Compositions for delivery of polynucleotides

Abstract

The present disclosure provides compositions for delivering polynucleotides and methods of using same to treat genetic diseases. The present disclosure also provides polynucleotide conjugates. The present disclosure further provides anti-transferrin receptor antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 293,614, filed December 23, 2021, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0002] Polynucleotides have become useful agents for the treatment of human diseases. Targeted delivery of polynucleotides to tissues other than the liver (including solid tumors, muscle, or immune cells) is an ongoing challenge for oligonucleotide therapeutics. Because their chemical composition includes a charged phosphate backbone and a polymeric size ranging from 6,000 to 18,000 daltons, delivery of polynucleotides to cells has typically relied on formulation techniques using nanoparticle carrier systems such as lipid nanoparticles, liposomes, dendriplexes, gold particles, and virus-like particles. Although these strategies have proven effective, clinical development has typically been limited to the liver as a result of the natural accumulation of most delivery carrier systems in hepatic tissue. Furthermore, these nanocarrier formulations often contain multiple molecular components with their own subsets of toxicities, which affect the dosing limits of most polynucleotide therapeutics.

[0003] In recent years, conjugation of oligonucleotides to targeting moieties such as peptides, proteins or antibodies has proven effective. Antibodies (mAbs) are a highly attractive platform for creating targeted oligonucleotide therapeutics. Antibody-drug conjugates (ADCs) are a clinically proven and versatile means of specifically targeting highly cytotoxic payloads to numerous cancer cell types, thereby improving the therapeutic index of promising anticancer toxic agents. Oligonucleotides bearing tris-galNAc moieties targeting the asialoglycoprotein receptor (ASGPR) in hepatocytes have shown great success in multiple clinical trials. These promising results point the way forward for the targeted delivery of polynucleotides to other non-hepatic tissues.

[0004] Some progress has been made through the bioconjugation of oligonucleotide molecules to peptide or protein targeting moieties, which have shown promise in preclinical models. However, there are several challenges that limit the application of this approach. These challenges include: 1) achieving uniform cellular distribution and penetration in dense tissues such as tumors and muscles; 2) protecting chemically unstable oligonucleotides from degradation caused by extracellular nucleases until they are taken up by cells; 3) having efficient endosomal escape of the conjugate to the cytosol after uptake; 4) maintaining the desired solubility of the protein component after conjugation of hydrophobic linkers that can aid in endosomal escape; 5) designing chemistry and processes that allow the correct ratio of oligo to protein and specific conjugation points to produce therapeutic effects with the appropriate consistency and quality to pass CMC regulatory standards for human use; and 6) stabilizing antibody oligoconjugates by appropriate formulation to enhance long-term stability and avoid aggregation when higher DAR is achieved.

[0005] Thus, there remains a need for new compositions for delivering therapeutic polynucleotides. Summary of the Invention

[0006] The first aspect of the present disclosure provides a composition for delivering a polynucleotide. In some embodiments, the composition comprises a hybrid polymer and a polynucleotide. In some embodiments, the hybrid polymer comprises a cationic portion and a neutral portion. In some embodiments, the cationic portion of the hybrid polymer interacts with the polynucleotide non-covalently, for example, via ionic interactions.

[0007] In some embodiments, the cationic portion of the hybrid polymer is a cationic polypeptide. In some embodiments, the cationic polypeptide is a poly-arginine polypeptide. In some embodiments, the cationic polypeptide is a poly-lysine polypeptide. In some embodiments, the cationic polypeptide comprises arginine and lysine residues. In some embodiments, the cationic polypeptide comprises protamine. In some embodiments, the cationic polypeptide comprises L-amino acid residues. In some embodiments, the cationic polypeptide comprises D-amino acid residues. In some embodiments, the cationic polypeptide comprises L-amino acid residues and D-amino acid residues. In some embodiments, the cationic polypeptide comprises 9-18 amino acid residues. In some embodiments, the cationic polypeptide comprises 12 amino acid residues. In some embodiments, the cationic polypeptide is selected from the group of cationic polypeptides disclosed in Table 1.

[0008] In some embodiments, the cationic portion of the hybrid polymer comprises a cationic polymer having a size of about 600 to about 2500 Daltons. In some embodiments, the cationic polymer is a linear polymer. In some embodiments, the cationic polymer is a branched polymer. In some embodiments, the cationic polymer is selected from the group consisting of gelatin, glucosamine, N-acetylglucosamine, chitosan, cationic dextran, cationic cyclodextrin, cationic cellulose, polyethyleneimine (PEI), polyamidoamine (PAA), poly(amino-co-ester) (PAE), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), or cationic lipids such as DOTAP (N-(1-(2,3-dioleyloxy)propyl)-N,N,N trimethylammonium) chloride, poly[N,N-diethylaminoethyl methacrylate] (PDEAEMA), cationic mucic acid polymer (cMAP), and DOPE (dioleylphosphatidylethanolamine). In some embodiments, the cationic polymer is linear PEI. In some embodiments, the cationic polymer is branched PEI (BPEI).

[0009] In some embodiments, the hybrid polymer is any of the polymers disclosed in Tables 5, 7, 8, and 9 below. Each hybrid polymer listed in Tables 5, 7, 8, and 9 is considered a separate embodiment. In some embodiments, the hybrid polymer is any of the polymers disclosed in Tables 5, 7, 8, and 9 below. Each hybrid polymer listed in Tables 5, 7, 8, and 9 is considered a separate embodiment. In some embodiments, the hybrid polymer is any of the polymers disclosed in Tables 5, 7, 8, and 9 below. 12PolyArg9d, PEG1000DaPolyArg12, PEG2000DaPolyArg12, PEG5000DaPolyArg12, PolyArg12Cbp1.5kDa, PolyArg12Cbp3.9kDa, PolyArg12Cbp16kDa, CPolyArg12Cbp1.5kDa, PolyArg12Cbp2kDa, PolyArg12bp2kDa, amide dextran, lysine dextran, PEG PEI 15kda, BPEI-G-PEG550, and BPEI-G-PEG5000.

[0010] In some embodiments, the neutral portion of the hybrid polymer comprises a polymer with a size of about 100 to about 100,000 Daltons. In some embodiments, the neutral portion of the hybrid polymer comprises poly(ethylene glycol) (PEG). In some embodiments, the neutral portion of the hybrid polymer comprises a linear poly(ethylene glycol) (PEG). In some embodiments, the neutral portion of the hybrid polymer comprises a branched poly(ethylene glycol) (PEG). In some embodiments, the hybrid polymer is a PEGylated cationic polypeptide. In some embodiments, the hybrid polymer comprises a PEG9 to PEG1000 polymer. In some embodiments, the hybrid polymer comprises a PEG12 to PEG24 polymer.

[0011] In some embodiments, the hybrid polymer and the polynucleotide do not form aggregates or nanoparticles.

[0012] In some embodiments, the charge ratio of the cationic polypeptide to the polynucleotide is 0.25:1 to 5:1. In some embodiments, the charge ratio of the cationic polypeptide to the polynucleotide is 0.5:1 to 5:1. In some embodiments, the charge ratio of the cationic polypeptide to the polynucleotide is 1:1 to 4:1. In some embodiments, the charge ratio of the cationic polypeptide to the polynucleotide is 1:1 to 2:1. In some embodiments, the charge ratio of the cationic polypeptide to the polynucleotide is 1:1 or 2:1.

[0013] In some embodiments, the polynucleotide is conjugated to a targeting molecule. In some embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof, or a binding protein. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, a Fab-Fc, an Fv, a single chain Fv (scFv), a diabody, a minibody, and a Nanobody® molecule. In some embodiments, the bispecific antibody is a Bispecific T-cell Engager (BiTE) or a Dual Affinity Retargeting Antibody (DART). In some embodiments, the Nanobody® is a Nanobody-HSA®. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG molecule or is derived from an IgG molecule. In some embodiments, the IgG molecule is an IgG1 molecule or an IgG4 molecule.

[0014] In some embodiments, the binding protein is a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor comprises the extracellular domain of the receptor. In some embodiments, the soluble receptor is an Fc fusion protein.

[0015] In some embodiments, the targeting molecule is a therapeutically or biologically active molecule.

