WO2022056117A1

WO2022056117A1 - Nucleic acid-polypeptide compositions and uses thereof

Info

Publication number: WO2022056117A1
Application number: PCT/US2021/049651
Authority: WO
Inventors: Jae B. KIM; Art LEVIN; Rob BURKE; Hanhua Huang
Original assignee: Avidity Biosciences, Inc.
Priority date: 2020-09-10
Filing date: 2021-09-09
Publication date: 2022-03-17

Abstract

Disclosed herein are compositions and pharmaceutical formulations that comprise a binding moiety conjugated to a polynucleic acid molecule and optionally to a polymer. Also described herein include methods for treating a SARS-CoV or SARS-CoV-2 infection and diseases associated therewith. Also described herein include methods for treating a COVID-19 and related diseases and conditions, including but not limited to cardiomyopathy and heart failure.

Description

NUCLEIC ACID-POLYPEPTIDE COMPOSITIONS AND USES THEREOF

CROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63/076,631 filed September 10, 2020, which is incorporated herein by reference in its entirety.

BACKGROUND OF THE DISCLOSURE

[0002] Gene suppression by oligonucleotide-induced gene silencing provides several levels of control, including but not limited to transcription inactivation, mRNA degradation, interruption of replication, splicing modulation, and transcriptional attenuation. In some instances, RNA interference (RNAi) provides long lasting effect over multiple cell divisions. As such, 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.

SUMMARY OF THE DISCLOSURE

[0003] Disclosed herein includes compositions and pharmaceutical formulations that comprise a binding moiety conjugated to a polynucleic acid molecule specific for viral genes and/or their gene products. In some aspects the composition includes a polymer, and/or a cell membrane penetrating moiety. In some aspects, also described herein include methods for treating a disease or condition (e.g., virus infection) that utilizes 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.

[0004] 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).

[0005] Disclosed herein, in certain aspects, is a molecule of Formula (I):

A-(X-B)_n

Formula I wherein,

A is a binding moiety;

B is a polynucleotide; n is an integer between 1 and 10.

In some aspects, 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. In some aspects, the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.

[0006] In some aspects, 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. In some aspects, the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some aspects, the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some aspects, the at least one inverted abasic moiety is at at least one terminus.

[0007] In some aspects, the polynucleotide comprises a single-stranded nucleic acid molecule. In some aspects the single strand is an antisense oligonucleotide, or a PMO. In some aspects, the polynucleotide comprises two or more strands. In some aspects, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some aspects, the second polynucleotide comprises at least one modification.

[0008] In some aspects, 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.

[0009] In some aspects, 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.

[0010] In some aspects, 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.

[0011] In some aspects, 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.

[0012] In some aspects, 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. In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0013] In some aspects, 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.

[0014] Disclosed herein, in certain aspects, is a molecule of Formula (II):

A-(X-B-Y-C)_n Formula II wherein,

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.

[0015] In some aspects, 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. In some aspects, the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some aspects, the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some aspects, the at least one inverted abasic moiety is at at least one terminus.

[0016] In some aspects, the polynucleotide comprises a single-stranded molecule. In some aspects the single strand is an antisense oligonucleotide, or a PMO. In some aspects, the polynucleotide comprises two or more strands. In some aspects, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some aspects, the second polynucleotide comprises at least one modification.

[0017] In some aspects, 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.

[0018] In some aspects, 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.

[0019] In some aspects, 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.

[0020] In some aspects, X and Y are independently 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, Y is a Ci- G> alkyl group. In some aspects, X is a homobifunctional linker or a heterobifunctional linker, optionally conjugated to a Ci-Ce alkyl group. In some aspects, Y is a homobifunctional linker or a heterobifunctional linker.

[0021] In some aspects, the binding moiety is an antibody or antigen binding fragment thereof. In some aspects, 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. In some aspects, 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.

[0022] In some aspects, C is polyethylene glycol. In some aspects, C has a molecular weight of about 5000 Da.

[0023] In some aspects, 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.

[0024] In some aspects, the molecule further comprises D. In some aspects, D is conjugated to C or to A.

[0025] In some aspects, D is conjugated to the molecule of Formula (I) or Formula (II) according to Formula (III):

A-(X-B)z-(Y-C)n-(L-D)_z

Formula III wherein,

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.

[0026] In some aspects, D is INF7 or melittin.

[0027] In some aspects, D is a cell-penetrating or endosomolytic polymer or peptide. [0028] In some aspects, L is a Ci-Ce alkyl group. In some aspects, L is a homobifunctional linker or a heterobifunctional linker.

[0029] In some aspects, the molecule further comprises at least a second binding moiety A. In some aspects, the at least second binding moiety A is conjugated to A, to B, or to C. In some aspects, the at least second binding moiety A is cholesterol. In some aspects, binding moiety A is an antibody or antigen binding fragment thereof. In some aspects 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.

[0030] In some aspects, the molecule further comprises at least an additional polynucleotide B. In some aspects, the at least an additional polynucleotide B is conjugated to A, to B, or to C.

[0031] In some aspects, the molecule further comprises at least an additional polymer C. In some aspects, the at least an additional polymer C is conjugated to A, to B, or to C.

[0032] Disclosed herein, in certain aspects, is a pharmaceutical composition comprising a molecule described above, and a pharmaceutically acceptable excipient. In some aspects, the pharmaceutical composition is formulated as a nanoparticle formulation, including lipid nanoparticle formulation. In some aspects, the pharmaceutical composition is formulated for parenteral, oral, intranasal, pulmonary, buccal, rectal, or transdermal administration.

[0033] Disclosed herein, in certain aspects, is a method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a composition comprising a molecule described herein. In some aspects, the disease or disorder is a virus infection. In some aspects, the disease is caused or exacerbated by the virus infection. In some aspects, the disease is cardiomyopathy. In some aspects, the cardiomyopathy is caused by SARS-CoV-2 infection. In some aspects, the cardiomyopathy is associated with COVID-19 or infection with SARS-CoV-2 resulting in de novo or exacerbated pre-existing cardiomyopathy.

[0034] Disclosed herein, in certain aspects, is a method of inhibiting the expression of a target nucleotide in a cell of a patient, comprising administering a molecule described herein. In some aspects, the method is an in vivo method. In some aspects, the patient is a human with or recovering from COVID-19, with or without cardiomyopathy.

[0035] Disclosed herein, in certain aspects, is a 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. In some embodiments, 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.

In some instances, 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.

[0036] In some instances, 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. In some instances, wherein 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.

[0037] In some instances, the sense strand and antisense strand form a double-stranded siRNA molecule. In some instances, the double-stranded region is 15-30 nucleotide pairs in length [0038] Disclosed herein, in certain aspects, is a kit comprising a molecule described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Various aspects of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative aspects, in which the principles of the disclosure are utilized, and the accompanying drawings below.

[0040] 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. [0041] 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.

[0042] 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.

[0043] FIGs. 4-8 illustrate conjugation schemes described herein.

DETAILED DESCRIPTION OF THE DISCLOSURE

[0044] Nucleic acid (e.g., RNAi) therapy is a targeted therapy with high selectivity and specificity. However, in some instances, nucleic acid therapy is also hindered by poor intracellular uptake, limited blood stability and non-specific immune stimulation. To address these issues, 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.

[0045] In some aspects, 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. For example, if 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) further effects intracellular uptake, stability, toxicity, efficacy, and/or non-specific immune stimulation.

[0046] In some aspects, described herein include a molecule those arrangement of the nucleic acid components effects intracellular uptake, stability, toxicity, efficacy, and/or non-specific immune stimulation. In some instances, the molecule comprises a binding moiety conjugated to a polynucleic acid molecule and optionally to a polymer. In some aspects, 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. In some aspects 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. In some instances, the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety. In some instances, the molecule of Formula (I) or Formula (II) further comprises D, a cell-penetrating or endosomolytic moiety.

[0047] In some aspects, a molecule described herein is further used to treat a disease or disorder. In some instances, 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. In some instances, the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety. In some instances, the molecule of Formula (I) further comprises D, a cell-penetrating or endosomolytic moiety.

[0048] In some aspects, 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. In such instances, 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. In some aspects, 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. In some instances, the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety. In some instances, the molecule of Formula (I) or Formula (II) further comprises D, a cell-penetrating or endosomolytic moiety.

[0049] In some aspects, 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. In some instance, 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. In some aspects, 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. In some instances, the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety. In some instances, the molecule of Formula (I) further comprises D, a cell-penetrating or endosomolytic moiety.

[0050] In additional aspects, described herein include a kit, which comprises one or more of the molecules described herein.

Therapeutic Molecule Platform

[0051] In some aspects, a molecule (e.g., a therapeutic molecule) described herein comprises a binding moiety conjugated to a polynucleic acid molecule and a polymer.

[0052] Disclosed herein, in certain aspects, is a molecule of Formula (I):

A-(X-B)_n Formula I wherein,

A is a binding moiety;

B is a polynucleotide; n is an integer between 1 and 10; and

In some aspects, 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. In some aspects, the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety. [0053] In some aspects, 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. In some aspects, the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some aspects, the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some aspects, the at least one inverted abasic moiety is at at least one terminus.

