynucleic acid molecule is about 12 nucleotides in length. In some instances, the polynucleic acid molecule is about 11 nucleotides in length. In some instances, the polynucleic acid molecule is about 10 nucleotides in length. In some instances, the polynucleic acid molecule is between about 10 and about 50 nucleotides in length. In some instances, the polynucleic acid molecule is between about 10 and about 45 nucleotides in length.
  • the polynucleic acid molecule is between about 10 and about 40 nucleotides in length. In some instances, the polynucleic acid molecule is between about 10 and about 35 nucleotides in length. In some instances, the polynucleic acid molecule is between about 10 and about 30 nucleotides in length. In some instances, the polynucleic acid molecule is between about 10 and about 25 nucleotides in length. In some instances, the polynucleic acid molecule is between about 10 and about 20 nucleotides in length. In some instances, the polynucleic acid molecule is between about 15 and about 25 nucleotides in length. In some instances, the polynucleic acid molecule is between about 15 and about 30 nucleotides in length. In some instances, the polynucleic acid molecule is between about 12 and about 30 nucleotides in length.
  • the polynucleic acid molecule comprises a first polynucleotide. In some instances, the polynucleic acid molecule comprises a second polynucleotide. In some instances, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the first polynucleotide is a sense strand or passenger strand. In some instances, the second polynucleotide is an antisense strand or guide strand.
  • the polynucleic acid molecule is a first polynucleotide.
  • the first polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the first polynucleotide is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, form about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.
  • the first polynucleotide is about 50 nucleotides in length. In some instances, the first polynucleotide is about 45 nucleotides in length. In some instances, the first polynucleotide is about 40 nucleotides in length. In some instances, the first polynucleotide is about 35 nucleotides in length. In some instances, the first polynucleotide is about 30 nucleotides in length. In some instances, the first polynucleotide is about 25 nucleotides in length. In some instances, the first polynucleotide is about 20 nucleotides in length.
  • the first polynucleotide is about 19 nucleotides in length. In some instances, the first polynucleotide is about 18 nucleotides in length. In some instances, the first polynucleotide is about 17 nucleotides in length. In some instances, the first polynucleotide is about 16 nucleotides in length. In some instances, the first polynucleotide is about 15 nucleotides in length. In some instances, the first polynucleotide is about 14 nucleotides in length. In some instances, the first polynucleotide is about 13 nucleotides in length. In some instances, the first polynucleotide is about 12 nucleotides in length.
  • the first polynucleotide is about 11 nucleotides in length. In some instances, the first polynucleotide is about 10 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 50 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 45 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 40 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 35 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 30 nucleotides in length.
  • the first polynucleotide is between about 10 and about 25 nucleotides in length. In some instances, the first polynucleotide is between about 10 and about 20 nucleotides in length. In some instances, the first polynucleotide is between about 15 and about 25 nucleotides in length. In some instances, the first polynucleotide is between about 15 and about 30 nucleotides in length. In some instances, the first polynucleotide is between about 12 and about 30 nucleotides in length.
  • the polynucleic acid molecule is a second polynucleotide.
  • the second polynucleotide is from about 10 to about 50 nucleotides in length. In some instances, the second polynucleotide is from about 10 to about 30, from about 15 to about 30, from about 18 to about 25, form about 18 to about 24, from about 19 to about 23, or from about 20 to about 22 nucleotides in length.
  • the second polynucleotide is about 50 nucleotides in length. In some instances, the second polynucleotide is about 45 nucleotides in length. In some instances, the second polynucleotide is about 40 nucleotides in length. In some instances, the second polynucleotide is about 35 nucleotides in length. In some instances, the second polynucleotide is about 30 nucleotides in length. In some instances, the second polynucleotide is about 25 nucleotides in length. In some instances, the second polynucleotide is about 20 nucleotides in length.
  • the second polynucleotide is about 19 nucleotides in length. In some instances, the second polynucleotide is about 18 nucleotides in length. In some instances, the second polynucleotide is about 17 nucleotides in length. In some instances, the second polynucleotide is about 16 nucleotides in length. In some instances, the second polynucleotide is about 15 nucleotides in length. In some instances, the second polynucleotide is about 14 nucleotides in length. In some instances, the second polynucleotide is about 13 nucleotides in length. In some instances, the second polynucleotide is about 12 nucleotides in length.
  • the second polynucleotide is about 11 nucleotides in length. In some instances, the second polynucleotide is about 10 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 50 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 45 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 40 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 35 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 30 nucleotides in length.
  • the second polynucleotide is between about 10 and about 25 nucleotides in length. In some instances, the second polynucleotide is between about 10 and about 20 nucleotides in length. In some instances, the second polynucleotide is between about 15 and about 25 nucleotides in length. In some instances, the second polynucleotide is between about 15 and about 30 nucleotides in length. In some instances, the second polynucleotide is between about 12 and about 30 nucleotides in length.
  • the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide.
  • the polynucleic acid molecule further comprises a blunt terminus, an overhang, or a combination thereof.
  • the blunt terminus is a 5’ blunt terminus, a 3’ blunt terminus, or both.
  • the overhang is a 5’ overhang, 3’ overhang, or both.
  • the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base pairing nucleotides.
  • the overhang comprises 1, 2, 3, 4, 5, or 6 non-base pairing nucleotides.
  • the overhang comprises 1, 2, 3, or 4 non-base pairing nucleotides. In some cases, the overhang comprises 1 non-base pairing nucleotide. In some cases, the overhang comprises 2 non-base pairing nucleotides. In some cases, the overhang comprises 3 non-base pairing nucleotides. In some cases, the overhang comprises 4 non-base pairing nucleotides. [00100] In some aspects, the sequence of the polynucleic acid molecule is at least 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 99.5% complementary to a target sequence described herein.
  • the sequence of the polynucleic acid molecule is at least 50% complementary to a target sequence described herein. In some aspects, the sequence of the polynucleic acid molecule is at least 60% complementary to a target sequence described herein. In some aspects, the sequence of the polynucleic acid molecule is at least 70% complementary to a target sequence described herein. In some aspects, the sequence of the polynucleic acid molecule is at least 80% complementary to a target sequence described herein. In some aspects, the sequence of the polynucleic acid molecule is at least 90% complementary to a target sequence described herein. In some aspects, the sequence of the polynucleic acid molecule is at least 95% complementary to a target sequence described herein. In some aspects, the sequence of the polynucleic acid molecule is at least 99% complementary to a target sequence described herein. In some instances, the sequence of the polynucleic acid molecule is 100% complementary to a target sequence described herein.
  • the sequence of the polynucleic acid molecule has 5 or less mismatches to a target sequence described herein. In some aspects, the sequence of the polynucleic acid molecule has 4 or less mismatches to a target sequence described herein. In some instances, the sequence of the polynucleic acid molecule may have 3 or less mismatches to a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule may have 2 or less mismatches to a target sequence described herein. In some cases, the sequence of the polynucleic acid molecule may have 1 or less mismatches to a target sequence described herein.
  • the specificity of the polynucleic acid molecule that hybridizes to a target sequence described herein is a 95%, 98%, 99%, 99.5% or 100% sequence complementarity of the polynucleic acid molecule to a target sequence.
  • the hybridization is a high stringent hybridization condition.
  • the polynucleic acid molecule has reduced off-target effect.
  • off-targef ’ or “off-target effects” refer to any instance in which a polynucleic acid polymer directed against a given target causes an unintended effect by interacting either directly or indirectly with another mRNA sequence, a DNA sequence or a cellular protein or other moiety.
  • an “off-target effect” occurs when there is a simultaneous degradation of other transcripts due to partial homology or complementarity between that other transcript and the sense and/or antisense strand of the polynucleic acid molecule.
  • the polynucleic acid molecule comprises natural or synthetic or artificial nucleotide analogues or bases. In some cases, the polynucleic acid molecule comprises combinations of DNA, RNA and/or nucleotide analogues. In some instances, the synthetic or artificial nucleotide analogues or bases comprise modifications at one or more of ribose moiety, phosphate moiety, nucleoside moiety, or a combination thereof.
  • nucleotide analogues or artificial nucleotide base comprise a nucleic acid with a modification at a 2’ hydroxyl group of the ribose moiety.
  • the modification includes an H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, wherein R is an alkyl moiety.
  • Exemplary alkyl moiety includes, but is not limited to, halogens, sulfurs, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols and oxygen. In some instances, the alkyl moiety further comprises a modification.
  • the modification comprises an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso, group, a nitrile group, a heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur containing group (e.g., sulfoxide, sulfone, sulfide, and disulfide).
  • the alkyl moiety further comprises a hetero substitution.
  • the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur.
  • the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.
  • the modification at the 2’ hydroxyl group is a 2’-O-methyl modification or a 2’ -O-m ethoxy ethyl (2’-0-M0E) modification.
  • the 2’-O-methyl modification adds a methyl group to the 2’ hydroxyl group of the ribose moiety whereas the 2’0- methoxyethyl modification adds a methoxyethyl group to the 2’ hydroxyl group of the ribose moiety.
  • Exemplary chemical structures of a 2’-O-methyl modification of an adenosine molecule and 2’O-methoxyethyl modification of a uridine are illustrated below.
  • the modification at the 2’ hydroxyl group is a 2’-O-aminopropyl modification in which an extended amine group comprising a propyl linker binds the amine group to the 2’ oxygen.
  • this modification neutralizes the phosphate derived overall negative charge of the oligonucleotide molecule by introducing one positive charge from the amine group per sugar and thereby improves cellular uptake properties due to its zwitterionic properties.
  • An exemplary chemical structure of a 2’-O-aminopropyl nucleoside phosphoramidite is illustrated below.
  • the modification at the 2’ hydroxyl group is a locked or bridged ribose modification (e.g., locked nucleic acid or LNA) in which the oxygen molecule bound at the 2’ carbon is linked to the 4’ carbon by a methylene group, thus forming a 2'-C,4'-C-oxy-methylene- linked bicyclic ribonucleotide monomer.
  • LNA locked nucleic acid
  • Exemplary representations of the chemical structure of LNA are illustrated below. The representation shown to the left highlights the chemical connectivities of an LNA monomer. The representation shown to the right highlights the locked 3'- endo ( 3 E) conformation of the furanose ring of an LNA monomer.
  • the modification at the 2’ hydroxyl group comprises ethylene nucleic acids (ENA) such as for example 2’-4’-ethylene-bridged nucleic acid, which locks the sugar conformation into a Cf-endo sugar puckering conformation.
  • ENA ethylene nucleic acids
  • the bridged nucleic acids class of modified nucleic acids that also comprises LNA. Exemplary chemical structures of the ENA and bridged nucleic acids are illustrated below.
  • additional modifications at the 2’ hydroxyl group include 2'-deoxy, 2’- deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O- N-methylacetamido (2'-0-NMA).
  • nucleotide analogues comprise modified bases such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6- methyladenine, 6-methylguanine, N, N, - dimethyladenine, 2-propyladenine, 2propylguanine, 2-aminoadenine, 1 -methylinosine, 3- methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides having a modification at the 5 position, 5- (2- amino) propyl uridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1- methyladenosine, 2-methyladenosine, 3 -methylcytidine, 6-methyluridine, 2- methylguanosine, 7- methylguanosine, 2, 2-dimethylguanosine, 5- methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides
  • Modified nucleotides also include those nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having sugars or analogs thereof that are not ribosyl.
  • the sugar moieties in some cases are or be based on, mannoses, arabinoses, glucopyranoses, galactopyranoses, 4'-thioribose, and other sugars, heterocycles, or carbocycles.
  • the term nucleotide also includes what are known in the art as universal bases.
  • universal bases include but are not limited to 3 -nitropyrrole, 5-nitroindole, or nebularine.
  • nucleotide analogues further comprise morpholinos (PMOs), peptide nucleic acids (PNAs or PPMOs), methylphosphonate nucleotides, thiolphosphonate nucleotides, 2’- fluoro N3-P5’-phosphoramidites, 1’, 5’- anhydrohexitol nucleic acids (HNAs), or a combination thereof.