[0016] In some embodiments, the polynucleotide is selected from the group consisting of siRNA, ncRNA mimic, short hairpin RNA (shRNA), dicer-dependent siRNA (di-siRNA), antisense oligonucleotide (ASO), gapmer, mixmer, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), RNA activating oligonucleotide (RNAa), aptamer, exon skipping oligonucleotide, miRNA, miRNA mimic, mRNA, and guide RNA. In some embodiments, the polynucleotide is a miRNA mimic. In some embodiments, the miRNA mimic mimics miR-30. In some embodiments, the polynucleotide is a miRNA mimic selected from the group consisting of M30m1, M30m2, M30m3, and M30m4. In some embodiments, the polynucleotide is M30m3.

[0017] In some embodiments, the polynucleotide is an ASO. In some embodiments, the ASO is a DUX4-targeted ASO. In some embodiments, the DUX4-targeted ASO is selected from the group consisting of DUX4-targeted ASOs disclosed in Table 4. In some embodiments, the DUX4-targeted ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32.

[0018] In some embodiments, the targeting molecule and the polynucleotide provide a synergistic therapeutic or biological effect.

[0019] In some embodiments, the polynucleotide is directly conjugated to the targeting molecule. In some embodiments, the polynucleotide is conjugated to the targeting molecule via a linker. In some embodiments, the linker is a hydrophobic linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is a polymer linker. In some embodiments, the chemical linker is linear. In some embodiments, the chemical linker is cyclic. In some embodiments, the polymer linker comprises PEG, a sugar, a fatty acid, a phosphate, a pyrophosphate, or a polysarcosine. In some embodiments, the linker is a high molecular weight PEG linker. In some embodiments, the linker is a low molecular weight PEG linker.

[0020] In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable. In some embodiments, the linker is cleavable in vivo. In some embodiments, the cleavable linker is selected from the group consisting of disulfide linkers, self-immolative peptide polymer hybrids, and sulfatase-promoted aryl sulfate linkers. In some embodiments, the self-immolative peptide polymer hybrids include glucuronic acid, para-amino-benzoyloxy (PAB), 7-amino-3-hydroxyethyl-coumarin (7-AHC), or Fe(II)-reactive 1,2,4-trioxolane scaffolds (TRX). In some embodiments, the cleavable linker can be cleaved via reduction, hydrolysis, proteolysis, photocleavage, chemical cleavage, enzymatic cleavage, and bioorthogonal cleavage. In some embodiments, the chemical cleavage is by Fe II-mediated beta-elimination of TRX. In some embodiments, the enzymatic cleavage is by non-proteolytic sulfatase, beta-galactosidase / glucuronidase, or pyrophosphate phosphatase. In some embodiments, the bioorthogonal cleavage is by CuI-BTTAA or free copper ion mediated cleavage.

[0021] In some embodiments, the linker is conjugated to a lysine residue, a cysteine ​​residue, a histidine residue, or a non-natural amino acid residue in the targeting molecule. In some embodiments, the linker is conjugated to the targeting molecule by chemical or enzymatic conjugation. In some embodiments, chemical conjugation includes acylation and click chemistry. In some embodiments, enzymatic conjugation is via a sortase or transferase enzyme.

[0022] In some embodiments, each targeting molecule is conjugated to 1-8 polynucleotide molecules (DAR 1-8). In some embodiments, each targeting molecule is conjugated to one polynucleotide molecule (DAR1). In some embodiments, each targeting molecule is conjugated to two polynucleotide molecules (DAR2). In some embodiments, each targeting molecule is conjugated to three polynucleotide molecules (DAR3). In some embodiments, each targeting molecule is conjugated to four polynucleotide molecules (DAR4). In some embodiments, each targeting molecule is conjugated to five polynucleotide molecules (DAR5). In some embodiments, each targeting molecule is conjugated to six polynucleotide molecules (DAR6). In some embodiments, each targeting molecule is conjugated to seven polynucleotide molecules (DAR7). In some embodiments, each targeting molecule is conjugated to eight polynucleotide molecules (DAR8).

[0023] In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 30 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 40 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 50 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 60 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 500 Da. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of 7,500 kDa or less.

[0024] In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-C9-M30m3 (DAR3), cetuximab-DBCO-C4 / P5-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR2), cetuximab-DBCO-PEG9-M30m3 (DAR4), cetuximab-DBCO-PEG9-M30m3 (DAR6), cetuximab-linear-PEG13-M30m3 (DAR4), Cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR1), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2.5), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4.5), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR6.5), cetuximab-SMCC-M30m3(DAR4)(SMCC), cetuximab-MCVCPABcPNP-M30m3(DAR4)(MCVCPABcPNP), cetuximab-MCPEG4VCPABcPNP-M30m3(DAR4)(MCPEG4VCPABcPNP), cetuximab-C4-azide-DBCO-C5-M30m3, cetuximab-PEG4-azide-DBCO-PEG4-m30m3(DAR4), cetuximab-MC-PEG4-ValCit-PABc-M30m3(DAR1), cetuximab-MC-PEG4-ValCit-PABc-M30m3(DAR2), cetuximab-MC-PEG4-ValCit-PABc- M30m3 (DAR3), cetuximab-MC-PEG4-ValCit-PABc-M30m3 (DAR4), 3tf12-DBCO-PEG8-NCD5 (DAR1), 3tf12-DBCO-PEG8-M30m3 (DAR1), Fv55-SMCC-M30m3 (DAR1), Fv55-PEG8-DBCO-M30m3 (DAR1), Fv55-PEG8-DBCO-M30m3 (DAR2), Fv55-linker-M30m3 (DAR2), Fv55-DBCO-PEG8-M30m1 (DAR1), Fv55-DBCO-PEG8-M30m1 (DAR2), and ASO-carbon 4-DBCO-carbon 5-3tf12 (DAR1). In some embodiments, the polynucleotide conjugate is selected from the group consisting of antibody-polynucleotide conjugates disclosed in Table 5 or Table 6. Each APC disclosed in Table 5 or Table 6 is considered a separate embodiment.

[0025] In some embodiments, the composition comprises: (a) cetuximab-DBCO-C9-M30m3(DAR3) and PEG12-poly-(D-Arg)12; (b) cetuximab-DBCO-C4 / P5-M30m3(DAR3) and PEG12-poly-(D-Arg)12; (c) cetuximab-DBCO-PEG9-M30m3(DAR3) and PEG12-poly-(D-Arg)12; (d) cetuximab-DBCO-PEG9 (e) cetuximab-DBCO-PEG9-M30m3 (DAR4) and PEG12-poly-(D-Arg)12, (f) cetuximab-DBCO-PEG9-M30m3 (DAR6) and PEG12-poly-(D-Arg)12, (g) cetuximab-linear-PEG13-M30m3 (DAR4) and PEG12-poly-(D-Arg)12, (h) 3 (i) tf12-DBCO-PEG8-NCD5 and poly(L-Arg)9, (i) 3tf12-DBCO-PEG8-M30m3 and poly(L-Arg)9, (j) Fv55-SMCC-M30m3 and PEG12-poly(L-Arg)12, (k) Fv55-PEG30-M30m3 and PEG12-poly(L-Arg)12, (l) cetuximab-PEG4-azide-DBCO-PEG5-M30m3(DAR2) and PEG 12PolyArg12{d}, (m) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2) and PolyArg12Cbp3.9kDa, (n) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PEG12PolyArg12{d}, (o) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12Cbp3.9 kDa, (p) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-PEG2000Da, (q) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-PEG5000Da, (r) cetuximab-PEG4-azide-DBCO-PEG5-M (s) cetuximab-SMCC-M30m3 (DAR4) and PEG12PolyArg12{d}, (t) cetuximab-MCVCPABcPNP-M30m3 (DAR4) and PEG12PolyArg12{d}, (u) cetuximab-MCPEG4VCPABcPNP- (v) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR 2.5) and PEG12PolyArg12{d}, (w) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR 4.5) and PEG12PolyArg12{d}, (x) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR 6.5) and PEG12PolyArg12{d}, (y) cetuximab-C4(azide-DBCO)C5-M30m3 and PEG12PolyArg12, or (z) any of the combinations of antibody-polynucleotide conjugates and hybrid polymers disclosed in Table 5. Each of the antibody-polynucleotide conjugates and related hybrid polymers disclosed in Table 5 is considered a separate embodiment.

[0026] In a second aspect, the disclosure provides a polynucleotide conjugate comprising a polynucleotide conjugated to a targeting molecule.

[0027] In some embodiments, the targeting molecule is an antibody or antigen-binding fragment thereof, or a binding protein. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, Fab-Fc, Fv, single chain Fv (scFv), a diabody, a minibody, a vNAR, a centrin, and a Nanobody® molecule. In some embodiments, the bispecific antibody is a bispecific T cell engager (BiTE) or a dual affinity retargeting antibody (DART). In some embodiments, the Nanobody® is Nanobody-HSA®. In some embodiments, the antibody or antigen-binding fragment thereof is an IgG molecule or is derived from an IgG molecule. In some embodiments, the IgG molecule is an IgG1 molecule or an IgG4 molecule.