[0054] In some aspects, the polynucleotide comprises a single-stranded molecule. In some aspects the single strand is an antisense oligonucleotide, or a PMO. In some aspects, the polynucleotide comprises two or more strands. In some aspects, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some aspects, the second polynucleotide comprises at least one modification.

[0055] In some aspects, 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.

[0056] In some aspects, 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.

[0057] In some aspects, 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.

[0058] In some aspects, 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.

[0059] In some aspects, 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. In some aspects, 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. In some aspects, 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.

[0060] In some aspects, A-X is conjugated to the 5’ end of B. In some aspects, A-X, is conjugated to an intemucleotide linkage group.

[0061] Disclosed herein, in certain aspects, is a molecule of Formula (II):

A-(X-B-Y-C)_n

Formula II wherein,

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

In some aspects, 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. In some aspects, the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety.

[0062] In some aspects, 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. In some aspects, the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). In some aspects, the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. In some aspects, the at least one inverted abasic moiety is at at least one terminus.

[0063] In some aspects, the polynucleotide comprises a single-stranded molecule. In some aspects the single strand is an antisense oligonucleotide, or a PMO. In some aspects, the polynucleotide comprises two or more strands. In some aspects, the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. In some aspects, the second polynucleotide comprises at least one modification.

[0064] In some aspects, 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.

[0065] In some aspects, 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.

[0066] In some aspects, 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.

[0067] In some aspects, X and Y are independently 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, Y is a Ci- G> alkyl group. In some aspects, X is a homobifunctional linker or a heterobifunctional linker, optionally conjugated to a Ci-Ce alkyl group. In some aspects, Y is a homobifunctional linker or a heterobifunctional linker.

[0068] In some aspects, the binding moiety is an antibody or binding fragment thereof. In some aspects, 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. In some aspects, 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.

[0069] In some aspects, C is polyethylene glycol. In some aspects, C has a molecular weight of about 5000 Da.

[0070] In some aspects, 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.

[0071] In some aspects, the molecule further comprises D. In some aspects, D is conjugated to C or to A. [0072] In some aspects, 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. In some instances, at least one A is conjugated at one terminus of B while at least one C is conjugated at the opposite terminus of B. In some instances, 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.

[0073] In some instances, at least one B and/or at least one C, and optionally at least one D are conjugated to a first A. In some instances, 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. In some cases, 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. In some instances, at least one additional A is further conjugated to the first A, to B, or to C. In additional instances, 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.

[0074] In some cases, two or more Bs and/or 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 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. In some instances, at least one additional A is further conjugated to the first A, to two or more Bs, or to two or more Cs. In additional instances, 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. In some cases, the at least one additional A is different than the first A. In some cases, the two or more Bs are different. In other cases, 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. If indirectly via the two or more Bs, 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. In some instances, 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. In additional instances, 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. In some cases, the at least one additional A is different than the first A. In some cases, the two or more Bs are different. In other cases, 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.

[0076] In some aspects, D is conjugated to the molecule of Formula (I) or Formula (II) according to Formula (III):

A-(X-B)z-(Y-C)n-(L-D)_z Formula III wherein,

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 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, 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.

[0077] In some aspects, D is INF7 or melittin. [0078] In some aspects, D is a cell-penetrating or endosomolytic polymer or peptide.

[0079] In some aspects, L is a Ci-Ce alkyl group. In some aspects, L is a homobifunctional linker or a heterobifunctional linker.

[0080] In some aspects, the molecule further comprises at least a second binding moiety A. In some aspects, the at least second binding moiety A is conjugated to A, to B, or to C. In some aspects, the at least second binding moiety A is cholesterol.

[0081] In some aspects, the molecule further comprises at least an additional polynucleotide B. In some aspects, the at least an additional polynucleotide B is conjugated to A, to B, or to C.

[0082] In some aspects, the molecule further comprises at least an additional polymer C. In some aspects, the at least an additional polymer C is conjugated to A, to B, or to C.

[0083] Disclosed herein, in certain aspects, is a pharmaceutical composition comprising a molecule described above, and a pharmaceutically acceptable excipient. In some aspects, the pharmaceutical composition is formulated as a nanoparticle formulation, including lipid nanoparticle formulation. In some aspects, the pharmaceutical composition is formulated for parenteral, oral, intranasal, pulmonary, buccal, rectal, or transdermal administration.

[0084] Disclosed herein, in certain aspects, is a method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a composition comprising a molecule described above. In some aspects, the disease or disorder is a virus infection. In some aspects, the disease is caused or exacerbated by the virus infection. In some aspects, the disease is cardiomyopathy. In some aspects, the cardiomyopathy is caused by SARS-CoV-2 infection. In some aspects, the cardiomyopathy is associated with COVID-19 or infection with SARS-CoV-2 resulting in de novo or exacerbated pre-existing cardiomyopathy.

[0085] Disclosed herein, in certain aspects, is 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. In some aspects, the method is an in vivo method. In some aspects, the patient is a human with or recovering from COVID-19, with or without cardiomyopathy.

[0086] Disclosed herein, in certain aspects, is a kit comprising a molecule described herein.

Polynucleic Acid Molecule Targets

[0087] In some aspects, 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). In some instances, the virus is SARS-CoV or SARS-CoV variant, and in some instances the virus is SARS- CoV-2 or SARS-CoV-2 variant.

Polynucleic Acid Molecule Sequences [0088] In some aspects, 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. In some instances, the polynucleic acid molecule B comprises a single antisense strand. In some instances, 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. In some instances, 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). In some instances, the virus is SARS-CoV or SARS-CoV variant, and in some instances the virus is SARS-CoV-2 or SARS-CoV-2 variant.

[0089] In some aspects, 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.

[0090] In some aspects, the polynucleic acid molecule B comprises a first polynucleotide and a second polynucleotide. In some instances, 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). In some instances, the virus is SARS- CoV or SARS-CoV variant, and in some instances the virus is SARS-CoV-2 or SARS-CoV-2 variant.

Polynucleic Acid Molecules

[0091] In some aspects, the polynucleic acid molecule described herein comprises RNA or DNA. In some cases, the polynucleic acid molecule comprises RNA. In some instances, 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). In some instances, RNA comprises shRNA. In some instances, RNA comprises miRNA. In some instances, RNA comprises dsRNA. In some instances, RNA comprises tRNA. In some instances, RNA comprises rRNA. In some instances, RNA comprises hnRNA. In some instances, the RNA comprises siRNA. In some instances, the polynucleic acid molecule comprises siRNA. In some cases, B comprises siRNA, antisense RNA, or PMO.

[0092] In some aspects, 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. In some instances, 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. In some instances, 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. In some instances, 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.

[0094] In some aspects, 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.

[0095] In some aspects, the polynucleic acid molecule is a first polynucleotide. In some aspects, 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.

[0096] In some instances, 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. In some instances, 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. In some instances, 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. In some instances, 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.

[0097] In some aspects, the polynucleic acid molecule is a second polynucleotide. In some aspects, 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.

[0098] In some instances, 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. In some instances, 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. In some instances, 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. In some instances, 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.

[0099] In some aspects, the polynucleic acid molecule comprises a first polynucleotide and a second polynucleotide. In some instances, the polynucleic acid molecule further comprises a blunt terminus, an overhang, or a combination thereof. In some instances, the blunt terminus is a 5’ blunt terminus, a 3’ blunt terminus, or both. In some cases, the overhang is a 5’ overhang, 3’ overhang, or both. In some cases, the overhang comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 non-base pairing nucleotides. In some cases, the overhang comprises 1, 2, 3, 4, 5, or 6 non-base pairing nucleotides. In some cases, 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. In some aspects, 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.

[00101] In some aspects, 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.

[00102] In some aspects, 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. In some instances, the hybridization is a high stringent hybridization condition.

[00103] In some aspects, the polynucleic acid molecule has reduced off-target effect. In some instances, “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. In some instances, 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.

[00104] In some aspects, 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.

[00105] In some aspects, nucleotide analogues or artificial nucleotide base comprise a nucleic acid with a modification at a 2’ hydroxyl group of the ribose moiety. In some instances, 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. In some instances, 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). In some instances, the alkyl moiety further comprises a hetero substitution. In some instances, the carbon of the heterocyclic group is substituted by a nitrogen, oxygen or sulfur. In some instances, the heterocyclic substitution includes but is not limited to, morpholino, imidazole, and pyrrolidino.

[00106] In some instances, the modification at the 2’ hydroxyl group is a 2’-O-methyl modification or a 2’ -O-m ethoxy ethyl (2’-0-M0E) modification. In some cases, 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.

2’-O-methyl-adenosine 2’-O-methoxyethyl uridine

[00107] In some instances, 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. In some instances, 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.

2’-O-aminopropyl nucleoside phosphoramidite

[00108] In some instances, 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. 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 (³E) conformation of the furanose ring of an LNA monomer.