  • Morpholino or phosphorodiamidate morpholino oligo (PMO) comprises synthetic molecules whose structure mimics natural nucleic acid structure by deviates from the normal sugar and phosphate structures.
  • the five-member ribose ring is substituted with a six member morpholino ring containing four carbons, one nitrogen and one oxygen.
  • the ribose monomers are linked by a phosphordiamidate group instead of a phosphate group.
  • the backbone alterations remove all positive and negative charges making morpholinos neutral molecules capable of crossing cellular membranes without the aid of cellular delivery agents such as those used by charged oligonucleotides.
  • peptide nucleic acid does not contain sugar ring or phosphate linkage and the bases are attached and appropriately spaced by oligoglycine-like molecules, therefore, eliminating a backbone charge.
  • modified internucleotide linkage include, but is not limited to, phosphorothioates, phosphorodithioates, methylphosphonates, 5'- alkylenephosphonates, 5'- methylphosphonate, 3 '-alkylene phosphonates, borontrifluoridates, borano phosphate esters and selenophosphates of 3'-5'linkage or 2'-5'linkage, phosphotriesters, thionoalkylphosphotriesters, hydrogen phosphonate linkages, alkyl phosphonates, alkylphosphonothioates, arylphosphonothioates, phosphoroselenoates, phosphorodiselenoates, phosphinates, phosphoramidates, 3'- alkylphosphoramidates, aminoalkylphosphoramidates, thionophosphoramidates,
  • the modification is a methyl or thiol modification such as methylphosphonate or thiolphosphonate modification.
  • exemplary thiolphosphonate nucleotide (left) and methylphosphonate nucleotide (right) are illustrated below.
  • a modified nucleotide includes, but is not limited to, 2’-fluoro N3-P5’- phosphoramidites illustrated as:
  • a modified nucleotide includes, but is not limited to, hexitol nucleic acid (or 1’, 5’ - anhydrohexitol nucleic acids (HNA)) illustrated as: Base
  • one or more modifications further optionally include modifications of the ribose moiety, phosphate backbone and the nucleoside, or modifications of the nucleotide analogues at the 3’ or the 5’ terminus.
  • the 3’ terminus optionally include a 3’ cationic group, or by inverting the nucleoside at the 3 ’-terminus with a 3 ’-3’ linkage.
  • the 3’-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3’ C5-aminoalkyl dT.
  • the 3’-terminus is optionally conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.
  • the 5’-terminus is conjugated with an aminoalkyl group, e.g., a 5’-O-alkylamino substituent.
  • the 5’-terminus is conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.
  • the polynucleic acid molecule comprises one or more of the artificial nucleotide analogues described herein. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogues described herein.
  • the artificial nucleotide analogues include 2’-O- methyl, 2 ’-O-m ethoxy ethyl (2’-0-M0E), 2’-O-aminopropyl, 2'-deoxy, 2’-deoxy-2'-fluoro, 2'-O- aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'- O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'- 0-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2’-fluoro N3-P5’-phosphoramidites, or a
  • the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of the artificial nucleotide analogues selected from 2’-O-methyl, 2’- O-methoxyethyl (2’-0-M0E), 2’-O-aminopropyl, 2'-deoxy, 2’-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-0-DMA0E), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphor,
  • the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2’-O-methyl modified nucleotides. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of 2’-O- methoxyethyl (2’-0-M0E) modified nucleotides. In some instances, the polynucleic acid molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 20, 25, or more of thiolphosphonate nucleotides.
  • the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modifications.
  • the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 1-20, 21-64, 65-108, 109-152, and 153-196.
  • the polynucleic acid molecule comprises at least about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22 or more modified nucleotides.
  • the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 1-20, 21-64, 65-108, 109-152, and 153-196.
  • the polynucleic acid molecule comprises at least one of: from about 5% to about 100% modification, from about 10% to about 100% modification, from about 20% to about 100% modification, from about 30% to about 100% modification, from about 40% to about 100% modification, from about 50% to about 100% modification, from about 60% to about 100% modification, from about 70% to about 100% modification, from about 80% to about 100% modification, and from about 90% to about 100% modification.
  • the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 1-20, 21-64, 65-108, 109-152, and 153-196.
  • polynucleic acid molecule comprise the artificial nucleotide analogues described herein. In some instances, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of the polynucleic acid molecule comprise the artificial nucleotide analogues described herein.
  • a polynucleic acid molecule of SEQ ID NOs: 1-20, 21-64, 65-108, 109-152, and 153-196 comprise the artificial nucleotide analogues described herein.
  • a polynucleic acid molecule of SEQ ID NOs: 1-20 comprise the artificial nucleotide analogues described herein.
  • about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of a polynucleic acid molecule of SEQ ID NOs: 21-64 comprise the artificial nucleotide analogues described herein.
  • a polynucleic acid molecule of SEQ ID NOs: 65-108 comprise the artificial nucleotide analogues described herein.
  • about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% of a polynucleic acid molecule of SEQ ID NOs: 109- 152 comprise the artificial nucleotide analogues described herein.
  • a polynucleic acid molecule of SEQ ID NOs: 153-196 comprise the artificial nucleotide analogues described herein.
  • the artificial nucleotide analogues include 2’-O-methyl, 2’ -O-m ethoxy ethyl (2’-0-M0E), 2’-O-aminopropyl, 2'-deoxy, 2 ’-deoxy -2' -fluoro, 2'- O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2’-fluoro N3-P5’-phosphoramidites,
  • one or more of the artificial nucleotide analogues described herein are resistant toward nucleases such as for example ribonuclease such as RNase H, deoxyribonuclease such as DNase, or exonuclease such as 5 ’-3’ exonuclease and 3 ’-5’ exonuclease when compared to natural polynucleic acid molecules.
  • nucleases such as for example ribonuclease such as RNase H, deoxyribonuclease such as DNase, or exonuclease such as 5 ’-3’ exonuclease and 3 ’-5’ exonuclease when compared to natural polynucleic acid molecules.
  • artificial nucleotide analogues comprising 2’-O-methyl, 2’ -O-m ethoxy ethyl (2’-0-M0E), 2’-O-aminopropyl, 2'-deoxy, 2 ’-deoxy -2' -fluoro, 2'- O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2’-fluoro N3-P5’-phosphoramidites,
  • 2’-O-methyl modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • 2’O-methoxyethyl (2’-0-M0E) modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’- 3’ exonuclease or 3’-5’ exonuclease resistance).
  • 2’-O-aminopropyl modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • 2'-deoxy modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • 2’-deoxy-2'-fluoro modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5 ’-3’ exonuclease or 3 ’-5’ exonuclease resistance).
  • 2'-O- aminopropyl (2'-O-AP) modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • 2'-O- dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • 2'- O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • 2’- O- dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • 2'-O-N-methylacetamido (2'-0-NMA) modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • LNA modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • ENA modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • HNA modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • Morpholinos may be nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • PNA modified polynucleic acid molecule is resistant to nucleases (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • methylphosphonate nucleotides modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5 ’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • thiolphosphonate nucleotides modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • polynucleic acid molecule comprising 2’-fluoro N3-P5’-phosphoramidites is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance).
  • the 5’ conjugates described herein inhibit 5 ’-3’ exonucleolytic cleavage.
  • the 3’ conjugates described herein inhibit 3’-5’ exonucleolytic cleavage.
  • one or more of the artificial nucleotide analogues described herein have increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • the one or more of the artificial nucleotide analogues comprising 2’-O-methyl, 2’- O-methoxyethyl (2’-0-M0E), 2’-O-aminopropyl, 2'-deoxy, 2’-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified, LNA, ENA, PNA, HNA, morpholin
  • 2’-O- methyl modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • 2’-O- methoxyethyl (2’-O-MOE) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • 2’-O-aminopropyl modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • 2'-deoxy modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • 2’- deoxy-2'-fluoro modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • 2'-O- aminopropyl (2'-O-AP) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • 2’-O- dimethylaminoethyloxy ethyl (2'-O- DMAEOE) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • 2'-O-N- methylacetamido (2'-0-NMA) modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • LNA modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • ENA modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • PNA modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • HNA modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • morpholino modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • methylphosphonate nucleotides modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • thiolphosphonate nucleotides modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • polynucleic acid molecule comprising 2’- fluoro N3-P5’-phosphoramidites has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule.
  • the increased affinity is illustrated with a lower Kd, a higher melt temperature (Tm), or a combination thereof.
  • a polynucleic acid molecule described herein is a chirally pure (or stereo pure) polynucleic acid molecule, or a polynucleic acid molecule comprising a single enantiomer.
  • the polynucleic acid molecule comprises L-nucleotide.
  • the polynucleic acid molecule comprises D-nucleotides.
  • a polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror enantiomer.
  • a polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture.
  • the polynucleic acid molecule is a polynucleic acid molecule described in: U.S. Patent Publication Nos: 2014/194610 and 2015/211006; and PCT Publication No.: WO2015107425.
  • a polynucleic acid molecule described herein is further modified to include an aptamer conjugating moiety.
  • the aptamer conjugating moiety is a DNA aptamer conjugating moiety.
  • the aptamer conjugating moiety is Alphamer (Centauri Therapeutics), which comprises an aptamer portion that recognizes a specific cell-surface target and a portion that presents a specific epitopes for attaching to circulating antibodies.
  • Alphamer Cosmetic Therapeutics
  • a polynucleic acid molecule described herein is further modified to include an aptamer conjugating moiety as described in: U.S. Patent Nos: 8,604,184, 8,591,910, and 7,850,975.
  • a polynucleic acid molecule described herein is modified to increase its stability.
  • the polynucleic acid molecule is RNA (e.g., siRNA), the polynucleic acid molecule is modified to increase its stability.
  • the polynucleic acid molecule is modified by one or more of the modifications described above to increase its stability.
  • the polynucleic acid molecule is modified at the 2’ hydroxyl position, such as by 2’-O-methyl, 2’-O-methoxyethyl (2’-0-M0E), 2’-O-aminopropyl, 2'-deoxy, 2’-deoxy-2'- fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O- dimethylaminopropyl (2'-O-DMAP), 2’-O- dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O- N-m ethylacetamido (2'-0-NMA) modification or by a locked or bridged ribose conformation (e.g., LNA or ENA).
  • a locked or bridged ribose conformation e.g., LNA or ENA
  • the polynucleic acid molecule is modified by 2’-O-methyl and/or 2’- O-methoxyethyl ribose. In some cases, the polynucleic acid molecule also includes morpholinos, PNAs, UNA, methylphosphonate nucleotides, thiolphosphonate nucleotides, and/or 2’-fluoro N3- P5’-phosphoramidites to increase its stability. In some instances, the polynucleic acid molecule is a chirally pure (or stereo pure) polynucleic acid molecule. In some instances, the chirally pure (or stereo pure) polynucleic acid molecule is modified to increase its stability. Suitable modifications to the RNA to increase stability for delivery will be apparent to the skilled person.
  • a polynucleic acid molecule describe herein has RNAi activity that modulates expression of RNA encoded by a gene described supra.
  • a polynucleic acid molecule describe herein is a double-stranded siRNA molecule that down- regulates expression of a gene, wherein one of the strands of the double-stranded siRNA molecule comprises a nucleotide sequence that is complementary to a nucleotide sequence of the gene or RNA encoded by the gene or a portion thereof, and wherein the second strand of the doublestranded siRNA molecule comprises a nucleotide sequence substantially similar to the nucleotide sequence of the gene or RNA encoded by the gene or a portion thereof.
  • a polynucleic acid molecule describe herein is a double-stranded siRNA molecule that down- regulates expression of a gene, wherein each strand of the siRNA molecule comprises about 15 to 25, 18 to 24, or 19 to about 23 nucleotides, and wherein each strand comprises at least about 14, 17, or 19 nucleotides that are complementary to the nucleotides of the other strand.
  • a polynucleic acid molecule describe herein is a double-stranded siRNA molecule that down- regulates expression of a gene, wherein each strand of the siRNA molecule comprises about 19 to about 23 nucleotides, and wherein each strand comprises at least about 19 nucleotides that are complementary to the nucleotides of the other strand.