[0028] In some embodiments, the binding protein is a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor comprises the extracellular domain of the receptor. In some embodiments, the soluble receptor is an Fc fusion protein.

[0029] In some embodiments, the targeting molecule is a therapeutically or biologically active molecule.

[0030] In some embodiments, the polynucleotide is selected from the group consisting of an oligonucleotide, an siRNA, an ncRNA mimic, a short hairpin RNA (shRNA), a dicer-dependent siRNA (di-siRNA), an antisense oligonucleotide (ASO), a gapmer, a mixmer, a double-stranded RNA (dsRNA), a single-stranded RNAi (ssRNAi), a DNA-directed RNA interference (ddRNAi), an RNA-activating oligonucleotide (RNAa), an aptamer, an exon-skipping oligonucleotide, an miRNA, an miRNA mimic, an mRNA, and a guide RNA. In some embodiments, the polynucleotide is an miRNA mimic. In some embodiments, the miRNA mimic mimics miR-30. In some embodiments, the polynucleotide is an miRNA mimic selected from the group consisting of M30m1, M30m2, M30m3, and M30m4. In some embodiments, the polynucleotide is M30m3.

[0031] In some embodiments, the polynucleotide is an ASO. In some embodiments, the ASO is a DUX4-targeted ASO. In some embodiments, the DUX4-targeted ASO is selected from the group consisting of DUX4-targeted ASOs disclosed in Table 4. In some embodiments, the DUX4-targeted ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32.

[0032] In some embodiments, the targeting molecule and the polynucleotide provide a synergistic therapeutic or biological effect.

[0033] In some embodiments, the polynucleotide is directly conjugated to the targeting molecule. In some embodiments, the polynucleotide is conjugated to the targeting molecule via a linker. In some embodiments, the linker is a hydrophobic linker. In some embodiments, the linker is a peptide linker. In some embodiments, the linker is a chemical linker. In some embodiments, the chemical linker is a polymer linker. In some embodiments, the chemical linker is linear. In some embodiments, the chemical linker is cyclic. In some embodiments, the polymer linker comprises PEG, a sugar, a fatty acid, a phosphate, a pyrophosphate, or a polysarcosine. In some embodiments, the linker is a high molecular weight PEG linker. In some embodiments, the linker is a low molecular weight PEG linker.

[0034] In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable. In some embodiments, the linker is cleavable in vivo. In some embodiments, the cleavable linker is selected from the group consisting of disulfide linkers, self-immolative peptide polymer hybrids, and sulfatase-promoted aryl sulfate linkers. In some embodiments, the self-immolative peptide polymer hybrids include glucuronic acid, para-amino-benzoyloxy (PAB), 7-amino-3-hydroxyethyl-coumarin (7-AHC), or Fe(II)-reactive 1,2,4-trioxolane scaffolds (TRX). In some embodiments, the cleavable linker can be cleaved via reduction, hydrolysis, proteolysis, photocleavage, chemical cleavage, enzymatic cleavage, and bioorthogonal cleavage. In some embodiments, the chemical cleavage is by Fe II-mediated beta-elimination of TRX. In some embodiments, the enzymatic cleavage is by non-proteolytic sulfatase, beta-galactosidase / glucuronidase, or pyrophosphate phosphatase. In some embodiments, the bioorthogonal cleavage is by CuI-BTTAA or free copper ion mediated cleavage.

[0035] In some embodiments, the linker is conjugated to a lysine residue, a cysteine ​​residue, a histidine residue, or a non-natural amino acid residue in the targeting molecule. In some embodiments, the linker is conjugated to the targeting molecule by chemical or enzymatic conjugation. In some embodiments, chemical conjugation includes acylation and click chemistry. In some embodiments, enzymatic conjugation is via a sortase or transferase enzyme.

[0036] In some embodiments, each targeting molecule is conjugated to one to eight polynucleotide molecules (DAR1-8). In some embodiments, each targeting molecule is conjugated to one polynucleotide molecule (DAR1). In some embodiments, each targeting molecule is conjugated to two polynucleotide molecules (DAR2). In some embodiments, each targeting molecule is conjugated to three polynucleotide molecules (DAR3). In some embodiments, each targeting molecule is conjugated to four polynucleotide molecules (DAR4). In some embodiments, each targeting molecule is conjugated to five polynucleotide molecules (DAR5). In some embodiments, each targeting molecule is conjugated to six polynucleotide molecules (DAR6). In some embodiments, each targeting molecule is conjugated to seven polynucleotide molecules (DAR7). In some embodiments, each targeting molecule is conjugated to eight polynucleotide molecules (DAR8).

[0037] In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 30 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 40 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 50 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 60 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of 7,500 kDa or less.

[0038] In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-C9-M30m3 (DAR3), cetuximab-DBCO-C4 / P5-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR2), cetuximab-DBCO-PEG9-M30m3 (DAR4), cetuximab-DBCO-PEG9-M30m3 (DAR6), cetuximab-linear-PEG13-M30m3 (DAR4), Cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR1), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2.5), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4.5), cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR6.5), cetuximab-SMCC-M30m3(DAR4)(SMCC), cetuximab-MCVCPABcPNP-M30m3(DAR4)(MCVCPABcPNP), cetuximab-MCPEG4VCPABcPNP-M30m3(DAR4)(MCPEG4VCPABcPNP), cetuximab-C4-azide-DBCO-C5-M30m3, cetuximab-PEG4-azide-DBCO-PEG4-m30m3(DAR4), cetuximab-MC-PEG4-ValCit-PABc-M30m3(DAR1), cetuximab-MC-PEG4-ValCit-PABc-M30m3(DAR2), cetuximab-MC-PEG4-ValCit-PABc- M30m3 (DAR3), cetuximab-MC-PEG4-ValCit-PABc-M30m3 (DAR4), 3tf12-DBCO-PEG8-NCD5 (DAR1), 3tf12-DBCO-PEG8-M30m3 (DAR1), Fv55-SMCC-M30m3 (DAR1), Fv55-PEG8-DBCO-M30m3 (DAR1), Fv55-PEG8-DBCO-M30m3 (DAR2), Fv55-linker-M30m3 (DAR2), Fv55-DBCO-PEG8-M30m1 (DAR1), Fv55-DBCO-PEG8-M30m1 (DAR2), and ASO-carbon 4-DBCO-carbon 5-3tf12 (DAR1). In some embodiments, the polynucleotide conjugate is selected from the group consisting of antibody-polynucleotide conjugates disclosed in Table 5 or Table 6. Each APC disclosed in Table 5 or Table 6 is considered a separate embodiment.

[0039] The third aspect of the present disclosure provides a method for treating a genetic disease in a subject in need of such treatment.In some embodiments, the method comprises administering to the subject a therapeutically effective amount of any of the compositions disclosed herein or a therapeutically effective amount of any of the polynucleotide conjugates disclosed herein.

[0040] In some embodiments, the genetic disease is a viral infection, hi some embodiments, the viral infection is caused by a virus selected from the group consisting of adenovirus, anellovirus, arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, herpesvirus, orthomyxovirus, papillomavirus, paramyxovirus, parvovirus, picornavirus, pneumovirus, polyomavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and togavirus. In some embodiments, the virus is an adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah Forest virus, Bunyamwera virus, Lacrosse bunyavirus, Snowshoe hare bunyavirus, Cercopithecine herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus A, Cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhoori virus, Dugbe virus, virus), Dubenhague virus, Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB virus C / G hepatitis virus, Hantavirus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis D virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68, Human enterovirus 70, human herpesvirus 1, human herpesvirus 2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, human immunodeficiency virus, human papillomavirus 1, human papillomavirus 2, human papillomavirus 16, human papillomavirus 18, human parainfluenza, human parvovirus B19, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus,Influenza B virus, Influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langat virus, Lassa virus, Rosedale virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, Measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray Valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus, Polio virus, Punta Toro phlebovirus, Puuma laviras, The virus is selected from the group consisting of rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Sarivirus A, Sicilian sandfly fever virus, Sapporo virus, SARS coronavirus 2, Semliki forest virus, Seoul virus, Simian foamy virus, Simian virus 5, Sindbis virus, Southampton virus, St. Louis encephalitis virus, Tick-borne Powassan virus, Torque teno virus, Toscana virus, Uukuniemi virus, Vaccinia virus, Varicella zoster virus, Smallpox virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow fever virus, and Zika virus. In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA that targets a viral gene. In some embodiments, the polynucleotide is conjugated to a targeting molecule that specifically binds to a viral protein or a protein on the surface of the virus' host cell. In some embodiments, the polynucleotide and the targeting molecule exhibit synergistic effects in treating viral infections.