LNA (Locked Nucleic Acids)

[00109] In some instances, 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 are part of 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.

[00110] In some aspects, 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).

[00111] In some aspects, 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 such as 7-deaza- adenosine, 6-azouridine, 6-azocytidine, 6-azothymidine, 5- methyl-2 -thiouridine, other thio bases such as 2-thiouridine and 4-thiouridine and 2-thiocytidine, dihydrouridine, pseudouridine, queuosine, archaeosine, naphthyl and substituted naphthyl groups, any O-and N-alkylated purines and pyrimidines such as N6-methyladenosine, 5- methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridine-4-one, pyridine-2-one, phenyl and modified phenyl groups such as aminophenol or 2,4, 6-trimethoxy benzene, modified cytosines that act as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyi nucleotides, and alkylcarbonylalkylated nucleotides. 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. For example, 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. By way of example, universal bases include but are not limited to 3 -nitropyrrole, 5-nitroindole, or nebularine.

[00112] In some aspects, 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. In some instances, the five-member ribose ring is substituted with a six member morpholino ring containing four carbons, one nitrogen and one oxygen. In some cases, the ribose monomers are linked by a phosphordiamidate group instead of a phosphate group. In such cases, 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.

Morpholino

[00113] In some aspects, peptide nucleic acid (PNA) 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.

PNA

[00114] In some aspects, one or more modifications optionally occur at the intemucleotide linkage. In some instances, 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, phosphoropiperazidates, phosphoroanilothioates, phosphoroanilidates, ketones, sulfones, sulfonamides, carbonates, carbamates, methylenehydrazos, methylenedimethylhydrazos, formacetals, thioformacetals, oximes, methyleneiminos, methylenemethyliminos, thioamidates, linkages with riboacetyl groups, aminoethyl glycine, silyl or siloxane linkages, alkyl or cycloalkyl linkages with or without heteroatoms of, for example, 1 to 10 carbons that can be saturated or unsaturated and/or substituted and/or contain heteroatoms, linkages with morpholino structures, amides, polyamides wherein the bases can be attached to the aza nitrogens of the backbone directly or indirectly, and combinations thereof.

[00115] In some instances, 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.

[00116] In some instances, a modified nucleotide includes, but is not limited to, 2’-fluoro N3-P5’- phosphoramidites illustrated as:

[00117] In some instances, a modified nucleotide includes, but is not limited to, hexitol nucleic acid (or 1’, 5’ - anhydrohexitol nucleic acids (HNA)) illustrated as: Base

HO HO

HNA

[00118] In some aspects, 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. For example, the 3’ terminus optionally include a 3’ cationic group, or by inverting the nucleoside at the 3 ’-terminus with a 3 ’-3’ linkage. In another alternative, the 3’-terminus is optionally conjugated with an aminoalkyl group, e.g., a 3’ C5-aminoalkyl dT. In an additional alternative, the 3’-terminus is optionally conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site. In some instances, the 5’-terminus is conjugated with an aminoalkyl group, e.g., a 5’-O-alkylamino substituent. In some cases, the 5’-terminus is conjugated with an abasic site, e.g., with an apurinic or apyrimidinic site.

[00119] In some aspects, 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. In some aspects, 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 combination thereof. 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 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, thiolphosphonate nucleotides, 2 ’-fluoro N3-P5’-phosphoramidites, or a combination thereof. 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-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.

[00120] In some aspects, 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. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 1-20, 21-64, 65-108, 109-152, and 153-196.

[00121] In some instances, 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. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 1-20, 21-64, 65-108, 109-152, and 153-196.

[00122] In some instances, 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. In some instances, the polynucleic acid molecule is a polynucleic acid molecule of SEQ ID NOs: 1-20, 21-64, 65-108, 109-152, and 153-196.

[00123] In some instances, about 5 to about 100% of the 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. 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 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. 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 a polynucleic acid molecule of SEQ ID NOs: 1-20 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 a polynucleic acid molecule of SEQ ID NOs: 21-64 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 a polynucleic acid molecule of SEQ ID NOs: 65-108 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 a polynucleic acid molecule of SEQ ID NOs: 109- 152 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 a polynucleic acid molecule of SEQ ID NOs: 153-196 comprise the artificial nucleotide analogues described herein. In some aspects, 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, or a combination thereof. [00124] In some cases, 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. In some instances, 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, or combinations thereof 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. In some instances, 2’-O-methyl modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance). In some instances, 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). In some instances, 2’-O-aminopropyl modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance). In some instances, 2'-deoxy modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance). In some instances, 2’-deoxy-2'-fluoro modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5 ’-3’ exonuclease or 3 ’-5’ exonuclease resistance). In some instances, 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). In some instances, 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). In some instances, 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). In some instances, 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). In some instances, 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). In some instances, LNA modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance). In some instances, ENA modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance). In some instances, 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). In some instances, PNA modified polynucleic acid molecule is resistant to nucleases (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance). In some instances, methylphosphonate nucleotides modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5 ’-3’ exonuclease or 3’-5’ exonuclease resistance). In some instances, thiolphosphonate nucleotides modified polynucleic acid molecule is nuclease resistance (e.g., RNase H, DNase, 5’-3’ exonuclease or 3’-5’ exonuclease resistance). In some instances, 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). In some instances, the 5’ conjugates described herein inhibit 5 ’-3’ exonucleolytic cleavage. In some instances, the 3’ conjugates described herein inhibit 3’-5’ exonucleolytic cleavage.

[00125] In some aspects, 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, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2’-fluoro N3-P5’-phosphoramidites can have increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2’-O- methyl modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 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. In some instances, 2’-O-aminopropyl modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2'-deoxy modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 2’- deoxy-2'-fluoro modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 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. In some instances, 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. In some instances, 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. In some instances, 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. In some instances, 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. In some instances, LNA modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, ENA modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, PNA modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, HNA modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, morpholino modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, methylphosphonate nucleotides modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, thiolphosphonate nucleotides modified polynucleic acid molecule has increased binding affinity toward their mRNA target relative to an equivalent natural polynucleic acid molecule. In some instances, 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. In some cases, the increased affinity is illustrated with a lower Kd, a higher melt temperature (Tm), or a combination thereof.

[00126] In some aspects, 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. In some instances, the polynucleic acid molecule comprises L-nucleotide. In some instances, the polynucleic acid molecule comprises D-nucleotides. In some instance, a polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of its mirror enantiomer. In some cases, a polynucleic acid molecule composition comprises less than 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or less of a racemic mixture. In some instances, 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. [00127] In some aspects, a polynucleic acid molecule described herein is further modified to include an aptamer conjugating moiety. In some instances, the aptamer conjugating moiety is a DNA aptamer conjugating moiety. In some instances, 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. In some instance, 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.

[00128] In additional aspects, a polynucleic acid molecule described herein is modified to increase its stability. In some embodiment, the polynucleic acid molecule is RNA (e.g., siRNA), the polynucleic acid molecule is modified to increase its stability. In some instances, the polynucleic acid molecule is modified by one or more of the modifications described above to increase its stability. In some cases, 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). In some cases, 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.

[00129] In some aspects, a polynucleic acid molecule describe herein has RNAi activity that modulates expression of RNA encoded by a gene described supra. In some instances, 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. In some cases, 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. In some cases, 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. In some instances, the gene is SARS-COV or SARS-COV-2 associated genes or gene products.

[00130] In some aspects, a polynucleic acid molecule described herein is constructed using chemical synthesis and/or enzymatic ligation reactions using procedures known in the art. For example, 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. "ENA oligonucleotides as therapeutics". Current opinion in molecular therapeutics 8 (2): 144-149 (2006); and Abramova et al., “Novel oligonucleotide analogues based on morpholino nucleoside subunits-anti sense technologies: new chemical possibilities,” Indian Joumal of Chemistry 48B: 1721-1726 (2009). Alternatively, 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).

Conjugation Chemistry

[00131] In some aspects, a polynucleic acid molecule is conjugated to a binding moiety. In some instances, 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. In some instances, the polynucleic acid molecule is further conjugated to a polymer, and optionally a cellpenetrating or endosomolytic moiety.

[00132] In some aspects, 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. Natl. Acad. Sci. USA 1999, 96, 10068-10073; or Wu, et al. “Building complex glycopeptides: Development of a cysteine-free native chemical ligation protocol,” Angew. Chem. Int. Ed. 2006, 45, 4116-4125. In some instances, the conjugation is as described in U.S. Patent No. 8,936,910. In some aspects, the polynucleic acid molecule is conjugated to the binding moiety either site-specifically or non- specifically via native ligation chemistry.

[00133] In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing a “traceless” coupling technology (Philochem). In some instances, 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. (see Casi et al., “Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery,” JACS 134(13): 5887-5892 (2012)) [00134] In some instances, 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. In some instances, the unnatural amino acid comprises /?-acetylphenylalanine (pAcPhe). In some instances, 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)).