  • the gene is SARS-COV or SARS-COV-2 associated genes or gene products.
  • a polynucleic acid molecule described herein is constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art.
  • a polynucleic acid molecule is chemically synthesized using naturally occurring nucleotides or variously modified nucleotides designed to increase the biological stability of the molecules or to increase the physical stability of the duplex formed between the polynucleic acid molecule and target nucleic acids.
  • Exemplary methods include those described in: U.S. Patent Nos. 5,142,047; 5,185,444; 5,889,136; 6,008,400; and 6,111,086; PCT Publication No. W02009099942; or European Publication No. 1579015.
  • Additional exemplary methods include those described in: Griffey et al., “2’-O-aminopropyl ribonucleotides: a zwitterionic modification that enhances the exonuclease resistance and biological activity of antisense oligonucleotides,” J. Med. Chem. 39(26):5100-5109 (1997)); Obika, et al. "Synthesis of 2'-O,4'-C-methyleneuridine and -cytidine. Novel bicyclic nucleosides having a fixed C3, -endo sugar puckering". Tetrahedron Letters 38 (50): 8735 (1997); Koizumi, M.
  • the polynucleic acid molecule is produced biologically using an expression vector into which a polynucleic acid molecule has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted polynucleic acid molecule will be of an antisense orientation to a target polynucleic acid molecule of interest).
  • a polynucleic acid molecule is conjugated to a binding moiety.
  • the binding moiety comprises amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, as well as analogs or derivatives of all of these classes of substances. Additional examples of binding moiety also include steroids, such as cholesterol, phospholipids, di-and triacylglycerols, fatty acids, hydrocarbons (e.g., saturated, unsaturated, or contains substitutions), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some instances, the binding moiety is an antibody or binding fragment thereof.
  • the polynucleic acid molecule is further conjugated to a polymer, and optionally a cellpenetrating or endosomolytic moiety.
  • the polynucleic acid molecule is conjugated to the binding moiety by a chemical ligation process. In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a native ligation. In some instances, the conjugation is as described in: Dawson, et al. “Synthesis of proteins by native chemical ligation,” Science 1994, 266, 776-779; Dawson, et al. “Modulation of Reactivity in Native Chemical Ligation through the Use of Thiol Additives,” J. Am. Chem. Soc. 1997, 119, 4325-4329; hackeng, et al. “Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology.,” Proc.
  • the polynucleic acid molecule is conjugated to the binding moiety either site-specifically or non- specifically via native ligation chemistry.
  • the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing a “traceless” coupling technology (Philochem).
  • the “traceless” coupling technology utilizes an N-terminal 1,2-aminothiol group on the binding moiety which is then conjugate with a polynucleic acid molecule containing an aldehyde group.
  • the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing an unnatural amino acid incorporated into the binding moiety.
  • the unnatural amino acid comprises /?-acetylphenylalanine (pAcPhe).
  • the keto group of pAcPhe is selectively coupled to an alkoxy-amine derivatived conjugating moiety to form an oxime bond, (see Axup et al., “Synthesis of site-specific antibodydrug conjugates using unnatural amino acids,” PNAS 109(40): 16101-16106 (2012)).
  • the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing an enzyme-catalyzed process.
  • the site-directed method utilizes SMARTagTM technology (Redwood).
  • the SMART agTM technology comprises generation of a formylglycine (FGly) residue from cysteine by formylglycine-generating enzyme (FGE) through an oxidation process under the presence of an aldehyde tag and the subsequent conjugation of FGly to an alkylhydraine-functionalized polynucleic acid molecule via hydrazino-Pictet-Spengler (HIPS) ligation,
  • FGE formylglycine-generating enzyme
  • HIPS hydrazino-Pictet-Spengler
  • the enzyme-catalyzed process comprises microbial transglutaminase (mTG).
  • mTG microbial transglutaminase
  • the polynucleic acid molecule is conjugated to the binding moiety utilizing a microbial transglutaminze catalyzed process.
  • mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine within the recognition sequence and a primary amine of a functionalized polynucleic acid molecule.
  • mTG is produced from Streptomyces mobarensis. (see Strop et al., “Location matters: site of conjugation modulates stability and pharmacokinetics of antibody drug conjugates,” Chemistry and Biology 20(2) 161-167 (2013))
  • the polynucleic acid molecule is conjugated to the binding moiety by a method as described in PCT Publication No. W02014/140317, which utilizes a sequence-specific transpeptidase.
  • the polynucleic acid molecule is conjugated to the binding moiety by a method as described in U.S. Patent Publication Nos. 2015/0105539 and 2015/0105540.
  • the binding moiety A is a polypeptide.
  • the polypeptide is an antibody or antigen fragment thereof.
  • the fragment is an antigen binding fragment.
  • the antibody or antigen binding fragment thereof comprises a humanized antibody or antigen binding fragment thereof, murine antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or antigen binding fragment thereof, monovalent Fab’, divalent Fab2, F(ab)'3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein (dsFv), single-domain antibody (sdAb), Ig NAR, camelid antibody or antigen binding fragment thereof, bispecific antibody or antigen biding fragment thereof, or a chemically modified derivative thereof.
  • A is an antibody or antigen binding fragment thereof.
  • A is a humanized antibody or antigen binding fragment thereof, murine antibody or antigen binding fragment thereof, chimeric antibody or antigen binding fragment thereof, monoclonal antibody or binding fragment thereof, monovalent Fab’, divalent Fab2, F(ab)'3 fragments, single-chain variable fragment (scFv), bis-scFv, (scFv)2, diabody, minibody, nanobody, triabody, tetrabody, disulfide stabilized Fv protein ("dsFv”), single-domain antibody (sdAb), Ig NAR, camelid antibody or antigen binding fragment thereof, bispecific antibody or biding fragment thereof, or a chemically modified derivative thereof.
  • A is a humanized antibody or antigen binding fragment thereof. In some instances, A is a murine antibody or antigen binding fragment thereof. In some instances, A is a chimeric antibody or antigen binding fragment thereof. In some instances, A is a monoclonal antibody or binding fragment thereof. In some instances, A is a monovalent Fab’. In some instances, A is a diavalent Fab2. In some instances, A is a single-chain variable fragment (scFv).
  • the binding moiety A is a bispecific antibody or antigen binding fragment thereof.
  • the bispecific antibody is a trifunctional antibody or a bispecific mini -antibody.
  • the bispecific antibody is a trifunctional antibody.
  • the trifunctional antibody is a full length monoclonal antibody comprising binding sites for two different antigens. Additional exemplary trifunctional antibodies include mAb 2 from F-star Biotechnology Ltd.
  • A is a bispecific trifunctional antibody.
  • the bispecific antibody is a bispecific mini-antibody.
  • the bispecific mini-antibody comprises divalent Fab2, F(ab)'3 fragments, bis-scFv, (scFv)2, diabody, minibody, triabody, tetrabody or a bi-specific T-cell engager (BiTE).
  • the bispecific T-cell engager is a fusion protein that contains two single-chain variable fragments (scFvs) in which the two scFvs target epitopes of two different antigens.
  • the binding moiety A is a bispecific mini-antibody.
  • A is a bispecific Fab2.
  • A is a bispecific F(ab)'3 fragment.
  • A is a bispecific bis-scFv.
  • A is a bispecific (scFv)2.
  • A is a bispecific diabody.
  • A is a bispecific minibody.
  • A is a bispecific triabody.
  • A is a bispecific tetrabody.
  • A is a bi-specific T-cell engager (BiTE).
  • A is a bispecific mini-antibody selected from: DART (dual-affinity re-targeting platform; MacroGenics), and domain antibodies (dAbs from Domantis/GSK).
  • the binding moiety A is a trispecific antibody.
  • the trispecific antibody comprises F(ab)'3 fragments or a triabody.
  • A is a trispecific F(ab)'3 fragment.
  • A is a triabody.
  • A is a trispecific antibody as described in Dimas, et al., “Development of a trispecific antibody designed to simultaneously and efficiently target three different antigens on tumor cells,” Mol. Pharmaceutics, 12(9): 3490-3501 (2015).
  • the binding moiety A is an antibody or antigen binding fragment thereof that recognizes a cell surface protein.
  • the cell surface protein comprises clusters of differentiation (CD) cell surface markers.
  • CD cell surface markers include, but are not limited to, CD1, CD2, CD3, CD4, CD5, CD6, CD7, CD8, CD9, CD10, CD1 la, CD1 lb, CDl lc, CDl ld, CDwl2, CD13, CD14, CD15, CD15s, CD16, CDwl7, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD45RO, CD45RA, CD45RB, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d
  • the binding moiety A is conjugated according to Formula (I), Formula (II), or Formula (III) to a polynucleic acid molecule (B), and optionally to a polymer (C), and optionally a cell-penetrating or endosomolytic moiety (D) as described herein.
  • the polynucleic acid molecule comprises a sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to a sequence selected from SEQ ID NOs: 1-20, 21-108, and 109-196.
  • the polymer C comprises polyalkylen oxide (e.g., polyethylene glycol).
  • the cell-penetrating or endosomolytic moiety D comprises INF7 or melittin, or their respective derivatives.
  • the binding moiety A is conjugated to a polynucleic acid molecule (B) non-specifically. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) via a lysine residue or a cysteine residue, in a non-site specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) via a lysine residue in a non-site specific manner. In some cases, the binding moiety A is conjugated to a polynucleic acid molecule (B) via a cysteine residue in a non-site specific manner.
  • the binding moiety A is conjugated to a polynucleic acid molecule (B) in a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) through a lysine residue, a cysteine residue, at the 5 ’-terminus, at the 3 ’-terminus, an unnatural amino acid, or an enzyme-modified or enzyme-catalyzed residue, via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) through a lysine residue via a site-specific manner.
  • the binding moiety A is conjugated to a polynucleic acid molecule (B) through a cysteine residue via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) at the 5’- terminus via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) at the 3 ’-terminus via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) through an unnatural amino acid via a site-specific manner. In some instances, the binding moiety A is conjugated to a polynucleic acid molecule (B) through an enzyme-modified or enzyme-catalyzed residue via a site-specific manner.
  • one or more polynucleic acid molecule (B) is conjugated to a binding moiety A.
  • about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or more polynucleic acid molecules are conjugated to one binding moiety A.
  • about 1 polynucleic acid molecule is conjugated to one binding moiety A.
  • about 2 polynucleic acid molecules are conjugated to one binding moiety A.
  • about 3 polynucleic acid molecules are conjugated to one binding moiety A.
  • about 4 polynucleic acid molecules are conjugated to one binding moiety A.
  • about 5 polynucleic acid molecules are conjugated to one binding moiety A.
  • about 6 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 7 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 8 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 9 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 10 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 11 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 12 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 13 polynucleic acid molecules are conjugated to one binding moiety A.
  • polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 15 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 16 polynucleic acid molecules are conjugated to one binding moiety A. In some cases, the one or more polynucleic acid molecules are the same. In other cases, the one or more polynucleic acid molecules are different.
  • the number of polynucleic acid molecule (B) conjugated to a binding moiety A forms a ratio.
  • the ratio is referred to as a DAR (drug-to-antibody) ratio, in which the drug as referred to herein is the polynucleic acid molecule (B).
  • the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or greater.
  • the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1 or greater.
  • the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 2 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 3 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 4 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 5 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 6 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 7 or greater.
  • the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 8 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 9 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 10 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 11 or greater. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 12 or greater.
  • the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 2. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 3. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 4.
  • the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 5. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 6. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 7. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 8. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 9. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 10.
  • the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 11. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 12. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 13. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 14. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 15. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 16.
  • the DAR ratio of the polynucleic acid molecule (B) to binding moiety is the DAR ratio of the polynucleic acid molecule (B) to binding moiety
  • the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 1. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 2. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 4. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 6. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 8. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is 12.
  • an antibody or its binding fragment is further modified using conventional techniques known in the art, for example, by using amino acid deletion, insertion, substitution, addition, and/or by recombination and/or any other modification (e.g. posttranslational and chemical modifications, such as glycosylation and phosphorylation) known in the art either alone or in combination.
  • the modification further comprises a modification for modulating interaction with Fc receptors.