[0041] In some embodiments, the genetic disease is cancer. In some embodiments, the cancer is characterized by overexpression of a cancer gene. In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA that targets the cancer gene. In some embodiments, the cancer is characterized by reduced expression of a tumor suppressor gene. In some embodiments, the polynucleotide comprises an mRNA molecule that encodes the tumor suppressor gene. In some embodiments, the polynucleotide comprises a guide RNA that restores expression of the tumor suppressor gene.

[0042] In some embodiments, the polynucleotide is conjugated to a targeting molecule that specifically binds to tumor cells of the cancer. In some embodiments, the targeting molecule specifically binds to epidermal growth factor receptor, and the polynucleotide is a miR-30 miRNA or a mimic thereof. In some embodiments, the targeting molecule specifically binds to TFR. In some embodiments, the targeting molecule is selected from the group consisting of FV55 scFv, Fv55 diabody, and 3TF12. In some embodiments, the targeting molecule specifically binds to ACVR1, and the polynucleotide is a miR-30 miRNA or a mimic thereof. In some embodiments, the targeting molecule specifically binds to ACVR1, and the polynucleotide is a miR-26 miRNA or a mimic thereof. In some embodiments, the polynucleotide and the targeting molecule exhibit synergistic effects in the treatment of cancer.

[0043] In some embodiments, the genetic disease is a neuromuscular disease. In some embodiments, the neuromuscular disease is a muscular dystrophy. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA targeting DUX4, DMPK, or CAPN3. In some embodiments, the polynucleotide is an ASO targeting DUX4. In some embodiments, the DUX4 targeting ASO is selected from the group consisting of DUX4 targeting ASOs disclosed in Table 4. In some embodiments, the DUX4 targeting ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32. In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy. In some embodiments, the polynucleotide is an mRNA, cDNA, or vector encoding dystrophin or utrophin. In some embodiments, the polynucleotide is a guide RNA that restores expression of dystrophin or utrophin.

[0044] In some embodiments, the polynucleotide is conjugated to a targeting molecule that specifically binds to a marker on the surface of skeletal muscle cells of the subject. In some embodiments, the targeting molecule specifically binds to ACVR1. In some embodiments, the targeting molecule specifically binds to ACVR1, and the polynucleotide is a DUX4 targeting ASO. In some embodiments, the polynucleotide and the targeting molecule show synergistic effects in treating muscular dystrophy.

[0045] A fourth aspect of the disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human transferrin receptor (TfR1). In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (VH) comprising the amino acid sequence QVQVQDSGGELVQPGGSLRVSCKASGFNIKDSYMHWVRQAPGKGLEWVAFIDPETGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSIYWYFDVWGKGTTVTVSS and a light chain variable region (VL) comprising the amino acid sequence DIQMTQSPSSLSASVGQRVTITCRASQSLLNSSNQKNSLGWYQQKPGKAPKLLIYFASTRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTKVDIKRC.

[0046] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a full length antibody, a Fab, a Fab-Fc, an Fv, a single chain Fv (scFv), a diabody, a minibody, and a Nanobody®. In some embodiments, the antibody or antigen-binding fragment thereof is an scFv. In some embodiments, the antibody or antigen-binding fragment thereof is a diabody.

[0047] In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence GGGGS. In some embodiments, the linker comprises the amino acid sequence (GGGGS) N and N is 1 to 3. In some embodiments, when the antibody or antigen-binding fragment thereof is an scFv, the linker comprises the amino acid sequence (GGGGS) 3 In some embodiments, when the antibody or antigen-binding fragment thereof is a diabody, the linker comprises the amino acid sequence (GGGGS) N where N is 1 or 2.

[0048] This patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee. [Brief description of the drawings]