[00135] In some instances, the polynucleic acid molecule is conjugated to the binding moiety by a site-directed method utilizing an enzyme-catalyzed process. In some instances, the site-directed method utilizes SMARTag™ technology (Redwood). In some instances, the SMART ag™ 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, (see Wu et al., “Sitespecific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag,” PNAS 106(9): 3000-3005 (2009); Agarwal, et al., “A Pictet- Spengler ligation for protein chemical modification,” PNAS 110(1): 46-51 (2013))

[00136] In some instances, the enzyme-catalyzed process comprises microbial transglutaminase (mTG). In some cases, the polynucleic acid molecule is conjugated to the binding moiety utilizing a microbial transglutaminze catalyzed process. In some instances, 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. In some instances, 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))

[00137] In some instances, 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.

[00138] In some instances, 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.

Binding Moiety

[00139] In some aspects, the binding moiety A is a polypeptide. In some instances, the polypeptide is an antibody or antigen fragment thereof. In some cases, the fragment is an antigen binding fragment. In some instances, 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.

[00140] In some instances, A is an antibody or antigen binding fragment thereof. In some instances, 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. In some instances, 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).

[00141] In some aspects, the binding moiety A is a bispecific antibody or antigen binding fragment thereof. In some instances, the bispecific antibody is a trifunctional antibody or a bispecific mini -antibody. In some cases, the bispecific antibody is a trifunctional antibody. In some instances, the trifunctional antibody is a full length monoclonal antibody comprising binding sites for two different antigens. Additional exemplary trifunctional antibodies include mAb² from F-star Biotechnology Ltd. In some instances, A is a bispecific trifunctional antibody.

[00142] In some cases, the bispecific antibody is a bispecific mini-antibody. In some instances, 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). In some aspects, 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.

[00143] In some aspects, the binding moiety A is a bispecific mini-antibody. In some instances, A is a bispecific Fab2. In some instances, A is a bispecific F(ab)'3 fragment. In some cases, A is a bispecific bis-scFv. In some cases, A is a bispecific (scFv)2. In some aspects, A is a bispecific diabody. In some aspects, A is a bispecific minibody. In some aspects, A is a bispecific triabody. In other aspects, A is a bispecific tetrabody. In other aspects, A is a bi-specific T-cell engager (BiTE). In additional aspects, A is a bispecific mini-antibody selected from: DART (dual-affinity re-targeting platform; MacroGenics), and domain antibodies (dAbs from Domantis/GSK).

[00144] In some aspects, the binding moiety A is a trispecific antibody. In some instances, the trispecific antibody comprises F(ab)'3 fragments or a triabody. In some instances, A is a trispecific F(ab)'3 fragment. In some cases, A is a triabody. In some aspects, 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).

[00145] In some aspects, the binding moiety A is an antibody or antigen binding fragment thereof that recognizes a cell surface protein. In some instances, the cell surface protein comprises clusters of differentiation (CD) cell surface markers. Exemplary 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, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CDw60, CD61, CD62E, CD62L (L-selectin), CD62P, CD63, CD64, CD65, CD66a, CD66b, CD66c, CD66d, CD66e, CD71, CD79 (e.g., CD79a, CD79b), CD90, CD95 (Fas), CD103, CD104, CD125 (IL5RA), CD134 (0X40), CD137 (4-1BB), CD152 (CTLA-4), CD221, CD274, CD279 (PD-1), CD319 (SLAMF7), CD326 (EpCAM), and the like.

[00146] In some aspects, 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. In some instances, 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. In some instances, the polymer C comprises polyalkylen oxide (e.g., polyethylene glycol). In some aspects, the cell-penetrating or endosomolytic moiety D comprises INF7 or melittin, or their respective derivatives.

[00147] In some aspects, 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.

[00148] In some aspects, 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. In some instances, 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.

[00149] In some aspects, one or more polynucleic acid molecule (B) is conjugated to a binding moiety A. In some instances, 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. In some instances, about 1 polynucleic acid molecule is conjugated to one binding moiety A. In some instances, about 2 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 3 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 4 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, about 5 polynucleic acid molecules are conjugated to one binding moiety A. In some instances, 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. In some instances, about 14 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.

[00150] In some aspects, the number of polynucleic acid molecule (B) conjugated to a binding moiety A forms a ratio. In some instances, 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). In some instances, 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. In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety A is about 1 or greater. In some instances, 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. In some instances, 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.

[00151] In some instances, 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. In some instances, 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. In some instances, 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.

[00152] In some instances, the DAR ratio of the polynucleic acid molecule (B) to binding moiety

A is 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 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.

[00153] In some aspects, 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. In some instances, the modification further comprises a modification for modulating interaction with Fc receptors. In some instances, 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.

[00154] In some instances, an antibody binding fragment further encompasses its derivatives and includes polypeptide sequences containing at least one CDR.

[00155] In some instances, the term “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.

[00156] In some instances, a bispecific single chain antibody construct relates to a construct comprising two antibody derived binding domains. In such embodiments, bi-specific single chain antibody construct is tandem bi-scFv or diabody. In some instances, a scFv contains a VH and VL domain connected by a linker peptide. In some instances, 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.

[00157] In some embodiments, binding to or interacting with as used herein defines a binding/interaction of at least two antigen-interaction-sites with each other. In some instances, 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. In some cases, the binding/interaction is also understood to define a specific recognition. In such cases, 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. For example, specific recognition relates to the specificity of the antibody molecule, or to its ability to discriminate between the specific regions of a target molecule. In additional instances, 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. In further embodiments, the binding is exemplified by the specificity of a "key -lock-principle". Thus in some instances, 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. In such cases, 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.

[00158] In some instances, specific interaction further refers to a reduced cross-reactivity of the antibody or its binding fragment or a reduced off-target effect. For example, 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.

[00159] In some embodiments, 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.

Additional Binding Moieties

[00160] In some aspects, the binding moiety is a plasma protein. In some instances, the plasma protein comprises albumin or transferrin. In some instances, the binding moiety A is albumin. In some instances, the binding moiety A is transferrin. In some instances, transferrin is conjugated by one or more of a conjugation chemistry described herein to a polynucleic acid molecule. In some instances, transferrin is conjugated by native ligation chemistry to a polynucleic acid molecule. In some instances, transferrin is conjugated by lysine conjugation to a polynucleic acid molecule.

[00161] In some instances, the binding moiety is a steroid. Exemplary steroids include cholesterol, phospholipids, di-and triacylglycerols, fatty acids, hydrocarbons that are saturated, unsaturated, comprise substitutions, or combinations thereof. In some instances, the steroid is cholesterol. In some instances, the binding moiety is cholesterol. In some instances, cholesterol is conjugated by one or more of a conjugation chemistry described herein to a polynucleic acid molecule. In some instances, cholesterol is conjugated by native ligation chemistry to a polynucleic acid molecule. In some instances, cholesterol is conjugated by lysine conjugation to a polynucleic acid molecule.

[00162] In some instances, the binding moiety is a polymer, including but not limited to poly nucleic acid molecule aptamers that bind to specific surface markers on cells. In this instance 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.

[00163] In some cases, the binding moiety is a peptide. In some cases, 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. In some instances, 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).

[00164] In additional cases, the binding moiety is a small molecule. In some instances, the small molecule is an antibody-recruiting small molecule. In some cases, 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. For example, in some instances, 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. In some instances, 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

[00165] In some aspects, polypeptides described herein (e.g., antibodies and antigen binding fragments) 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.

[00166] In some instances, 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.

[00167] Alternatively, 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.

[00168] In some instances, 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). Alternatively, 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).

[00169] In some aspects, 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. [00170] In some aspects, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide. Techniques for the assembly of functional Fv fragments in A", coll are also optionally used (Skerra et al., 1988, Science 242: 1038-1041).

[00171] In some aspects, 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. In specific aspects, the expression of the antibody is regulated by a constitutive, an inducible or a tissue, specific promoter.

[00172] In some aspects, a variety of host-expression vector systems is utilized to express an antibody or its binding fragment described herein. Such 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. These include, but are not limited to, 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 promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g. the adenovirus late promoter; the vaccinia virus 7.5K promoter).

[00173] In some instances, 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.

Polymer Conjugating Moiety

[00174] In some aspects, 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.

[00175] In some instances, 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. In some instances, the polymer moiety C includes a polysaccharide, lignin, rubber, or polyalkylen oxide (e.g., polyethylene glycol). In some instances, 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. As used herein, a mixture refers to the use of different polymers within the same compound as well as in reference to block copolymers. In some cases, block copolymers are polymers wherein at least one section of a polymer is build up from monomers of another polymer. In some instances, the polymer moiety C comprises polyalkylene oxide. In some instances, the polymer moiety C comprises PEG. In some instances, the polymer moiety C comprises polyethylene imide (PEI) or hydroxy ethyl starch (HES).