  • the one or more modifications include those described in, for example, International Publication No. WO97/34631, which discloses amino acid residues involved in the interaction between the Fc domain and the FcRn receptor. Methods for introducing such modifications in the nucleic acid sequence underlying the amino acid sequence of an antibody or its binding fragment is well known to the person skilled in the art.
  • an antibody binding fragment further encompasses its derivatives and includes polypeptide sequences containing at least one CDR.
  • single-chain as used herein means that the first and second domains of a bi-specific single chain construct are covalently linked, preferably in the form of a colinear amino acid sequence encodable by a single nucleic acid molecule.
  • a bispecific single chain antibody construct relates to a construct comprising two antibody derived binding domains.
  • bi-specific single chain antibody construct is tandem bi-scFv or diabody.
  • a scFv contains a VH and VL domain connected by a linker peptide.
  • linkers are of a length and sequence sufficient to ensure that each of the first and second domains can, independently from one another, retain their differential binding specificities.
  • binding to or interacting with as used herein defines a binding/interaction of at least two antigen-interaction-sites with each other.
  • antigen-interaction-site defines a motif of a polypeptide that shows the capacity of specific interaction with a specific antigen or a specific group of antigens.
  • the binding/interaction is also understood to define a specific recognition.
  • specific recognition refers to that the antibody or its binding fragment is capable of specifically interacting with and/or binding to at least two amino acids of each of a target molecule.
  • specific recognition relates to the specificity of the antibody molecule, or to its ability to discriminate between the specific regions of a target molecule.
  • the specific interaction of the antigen-interaction-site with its specific antigen results in an initiation of a signal, e.g. due to the induction of a change of the conformation of the antigen, an oligomerization of the antigen, etc.
  • the binding is exemplified by the specificity of a "key -lock-principle".
  • specific motifs in the amino acid sequence of the antigen-interaction-site and the antigen bind to each other as a result of their primary, secondary or tertiary structure as well as the result of secondary modifications of said structure.
  • the specific interaction of the antigen-interaction-site with its specific antigen results as well in a simple binding of the site to the antigen.
  • specific interaction further refers to a reduced cross-reactivity of the antibody or its binding fragment or a reduced off-target effect.
  • the antibody or its binding fragment that bind to the polypeptide/protein of interest but do not or do not essentially bind to any of the other polypeptides are considered as specific for the polypeptide/protein of interest.
  • Examples for the specific interaction of an antigen-interaction-site with a specific antigen comprise the specificity of a ligand for its receptor, for example, the interaction of an antigenic determinant (epitope) with the antigenic binding site of an antibody.
  • the antibody or binding fragment thereof is an anti-TfR.1 antibody, an anti-ACE2 antibody, an anti-TMPRSS2 antibody, or antigen binding fragment thereof.
  • the binding moiety is a plasma protein.
  • the plasma protein comprises albumin or transferrin.
  • the binding moiety A is albumin.
  • the binding moiety A is transferrin.
  • transferrin is conjugated by one or more of a conjugation chemistry described herein to a polynucleic acid molecule.
  • transferrin is conjugated by native ligation chemistry to a polynucleic acid molecule.
  • transferrin is conjugated by lysine conjugation to a polynucleic acid molecule.
  • the binding moiety is a steroid.
  • steroids include cholesterol, phospholipids, di-and triacylglycerols, fatty acids, hydrocarbons that are saturated, unsaturated, comprise substitutions, or combinations thereof.
  • the steroid is cholesterol.
  • the binding moiety is cholesterol.
  • cholesterol is conjugated by one or more of a conjugation chemistry described herein to a polynucleic acid molecule.
  • cholesterol is conjugated by native ligation chemistry to a polynucleic acid molecule.
  • cholesterol is conjugated by lysine conjugation to a polynucleic acid molecule.
  • the binding moiety is a polymer, including but not limited to poly nucleic acid molecule aptamers that bind to specific surface markers on cells.
  • the binding moiety is a polynucleic acid that does not hybridize to a target gene or mRNA, but instead is capable of selectively binding to a cell surface marker similarly to an antibody binding to its specific epitope of a cell surface marker.
  • the binding moiety is a peptide.
  • the peptide comprises between about 1 and about 3 kDa. In some cases, the peptide comprises between about 1.2 and about 2.8 kDa, about 1.5 and about 2.5 kDa, or about 1.5 and about 2 kDa.
  • the peptide is a bicyclic peptide. In some cases, the bicyclic peptide is a constrained bicyclic peptide. In some instances, the binding moiety is a bicyclic peptide (e.g., bicycles from Bicycle Therapeutics).
  • the binding moiety is a small molecule.
  • the small molecule is an antibody-recruiting small molecule.
  • the antibody-recruiting small molecule comprises a target-binding terminus and an antibody-binding terminus, in which the target-binding terminus is capable of recognizing and interacting with a cell surface receptor.
  • the target-binding terminus comprising a glutamate urea compound enables interaction with PSMA, thereby, enhances an antibody interaction with a cell (e.g., a cancerous cell) that expresses PSMA.
  • a binding moiety is a small molecule described in Zhang et al., “A remote arene-binding site on prostate specific membrane antigen revealed by antibody-recruiting small molecules,” J Am Chem Soc. 132(36): 12711-12716 (2010); or McEnaney, et al., “Antibody-recruiting molecules: an emerging paradigm for engaging immune function in treating human disease,” ACS Chem Biol. 7(7): 1139-1151 (2012). Production of Antibodies or Binding Fragments Thereof
  • polypeptides described herein are produced using any method known in the art to be useful for the synthesis of polypeptides (e.g., antibodies), in particular, by chemical synthesis or by recombinant expression, and are preferably produced by recombinant expression techniques.
  • an antibody or its binding fragment thereof is expressed recombinantly, and the nucleic acid encoding the antibody or its antigen binding fragment is assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242), which involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • chemically synthesized oligonucleotides e.g., as described in Kutmeier et al., 1994, BioTechniques 17:242
  • a nucleic acid molecule encoding an antibody is optionally generated from a suitable source (e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence.
  • a suitable source e.g., an antibody cDNA library, or cDNA library generated from any tissue or cells expressing the immunoglobulin
  • an antibody or its binding is optionally generated by immunizing an animal, such as a rabbit, to generate polyclonal antibodies or, more preferably, by generating monoclonal antibodies, e.g., as described by Kohler and Milstein (1975, Nature 256:495-497) or, as described by Kozbor et al. (1983, Immunology Today 4:72) or Cole et al. (1985 in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • a clone encoding at least the Fab portion of the antibody is optionally obtained by screening Fab expression libraries (e.g., as described in Huse et al., 1989, Science 246: 1275-1281) for clones of Fab fragments that bind the specific antigen or by screening antibody libraries (See, e.g., Clackson et al., 1991, Nature 352:624; Hane et al., 1997 Proc. Natl. Acad. Sci. USA 94:4937).
  • chimeric antibodies techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Natl. Acad. Sci. 81 :851-855; Neuberger et al., 1984, Nature 312:604- 608; Takeda et al., 1985, Nature 314:452-454) by splicing genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity are used.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
  • an expression vector comprising the nucleotide sequence of an antibody or the nucleotide sequence of an antibody is transferred to a host cell by conventional techniques (e.g., electroporation, liposomal transfection, and calcium phosphate precipitation), and the transfected cells are then cultured by conventional techniques to produce the antibody.
  • the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.
  • host-expression vector systems is utilized to express an antibody or its binding fragment described herein.
  • host-expression systems represent vehicles by which the coding sequences of the antibody is produced and subsequently purified, but also represent cells that are, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or its binding fragment in situ.
  • host-expression systems represent vehicles by which the coding sequences of the antibody is produced and subsequently purified, but also represent cells that are, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody or its binding fragment in situ.
  • microorganisms such as bacteria (e.g., E. coli and B.
  • siibtiHs transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing an antibody or its binding fragment coding sequences; yeast (e.g., Saccharomyces Pichia) transformed with recombinant yeast expression vectors containing an antibody or its binding fragment coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing an antibody or its binding fragment coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus (CaMV) and tobacco mosaic virus (TMV)) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing an antibody or its binding fragment coding sequences; or mammalian cell systems (e.g., COS, CHO, BH, 293, 293T, 3T3 cells) harboring recombinant expression constructs containing promoter
  • any method known in the art for purification of an antibody is used, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • a polymer moiety C is further conjugated to a polynucleic acid molecule described herein, a binding moiety described herein, or in combinations thereof. In some instances, a polymer moiety C is conjugated a polynucleic acid molecule. In some cases, a polymer moiety C is conjugated to a binding moiety. In other cases, a polymer moiety C is conjugated to a polynucleic acid molecule-binding moiety molecule. In additional cases, a polymer moiety C is conjugated, as illustrated in Figure 1, and as discussed under the Therapeutic Molecule Platform section.
  • the polymer moiety C is a natural or synthetic polymer, consisting of long chains of branched or unbranched monomers, and/or cross-linked network of monomers in two or three dimensions.
  • the polymer moiety C includes a polysaccharide, lignin, rubber, or polyalkylen oxide (e.g., polyethylene glycol).
  • the at least one polymer moiety C includes, but is not limited to, alpha-, omega-dihydroxylpolyethyleneglycol, biodegradable lactone-based polymer, e.g.
  • polyacrylic acid polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefin, polyamide, polycyanoacrylate, polyimide, polyethylenterephthalat (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethane as well as mixtures thereof.
  • a mixture refers to the use of different polymers within the same compound as well as in reference to block copolymers.
  • block copolymers are polymers wherein at least one section of a polymer is build up from monomers of another polymer.
  • the polymer moiety C comprises polyalkylene oxide.
  • the polymer moiety C comprises PEG.
  • the polymer moiety C comprises polyethylene imide (PEI) or hydroxy ethyl starch (HES).
  • C is a PEG moiety.
  • the PEG moiety is conjugated at the 5’ terminus of the polynucleic acid molecule while the binding moiety is conjugated at the 3’ terminus of the polynucleic acid molecule.
  • the PEG moiety is conjugated at the 3’ terminus of the polynucleic acid molecule while the binding moiety is conjugated at the 5’ terminus of the polynucleic acid molecule.
  • the PEG moiety is conjugated to an internal site of the polynucleic acid molecule.
  • the PEG moiety, the binding moiety, or a combination thereof are conjugated to an internal site of the polynucleic acid molecule.
  • the conjugation is a direct conjugation. In some instances, the conjugation is via native ligation.
  • the polyalkylene oxide e.g., PEG
  • the polydispers material comprises disperse distribution of different molecular weight of the material, characterized by mean weight (weight average) size and dispersity.
  • the monodisperse PEG comprises one size of molecules.
  • C is poly- or monodispersed polyalkylene oxide (e.g., PEG) and the indicated molecular weight represents an average of the molecular weight of the polyalkylene oxide, e.g., PEG, molecules.
  • the molecular weight of the polyalkylene oxide is about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.
  • PEG polyalkylene oxide
  • C is polyalkylene oxide (e.g., PEG) and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da.
  • PEG polyalkylene oxide
  • C is PEG and has a molecular weight of about 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, 2500, 2600, 2700, 2800, 2900, 3000, 3250, 3350, 3500, 3750, 4000, 4250, 4500, 4600, 4750, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 10,000, 12,000, 20,000, 35,000, 40,000, 50,000, 60,000, or 100,000 Da. In some instances, the molecular weight of C is about 200 Da.
  • the molecular weight of C is about 300 Da. In some instances, the molecular weight of C is about 400 Da. In some instances, the molecular weight of C is about 500 Da. In some instances, the molecular weight of C is about 600 Da. In some instances, the molecular weight of C is about 700 Da. In some instances, the molecular weight of C is about 800 Da. In some instances, the molecular weight of C is about 900 Da. In some instances, the molecular weight of C is about 1000 Da. In some instances, the molecular weight of C is about 1100 Da. In some instances, the molecular weight of C is about 1200 Da. In some instances, the molecular weight of C is about 1300 Da.