[0049] [Figure 1A] Representative hybrid polymers and conjugates containing them are shown. Panels A to D show the structures of representative hybrid polymers. [Figure 1B] Representative hybrid polymers and conjugates containing them are shown. Panels A to D show the structures of representative hybrid polymers. [Figure 1C] Representative hybrid polymers and conjugates containing them are shown. Panels A to D show the structures of representative hybrid polymers. [Figure 1D] Representative hybrid polymers and conjugates containing them are shown. Panels A to D show the structures of representative hybrid polymers. [Figure 1E] Representative hybrid polymers and conjugates containing them are shown. Panel E shows an antibody-oligonucleotide conjugate coated with a representative hybrid polymer. [Figure 1F] Representative hybrid polymers and conjugates containing them are shown. Panel F shows a diabody-siRNA conjugate coated with a representative hybrid polymer. [Figure 1G] Representative hybrid polymers and conjugates containing them are shown. Panel G shows a nanobody-ASO conjugate coated with a representative hybrid polymer. [Figure 1H] Representative hybrid polymers and conjugates containing them are shown. Panel H shows an antibody-mRNA conjugate coated with a representative hybrid polymer. [Figure 1I]Representative hybrid polymers and conjugates containing them are shown. Panel I shows a cytokine-gRNA conjugate coated with a representative hybrid polymer. [Figure 1J] Representative hybrid polymers and conjugates containing them are shown. Panel J shows an antibody-DNA plasmid conjugate coated with a representative hybrid polymer. [Figure 1K] Representative hybrid polymers and conjugates containing them are shown. Panel K shows an antibody-cDNA conjugate coated with a representative hybrid polymer. [Figure 2A] 1 shows a representative synthesis scheme of antibody-oligonucleotide conjugates via click chemistry. [Figure 2B] 1 shows a representative synthesis scheme for antibody-oligonucleotide conjugates via acylation chemistry. [Figure 2C] 1 shows a representative synthesis scheme of an antibody-oligonucleotide conjugate via site-specific cysteine ​​conjugation with a cleavable linker. [Figure 2D.I] 1 shows a representative manufacturing process for a hybrid polymer-coated antibody-oligonucleotide conjugate. [Figure 2D.II] 1 shows a representative manufacturing process for a hybrid polymer-coated antibody-oligonucleotide conjugate. [Diagram 3] A and B show a representative purification process of diabody-oligonucleotide conjugates using SEC FPLC. [Figure 4] A and B show a representative purification process of an antibody-oligonucleotide conjugate using SEC FPLC. [Diagram 5] A representative purification process of antibody-miRNA conjugates with different DARs using SEC FPLC is shown. [Figure 6]A and B show the precipitation and aggregation states of Fv55-DBCO-PEG8-miRNA-AF488 conjugates with various Arg12 peptides. [Figure 7] FIG. 1 shows ELISA antigen affinity of various diabody-miRNA conjugates. [Figure 8A] 1 shows the structure of a representative hybrid polymer-coated antibody-miRNA conjugate. [Figure 8B] Figure 1 shows ELISA antigen affinity of mAb-miRNA conjugates coated with hybrid polymers. [Figure 9A] The stability of miRNA mimics alone or together with cationic peptides or hybrid polymers in human serum is shown. [Figure 9B] The stability of miRNA mimics alone or together with cationic peptides or hybrid polymers in human serum is shown. [Figure 9C] The stability of miRNA mimics alone or together with cationic peptides or hybrid polymers in human serum is shown. [Figure 9D] The stability of miRNA mimics alone or together with cationic peptides or hybrid polymers in human serum is shown. [Figure 10] 1 shows the stability of cetuximab-miRNA conjugates alone or with cationic peptides or hybrid polymers in human serum. [Figure 11] 1 shows the stability of diabody-ScFv-miRNA conjugates alone or together with the hybrid polymer in human serum. [Figure 12] Figure 2 shows the stability of ScFv-duplex conjugates with different linkers in human serum. [Figure 13A]Figure 1 shows the in vitro delivery efficiency of cetuximab-miRNA conjugates in human cancer cells in the presence (left bar) and absence (right bar) of the hybrid polymer Preg12PolyArg12{d} at three different DARs. The mean fluorescence intensity was averaged based on the ASD647 signal of each cell. * indicates p<0.005 by Student's t-test compared to no delivery. Error bars represent standard deviation. [Figure 13B] Representative images of the summarized transfection experiments are provided in FIG. 13A. [Figure 13C] The results of reporter assays are provided using luciferase reporters that contain miR-30 target sites within the 3' untranslated region (UTR) of the luciferase transcript. Reduction in luminescence indicates knockdown of the reporter construct by the tested miR-30 microRNA mimic. Data are reported as the mean + / - SEM of three replicates. [Figure 14A] The structure of Preg12PolyArg12{d} is shown. [Figure 14B] The structure of PolyArg12Cbp (3.9 kDa) is shown. [Figure 14C] This figure provides the results of reporter assays using luciferase reporters containing miR-30 target sites within the 3' untranslated region (UTR) of the luciferase transcript using different antibody polynucleotide conjugates (APCs) and different hybrid polymers. Data are reported as the mean + / - SEM of three replicates. [Figure 14D] This figure provides the results of reporter assays using luciferase reporters containing miR-30 target sites within the 3' untranslated region (UTR) of the luciferase transcript using different antibody polynucleotide conjugates (APCs) and different hybrid polymers. Data are reported as the mean + / - SEM of three replicates. [Figure 14E]This figure provides the results of reporter assays using luciferase reporters containing miR-30 target sites within the 3' untranslated region (UTR) of the luciferase transcript using different antibody polynucleotide conjugates (APCs) and different hybrid polymers. Data are reported as the mean + / - SEM of three replicates. [Figure 14F] 13 provides the results of qPCR of miR30 downstream genes after treatment with various APCs in the presence or absence of the hybrid polymer Preg12PolyArg12{d}. [Figure 14G] This figure provides the results of reporter assays using luciferase reporters containing miR-30 target sites within the 3' untranslated region (UTR) of the luciferase transcript using different antibody polynucleotide conjugates (APCs) and different hybrid polymers. Data are reported as the mean + / - SEM of three replicates. [Figure 15] 3 shows the in vitro cellular activity of 3tf12-M30m3 XTT and Lum. [Figure 16] 1 shows the in vitro activity of fv55-SMCC-M30m3 by the hybrid polymer peptide. [Figure 17A] Figure 1 shows the in vitro efficacy of various cetuximab-miRNA conjugates in the presence and absence of hybrid polymers. Data are reported as mean + / - SEM of triplicate experiments. [Figure 17B] Figure 1 shows the in vitro efficacy of various cetuximab-miRNA conjugates in the presence and absence of hybrid polymers. Data are reported as mean + / - SEM of triplicate experiments. [Figure 17C] Figure 1 shows the in vitro efficacy of various cetuximab-miRNA conjugates in the presence and absence of hybrid polymers. Data are reported as mean + / - SEM of triplicate experiments. [Figure 18A] Shows the in vitro efficacy of ScFv-ASO conjugates. [Figure 18B] Shows the in vitro efficacy of ScFv-ASO conjugates. [Figure 19A.I] 1 shows that cetuximab-miRNA conjugates with peptide hybrid coating improve delivery to tumors. AI includes a graphic image of the photographic image. [Figure 19A.II] Figure 2 shows that cetuximab-miRNA conjugates with peptide hybrid coating improve delivery to tumors. A.II provides photographic images showing tumor delivery of cetuximab-miRNA conjugates. [Figure 19B.I] BI includes a graphic image of the photographic image showing that cetuximab-miRNA conjugates with peptide hybrid coating improve delivery to tumors. [Figure 19B.II] Figure 2 shows that cetuximab-miRNA conjugates with peptide hybrid coating improve delivery to tumors.B.II provides photographic images showing tumor delivery of cetuximab-miRNA conjugates. [Figure 19C.I] CI shows that cetuximab-miRNA conjugates with peptide hybrid coating improve delivery to tumors. CI includes a graphic image of the photographic image. [Figure 19C.II] C.II shows that cetuximab-miRNA conjugates with peptide hybrid coating improve delivery to tumors. C.II provides photographic images showing tumor delivery of cetuximab-miRNA conjugates. [Figure 19D] 1 shows that cetuximab-miRNA conjugates with a peptide hybrid coating improve delivery to tumors. [Figure 20A.I]Figures 20A.I, 20A.II, 20B.I, 20B.II, and 20C show that IR750-FV55 diabody-miRNA conjugates with peptide hybrid coating improve delivery to tumors. Figures 20A.II and 20B.II provide photographic images showing tumor delivery of IR2750-FV55 diabody-miRNA conjugates. Figures 20A.I and 20B.I each include a graphic image of those photographic images. [Figure 20A.II] Figures 20A.I, 20A.II, 20B.I, 20B.II, and 20C show that IR750-FV55 diabody-miRNA conjugates with peptide hybrid coating improve delivery to tumors. Figures 20A.II and 20B.II provide photographic images showing tumor delivery of IR2750-FV55 diabody-miRNA conjugates. Figures 20A.I and 20B.I each include a graphic image of those photographic images. [Fig. 20B.I] Figures 20A.I, 20A.II, 20B.I, 20B.II, and 20C show that IR750-FV55 diabody-miRNA conjugates with peptide hybrid coating improve delivery to tumors. Figures 20A.II and 20B.II provide photographic images showing tumor delivery of IR2750-FV55 diabody-miRNA conjugates. Figures 20A.I and 20B.I each include a graphic image of those photographic images. [Fig. 20B.II] Figures 20A.I, 20A.II, 20B.I, 20B.II, and 20C show that IR750-FV55 diabody-miRNA conjugates with peptide hybrid coating improve delivery to tumors. Figures 20A.II and 20B.II provide photographic images showing tumor delivery of IR2750-FV55 diabody-miRNA conjugates. Figures 20A.I and 20B.I each include a graphic image of those photographic images. [Figure 20C]Figures 20A.I, 20A.II, 20B.I, 20B.II, and 20C show that IR750-FV55 diabody-miRNA conjugates with peptide hybrid coating improve delivery to tumors. Figures 20A.II and 20B.II provide photographic images showing tumor delivery of IR2750-FV55 diabody-miRNA conjugates. Figures 20A.I and 20B.I each include a graphic image of those photographic images. [Figure 21A.I] Figure 21A.II shows that conjugation of Cetuximab-C9-M30m3 conjugate with hybrid polymer PEG12PolyArg12{d} improves antitumor efficacy. Figure 21A.II provides photographic images showing tumor delivery of Cetuximab-C9-M30m3 conjugate. Figure 21A.I includes graphic images of those photographic images. [Figure 21A.II] Figure 21A.II shows that conjugation of Cetuximab-C9-M30m3 conjugate with hybrid polymer PEG12PolyArg12{d} improves antitumor efficacy. Figure 21A.II provides photographic images showing tumor delivery of Cetuximab-C9-M30m3 conjugate. Figure 21A.I includes graphic images of those photographic images. [Figure 21B] We show that conjugation of the cetuximab-C9-M30m3 conjugate with the hybrid polymer PEG12PolyArg12{d} improves the antitumor efficacy. [Figure 21C] 1 shows UMSCC109 tumor growth following treatment with cetuximab-MCVCPABcPNP-M30m3 DAR4, cetuximab-SMCC-M30m3 DAR4, cetuximab control, and phosphate buffered saline (PBS). [Figure 21D] FIG. 1 shows the growth of various tumors following treatment with Cetuximab-PEG4-Azide-DBCO-PEG5-M30m3 DAR2(APC1), Cetuximab control, and PBS. [Figure 21E]13 shows UMSCC109 tumor growth after treatment with PBS and Cetuximab-PEG4-Azide-DBCO-PEG5-M30m3 at DAR 2.5, 4.5 and 6.5 in combination with Preg12PolyArg12{d} at a charge:charge ratio of 1:1. [Figure 21F] ELISA results showing delivery of M30m3 in various mouse tissues following treatment with PBS control, unconjugated control mixture (cetuximab, M30m3, and Preg12PolyArg12{d}), APC2 (cetuximab-PEG4-azide-DBCO-PEG5-M30m3 at DAR2) in the presence or absence of Preg12PolyArg12{d} or R12Cbp3.9kda at a charge:charge ratio of 0.5:1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0050] overview The practice of the methods disclosed herein, and the preparation and use of the compositions disclosed herein employ, unless otherwise indicated, conventional techniques of molecular biology, biochemistry, chromatin structure and analysis, computational chemistry, cell culture, recombinant DNA, and related fields within the art, which are fully explained in the literature. For example, Sambrook et al.MOLECULAR CLONING:A LABORATORY MANUAL,Second edition,Cold Spring Harbor Laboratory Press,1989 and Third edition,2001;Ausubel et al.,CURRENT PROTOCOLS IN MOLECULAR BIOLOGY,John Wiley & Sons,New York,1987 and periodic updates;the series METHODS IN ENZYMOLOGY,Academic Press,San Diego;Wolffe,CHROMATIN STRUCTURE AND FUNCTION,Third edition,Academic Press,San Diego,1998;METHODS IN ENZYMOLOGY,Vol.304,“Chromatin”(PMWassarman and APWolffe,eds.),Academic Press,San Diego,1999;andMETHODS IN MOLECULAR BIOLOGY,Vol.119,Chromatin Protocols” (PB Becker, ed.) Humana Press, Totowa, 1999.