[00176] In some instances, C is a PEG moiety. In some instances, 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. In some instances, 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. In some instances, the PEG moiety is conjugated to an internal site of the polynucleic acid molecule. In some instances, the PEG moiety, the binding moiety, or a combination thereof, are conjugated to an internal site of the polynucleic acid molecule. In some instances, the conjugation is a direct conjugation. In some instances, the conjugation is via native ligation. [00177] In some aspects, the polyalkylene oxide (e.g., PEG) is a polydispers or monodispers compound. In some instances, polydispers material comprises disperse distribution of different molecular weight of the material, characterized by mean weight (weight average) size and dispersity. In some instances, the monodisperse PEG comprises one size of molecules. In some aspects, 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.

[00178] In some aspects, the molecular weight of the polyalkylene oxide (e.g., PEG) 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.

[00179] In some aspects, 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. In some aspects, 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. In some instances, 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. In some instances, 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. In some instances, 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. In some instances, 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. In some instances, 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):

Formula IV

[00181] wherein m is independently at each occurrence 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, preferably 4- 6 or 5; and n is independently at each occurrence 1, 2, 3, 4, or 5. In some aspects, m and n are, for example, about 10. [00182] In some instances, 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. In some instances, the PEG moiety is in a range of from about 500 Da to about 50,000 Da. In some instances, 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. [00183] In some instances, the polymer moiety C is cMAP-PEG copolymer, an mPEG-cMAP- PEGm triblock polymer, or a cMAP-PEG-cMAP triblock polymer. In some cases, the polymer moiety C is cMAP-PEG copolymer. In other cases, the polymer moiety C is an mPEG-cMAP- PEGm triblock polymer. In additional cases, the polymer moiety C is a cMAP-PEG-cMAP triblock polymer.

Endosomolytic Moiety

[00184] In some aspects, a molecule of Formula (I), Formula (II), or Formula (III) further comprises an additional conjugating moiety. In some instances, the additional conjugating moiety is a cell -penetrating or endosomolytic moiety. In some cases, 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. In some cases, 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. In some cases, the cell-penetrating or endosomolytic moiety comprises a cell-penetrating or endosomolytic polypeptide. In other cases, the cell-penetrating or endosomolytic moiety comprises a cellpenetrating or endosomolytic polymer.

Cell Penetrating and Endosomolytic Polypeptides

[00185] In some aspects, a molecule of Formula (I), Formula (II), or Formula (III) is further conjugated with a cell-penetrating or endosomolytic polypeptide. In some cases, the cellpenetrating or endosomolytic polypeptide is a pH-dependent membrane active peptide. In some cases, the cell-penetrating or endosomolytic polypeptide is an amphipathic polypeptide. In additional cases, the cell-penetrating or endosomolytic polypeptide is a peptidomimetic. In some instances, the cell-penetrating or endosomolytic polypeptide comprises INF, melittin, meucin, or their respective derivatives thereof. In some instances, 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.

Cell Penetrating and Endosomolytic Polymers

[00186] In some aspects, a molecule of Formula (I), Formula (II), or Formula (III) is further conjugated with a cell-penetrating or endosomolytic polymer. As used herein, a cell-penetrating or endosomolytic polymer comprises a linear, a branched network, a star, a comb, or a ladder type of polymer. In some instances, a cell-penetrating or endosomolytic polymer is a homopolymer or a copolymer comprising two or more different types of monomers. In some cases, a cell-penetrating or endosomolytic polymer is a polycation polymer. In other cases, a cell-penetrating or endosomolytic polymer is a polyanion polymer.

[00187] In some instances, a polycation polymer comprises monomer units that are charge positive, charge neutral, or charge negative, with a net charge being positive. In other cases, 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).

[00188] In some cases, a polyanion polymer comprises monomer units that are charge positive, charge neutral, or charge negative, with a net charge being negative. In other cases, 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).

[00189] In some aspects, 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.

[00190] In some instances, a cell-penetrating or endosomolytic moiety described herein is a membrane-disruptive polymer. In some cases, the membrane-disruptive polymer comprises a cationic polymer, a neutral or hydrophobic polymer, or an anionic polymer. In some instances, the membrane-disruptive polymer is a hydrophilic polymer. [00191] In some instances, 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.

[00192] In some instances, p(alkylacrylic acids) include poly(propylacrylic acid) (polyP AA), poly(methacrylic acid) (PMAA), poly(ethylacrylic acid) (PEAA), and poly(propyl acrylic acid) (PPAA). In some instances, a p(alkylacrylic acid) include a p(alkylacrylic acid) described in Jones, et al., Biochemistry Journal 372: 65-75 (2003).

[00193] In some aspects, 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))

[00194] In some aspects, a pH-responsive membrane-disruptive polymer comprises p(styrene-alt- maleic anhydride), (see Henry, et al., Biomacromolecules 7: 2407-2414 (2006))

[00195] In some aspects, 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))

[00196] In some aspects, a pH-responsive membrane-disruptive polymer comprises a lytic polymer comprising the base structure of:

[00197] In some instances, 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).

[00198] In some instances, the additional conjugate comprises a detergent (e.g., Triton X-100). In some instances, 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). In some instances, 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)).

Cell Penetrating and Endosomolytic Lipids

[00199] In some aspects, the cell-penetrating or endosomolytic moiety is a lipid (e.g., a fusogenic lipid). In some aspects, 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).

[00200] In some instances, a cell-penetrating or endosomolytic moiety is a lipid (e.g., a fusogenic lipid) described in PCT Publication No. WO09/126,933.

Cell Penetrating and Endosomolytic Small Molecules

[00201] In some aspects, the cell-penetrating or endosomolytic moiety is a small molecule. In some aspects, 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. In some instances, 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-butylamino)quinoline); 4-(4-diethyl-amino- l-butylamino)quinoline; 7-hydroxy-4-(4-diethylamino-l-butylamino)quinoline; 7-chloro-4-(l- carboxy-4-diethylamino-l-butylamino)quinoline; 7-fluoro-4-(l -carboxy -4-diethyl-amino-l- butylamino)quinoline; 4-(l-carboxy-4-di ethylamino- 1 -butylamino) quinoline; 7-hydroxy-4-(l- carboxy-4-diethylamino-l-butylamino)quinoline; 7-chloro-4-(l -carboxy -4-di ethylamino- 1- methylbutylamino)quinoline; 7-fluoro-4-( 1 -carboxy -4-di ethyl-amino- 1 - methylbutylamino)quinoline; 4-(l-carboxy-4-diethylamino-l-methylbutylamino)quinoline; 7- hydroxy-4-(l -carboxy -4-di ethylamino- l-methylbutylamino)quinoline; 7-fluoro-4-(4-ethyl-(2- hydroxyethyl)-amino-l-methylbutylamino)quinoline; 4-(4-ethyl-(2-hydroxy-ethyl)-amino-l- methylbutylamino-)quinoline; 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino- 1 - methylbutylamino)quinoline; hydroxychloroquine phosphate; 7-chloro-4-(4-ethyl-(2-hydroxyethyl- l)-amino-l-butylamino)quinoline (desmethylhydroxychloroquine); 7-fluoro-4-(4-ethyl-(2- hydroxyethyl)-amino- 1 -butylamino)quinoline; 4-(4-ethyl-(2-hydroxyethyl)-amino- 1 - butylamino)quinoline; 7-hydroxy-4-(4-ethyl-(2-hydroxyethyl)-amino-l-butylamino) quinoline; 7- chloro-4-(l -carboxy -4-ethyl-(2-hydroxyethyl)-amino-l-butylamino)quinoline; 7-fluoro-4-(l- carboxy-4-ethyl-(2-hydroxyethyl)-amino-l-butylamino)quinoline; 4-(l -carboxy -4-ethyl-(2- hydroxyethyl)-amino- 1 -butylamino)quinoline; 7-hydroxy-4-(l -carboxy-4-ethyl-(2-hy droxy ethylamino- l-butylamino)quinoline; 7-chloro-4-(l -carboxy -4-ethyl-(2-hy droxy ethyl)-amino-l- methylbutylamino)quinoline; 7-fluoro-4-(l -carboxy -4-ethyl-(2 -hydroxy ethyl)-amino-l- methylbutylamino)quinoline; 4-(l -carboxy -4-ethyl -(2 -hydroxy ethyl)-amino- 1 - methylbutylamino)quinoline; 7-hydroxy-4-(l -carboxy-4-ethyl-(2 -hydroxy ethyl)-amino- 1 - methylbutylamino)quinoline; 8-[(4-aminopentyl)amino-6-methoxy dihydrochloride quinoline; 1- acetyl-l,2,3,4-tetrahydroquinoline; 8-[(4-aminopentyl)amino]-6-m ethoxy quinoline dihydrochloride; 1 -butyryl- 1,2, 3, 4-tetrahydroquinoline; 3-chloro-4-(4-hydroxy-alpha,alpha'-bis(2- methyl-l-pyrrolidinyl)-2,5-xylidinoquinoline, 4-[(4-di ethyl -amino)-l -methylbutyl-amino]-6- methoxy quinoline; 3-fluoro-4-(4-hydroxy-alpha,alpha'-bis(2-methyl-l-pyrrolidinyl)-2,5- xylidinoquinoline, 4- [(4-di ethylamino)- 1 -methylbutyl-amino]-6-m ethoxy quinoline; 4-(4-hy droxy - alpha,alpha'-bis(2-methyl-l-pyrrolidinyl)-2,5-xylidinoquinoline; 4-[(4-diethylamino)-l- methylbutyl-amino]-6-methoxy quinoline; 3,4-dihydro-l-(2H)-quinolinecarboxyaldehyde; 1,1'- pentamethylene diquinoleinium diiodide; 8-quinolinol sulfate and amino, aldehyde, carboxylic, hydroxyl, halogen, keto, sulfhydryl and vinyl derivatives or analogs thereof. In some instances, 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.