  • the molecular weight of C is about 1400 Da. In some instances, the molecular weight of C is about 1450 Da. In some instances, the molecular weight of C is about 1500 Da. In some instances, the molecular weight of C is about 1600 Da. In some instances, the molecular weight of C is about 1700 Da. In some instances, the molecular weight of C is about 1800 Da. In some instances, the molecular weight of C is about 1900 Da. In some instances, the molecular weight of C is about 2000 Da. In some instances, the molecular weight of C is about 2100 Da. In some instances, the molecular weight of C is about 2200 Da. In some instances, the molecular weight of C is about 2300 Da.
  • the molecular weight of C is about 2400 Da. In some instances, the molecular weight of C is about 2500 Da. In some instances, the molecular weight of C is about 2600 Da. In some instances, the molecular weight of C is about 2700 Da. In some instances, the molecular weight of C is about 2800 Da. In some instances, the molecular weight of C is about 2900 Da. In some instances, the molecular weight of C is about 3000 Da. In some instances, the molecular weight of C is about 3250 Da. In some instances, the molecular weight of C is about 3350 Da. In some instances, the molecular weight of C is about 3500 Da. In some instances, the molecular weight of C is about 3750 Da.
  • the molecular weight of C is about 4000 Da. In some instances, the molecular weight of C is about 4250 Da. In some instances, the molecular weight of C is about 4500 Da. In some instances, the molecular weight of C is about 4600 Da. In some instances, the molecular weight of C is about 4750 Da. In some instances, the molecular weight of C is about 5000 Da. In some instances, the molecular weight of C is about 5500 Da. In some instances, the molecular weight of C is about 6000 Da. In some instances, the molecular weight of C is about 6500 Da. In some instances, the molecular weight of C is about 7000 Da. In some instances, the molecular weight of C is about 7500 Da.
  • the molecular weight of C is about 8000 Da. In some instances, the molecular weight of C is about 10,000 Da. In some instances, the molecular weight of C is about 12,000 Da. In some instances, the molecular weight of C is about 20,000 Da. In some instances, the molecular weight of C is about 35,000 Da. In some instances, the molecular weight of C is about 40,000 Da. In some instances, the molecular weight of C is about 50,000 Da. In some instances, the molecular weight of C is about 60,000 Da. In some instances, the molecular weight of C is about 100,000 Da. [00180] In some aspects, the polymer moiety C comprises a cationic mucic acid-based polymer (cMAP). In some instances, cMPA comprises one or more subunit of at least one repeating subunit, and the subunit structure is represented as Formula (IV):
  • cMAP is further conjugated to a PEG moiety, generating a cMAP-PEG copolymer, an mPEG-cMAP-PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer.
  • the PEG moiety is in a range of from about 500 Da to about 50,000 Da.
  • the PEG moiety is in a range of from about 500 Da to about 1000 Da, greater than 1000 Da to about 5000 Da, greater than 5000 Da to about 10,000 Da, greater than 10,000 to about 25,000 Da, greater than 25,000 Da to about 50,000 Da, or any combination of two or more of these ranges.
  • the polymer moiety C is cMAP-PEG copolymer, an mPEG-cMAP- PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer.
  • the polymer moiety C is cMAP-PEG copolymer.
  • the polymer moiety C is an mPEG-cMAP- PEGm triblock polymer.
  • the polymer moiety C is a cMAP-PEG-cMAP triblock polymer.
  • a molecule of Formula (I), Formula (II), or Formula (III) further comprises an additional conjugating moiety.
  • the additional conjugating moiety is a cell -penetrating or endosomolytic moiety.
  • the cell-penetrating or endosomolytic moiety is a cellular compartmental release component, such as a compound capable of releasing from any of the cellular compartments known in the art, such as the endosome, lysosome, endoplasmic reticulum (ER), Golgi apparatus, microtubule, peroxisome, or other vesicular bodies with the cell.
  • the cell-penetrating or endosomolytic moiety comprises a cellpenetrating or endosomolytic polypeptide, a cell-penetrating or endosomolytic polymer, a cellpenetrating or endosomolytic lipid, or a cell-penetrating or endosomolytic small molecule.
  • the cell-penetrating or endosomolytic moiety comprises a cell-penetrating or endosomolytic polypeptide.
  • the cell-penetrating or endosomolytic moiety comprises a cellpenetrating or endosomolytic polymer.
  • a molecule of Formula (I), Formula (II), or Formula (III) is further conjugated with a cell-penetrating or endosomolytic polypeptide.
  • the cellpenetrating or endosomolytic polypeptide is a pH-dependent membrane active peptide.
  • the cell-penetrating or endosomolytic polypeptide is an amphipathic polypeptide.
  • the cell-penetrating or endosomolytic polypeptide is a peptidomimetic.
  • the cell-penetrating or endosomolytic polypeptide comprises INF, melittin, meucin, or their respective derivatives thereof.
  • the cell-penetrating or endosomolytic polypeptide comprises INF or its derivatives thereof. In other cases, the cell-penetrating or endosomolytic polypeptide comprises melittin or its derivatives thereof. In additional cases, the cell-penetrating or endosomolytic polypeptide comprises meucin or its derivatives thereof.
  • a molecule of Formula (I), Formula (II), or Formula (III) is further conjugated with a cell-penetrating or endosomolytic polymer.
  • a cell-penetrating or endosomolytic polymer comprises a linear, a branched network, a star, a comb, or a ladder type of polymer.
  • a cell-penetrating or endosomolytic polymer is a homopolymer or a copolymer comprising two or more different types of monomers.
  • a cell-penetrating or endosomolytic polymer is a polycation polymer.
  • a cell-penetrating or endosomolytic polymer is a polyanion polymer.
  • a polycation polymer comprises monomer units that are charge positive, charge neutral, or charge negative, with a net charge being positive.
  • a polycation polymer comprises a non-polymeric molecule that contains two or more positive charges.
  • Exemplary cationic polymers include, but are not limited to, poly(L-lysine) (PLL), poly(L-arginine) (PLA), polyethyleneimine (PEI), poly[a-(4-aminobutyl)-L-glycolic acid] (PAGA), 2-(dimethylamino)ethyl methacrylate (DMAEMA), or N,N-Diethylaminoethyl. Methacrylate (DEAEMA).
  • a polyanion polymer comprises monomer units that are charge positive, charge neutral, or charge negative, with a net charge being negative.
  • a polyanion polymer comprises a non-polymeric molecule that contains two or more negative charges.
  • Exemplary anionic polymers include p(alkylacrylates) (e.g., poly(propyl acrylic acid) (PPAA)) or poly(N-isopropylacrylamide) (NIP AM).
  • Additional examples include PP75, a L-phenylalanine- poly(L-lysine isophthalamide) polymer described in Khormaee, et al., “Edosomolytic anionic polymer for the cytoplasmic delivery of siRNAs in localized in vivo applications,” Advanced Functional Materials 23: 565-574 (2013).
  • a cell-penetrating or endosomolytic polymer described herein is a pH- responsive endosomolytic polymer.
  • a pH-responsive polymer comprises a polymer that increases in size (swell) or collapses depending on the pH of the environment.
  • Polyacrylic acid and chitosan are examples of pH-responsive polymers.
  • a cell-penetrating or endosomolytic moiety described herein is a membrane-disruptive polymer.
  • the membrane-disruptive polymer comprises a cationic polymer, a neutral or hydrophobic polymer, or an anionic polymer.
  • the membrane-disruptive polymer is a hydrophilic polymer.
  • a cell-penetrating or endosomolytic moiety described herein is a pH- responsive membrane-disruptive polymer.
  • Exemplary pH-responsive membrane-disruptive polymers include p(alkylacrylic acids), poly(N-isopropylacrylamide) (NIP AM) copolymers, succinylated p(glycidols), and p(P-malic acid) polymers.
  • p(alkylacrylic acids) include poly(propylacrylic acid) (polyP AA), poly(methacrylic acid) (PMAA), poly(ethylacrylic acid) (PEAA), and poly(propyl acrylic acid) (PPAA).
  • a p(alkylacrylic acid) include a p(alkylacrylic acid) described in Jones, et al., Biochemistry Journal 372: 65-75 (2003).
  • a pH-responsive membrane-disruptive polymer comprises p(butyl acrylate-co-methacrylic acid), (see Bulmus, et al., Journal of Controlled Release 93: 105-120 (2003); and Yessine, et al., Biochimica et Biophysica Acta 1613: 28-38 (2003))
  • a pH-responsive membrane-disruptive polymer comprises p(styrene-alt- maleic anhydride), (see Henry, et al., Biomacromolecules 7: 2407-2414 (2006))
  • a pH-responsive membrane-disruptive polymer comprises pyridyldisulfide acrylate (PDSA) polymers such as poly(MAA-co-PDSA), poly(EAA-co-PDSA), poly(PAA-co-PDSA), poly(MAA-co-B A-co-PDS A), poly(EAA-co-BA-co-PDSA), or poly(PAA- co-BA-co-PDSA) polymers, see El-Sayed, et al., “Rational design of composition and activity correlations for pH-responsive and glutathione-reactive polymer therapeutics,” Journal of Controlled Release 104: 417-427 (2005); or Flanary et al., “Antigen delivery with poly(propylacrylic acid) conjugation enhanced MHC-1 presentation and T-cell activation,” Bioconjugate Chem. 20: 241-248 (2009))
  • PDSA pyridyldisulfide acrylate
  • a pH-responsive membrane-disruptive polymer comprises a lytic polymer comprising the base structure of:
  • a cell-penetrating or endosomolytic moiety described herein is further conjugated to an additional conjugate, e.g., a polymer (e.g., PEG), or a modified polymer (e.g., cholesterol-modified polymer).
  • an additional conjugate e.g., a polymer (e.g., PEG), or a modified polymer (e.g., cholesterol-modified polymer).
  • the additional conjugate comprises a detergent (e.g., Triton X-100).
  • a cell-penetrating or endosomolytic moiety described herein comprises a polymer (e.g., a poly(amidoamine)) conjugated with a detergent (e.g., Triton X-100).
  • a cell-penetrating or endosomolytic moiety described herein comprises poly(amidoamine)-Triton X- 100 conjugate (Duncan, et al., “A polymer-Triton X-100 conjugate capable of pH-dependent red blood cell lysis: a model system illustrating the possibility of drug delivery within acidic intracellular compartments,” Journal of Drug Targeting . 341-347 (1994)).
  • the cell-penetrating or endosomolytic moiety is a lipid (e.g., a fusogenic lipid).
  • a molecule of Formula (I), Formula (II), or Formula (III) is further conjugated with a cell-penetrating or endosomolytic lipid (e.g., fusogenic lipid).
  • Exemplary fusogenic lipids include l,2-dileoyl-sn-3 -phosphoethanolamine (DOPE), phosphatidylethanolamine (POPE), palmitoyloleoylphosphatidylcholine (POPC), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- tetraen-19-ol (Di-Lin), N-methyl(2,2-di((9Z,12Z)-octadeca-9,12-dienyl)-l,3-dioxolan-4- yl)m ethanamine (DLin-k-DMA) and N-methyl-2-(2,2-di((9Z,12Z)-octadeca-9, 12-dienyl)- 1,3- dioxolan-4-yl)ethanamine (XTC).
  • DOPE l,2-dileoyl-sn-3 -phosphoethanolamine
  • POPE
  • a cell-penetrating or endosomolytic moiety is a lipid (e.g., a fusogenic lipid) described in PCT Publication No. WO09/126,933.
  • the cell-penetrating or endosomolytic moiety is a small molecule.
  • a molecule of Formula (I), Formula (II), or Formula (III) is further conjugated with a cell-penetrating or endosomolytic small molecule.
  • Exemplary small molecules suitable as endosomolytic moieties include, but are not limited to, quinine, chloroquine, hydroxychloroquines, amodiaquins (camoquines), amopyroquines, primaquines, mefloquines, nivaquines, halofantrines, quinone imines, or a combination thereof.