[0051] definition The term "herein" means the entire application.

[0052] Unless otherwise defined herein, scientific and technical terms used in this application shall have the meanings commonly understood by those of ordinary skill in the art. In general, the nomenclature used in connection with the compounds, compositions, and methods described herein is that which is well known and commonly used in the art.

[0053] It is to be understood that any of the embodiments described herein, including those described under different aspects of the disclosure and different parts of this specification (including those described in the examples only), may be combined with one or more other embodiments of the invention, unless expressly disclaimed or inappropriate, and that the combination of embodiments is not limited to the specific combinations claimed via multiple dependent claims.

[0054] All of the above, as well as any other publications, patents, and published patent applications mentioned in this application are specifically incorporated herein by reference. In case of conflict, the present specification, including its specific definitions, will control.

[0055] It is understood that throughout this specification the term "comprise" or variations such as "comprises" or "comprising" implies the inclusion of a stated integer (or component) or group of integers (or components) but not the exclusion of any other integer (or component) or group of integers (or components).

[0056] Throughout this specification, when a composition is described as having, including, or comprising certain components (or variations thereof), it is contemplated that the composition can also consist essentially of or consist of the recited components. Similarly, when a method or process is described as having, including, or comprising certain process steps, the process can also consist essentially of or consist of the recited process steps. Further, it should be understood that the order of steps or order for performing certain actions is immaterial so long as the components and methods described herein are operable. Moreover, two or more steps or actions may be performed simultaneously.

[0057] The term "including" is used to mean "including but not limited to." "Including" and "including but not limited to" are used interchangeably.

[0058] As used herein, the term "about" or "approximately" means within an acceptable range of error for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system.

[0059] In the context of describing elements (particularly in the context of the claims which follow), the terms "a," "an," and "the," and similar referents, should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

[0060] As used herein, the term "or" should be understood to mean "and / or" unless the context clearly dictates otherwise.

[0061] The recitation of ranges of values ​​herein is intended to serve merely as a shorthand method of individually referring to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein, unless otherwise indicated herein. All methods described herein may be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or illustrative language (e.g., "such as") provided herein is intended merely to facilitate understanding of the embodiments, and does not limit the scope of the claims, unless otherwise indicated. No language in this specification should be construed as indicating any non-claimed element as essential.

[0062] The terms "nucleic acid", "polynucleotide" and "oligonucleotide" are used interchangeably and refer to deoxyribonucleotide or ribonucleotide polymers in linear or circular structure and in either single- or double-stranded form. For the purposes of this disclosure, these terms should not be construed as limiting with respect to the length of the polymer. These terms can include known analogs of natural nucleotides, as well as nucleotides that are modified at the base, sugar, and / or phosphate moieties (e.g., phosphorothioate backbones, 2'-deoxy-, 2'-O-methyl-, 2'-deoxy-2'-fluoro-modified nucleotides, and end cap molecules at the 3', 5', or both the 3' and 5' ends). In general, analogs of a particular nucleotide have the same base-pairing specificity, i.e., an analog of A will base pair with T. The terms also include polymers that include one or more chemically modified nucleotides. Non-limiting examples of polynucleotides include small interfering RNA (siRNA), microRNA (miRNA), miRNA mimics, short hairpin RNA (shRNA), double-stranded RNA (dsRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), heterogeneous nuclear RNA (hnRNA), antisense oligonucleotides (ASOs, including exon skipping ASOs), messenger RNA (mRNA), complementary DNA (cDNA), plasmids and vectors, and guide RNA (gRNA).

[0063] The terms "polypeptide," "peptide," and "protein" are used interchangeably to refer to a polymer of amino acid residues. The terms also apply to amino acid polymers in which one or more amino acids are chemical analogues or modified derivatives of a corresponding naturally occurring amino acid.

[0064] As used herein, the term "residue" refers to a position in a protein and the associated amino acid identity.

[0065] As used herein, the terms "Fc", "Fc region" or "Fc domain" are used interchangeably herein and refer to a polypeptide comprising the constant region of an antibody, in some cases excluding the first constant region immunoglobulin domain (e.g., CH1) or a portion thereof, and in some cases, a portion of the hinge. Thus, Fc can refer to the last two constant region immunoglobulin domains (e.g., CH2 and CH3) of IgA, IgD and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. In the case of IgA and IgM, Fc can include the J chain. In the case of IgG, the Fc domain includes immunoglobulin domains Cγ2 and Cγ3 (Cγ2 and Cγ3), and the lower hinge region between Cγ1 (Cγ1) and Cγ2 (Cγ2). In some embodiments, Fc refers to the truncated CH1 domain, and CH2 and CH3 of an immunoglobulin. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues E216 or C226 or P230 at its carboxyl terminus, where the numbering is according to EU numbering. In some embodiments, the Fc domain is derived from a human IgG1 heavy chain Fc domain. In some embodiments, the Fc domain is derived from a human IgG2 heavy chain Fc domain. "EU format as per Edelman" or "EU numbering" or "EU index" refers to the residue numbering of the human Fc domain as set forth in Edelman GM et al. (Proc. Natl. Acad. USA (1969), 63, 78-85, incorporated herein by reference in its entirety).

[0066] As used herein, the term "Fc fusion protein" generally refers to a protein comprising an Fc region attached (optionally via a linker moiety) to a different protein.

[0067] As used herein, the term "antibody" or "Ab" refers to an immunoglobulin molecule (e.g., a complete antibody, an antibody fragment, or a modified antibody) that can recognize and bind to a specific target or antigen, such as, for example, a carbohydrate, a polynucleotide, a lipid, or a polypeptide, via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" can encompass any type of antibody, including monoclonal antibodies, polyclonal antibodies, human antibodies, engineered antibodies (including humanized antibodies, fully human antibodies, chimeric antibodies, single chain antibodies, artificially selected antibodies, CDR-bearing antibodies, etc.) that specifically bind to a given antigen. In some embodiments, "antibody" and / or "immunoglobulin" (Ig) refers to a polypeptide that includes at least two heavy (H) chains (about 50-70 kDa) and two light (L) chains (about 25 kDa), optionally interconnected by disulfide bonds. There are two types of light chains: λ and κ. In humans, the λ and κ light chains are similar, but only one type exists for each antibody. Heavy chains are classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. See generally, Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)), incorporated by reference in its entirety.

[0068] An "antigen-binding fragment" of an antibody refers to a fragment of a full-length antibody that retains the ability to specifically bind to an antigen (preferably with substantially the same binding affinity). Examples of antigen-binding fragments include (i) a Fab fragment, i.e., a monovalent fragment consisting of the VL, VH, CL and CH1 domains, (ii) a F(ab')2 fragment, i.e., a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the VH and CH1 domains, (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment consisting of the VH domain (Ward et al., (1989) Nature 341:544-546), and (vi) an isolated complementarity determining region (CDR), a disulfide-linked Fv (dsFv), an anti-idiotypic (anti-Id) antibody and an intrabody. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker that allows them to be produced as a single protein chain using recombinant methods, with the VL and VH regions pairing to form a monovalent molecule (known as a single-chain Fv (scFv)). See, for example, Bird et al. Science 242:423-426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988). Other forms of single-chain antibodies, such as diabodies, are also encompassed. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but these domains are allowed to pair with complementary domains and create two antigen-binding sites by using a linker that is too short to allow pairing between the two domains on the same chain (see, e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123).

[0069] The terms "microRNA", "miRNA" and "miR" are used interchangeably herein to refer to single-stranded non-coding RNAs that function in RNA silencing and post-transcriptional regulation of gene expression. miRNAs function by complementary base pairing with mRNA molecules, which silence mRNAs by, among other things, one or more of the following: (a) cleavage of the mRNA strand into two parts, (b) destabilization of the mRNA by shortening the poly(A) tail, and (c) decreasing the efficiency of translation of mRNA into protein by ribosomes.