Linkers

[00202] In some aspects, a linker described herein is a cleavable linker or a non-cleavable linker. In some instances, the linker is a cleavable linker. In some instances, the linker is an acid cleavable linker. In some instances, the linker is a non-cleavable linker. In some instances, the linker includes a Ci-Ce alkyl group (e.g., a Cs, C4, C3, C2, or Ci alkyl group). In some instances, the linker includes homobifunctional cross linkers, heterobifunctional cross linkers, and the like. In some instances, 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-3 '- (2'-pyridyldithio)propionamido)butane (DPDPB), bismaleimidohexane (BMH), aryl halide- containing compound (DFDNB), such as e.g. l,5-difluoro-2,4-dinitrobenzene or l,3-difluoro-4,6- dinitrobenzene, 4, 4'-difluoro-3, 3 '-dinitrophenyl sulfone (DFDNPS), bis-[|3-(4- azidosalicylamido)ethyl]disulfide (BASED), 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).

[00203] In some aspects, 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-LC-sMPT), succinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate (sMCC), sulfosuccinimidyl-4-(N- maleimidomethyl)cyclohexane- 1 -carboxylate (sulfo-sMCC), m-maleimidobenzoyl-N- hydroxysuccinimide ester (MBs), m-maleimidobenzoyl-N-hydroxysulfosuccinimide ester (sulfo- MBs), N-succinimidyl(4-iodoacteyl)aminobenzoate (sIAB), sulfosuccinimidyl(4- iodoacteyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y- maleimidobutyryloxy)succinimide ester (GMBs), N-(y-maleimidobutyryloxy)sulfosuccinimide ester (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino)hexanoate (sIAX), succinimidyl 6-[6- (((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4- (((iodoacetyl)amino)methyl)cyclohexane-l -carboxylate (sIAC), succinimidyl 6-((((4- iodoacetyl)amino)methyl)cyclohexane-l-carbonyl)amino) hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl -reactive and sulfhydryl-reactive cross-linkers such as 4-(4-N- maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-l- carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive cross-linkers such as N-hydroxysuccinimidyl-4-azidosalicylic acid (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylic acid (sulfo-NHs-AsA), sulfosuccinimidyl-(4- azidosalicylamido)hexanoate (sulfo-NHs-LC-AsA), sulfosuccinimidyl-2-(p- azidosalicylamido)ethyl- 1 ,3 '-dithiopropionate (sAsD), N-hydroxy succinimidyl-4-azidobenzoate (HsAB), N-hydroxysulfosuccinimidyl-4-azidobenzoate (sulfo-HsAB), N-succinimidyl-6-(4'-azido- 2'-nitrophenylamino)hexanoate (sANPAH), sulfosuccinimidyl-6-(4'-azido-2'- nitrophenylamino)hexanoate (sulfo-sANPAH), N-5-azido-2-nitrobenzoyloxysuccinimide (ANB- NOs), sulfosuccinimidyl-2-(m-azido-o-nitrobenzamido)-ethyl-l,3'-dithiopropionate (sAND), N- succinimidyl-4(4-azidophenyl) 1,3 '-dithiopropionate (sADP), N-sulfosuccinimidyl(4-azidophenyl)- 1,3 '-dithiopropionate (sulfo-sADP), sulfosuccinimidyl 4-(p-azidophenyl)butyrate (sulfo-sAPB), sulfosuccinimidyl 2-(7-azido-4-methylcoumarin-3-acetamide)ethyl-l, 3 '-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumain-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydrylreactive and photoreactive cross-linkers such asl-(p-Azidosalicylamido)-4-(iodoacetamido)butane (AsIB), N-[4-(p-azidosalicylamido)butyl]-3 '-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimide carbonyl -reactive and photoreactive cross-linkers such as p-azidobenzoyl hydrazide (ABH), carboxylate-reactive and photoreactive cross-linkers such as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive cross-linkers such as p-azidophenyl glyoxal (APG).

[00204] In some instances, the linker comprises a reactive functional group. In some cases, the reactive functional group comprises a nucleophilic group that is reactive to an electrophilic group present on a binding moiety. Exemplary electrophilic groups include carbonyl groups — such as aldehyde, ketone, carboxylic acid, ester, amide, enone, acyl halide or acid anhydride. In some aspects, the reactive functional group is aldehyde. Exemplary nucleophilic groups include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide. [00205] In some aspects, the linker comprises a maleimide group. In some instances, 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). In other instances, 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.

[00206] In some aspects, the maleimide group is a self-stabilizing maleimide. In some instances, 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. In some instances, 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). In some instances, the linker comprises a self-stabilizing maleimide. In some instances, the linker is a self-stabilizing maleimide.

[00207] In some aspects, the linker comprises a peptide moiety. In some instances, the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some instances, the peptide moiety is a cleavable peptide moiety (e.g., either enzymatically or chemically). In some instances, the peptide moiety is a non-cleavable peptide moiety. In some instances, 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. In some instances, 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. In some cases, the linker comprises Val-Cit. In some cases, the linker is Val-Cit.

[00208] In some aspects, the linker comprises a benzoic acid group, or its derivatives thereof. In some instances, the benzoic acid group or its derivatives thereof comprise paraaminobenzoic acid (PABA). In some instances, the benzoic acid group or its derivatives thereof comprise gamma- aminobutyric acid (GABA).

[00209] In some aspects, 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.

[00210] In some aspects, 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.

[00211] In some aspects, the linker is a dendritic type linker. In some instances, the dendritic type linker comprises a branching, multifunctional linker moiety. In some instances, the dendritic type linker is used to increase the molar ratio of polynucleotide B to the binding moiety A. In some instances, the dendritic type linker comprises PAMAM dendrimers.

[00212] In some aspects, 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. 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. In some cases, 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). In some instances, the linker is a traceless linker described in Blaney, et al., “Traceless solid-phase organic synthesis,” Chem. Rev. 102: 2607-2024 (2002). In some instances, a linker is a traceless linker as described in U.S. Patent No. 6,821,783.

[00213] In some instances, 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). In some instances, 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. In some cases, a linker that exhibits steric hinderance comprises SMCC and SPDB.

[00214] In some instances, the linker is an acid cleavable linker. In some instances, the acid cleavable linker comprises a hydrazone linkage, which is susceptible to hydrolytic cleavage. In some cases, the acid cleavable linker comprises a thiomaleamic acid linker. In some cases, 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).

[00215] In some instances, 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. [00216] In some aspects, 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.

[00217] In some instances, 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. In some instances, 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.

[00218] In some instances, 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. In some instances, Y comprises a benzoic acid group, such as PABA. In additional instances, Y comprises a combination of a maleimide group, a peptide moiety, and/or a benzoic acid group. In additional instances, Y comprises a me group. In additional instances, Y comprises a mc-val-cit group. In additional instances, Y comprises a val-cit-PABA group. In additional instances, Y comprises a mc-val-cit-PABA group.

[00219] In some instances, 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. In some instances, L comprises a benzoic acid group, such as PABA. In additional instances, L comprises a combination of a maleimide group, a peptide moiety, and/or a benzoic acid group. In additional instances, L comprises a me group. In additional instances, L comprises a mc-val-cit group. In additional instances, L comprises a val-cit-PABA group. In additional instances, L comprises a mc-val-cit-PABA group.

Methods of Use

[00220] In some aspects, 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. In some instances, the disease or disorder is COVID-19. In some instances, the disease or disorder is a cardiomyopathy or heart failure associated with COVID-19. In some aspects, 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.). In some aspects, 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.

[00221] In some aspects, 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. In some cases, 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.

[00222] In some aspects, the composition or a pharmaceutical formulation described herein is used in conjunction with a vaccine. In some instances, the vaccine is an in situ vaccination. In some instances, the vaccine is a cell-based vaccine. In some instances, the vaccine is a non-cell based vaccine. In some instances, 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). In some instances, 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).

Pharmaceutical Formulation

[00223] In some aspects, 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. In some instances, the pharmaceutical composition describe herein is formulated for parenteral (e.g., intravenous, subcutaneous, intramuscular) administration. In other instances, the pharmaceutical composition describe herein is formulated for oral administration. In still other instances, the pharmaceutical composition describe herein is formulated for intranasal or pulmonary inhalation administration.

[00224] In some aspects, 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.