  • quinoline endosomolytic moieties include, but are not limited to, 7-chloro-4-(4-diethylamino-l-methylbutyl-amino)quinoline (chloroquine); 7-chloro-4-(4-ethyl-(2-hydroxyethyl)-amino-l-methylbutyl-amino)quinoline (hydroxychloroquine); 7-fluoro-4-(4-diethylamino-l-methylbutyl-amino)quinoline; 4-(4- di ethylamino- 1 -methylbutylamino) quinoline; 7-hydroxy-4-(4-diethyl-amino-l- methylbutylamino)quinoline; 7-chloro-4-(4-diethylamino-l-butylamino)quinoline (desmethylchloroquine); 7-fluoro-4-(4-diethylamino-l-butyla
  • a cell-penetrating or endosomolytic moiety is a small molecule described in Naisbitt et al (1997, J Pharmacol Exp Therapy 280:884-893) and in U.S. Patent No. 5,736,557.
  • a linker described herein is a cleavable linker or a non-cleavable linker.
  • the linker is a cleavable linker.
  • the linker is an acid cleavable linker.
  • the linker is a non-cleavable linker.
  • the linker includes a Ci-Ce alkyl group (e.g., a Cs, C4, C3, C2, or Ci alkyl group).
  • the linker includes homobifunctional cross linkers, heterobifunctional cross linkers, and the like.
  • the linker comprises a homobifunctional linker.
  • Exemplary homobifuctional linkers include, but are not limited to, Lomant's reagent dithiobis (succinimidylpropionate) DSP, 3'3'-dithiobis(sulfosuccinimidyl proprionate (DTSSP), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo DST), ethylene glycobis(succinimidylsuccinate) (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), l,4-di
  • DFDNPS bis-[
  • BASED bis-[
  • formaldehyde glutaraldehyde
  • 1,4-butanediol diglycidyl ether adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3 '-dimethylbenzidine, benzidine, a,a'-p-diaminodiphenyl, diiodo-p-xylene sulfonic acid, N,N'-ethylene- bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).
  • the linker comprises a heterobifunctional linker.
  • exemplary heterobifunctional linker include, but are not limited to, amine-reactive and sulfhydryl cross-linkers such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2- pyridyldithio)propi onate (LC-sPDP), water-soluble-long-chain N-succinimidyl 3-(2-pyridyldithio) propionate (sulfo-LC-sPDP), succinimidyloxycarbonyl-a-methyl-a-(2-pyridyldithio)toluene (sMPT), sulfosuccinimidyl-6-[a-methyl-a-(2-pyridyldithio)toluamido]hexanoate (sulfo
  • the linker comprises a reactive functional group.
  • the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on a binding moiety.
  • electrophilic groups include carbonyl groups — such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or acid anhydride.
  • the reactive functional group is aldehyde.
  • nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide.
  • the linker comprises a maleimide group.
  • the maleimide group is also referred to as a maleimide spacer. In some instances, the maleimide group further encompasses a caproic acid, forming maleimidocaproyl (me). In some cases, the linker comprises maleimidocaproyl (me). In some cases, the linker is maleimidocaproyl (me).
  • the maleimide group comprises a maleimidomethyl group, such as succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sMCC) or sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sulfo-sMCC) described above.
  • a maleimidomethyl group such as succinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sMCC) or sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane-l -carboxylate (sulfo-sMCC) described above.
  • the maleimide group is a self-stabilizing maleimide.
  • the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) to incorporate a basic amino group adjacent to the maleimide to provide intramolecular catalysis of tiosuccinimide ring hydrolysis, thereby eliminating maleimide from undergoing an elimination reaction through a retro- Michael reaction.
  • the self-stabilizing maleimide is a maleimide group described in Lyon, et al., “Self-hydrolyzing maleimides improve the stability and pharmacological properties of antibody-drug conjugates,” Nat. Biotechnol. 32(10): 1059-1062 (2014).
  • the linker comprises a self-stabilizing maleimide.
  • the linker is a self-stabilizing maleimide.
  • the linker comprises a peptide moiety.
  • the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues.
  • the peptide moiety is a cleavable peptide moiety (e.g., either enzymatically or chemically).
  • the peptide moiety is a non-cleavable peptide moiety.
  • the peptide moiety comprises Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala-Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu- Ala-Leu, or Gly-Phe-Leu-Gly.
  • Val-Cit valine-citrulline
  • the linker comprises a peptide moiety such as: Val-Cit (valine-citrulline), Gly-Gly-Phe-Gly, Phe-Lys, Val-Lys, Gly-Phe-Lys, Phe-Phe-Lys, Ala- Lys, Val-Arg, Phe-Cit, Phe-Arg, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe- Leu-Gly.
  • the linker comprises Val-Cit.
  • the linker is Val-Cit.
  • the linker comprises a benzoic acid group, or its derivatives thereof.
  • the benzoic acid group or its derivatives thereof comprise paraaminobenzoic acid (PABA).
  • the benzoic acid group or its derivatives thereof comprise gamma- aminobutyric acid (GABA).
  • the linker comprises one or more of a maleimide group, a peptide moiety, and/or a benzoic acid group, in any combination. In some aspects, the linker comprises a combination of a maleimide group, a peptide moiety, and/or a benzoic acid group. In some instances, the maleimide group is maleimidocaproyl (me). In some instances, the peptide group is val-cit. In some instances, the benzoic acid group is PABA. In some instances, the linker comprises a mc-val-cit group. In some cases, the linker comprises a val-cit-PABA group. In additional cases, the linker comprises a mc-val-cit-PABA group.
  • the linker is a self-immolative linker or a self-elimination linker. In some cases, the linker is a self-immolative linker. In other cases, the linker is a self-elimination linker (e.g., a cyclization self-elimination linker). In some instances, the linker comprises a linker described in U.S. Patent No. 9,089,614 or PCT Publication No. WO2015038426.
  • the linker is a dendritic type linker.
  • the dendritic type linker comprises a branching, multifunctional linker moiety.
  • the dendritic type linker is used to increase the molar ratio of polynucleotide B to the binding moiety A.
  • the dendritic type linker comprises PAMAM dendrimers.
  • the linker is a traceless linker or a linker in which after cleavage does not leave behind a linker moiety (e.g., an atom or a linker group) to a binding moiety A, a polynucleotide B, a polymer C, or a cell-penetrating or endosomolytic moiety D.
  • a linker moiety e.g., an atom or a linker group
  • Exemplary traceless linkers include, but are not limited to, germanium linkers, silicium linkers, sulfur linkers, selenium linkers, nitrogen linkers, phosphorus linkers, boron linkers, chromium linkers, or phenylhydrazide linker.
  • the linker is a traceless aryl-triazene linker as described in Hejesen, et al., “A traceless aryl-triazene linker for DNA-directed chemistry,” Or g Biomol Chem 11(15): 2493-2497 (2013).
  • the linker is a traceless linker described in Blaney, et al., “Traceless solid-phase organic synthesis,” Chem. Rev. 102: 2607-2024 (2002).
  • a linker is a traceless linker as described in U.S. Patent No. 6,821,783.
  • the linker comprises a functional group that exerts steric hinderance at the site of bonding between the linker and a conjugating moiety (e.g., A, B, C, or D described herein).
  • the steric hinderance is a steric hindrance around a disulfide bond.
  • Exemplary linkers that exhibit steric hinderance comprises a heterobifunctional linker, such as a heterobifunctional linker described above.
  • a linker that exhibits steric hinderance comprises SMCC and SPDB.
  • the linker is an acid cleavable linker.
  • the acid cleavable linker comprises a hydrazone linkage, which is susceptible to hydrolytic cleavage.
  • the acid cleavable linker comprises a thiomaleamic acid linker.
  • the acid cleavable linker is a thiomaleamic acid linker as described in Castaneda, et al, “Acid-cleavable thiomaleamic acid linker for homogeneous antibody-drug conjugation,” Chem. Commun. 49: 8187- 8189 (2013).
  • the linker is a linker described in U.S. Patent Nos. 6,884,869; 7,498,298; 8,288,352; 8,609,105; or 8,697,688; U.S. Patent Publication Nos. 2014/0127239; 2013/028919; 2014/286970; 2013/0309256; 2015/037360; or 2014/0294851; or PCT Publication Nos. WO2015057699; W02014080251; WO2014197854; W02014145090; or WO2014177042.
  • X, Y, and L are independently a bond or a linker. In some instances, X, Y, and L are independently a bond. In some cases, X, Y, and L are independently a linker.
  • X is a bond or a linker. In some instances, X is a bond. In some instances, X is a linker. In some instances, the linker is a Ci-Ce alkyl group. In some cases, X is a Ci-Ce alkyl group, such as for example, a Cs, C4, C3, C2, or Ci alkyl group. In some cases, the Ci- G> alkyl group is an unsubstituted Ci-Ce alkyl group. As used in the context of a linker, and in particular in the context of X, alkyl means a saturated straight or branched hydrocarbon radical containing up to six carbon atoms.
  • X is a non-polymeric linker. In some instances, X includes a homobifuctional linker or a heterobifunctional linker described supra. In some cases, X includes a heterobifunctional linker. In some cases, X includes sMCC. In other instances, X includes a heterobifunctional linker optionally conjugated to a Ci-Ce alkyl group. In other instances, X includes sMCC optionally conjugated to a Ci-Ce alkyl group. In additional instances, X does not include a homobifuctional linker or a heterobifunctional linker described supra.
  • Y is a bond or a linker. In some instances, Y is a bond. In other cases, Y is a linker. In some aspects, Y is a Ci-Ce alkyl group. In some instances, Y is a homobifuctional linker or a heterobifunctional linker described supra. In some instances, Y is a homobifuctional linker described supra. In some instances, Y is a heterobifunctional linker described supra. In some instances, Y comprises a maleimide group, such as maleimidocaproyl (me) or a selfstabilizing maleimide group described above. In some instances, Y comprises a peptide moiety, such as Val-Cit.
  • Y comprises a benzoic acid group, such as PABA.
  • Y comprises a combination of a maleimide group, a peptide moiety, and/or a benzoic acid group.
  • Y comprises a me group.
  • Y comprises a mc-val-cit group.
  • Y comprises a val-cit-PABA group.
  • Y comprises a mc-val-cit-PABA group.
  • L is a bond or a linker. In some cases, L is a bond. In other cases, L is a linker. In some aspects, L is a Ci-Ce alkyl group. In some instances, L is a homobifuctional linker or a heterobifunctional linker described supra. In some instances, L is a homobifuctional linker described supra. In some instances, L is a heterobifunctional linker described supra. In some instances, L comprises a maleimide group, such as maleimidocaproyl (me) or a selfstabilizing maleimide group described above. In some instances, L comprises a peptide moiety, such as Val-Cit.
  • L comprises a benzoic acid group, such as PABA.
  • L comprises a combination of a maleimide group, a peptide moiety, and/or a benzoic acid group.
  • L comprises a me group.
  • L comprises a mc-val-cit group.
  • L comprises a val-cit-PABA group.
  • L comprises a mc-val-cit-PABA group.
  • a composition or a pharmaceutical formulation described herein comprising a binding moiety conjugated to a polynucleic acid molecule and a polymer is used for the treatment of a disease or disorder mediated by SARS-CoV or SARS-CoV-2.
  • the disease or disorder is COVID-19.
  • the disease or disorder is a cardiomyopathy or heart failure associated with COVID-19.
  • a composition or a pharmaceutical formulation described herein is used as a therapy for the treatment of COVID-19 patients (e.g., a patient infected with COVID-19, SARS-CoV or SARS-CoV-2, a patient suffered from symptoms, diseases or disorders related to COVID-19, etc.).
  • a composition or a pharmaceutical formulation described herein is used as a therapy for the prevention or treatment of SARS-CoV or SARS-CoV-2 infections.
  • a molecule of Formula (I), Formula (II), or Formula (III) in combination with a one or more other drugs is used for the treatment of a disease or disorder mediated by SARS- CoV or SARS-CoV-2 including but not limited to COVID-19, cardiomyopathy, or heart failure.
  • a molecule of Formula (I), Formula (II), or Formula (III) is used in combination with another drug is used for the treatment of an autoimmune or inflammatory disease or disorder.
  • the composition or a pharmaceutical formulation described herein is used in conjunction with a vaccine.
  • the vaccine is an in situ vaccination.
  • the vaccine is a cell-based vaccine.
  • the vaccine is a non-cell based vaccine.