[0070] The terms "subject," "patient," and "individual" are used interchangeably and refer to mammals, including, but not limited to, human patients and non-human primates, as well as laboratory animals such as rabbits, dogs, cats, rats, mice, and other animals. Thus, as used herein, the term "subject" or "patient" refers to any mammalian patient or subject to which the compositions of the present disclosure can be administered. Subjects of the present invention include those with disorders.

[0071] As used herein, the term "genetic disease" refers to a disease or disorder that can be treated with polynucleotide therapeutics.Examples of genetic disease include any disease or disorder caused by gene mutation, cancer, and viral infection, disease or disorder caused by mutation that can be corrected using gene editing (e.g., CRISPR / Cas9, or zinc finger nuclease), disease or disorder caused by overexpression of genes, and disease or disorder caused by reduced or absent expression of genes.

[0072] "Charge ratio", "N / P", "N / O" and "N + / O -The term "ratio" is used interchangeably and refers to the ratio of positively charged amine (N or nitrogen) groups in the cationic polymer to negatively charged phosphate (P or O) groups in the polynucleotide. "N" refers to nitrogen (N) in the cationic polymer. "P" and "O" are used interchangeably and refer to phosphate (P) groups or oxygen (O) in a phosphate group in the polynucleotide.

[0073] As used herein, the term "targeting molecule" refers to a molecule that binds to or localizes to a particular target or location. The molecule can be, for example, an antibody or an antigen-binding fragment thereof, or a binding protein.

[0074] As used herein, the term "hybrid polymer" refers to a polymer that includes at least two portions that differ from each other in composition. In some embodiments, at least one portion of the hybrid polymer is a cationic polymer and at least one portion of the hybrid polymer is a neutral polymer.

[0075] As used herein, the term "aggregate" refers to the non-covalent association of molecules to form a solid or mesophase complex structure. The onset of aggregation and the onset of bimolecular attraction are not the same. The aggregated phase involves interactions between multiple molecules, whereas the molecules in the dilute phase interact very weakly with each other. Aggregation begins when the free energy per molecule is equal in the dilute and aggregated phases. In the context of targeting molecule-conjugated polynucleotides, aggregation requires at least 10 non-covalently bound conjugates. In the context of unconjugated polynucleotides, aggregation depends on the length of the polynucleotide. For example, for shorter polynucleotides (e.g., oligonucleotides), aggregates require at least 10 non-covalently bound polynucleotides, whereas gene-length polynucleotides (e.g., average 10 kb to 15 kb for human genes) require fewer polynucleotides for aggregation.

[0076] The term "pharmaceutical effective amount", "therapeutically effective amount" or "therapeutically effective dose" refers to an amount effective to treat a patient's disease, e.g., to effect beneficial and / or desirable changes in the overall health of a patient suffering from a disease, treatment, cure, suppression or remission of a disease (e.g., a genetic disease as described herein), physiological response or condition, etc. A complete therapeutic effect does not necessarily occur by administration of a single dose, but may occur only after administration of a series of doses. Thus, a therapeutically effective amount can be administered in one or more administrations. The exact effective amount required for a subject depends, for example, on the subject's size, health and age, the nature and extent of the disease, the therapeutic agent or combination of therapeutic agents selected for administration, and the mode of administration. Those skilled in the art can easily determine the effective amount for a given situation by routine experimentation. Those skilled in the art will recognize that, for example, treating cancer includes, but is not limited to, killing cancer cells, preventing the growth of new cancer cells, causing tumor regression (reducing tumor size), causing a reduction in metastasis, improving the patient's vital functions, improving the patient's health, reducing pain, improving appetite, improving the patient's weight, and any combination thereof. The term "pharmaceutical effective amount", "therapeutically effective amount" or "therapeutically effective dose" also refers to the amount required to improve the clinical symptoms of a patient. The methods of treatment or methods of treatment described herein should not be construed as or limited to "curing" a disease.

[0077] As used herein, the term "treat" or "treatment" includes reversing, reducing, or arresting the symptoms, clinical signs, and underlying pathology of a condition in a manner that improves or stabilizes the condition of a subject. As used herein and as is well understood in the art, "treatment" is an approach to obtain beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation, remission, or delay in progression of one or more symptoms or conditions associated with a condition, either detectable or undetectable; reduction in the extent of disease; stable (i.e., not worsening) disease status; delay or slowing of disease progression; remission or alleviation, and remission (either partial or total) of disease status. "Treatment" can also mean extending survival compared to the expected survival in the absence of treatment. Exemplary beneficial clinical results are described herein.

[0078] "Administering" or "administration" of the compositions disclosed herein to a subject can be performed using one of a variety of methods known to those skilled in the art. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods of time. In some embodiments, administration includes both direct administration, including self-administration, and indirect administration, including the act of prescribing a drug. For example, as used herein, a physician who has instructed a patient to self-administer a drug or have another physician administer the drug, and / or who has provided a patient with a prescription for a drug, has administered the drug to the patient. If the method is part of a treatment regimen that includes multiple pharmaceutical compositions or treatment modalities, the present disclosure contemplates that the pharmaceutical compositions can be administered at the same or different times and via the same or different routes of administration.

[0079] Compositions for delivery of polynucleotides A first aspect of the present disclosure provides a composition for delivering a polynucleotide. In some embodiments, the composition comprises a hybrid polymer and a polynucleotide. In some embodiments, the hybrid polymer comprises a cationic portion and a neutral portion. In some embodiments, the cationic portion of the hybrid polymer interacts with the polynucleotide non-covalently. In some embodiments, the cationic portion of the hybrid polymer interacts with the polynucleotide via ionic interactions. In some embodiments, the cationic portion of the hybrid polymer and the polynucleotide form an ionic complex. In some embodiments, the hybrid polymer protects the polynucleotide from biological degradation in vivo. Cationic polymers (including cationic polypeptides) are known in the art. See, for example, WO2017 / 173408, which is incorporated herein by reference in its entirety. Without wishing to be bound by theory, the cationic portion of the hybrid polymer (e.g., cationic polypeptide) binds to the polynucleotide and brings it into close association with the cell membrane for uptake by the cell.

[0080] In some embodiments, the cationic portion of the hybrid polymer is a cationic polypeptide. In some embodiments, the cationic polypeptide comprises a positively charged amino acid residue. In some embodiments, the cationic polypeptide comprises a positively charged amino acid residue and a non-positively charged amino acid residue. In some embodiments, the non-positively charged amino acid residue is a neutral amino acid residue. In some embodiments, the non-positively charged amino acid residue is a negatively charged amino acid residue. In some embodiments, the non-positively charged amino acid residue is a neutral amino acid residue and a negatively charged amino acid residue. In some embodiments, the cationic peptide does not insert into or disrupt a cell membrane. In some embodiments, no more than 40% of the amino acid residues in the cationic peptide are hydrophobic residues. In some embodiments, no more than 35% of the amino acid residues in the cationic peptide are hydrophobic residues. In some embodiments, no more than 30% of the amino acid residues in the cationic peptide are hydrophobic residues. In some embodiments, no more than 25% of the amino acid residues in the cationic peptide are hydrophobic residues. In some embodiments, no more than 20% of the amino acid residues in the cationic peptide are hydrophobic residues. In some embodiments, no more than 15% of the amino acid residues in the cationic peptide are hydrophobic residues. In some embodiments, no more than 10% of the amino acid residues in the cationic peptide are hydrophobic residues. In some embodiments, no more than 5% of the amino acid residues in the cationic peptide are hydrophobic residues. In some embodiments, the cationic peptide does not contain any hydrophobic residues.

[0081] In some embodiments, at least 25% of the amino acid residues in the cationic polypeptide are positively charged amino acid residues. In some embodiments, at least 30% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 35% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 40% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 45% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 50% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 55% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 60% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 65% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 70% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 75% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 80% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 85% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 90% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, at least 95% of the amino acid residues in the cationic peptide are positively charged amino acid residues. In some embodiments, all amino acid residues in the cationic peptide are positively charged amino acid residues.