[00225] In some instances, the pharmaceutical formulation includes multiparticulate formulations. In some instances, the pharmaceutical formulation includes nanoparticle formulations. In some instances, nanoparticles comprise cMAP, cyclodextrin, or lipids. In some cases, nanoparticles comprise solid lipid nanoparticles, polymeric nanoparticles, self-emulsifying nanoparticles, liposomes, microemulsions, or micellar solutions.

[00226] In some instances, a nanoparticle includes a core or a core and a shell, as in a core-shell nanoparticle. In some instances, 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).

[00227] 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. 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.

[00228] 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.

Therapeutic Regimens

[00229] In some aspects, the pharmaceutical compositions described herein are administered for therapeutic applications. In some aspects, the pharmaceutical composition is administered once per day, twice per day, three times per day or more. In some aspects, 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. In some aspects 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. [00230] In some aspects, 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).

[00231] In some aspects, 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.

[00232] In the case wherein the patient’s status does improve, upon the doctor’s discretion 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”). In some instances, 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%.

[00233] Once improvement of the patient's conditions has occurred, 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.

[00234] In some aspects, 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. In some instances, 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.

[00235] The foregoing ranges are merely suggestive, as the number of variables in regard to an individual treatment regime is large, and considerable excursions from these recommended values are not uncommon. Such dosages is altered depending on a number of variables, not limited to the activity of the compound used, the disease or condition to be treated, the mode of administration, the requirements of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

[00236] In some aspects, 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/Article of Manufacture

[00237] Disclosed herein, in certain aspects, are 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. In one embodiment, the containers are formed from a variety of materials such as glass or plastic. [00238] 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.

[00239] For example, the container(s) include a molecule of Formula (I), Formula (II), or Formula (III) as disclosed herein. Such kits optionally include an identifying description or label or instructions relating to its use in the methods described herein. [00240] 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.

[00241] In one embodiment, a label is on or associated with the container. In one embodiment, 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. In one embodiment, 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.

[00242] In certain aspects, 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. In one embodiment, the pack or dispenser device is accompanied by instructions for administration. In one embodiment, 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. In one embodiment, 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.

Certain Terminology

[00243] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of any subject matter claimed. In this application, the use of the singular includes the plural unless specifically stated otherwise. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. In this application, the use of “or” means “and/or” unless stated otherwise. Furthermore, use of the term “including” as well as other forms, such as “include”, “includes,” and “included,” is not limiting. [00244] As used herein, 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.

[00245] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[00246] As used herein, the terms “individual(s)”, “subject(s)” and “patient(s)” mean any mammal. In some aspects, the mammal is a human. In some aspects, 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).

[00247] As used herein, 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. [00248] The term “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.

[00249] “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. EXAMPLES

[00250] These examples are provided for illustrative purposes only and not to limit the scope of the claims provided herein.

Example 1. Sequences

[00251] 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.

TABLE 1: Target SARS-CoV and SARS-CoV-2 sequences for selection of oligonucleotides for RNAi (antisense RNA, siRNA, or PMO)

[00252] Comparative qPCR assay for determination of mRNA knockdown

[00253] 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) is used as an internal RNA loading control, with all TaqMan primers and probes for PPIB purchased from Applied Biosystems as prevalidated gene expression assays. 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).

[00254] 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.

Example 2: Synthesis, purification and analysis of conjugates of Formula (I)

[00255] Step 1: Antibody conjugation with maleimide-PEG-NHS followed by SH-TFR1

[00256] 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, is exchanged with IX Phosphate buffer (pH 7.4) and made up to 5mg/ml concentration. To this solution, 2 equivalents of SMCC linker or maleimide-PEGxkDa-NHS (x = 1, 5, 10, 20) is added and rotated for 4 hours at room temperature. 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.

[00257] Step 2: Purification

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.

Example 3: Synthesis, purification and analysis of conjugates of Formula (II)

[00258] Step 1: Antibody conjugation with SMCC linker followed by SH-TFRl-PEG5kDa [00259] 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. To this solution, 2 equivalents of SMCC linker (succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate) is added and rotated for 4 hours at room temperature. 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.

[00260] Step 2: Purification

[00261] 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.

Example 4: Synthesis, purification and analysis of additional conjugates of Formula (II) [00262] Step 1: Antibody conjugation with SPDP linker followed by SH-siRNA-PEG5kDa [00263] 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. To this solution, 2 equivalents of SPDP linker (succinimidyl 3-(2- pyridyldithio)propi onate) is added and rotated for 4 hours at room temperature. 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.

[00264] Step 2: Purification

[00265] 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.

Example 5: Synthesis, purification and analysis of conjugates of Formula (III)

[00266] Step 1: Antibody conjugation with SMCC linker or maleimide-PEG-NHS followed by SH-Cys-Peptide-

[00267] Conjugation scheme-4 for generating Antibody-siRNA conjugates is depicted in FIG. 7. 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 lOmg/ml concentration. To this solution, 3 equivalents of SMCC linker (succinimidyl 4-(N- maleimidomethyl)cyclohexane-l -carboxylate) or maleimide-PEGlkDa-NHS is added and rotated for 1.5 hours at room temperature. Unreacted SMCC linker or PEG linker is removed by spin filtration using 50 kDa MWCO Amicon spin filters and PBS buffer pH 7.4 (25mM MES pH=6.1 for Melittin conjugates). The retentate is collected and 3 equivalents of SH-Cys-Peptide-CONH2 is added at RT and rotated overnight. The reaction mixture is then purified by either HIC chromatography or cation exchange chromatography to isolate the antibody-Peptide or antibody- PEGlk-Peptide.

[00268] Step 2: Purification

[00269] The crude reaction mixture is purified by AKTA explorer FPLC using either hydrophobic interaction chromatography (HIC) method- 1 or cation exchange chromatography method- 1. Fractions containing the antibody-peptide conjugates are pooled, concentrated and buffer exchanged with PBS, pH 7.4 (10 mM Acetate pH=6.0 for Melittin conjugates).

Example 6: Synthesis, purification and analysis of additional conjugates of Formula (III) [00270] Step 1: Conjugation of PEG24 linker followed by SH-Cys-Peptide- to TFR1-

Ab-siRNA-PEG

[00271] 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.

[00272] Step 2: Purification

[00273] 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.

[00274] Step-3: Analysis of the purified conjugate

[00275] 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

[00276] 19 mer oligo sequences based on SASR-CoV-2 (NC_045512v2) were compared with

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).

TABLE 2: Selected SARS-CoV-2 siRNAs

TABLE 3: Modified selected SARS-CoV-2 siRNAs

upper case (N) = 2'-OH (ribo); lower case (n) = 2'-O-Me (methyl); Nf = 2'-F (fluoro); s = phosphorothioate backbone modification .

Example 8: In vitro screening of 44 SARS-CoV-2 siRNAs

[00277] SARS-CoV-2, strain USA-WA1/2020, was provided by the World Reference Center for

Emerging Viruses and Arboviruses (WRCEVA) and passaged three times in Vero 76 cells to prepare stock. The antiviral assay was performed in Vero E6 cells using test media of MEM supplemented with 2% FBS and 50 pg/mL gentamicin.

[00278] Stocks of modified siRNA in 50 pM solutions were serially diluted in Opti-MEM, so that the final high concentration of 100 nM for each siRNA, which were transfected into the VERO E6 cells before viral infections. On day 3 post-infection, once untreated virus control wells reached maximum CPE, plates were stained with neutral red dye for 2 hours. In vitro antiviral results are shown in Table 4.

TABLE 4. In vitro antiviral activity of siRNA samples against SARS-CoV-2. NA = not available

[00279] Results

[00280] Sample DI (COV2-37) showed the most activity against SARS-CoV-2 (SI>3) at a low concentration as shown in Table 4. Samples Al (COV2-1) and B5 (COV2-17) exhibited some inhibition of virus at higher concentrations Other samples did not exhibit any in vitro antiviral activity against SARS-CoV-2. The positive control compound performed as expected.

Example 9: In Vitro Antiviral Assay against SARS-CoV-2 Virus

[00281] SARS-CoV-2, strain USA-WA1/2020, was provided by the World Reference Center for Emerging Viruses and Arboviruses (WRCEVA) and passaged three times in Vero 76 cells to prepare stock. The antiviral assay was performed in Vero E6 cells using test media of MEM supplemented with 2% FBS and 50 pg/mL gentamicin.

[00282] Stocks of the modified siRNA COV2-17 and COV2-37 in 50 pM solutions were serially diluted in Opti-MEM, so that the final high concentration for each modified-siRNA is 500 nM or 50 nM. 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), and 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). The Lipofectamine 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.

[00283] Three wells of each dilution were infected with SARS-CoV-2 (MOI=0.001), and two wells remained uninfected as toxicity controls. Six wells on each plate were infected and untreated as virus controls, and six wells were uninfected and untreated as cell controls. Remdesivir was tested in parallel as a positive control, treating and infecting cells without transfection methods. Plates were incubated at 37±2°C, 5% CO2. [00284] On day 3 post-infection, once untreated virus control wells reached maximum CPE, plates were stained with a neutral red dye for approximately 2 hours (±15 minutes). The supernatant dye was removed, and wells were rinsed with PBS. 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.