  • a molecule of Formula (I), Formula (II), or Formula (III) in combination with dendritic cell-based vaccine is used for the treatment of a COVID-19 mediated disease or disorder (e.g., cardiomyopathy, heart failure).
  • a molecule of Formula (I), Formula (II), or Formula (III) in combination with an RNA, DNA, vectorized, subunit, purified protein, nanoparticle, LNP -formulated, or inactivated virus vaccine is used for the treatment of a COVID-19 mediated disease or disorder (e.g., cardiomyopathy, heart failure).
  • a COVID-19 mediated disease or disorder e.g., cardiomyopathy, heart failure.
  • the pharmaceutical formulations described herein are administered to a subject by multiple administration routes, including but not limited to, parenteral (e.g., intravenous, subcutaneous, intramuscular), oral, intranasal, pulmonary, buccal, rectal, or transdermal administration routes.
  • parenteral e.g., intravenous, subcutaneous, intramuscular
  • oral e.g., intranasal, pulmonary, buccal, rectal, or transdermal administration routes.
  • the pharmaceutical composition describe herein is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular) administration.
  • the pharmaceutical composition describe herein is formulated for oral administration.
  • the pharmaceutical composition describe herein is formulated for intranasal or pulmonary inhalation administration.
  • the pharmaceutical formulations include, but are not limited to, aqueous liquid dispersions, self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.
  • aqueous liquid dispersions self-emulsifying dispersions, solid solutions, liposomal dispersions, aerosols, solid dosage forms, powders, immediate release formulations, controlled release formulations, fast melt formulations, tablets, capsules, pills, delayed release formulations, extended release formulations, pulsatile release formulations, multiparticulate formulations (e.g., nanoparticle formulations), and mixed immediate and controlled release formulations.
  • the pharmaceutical formulation includes multiparticulate formulations.
  • the pharmaceutical formulation includes nanoparticle formulations.
  • nanoparticles comprise cMAP, cyclodextrin, or lipids.
  • nanoparticles comprise solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions.
  • a nanoparticle includes a core or a core and a shell, as in a core-shell nanoparticle.
  • a nanoparticle is further coated with molecules for attachment of functional elements (e.g., with one or more of a polynucleic acid molecule B or binding moiety A as described herein).
  • Surfactants include compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and the like.
  • compounds such as sodium lauryl sulfate, sodium docusate, Tween 60 or 80, triacetin, vitamin E TPGS, sorbitan monooleate, polyoxyethylene sorbitan monooleate, polysorbates, polaxomers, bile salts, glyceryl monostearate, copolymers of ethylene oxide and propylene oxide, e.g., Pluronic® (BASF), and the like.
  • Pluronic® Pluronic®
  • Additional surfactants include polyoxyethylene fatty acid glycerides and vegetable oils, e.g., polyoxyethylene (60) hydrogenated castor oil; and polyoxyethylene alkylethers and alkylphenyl ethers, e.g., octoxynol 10, octoxynol 40. Sometimes, surfactant is included to enhance physical stability or for other purposes.
  • Viscosity enhancing agents include, e.g., methyl cellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, hydroxypropylmethyl cellulose acetate stearate, hydroxypropylmethyl cellulose phthalate, carbomer, polyvinyl alcohol, alginates, acacia, chitosans and combinations thereof.
  • the pharmaceutical compositions described herein are administered for therapeutic applications.
  • the pharmaceutical composition is administered once per day, twice per day, three times per day or more.
  • the pharmaceutical composition is administered daily, every day, every alternate day, five days a week, once a week, every other week, two weeks per month, three weeks per month, once a month, twice a month, three times per month, or more.
  • the pharmaceutical composition is administered for at least 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 18 months, 2 years, 3 years, or more.
  • one or more pharmaceutical compositions are administered simultaneously, sequentially, or at an interval period of time. In some aspects, one or more pharmaceutical compositions are administered simultaneously. In some cases, one or more pharmaceutical compositions are administered sequentially. In additional cases, one or more pharmaceutical compositions are administered at an interval period of time (e.g., the first administration of a first pharmaceutical composition is on day one followed by an interval of at least 1, 2, 3, 4, 5, or more days prior to the administration of at least a second pharmaceutical composition).
  • two or more different pharmaceutical compositions are coadministered. In some instances, the two or more different pharmaceutical compositions are coadministered simultaneously. In some cases, the two or more different pharmaceutical compositions are coadministered sequentially without a gap of time between administrations. In other cases, the two or more different pharmaceutical compositions are coadministered sequentially with a gap of about 0.5 hour, 1 hour, 2 hour, 3 hour, 12 hours, 1 day, 2 days, or more between administrations.
  • the administration of the composition is given continuously; alternatively, the dose of the composition being administered is temporarily reduced or temporarily suspended for a certain length of time (i.e., a “drug holiday”).
  • the length of the drug holiday varies between 2 days and 1 year, including by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days.
  • the dose reduction during a drug holiday is from 10%-100%, including, by way of example only, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
  • a maintenance dose is administered if necessary. Subsequently, the dosage or the frequency of administration, or both, can be reduced, as a function of the symptoms, to a level at which the improved disease, disorder or condition is retained.
  • the amount of a given agent that correspond to such an amount varies depending upon factors such as the particular compound, the severity of the disease, the identity (e.g., weight) of the subject or host in need of treatment, but nevertheless is routinely determined in a manner known in the art according to the particular circumstances surrounding the case, including, e.g., the specific agent being administered, the route of administration, and the subject or host being treated.
  • the desired dose is conveniently presented in a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, for example as two, three, four or more sub-doses per day.
  • toxicity and therapeutic efficacy of such therapeutic regimens are determined by standard pharmaceutical procedures in cell cultures or experimental animals, including, but not limited to, the determination of the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between the toxic and therapeutic effects is the therapeutic index and it is expressed as the ratio between LD50 and ED50.
  • Compounds exhibiting high therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used in formulating a range of dosage for use in human.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. The dosage varies within this range depending upon the dosage form employed and the route of administration utilized.
  • kits and articles of manufacture for use with one or more of the compositions and methods described herein.
  • Such kits include a carrier, package, or container that is compartmentalized to receive one or more containers such as vials, tubes, and the like, each of the container(s) comprising one of the separate elements to be used in a method described herein.
  • Suitable containers include, for example, bottles, vials, syringes, and test tubes.
  • the containers are formed from a variety of materials such as glass or plastic.
  • the articles of manufacture provided herein contain packaging materials. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, bags, containers, bottles, and any packaging material suitable for a selected formulation and intended mode of administration and treatment.
  • the container(s) include a molecule of Formula (I), Formula (II), or Formula (III) as disclosed herein.
  • kits optionally include an identifying description or label or instructions relating to its use in the methods described herein.
  • a kit typically includes labels listing contents and/or instructions for use, and package inserts with instructions for use. A set of instructions will also typically be included.
  • a label is on or associated with the container.
  • a label is on a container when letters, numbers or other characters forming the label are attached, molded or etched into the container itself; a label is associated with a container when it is present within a receptacle or carrier that also holds the container, e.g., as a package insert.
  • a label is used to indicate that the contents are to be used for a specific therapeutic application. The label also indicates directions for use of the contents, such as in the methods described herein.
  • the pharmaceutical compositions are presented in a pack or dispenser device which contains one or more unit dosage forms containing a compound provided herein.
  • the pack for example, contains metal or plastic foil, such as a blister pack.
  • the pack or dispenser device is accompanied by instructions for administration.
  • the pack or dispenser is also accompanied with a notice associated with the container in form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the drug for human or veterinary administration. Such notice, for example, is the labeling approved by the U.S. Food and Drug Administration for prescription drugs, or the approved product insert.
  • compositions containing a compound provided herein formulated in a compatible pharmaceutical carrier are also prepared, placed in an appropriate container, and labeled for treatment of an indicated condition.
  • ranges and amounts can be expressed as “about” a particular value or range. About also includes the exact amount. Hence “about 5 pL” means “about 5 pL” and also “5 pL.” Generally, the term “about” includes an amount that would be expected to be within experimental error.
  • the terms “individual(s)”, “subject(s)” and “patient(s)” mean any mammal.
  • the mammal is a human.
  • the mammal is a non-human. None of the terms require or are limited to situations characterized by the supervision (e.g. constant or intermittent) of a health care worker (e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker).
  • a health care worker e.g. a doctor, a registered nurse, a nurse practitioner, a physician’s assistant, an orderly or a hospice worker.
  • the terms “treat,” “treating”, or “treatment,” and other grammatical equivalents include ameliorating or preventing the underlying causes of one or more symptoms of a disease or condition; alleviating, abating, or ameliorating one or more symptoms of a disease or condition; ameliorating, preventing, or reducing the appearance, severity, or frequency of one or more symptoms of a disease or condition; inhibiting the disease or condition, such as, for example, arresting the development of the disease or condition, relieving the disease or condition, causing regression of the disease or condition, relieving a condition caused by the disease or condition, or inhibiting the symptoms of the disease or condition either prophylactically and/or therapeutically.
  • pharmaceutically acceptable denotes an attribute of a material which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use.
  • “Pharmaceutically acceptable” can refer to a material, such as a carrier, or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, e.g., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.
  • “Pharmaceutically acceptable excipient” as used herein refers to any pharmaceutically acceptable ingredient in a pharmaceutical composition having no therapeutic activity and being non-toxic to the subject administered, such as disintegrators, binders, fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, diluents, excipients, preservatives or lubricants used in formulating pharmaceutical products.
  • Table 1 illustrates target SARS-CoV and SARS-CoV-2 sequences for selection of oligonucleotides for RNAi (antisense RNA, siRNA, or PMO), or inhibitory oligonucleotide described herein.
  • Strategies for targeting SARS-CoV-2 sequences include, but are not limited to, designing siRNAs specific for conserved sequences in structural proteins such as Spike, E, N; targeting sequences of non-structural proteins, such as 3CLpro and RdRp; utilize multiple oligonucleotides targeting multiple sequences in combination for mutations/resistance and synergistic activity; design oligonucleotides such as siRNA for conserved leader sequences; design oligonucleotides such as PMOs for interfering with discontinuous transcription junctions to induce errors in processing of the transcript such as skipping and reduction of sgRNA production; and potentially using dualstrand active siRNAs.
  • RNAi antisense RNA, siRNA, or PMO
  • Tissue samples are homogenized in Trizol.
  • Total RNA is isolated using RNeasy RNA isolation 96-well plates (Qiagen), then 500 ng RNA is reverse transcribed with a High Capacity RNA to cDNA kit (ThermoFisher).
  • Virus genome and mRNA is quantified by TaqMan qPCR analysis performed with a ViiA 7 Real-Time PCR System.
  • the TaqMan primers and probes for the viruses are designed and validated.
  • PPIB housekeeping gene
  • Results are calculated by the comparative Ct method, where the difference between the target gene Ct value and the PPIB Ct value (ACt) is calculated and then further normalized relative to the PBS control group by taking a second difference (AACt).
  • SARS-CoV-2 The genome of SARS-CoV-2 has been sequenced and is a single strand of RNA with a length of 29,902 nucleotides. The published sequence is found at GenBank Accession Number: MN908947.3.
  • Step 1 Antibody conjugation with maleimide-PEG-NHS followed by SH-TFR1
  • Conjugation scheme-1 for generating Antibody-siRNA conjugates is depicted in FIG. 4.
  • Anti-TFRl antibody TFRl-Ab
  • anti-ACE2 antibody anti-TMPRSS2 antibody, or antigen binding fragment thereof
  • IX Phosphate buffer pH 7.4
  • Unreacted maleimide-PEG is removed by spin filtration using 50 kDa MWCO Amicon spin filters and PBS pH 7.4.
  • the antibody-PEG-Mal conjugate is collected and transferred into a reaction vessel.
  • SH-C6-TFR1 (2 equivalents) is added at RT to the antibody -PEG-mal eimide in PBS and rotated overnight.
  • the reaction mixture is analyzed by analytical SAX column chromatography and conjugate along with unreacted antibody and siRNA is seen.
  • the crude reaction mixture is purified by AKTA explorer FPLC using anion exchange chromatography method. Fractions containing conjugate are pooled, concentrated and buffer exchanged with PBS, pH 7.4.
  • Antibody siRNA conjugates with SMCC linker, PEGlkDa, PEG5kDa and PEGlOkDa are separated based on the siRNA loading.
  • the same or substantially similar conjugation scheme can be used to generate antibody conjugate with inhibitory oligonucleotide other than siRNA e.g., antisense oligonucleotide.
  • Step 1 Antibody conjugation with SMCC linker followed by SH-TFRl-PEG5kDa
  • Conjugation scheme-2 for generating Antibody-siRNA conjugates is depicted in FIG. 5.
  • Anti-TFRl antibody (TFRl-Ab) anti-ACE2 antibody, or anti-TMPRSS2 antibody, or binding fragment thereof, is exchanged with IX Phosphate buffer (pH 7.4) and made up to 5mg/ml concentration.
  • 2 equivalents of SMCC linker succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate
  • Unreacted SMCC linker is removed by spin filtration using 50 kDa MWCO Amicon spin filters and PBS buffer pH 7.4. The retentate is collected and 2 equivalents of SH-C6-Ab- PEG5kDa is added at RT and rotated overnight. The reaction mixture is analyzed by analytical SAX column chromatography and the conjugate along with unreacted antibody and siRNA is observed.
  • the crude reaction mixture is purified by AKTA explorer FPLC using anion exchange chromatography method. Fractions containing the conjugates are pooled, concentrated and buffer exchanged with PBS, pH 7.4. The same or substantially similar conjugation scheme can be used to generate antibody conjugate with inhibitory oligonucleotide other than siRNA e.g., antisense oligonucleotide.
  • Step 1 Antibody conjugation with SPDP linker followed by SH-siRNA-PEG5kDa
  • Conjugation scheme-3 for generating Antibody-siRNA conjugates is depicted in FIG. 6.
  • Anti-TFRl antibody (TFRl-Ab), anti-ACE2 antibody, or anti-TMPRSS2 antibody, or binding fragment thereof, is exchanged with IX Phosphate buffer (pH 7.4) and made up to 5mg/ml concentration.
  • SPDP linker succinimidyl 3-(2- pyridyldithio)propi onate
  • SPDP linker succinimidyl 3-(2- pyridyldithio)propi onate
  • Unreacted SPDP linker is removed by spin filtration using 50 kDa MWCO Amicon spin filters and pH 7.4 PBS buffer.
  • the retentate is collected and 2 equivalents of SH-C6-siRNA-PEG5kDa is added at room temperature and rotated overnight.
  • the reaction mixture is analyzed by analytical SAX column chromatography and conjugate along with unreacted antibody and siRNA is seen.
  • the crude reaction mixture is purified by AKTA explorer FPLC using anion exchange chromatography method- 1. Fractions containing the antibody -PEG-siRNA conjugate are pooled, concentrated and buffer exchanged with PBS, pH 7.4. The same or substantially similar conjugation scheme can be used to generate antibody conjugate with inhibitory oligonucleotide other than siRNA e.g., antisense oligonucleotide.
  • Step 1 Antibody conjugation with SMCC linker or maleimide-PEG-NHS followed by SH-Cys-Peptide-
  • Conjugation scheme-4 for generating Antibody-siRNA conjugates is depicted in FIG. 7.
  • Anti-TFRl antibody TFRl-Ab
  • anti-ACE2 antibody anti-TMPRSS2 antibody, or binding fragment thereof
  • IX Phosphate buffer pH 7.4
  • 3 equivalents of SMCC linker succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate
  • maleimide-PEGlkDa-NHS is added and rotated for 1.5 hours at room temperature.
  • HIC hydrophobic interaction chromatography
  • Step 1 Conjugation of PEG24 linker followed by SH-Cys-Peptide- to TFR1- Ab-siRNA-PEG
  • Conjugation scheme-5 for generating Antibody-siRNA conjugates is depicted in FIG. 8.
  • Ab-siRNA-PEG conjugate with a siRNA loading of 1 is conjugated with 4 equivalents of PEGlk linker (succinimidyl 4-(N-maleimidomethyl)cyclohexane-l -carboxylate) in PBS, pH 7.4 buffer and rotated for 1.5 hours at room temperature.
  • Unreacted PEGlk linker is removed by spin filtration using 50 kDa MWCO Amicon spin filters and PBS buffer pH 7.4. The retentate is collected and 4 equivalents of SH-Cys-Peptide-CONH2 is added at RT and rotated overnight.
  • the reaction mixture is then purified by repeated spin filtration using PBS buffer pH7.4 and 50 kDa Amicon spin filters until the unreacted peptide is removed as monitored by HPLC.
  • the product contains a mixture of conjugates with 0, 1, 2, 3 or more peptides conjugated to the antibody backbone.
  • the same or substantially similar conjugation scheme can be used to generate antibody conjugate with inhibitory oligonucleotide other than siRNA e.g., antisense oligonucleotide.
  • Step-3 Analysis of the purified conjugate
  • the isolated conjugate is characterized by either mass spec or SDS-PAGE.
  • the purity and the peptide loading of the conjugate is assessed by analytical HPLC using either HIC method-2 or cation exchange chromatography method-2.
  • Example 7 In silico siRNA screening
  • SARS-CoV SARS-CoV (NC_004718.3) and MERS (NC_038294.1) genomes. Sequences with CpG motifs were avoided. Off-target analysis was performed against mature human reference RNAs and primary transcripts. Sequences with fewer than 3 mismatches were avoided. miRbase searched for matches with known human microRNAs with the same seed region, which were excluded. 44 oligonucleotides were selected based on the criteria and can target at least 99% of known SARS- CoV-2 variants (Table 2). The modified siRNAs were synthesized with cholesterol on the 5’ ends of their passenger strands (Table 3).
  • SARS-CoV-2 strain USA-WA1/2020, was provided by the World Reference Center for SARS-CoV-2, strain USA-WA1/2020, was provided by the World Reference Center for SARS-CoV-2, strain USA-WA1/2020, was provided by the World Reference Center for SARS-CoV-2, strain USA-WA1/2020, was provided by the World Reference Center for SARS-CoV-2, strain USA-WA1/2020, was provided by the World Reference Center for SARS-CoV-2, strain USA-WA1/2020, was provided by the World Reference Center for
  • Sample DI (COV2-37) showed the most activity against SARS-CoV-2 (SI>3) at a low concentration as shown in Table 4.
  • the positive control compound performed as expected.
  • SARS-CoV-2 strain USA-WA1/2020
  • WRCEVA World Reference Center for Emerging Viruses and Arboviruses
  • the modified siRNA COV2-17 is a double-stranded siRNA molecule with SEQ ID NO: 125 as the passenger (sense strand) and SEQ ID NO: 169 as the guide strand (antisense strand)
  • the modified siRNA COV2-37 is a double-stranded siRNA molecule with SEQ ID NO: 145 as the passenger strand (sense strand) and SEQ ID NO: 189 as the guide strand (antisense strand).
  • RNAiMAX transfection reagent (Invitrogen, ThermoFisher Scientific) diluted in Opti-MEM (18:300) was added in equal volume to each test concentration of siRNA. These complexes were incubated at room temperature for 5 minutes, then added in a 1/5 dilution to five wells each of a 96-well plate containing 80-100% confluent Vero E6 cells.
  • the incorporated dye was extracted in 50:50 Sorensen citrate buffer/ethanol for >30 minutes, and the optical density was read on a spectrophotometer at 540 nm. Optical densities were converted to percent of cell controls and normalized to the virus control, and the concentration of test compound required to inhibit CPE by 50% (EC50) was calculated by regression analysis. The concentration of compound that would cause 50% cell death in the absence of virus was similarly calculated (CC50).
  • the selective index (SI) is the CC50 divided by EC50.
  • SARS-CoV-2 infected cells transfected with the modified siRNA COV2-17 and COV2- 37 showed increased cell viability compared to control cells (FIGs. 1 and 2) indicating that the 2 siRNA reduced the activity of the virus (FIGs. 1 and 2).
  • Both the COV2-17 and COV2-37 siRNA decreased activity of the virus in a dose dependent manner with the with final concentration of 500 nM (FIG. 1) or 50 nM (FIG. 2).
  • the modified COV2-37 siRNA showed greater antiviral activity than the modified COV2-17 siRNA.
  • the modified siRNA COV2-17 and COV2-37 showed potent antiviral activity against SARS-CoV-2.
  • Example 10 Two modified siRNAs decrease Virus yield
  • the supernatant fluid from the in vitro antiviral assay against SARS-CoV-2 virus for concentrations of the 2 modified siRNA (COV2-17 and COV2-37) and remdesivir was collected on day 3 post infection, before neutral red staining (3 wells pooled).
  • the supernatant fluid was tested for virus titer using a standard endpoint dilution CCID50 assay, and titer calculations using the Reed-Muench (1948) equation.
  • the concentrations of the 2 modified siRNAs required to reduce virus yield by 1-3 loglO (EC90, EC99, EC99.9) were calculated by regression analysis.
  • Virus titers in cell culture supernatants were determined with a standard endpoint dilution CCID50 assay. Percent cytotoxicity, CPE, and virus titer reduction at each concentration are summarized in Table 5 and FIG. 3.
  • Sample A5 modified siRNA COV2-17) and DI (modified siRNA COV2-37) exhibited activity by virus yield reduction against SARS-CoV-2, (SI>10) (Table 5).
  • the modified COV2-37 siRNA provided the most reduction of the virus yield as determined by the values of the numerous ECs.
  • the modified siRNA COV2-17 and COV2-37 decreased antiviral activity against SARS-CoV-2 by reducing the virus yield.

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Abstract

L'invention divulgue des compositions et des formulations pharmaceutiques qui comprennent un fragment de liaison conjugué à une molécule d'acide polynucléique et éventuellement à un polymère. L'invention concerne également des méthodes de traitement d'une infection par le SARS-CoV ou le SARS-CoV-2 et de maladies qui leur sont associées. L'invention concerne en outre des méthodes de traitement de la COVID-19 et de maladies et de pathologies associés, comprenant, mais sans y être limitées, la cardiomyopathie et l'insuffisance cardiaque.
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WO2006113431A2 (fr) * 2005-04-13 2006-10-26 University Of Massachusetts Oligonucleotides duels fonctionnels destines a s'utiliser en tant qu'agents antiviraux
US20070037763A1 (en) * 2003-12-24 2007-02-15 Avi Biopharma, Inc. Oligonucleotide compound and method for treating nidovirus infections
US20070270360A1 (en) * 2003-04-15 2007-11-22 Sirna Therapeutics, Inc. Rna Interference Mediated Inhibition of Severe Acute Respiratory Syndrome (Sars) Gene Expression Using Short Interfering Nucleic Acid
WO2010105372A1 (fr) * 2009-03-20 2010-09-23 Protiva Biotherapeutics, Inc. Compositions et procedes d'inactivation de l'expression du virus de l'hepatite c

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US20070270360A1 (en) * 2003-04-15 2007-11-22 Sirna Therapeutics, Inc. Rna Interference Mediated Inhibition of Severe Acute Respiratory Syndrome (Sars) Gene Expression Using Short Interfering Nucleic Acid
US20050100885A1 (en) * 2003-04-28 2005-05-12 Crooke Stanley T. Compositions and methods for the treatment of severe acute respiratory syndrome (SARS)
US20070037763A1 (en) * 2003-12-24 2007-02-15 Avi Biopharma, Inc. Oligonucleotide compound and method for treating nidovirus infections
WO2006113431A2 (fr) * 2005-04-13 2006-10-26 University Of Massachusetts Oligonucleotides duels fonctionnels destines a s'utiliser en tant qu'agents antiviraux
WO2010105372A1 (fr) * 2009-03-20 2010-09-23 Protiva Biotherapeutics, Inc. Compositions et procedes d'inactivation de l'expression du virus de l'hepatite c

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WO2024249944A1 (fr) * 2023-06-02 2024-12-05 Avidity Biosciences, Inc. Compositions conjuguées comprenant des liaisons guanidine phosphoryle et leurs utilisations

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