[00285] Results

[00286] 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. Overall, the modified siRNA COV2-17 and COV2-37 showed potent antiviral activity against SARS-CoV-2.

Example 10: Two modified siRNAs decrease Virus yield

[00287] For virus yield reduction assays, 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.

[00288] TABLE 5. In vitro antiviral activity of siRNA samples against SARS-CoV-2.

Units are in nM for siRNA and pM for Remdesivir

ECso: 50% effective antiviral concentration

CCso: 50% cytotoxic concentration of compound without virus added EC90.99.999: Calculated concentration to reduce virus yield ay 1-3 logs SI = CCso/EC

[00289] Results

[00290] 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. Overall, the modified siRNA COV2-17 and COV2-37 decreased antiviral activity against SARS-CoV-2 by reducing the virus yield.

[00291] The examples and aspects described herein are for illustrative purposes only and various modifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

CLAIMS WHAT IS CLAIMED IS:

1. A molecule of Formula (I):

A-(X-B)n

Formula I wherein,

A is a binding moiety;

B is a polynucleotide; n is an integer between 1 and 10; and wherein 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, and wherein the polynucleotide comprises at least one 2’ modified nucleotide, at least one modified intemucleotide linkage, or at least one inverted abasic moiety.

2. The molecule of claim 1, wherein the at least one 2’ modified nucleotide comprises 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- dimethylaminoethyloxy ethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified nucleotide.

3. The molecule of claim 1 or 2, wherein the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA).

4. The molecule of any one of the claims 1-3, wherein the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.

5. The molecule of claim 1, wherein the at least one inverted abasic moiety is at at least one terminus.

6. The molecule of any one of the claims 1-5, wherein the polynucleotide is a single-stranded nucleic acid.

7. The molecule of any one of the claims 1-5, wherein the polynucleotide comprises two or more strands.

8. The molecule of claim 7, wherein the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule.

9. The molecule of claim 8, wherein the second polynucleotide comprises at least one modification.

-74- The molecule of any one of the claims 8-9, wherein the first polynucleotide and the second polynucleotide are RNA molecules. The molecule of any one of the claims 8-10, wherein the first polynucleotide and the second polynucleotide form a double-stranded siRNA molecule. The molecule of any one of the claims 8-11, wherein 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 at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 21-64, and 109-152. The molecule of any one of the claims 8-11, wherein the second polynucleotide consists of a sequence selected from the published sequence at GenBank Accession Number: MN908947.3, or a sequence selected from SEQ ID NOs: 1-20, 65-108, and 153-196. The molecule of any one of the claims 1-13, wherein X and Y are independently a bond or a non-polymeric linker group. The molecule of any one of the claims 1-14, wherein X is a bond, Ci-Ce alkyl group, a homobifuctional linker, or a heterobifunctional linker, optionally conjugated to a Ci-Ce alkyl group. The molecule of any one of the claims 1-15, wherein Y is a Ci-Ce alkyl group, a homobifuctional linker or a heterobifunctional linker. The molecule of any one of the claims 1-16, wherein the binding moiety is an antibody or antigen binding fragment thereof. The molecule of claim 17, wherein 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, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or antigen binding fragment thereof. The molecule of claim 17 or 18, wherein 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. A molecule of Formula (II):

A-(X-B-Y-C)_n

Formula II wherein,

A is a binding moiety;

-75- 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 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, 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 molecule of claim 20, wherein the at least one 2’ modified nucleotide comprises 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- dimethylaminoethyloxy ethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-0-NMA) modified nucleotide. The molecule of claim 20 or 21, wherein the at least one 2’ modified nucleotide comprises locked nucleic acid (LNA) or ethylene nucleic acid (ENA). The molecule of any one of the claims 20-22, wherein the at least one modified intemucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage. The molecule of claim 20, wherein the at least one inverted abasic moiety is at at least one terminus. The molecule of any one of the claims 20-24, wherein the polynucleotide is a single-strand molecule. The molecule of any one of the claims 20-24, wherein the polynucleotide comprises two or more strands. The molecule of claim 26, wherein the polynucleotide comprises a first polynucleotide and a second polynucleotide hybridized to the first polynucleotide to form a double-stranded polynucleic acid molecule. The molecule of claim 27, wherein the second polynucleotide comprises at least one modification. The molecule of any one of the claims 27-28, wherein the first polynucleotide and the second polynucleotide are RNA molecules. The molecule of any one of the claims 27-29, wherein the first polynucleotide and the second polynucleotide form a double-stranded siRNA molecule.

-76- The molecule of any one of the claims 27-30, wherein 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 at GenBank Accession Number: MN908947.3 or a sequence selected from SEQ ID NOs: 1-20, 21-64, and 109-152. The molecule of any one of the claims 27-31, wherein 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 at GenBank Accession Number: MN908947.3 or a sequence selected from SEQ ID NOs: 1-20, 65-108, and 153-196. The molecule of any one of the claims 20-32, wherein X and Y are independently a bond or a non-polymeric linker group. The molecule of any one of the claims 23-33, wherein X is a bond, a Ci-Ce alkyl group, a homobifuctional linker or a heterobifunctional linker, optionally conjugated to a Ci-Ce alkyl group. The molecule of any one of the claims 23-33, wherein Y is a Ci-Ce alkyl group, a homobifuctional linker or a heterobifunctional linker. The molecule of any one of the claims 23-35, wherein the binding moiety is an antibody or antigen binding fragment thereof. The molecule of claim 36, wherein 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, single-chain variable fragment (scFv), diabody, minibody, nanobody, single-domain antibody (sdAb), or camelid antibody or antigen binding fragment thereof. The molecule of claim 36 or 37, wherein the antibody or antigen binding fragment thereof is an anti-TfR.1 antibody, an anti-ACE2 antibody, an anti-TMPRSS2 antibody, or an antigen binding fragment thereof. The molecule of any one of the claims 20-38, wherein C is polyethylene glycol. The molecule of any one of the claims 20-39, wherein C has a molecular weight of about 5000 Da. The molecule of any one of the claims 20-40, wherein A-X is conjugated to the 5’ end of B and Y-C is conjugated to the 3’ end of B. The molecule of any one of the claims 20-41, wherein Y-C is conjugated to the 5’ end of B and A-X is conjugated to the 3’ end of B.

-77- The molecule of any one of the claims 20-42, wherein A-X, Y-C or a combination thereof is conjugated to an intemucleotide linkage group. The molecule of any one of claims 1-43, further comprising L-D, wherein L-D is conjugated to the molecule of Formula (I) or the molecule of Formula (II): wherein,

L is a bond or linker;

D is a cell-penetrating or endosomolytic moiety; and c is an integer between 1 and 10. The molecule of claim 44, wherein D is INF7 or melittin. The molecule of claim 44, wherein D is a cell-penetrating or endosomolytic polymer. The molecule of claim 44, wherein L is a Ci-Ce alkyl group, a homobifuctional linker, or a heterobifunctional linker. The molecule of any one of the claims 1-47, further comprising at least a second binding moiety A. The molecule of claim 48, wherein the at least second binding moiety A is conjugated to A, to B, or to C. The molecule of claim 48 or 49, wherein the at least second binding moiety A is cholesterol. The molecule of any one of the claims 1-50, further comprising at least an additional polynucleotide B. The molecule of claim 51, wherein the at least an additional polynucleotide B is conjugated to A, to B, or to C. The molecule of any one of the claims 1-52, further comprising at least an additional polymer C. The molecule of claim 53, wherein the at least an additional polymer C is conjugated to A, to B, or to C. A pharmaceutical composition comprising:

• a molecule of claims 1-54; and

• a pharmaceutically acceptable excipient. The pharmaceutical composition of claim 55, wherein the pharmaceutical composition is formulated as a nanoparticle formulation. The pharmaceutical composition of claim 55 or 56, wherein the pharmaceutical composition is formulated for parenteral, oral, intranasal, buccal, rectal, or transdermal administration. A method of treating a disease or disorder in a patient in need thereof, comprising administering to the patient a composition comprising a molecule of claims 1-54.

-78- The method of claim 58, wherein the disease or disorder is a COVID-19, COVID-19 associated cardiomyopathy, or COVID-19 associated heart failure. A method of inhibiting the replication, transcription, or expression of a target SARS-CoV or SARS-CoV-2 gene or gene product in a primary cell of a patient, comprising administering a molecule of claims 1-54 to the patient. The method of claim 60, wherein the primary cells are cardiomyocytes. The method of any one of the claims 58-61, wherein the patient is a human. A kit comprising a molecule of claims 1-54. A 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, wherein 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 polynucleic acid molecule of claim 64, wherein 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 polynucleic acid molecule of claim 64 or claim 65, wherein 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 polynucleic acid molecule of any one of claims 64-66, wherein 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 polynucleic acid molecule of any one of claims 64-67, wherein the sense strand and antisense strand form a double-stranded siRNA molecule. The polynucleic acid molecule of any one of claims 64-68, wherein the double-stranded region is 15-30 nucleotide pairs in length.

-79-