[0082] In some embodiments, the cationic polypeptide comprises at least 6 positively charged amino acid residues. In some embodiments, the cationic polypeptide comprises at least 7 positively charged amino acid residues. In some embodiments, the cationic polypeptide comprises at least 8 positively charged amino acid residues. In some embodiments, the cationic polypeptide comprises 6-20 positively charged amino acid residues. In some embodiments, the cationic polypeptide comprises 7-18 positively charged amino acid residues. In some embodiments, the cationic polypeptide comprises 8-18 positively charged amino acid residues. In some embodiments, the cationic polypeptide has at least 6 net positive charges. In some embodiments, the cationic polypeptide has at least 7 net positive charges. In some embodiments, the cationic polypeptide has at least 8 net positive charges. In some embodiments, the cationic polypeptide has 6-20 net positive charges. In some embodiments, the cationic polypeptide has 7-18 net positive charges. In some embodiments, the cationic polypeptide has 8-18 net positive charges. The net charge of a peptide is determined by subtracting the number of negatively charged amino acid residues from the number of positively charged amino acid residues.As an example, a cationic polypeptide that contains 10 positively charged amino acid residues and 3 negatively charged amino acid residues has a net positive charge of 7.Similarly, a cationic polypeptide that contains 7 positively charged amino acid residues and 10 neutral amino acid residues has a net positive charge of 7.

[0083] Positively charged amino acid residues include arginine ("Arg" or R), lysine ("Lys" or K), and histidine ("His" or H). In some embodiments, the cationic polypeptide comprises arginine, lysine, and / or histidine. In some embodiments, the cationic polypeptide comprises an arginine residue. In some embodiments, the cationic polypeptide comprises a lysine residue. In some embodiments, the cationic polypeptide comprises a histidine residue. In some embodiments, the cationic polypeptide comprises an arginine residue and a lysine residue. In some embodiments, the cationic polypeptide comprises an arginine residue and a histidine residue. In some embodiments, the cationic polypeptide comprises a histidine residue and a lysine residue. In some embodiments, the cationic polypeptide comprises an arginine residue, a lysine residue, and a histidine residue. The cationic polypeptide can be a poly-arginine polypeptide. In some embodiments, the cationic polypeptide is a poly-lysine polypeptide. Optionally, the cationic polypeptide is a poly-histidine polypeptide. Poly-arginine and poly-lysine have been shown to be less immunogenic than other cationic polypeptides, including cationic polypeptides that contain neutral and / or negatively charged amino acid residues. In fact, the FDA has approved poly-arginine for use as a coagulant and as a vaccine adjuvant. Those skilled in the art will recognize that arginine residues have a stronger affinity for polynucleotides than lysine or histidine residues. Furthermore, lysine residues have a stronger affinity for polynucleotides than histidine residues. Thus, the poly-lysine polypeptides used in the compositions and methods of the present disclosure are typically longer than the poly-arginine polypeptides used in the compositions and methods of the present disclosure. Similarly, the poly-histidine polypeptides used in the compositions and methods of the present disclosure are typically longer than the poly-arginine or poly-lysine polypeptides used in the compositions and methods of the present disclosure. In some embodiments, the cationic polypeptides include cross-linking amino acid residues.In some embodiments, the bridging amino acid residue is cysteine. In some embodiments, the cationic polypeptide comprises one or more hydrophobic amino acid residues. In some embodiments, the one or more hydrophobic amino acid residues are selected from the group consisting of phenylalanine, tryptophan, leucine, alanine, isoleucine, and combinations thereof. It is within the skill of the art to determine the exact length of a cationic polypeptide based on its composition (i.e., its number and type of amino acid residues).

[0084] In some embodiments, the cationic polypeptide comprises L-amino acid residues. The cationic polypeptide may comprise D-amino acid residues. In some embodiments, the cationic polypeptide comprises L-amino acid residues and D-amino acid residues. The cationic polypeptide may comprise 9-18 amino acid residues. In some embodiments, the cationic polypeptide comprises 12 amino acid residues. For the formation of nanoparticles, cationic polypeptides longer than 18 residues and comprising only cationic amino acid residues are preferred. Thus, to avoid the formation of nanoparticles, in some embodiments, when the cationic polypeptide comprises only cationic amino acid residues, the cationic polypeptide comprises 18 or fewer amino acid residues.

[0085] The cationic polypeptide may include protamine. In some embodiments, the cationic peptide is an arginine-glycine-aspartic acid (RGD) polypeptide. In some embodiments, the cationic polypeptide is a cell penetrating peptide (CPP) having a cationic charge. Non-limiting examples of suitable CPPs are shown in Table 1 below. Each CPP in Table 1 is considered a separate embodiment. TIFF2025503504000002.tif119159

[0086] In some embodiments, the cationic polypeptide is selected from the group of cationic polypeptides disclosed below in Table 2. In some embodiments, the hybrid polymer is selected from the group of hybrid polymers disclosed below in Table 2. Each cationic peptide and hybrid polymer in Table 2 is considered a separate embodiment. TIFF2025503504000003.tif203159

[0087] In some embodiments, the cationic polymer is a linear polymer. In some embodiments, the cationic polymer is a branched polymer. For the formation of nanoparticles, high molecular weight cationic polymers are preferred. Thus, in some embodiments, the cationic portion of the hybrid polymer comprises a low molecular weight polymer. In some embodiments, the low molecular weight polymer has a molecular weight of about 600 to about 2,000 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 700 to about 1,200 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 800 to about 1,000 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 600 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 700 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 800 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 900 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1000 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1100 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1200 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1300 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1400 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1500 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1600 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1700 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1800 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 1900 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of about 2000 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 600-2,000 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 700-1,200 Daltons.In some embodiments, the low molecular weight polymer has a molecular weight of 800-1,000 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 600 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 700 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 800 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 900 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1000 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1100 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1200 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1300 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1400 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1500 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1600 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1700 daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1800 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 1900 Daltons. In some embodiments, the low molecular weight polymer has a molecular weight of 2000 Daltons. In some embodiments, the low molecular weight polymer is selected from the group consisting of gelatin, glucosamine, N-acetylglucosamine, chitosan, cationic dextran, cationic cyclodextrin, cationic cellulose, polyethyleneimine (PEI), polyamidoamine (PAA), poly(amino-co-ester) (PAE), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), or cationic lipids such as DOTAP (N-(1-(2,3-dioleyloxy)propyl)-N,N,N trimethylammonium) chloride, cationic mucic acid polymer (cMAP), and DOPE (dioleylphosphatidylethanolamine). In some embodiments, the cationic polymer is linear PEI.In some embodiments, the cationic polymer is branched PEI (BPEI).

[0088] In some embodiments, the hybrid polymer is any of the polymers disclosed in Tables 5, 7, 8, and 9 below. Each hybrid polymer listed in Tables 5, 7, 8, and 9 is considered a separate embodiment. In some embodiments, the hybrid polymer is any of the polymers disclosed in Tables 5, 7, 8, and 9 below. Each hybrid polymer listed in Tables 5, 7, 8, and 9 is considered a separate embodiment. In some embodiments, the hybrid polymer is any of the polymers disclosed in Tables 5, 7, 8, and 9 below. Each hybrid polymer listed in Tables 5, 7, 8, and 9 is considered a separate embodiment. In some embodiments, the hybrid polymer is any of the polymers disclosed in Tables 5, 7, 8, and 9 below. In some embodiments, the hybrid polymer is selected from the group consisting of PEG12PolyArg9d, PEG1000DaPolyArg12, PEG2000DaPolyArg12, PEG5000DaPolyArg12, PolyArg12Cbp1.5kDa, PolyArg12Cbp3.9kDa, PolyArg12Cbp16kDa, CPolyArg12Cbp1.5kDa, PolyArg12Cbp2kDa, PolyArg12bp2kDa, amide dextran, lysine dextran, PEG PEI 15kda, BPEI-G-PEG550, and BPEI-G-PEG5000. In some embodiments, the hybrid polymer is PEG12PolyArg12{d}. In some embodiments, the hybrid polymer is PEG12PolyArg6. In some embodiments, the hybrid polymer is PEG12PolyArg6C. In some embodiments, the hybrid polymer is PEG24PolyArg12C. In some embodiments, the hybrid polymer is PEG24PolyArg12. In some embodiments, the hybrid polymer is PEG24PolyArg9. In some embodiments, the hybrid polymer is PolyArg12C-PEG2000Da. In some embodiments, the hybrid polymer is PolyArg12C-PEG5000Da. In some embodiments, the hybrid polymer is PolyArg12C-Dextran5000Da.In some embodiments, the hybrid polymer is PEG12PolyArg12. In some embodiments, the hybrid polymer is PEG12PolyArg9d. In some embodiments, the hybrid polymer is PEG1000DaPolyArg12. In some embodiments, the hybrid polymer is PEG2000DaPolyArg12. In some embodiments, the hybrid polymer is PEG5000DaPolyArg12. In some embodiments, the hybrid polymer is PolyArg12Cbp1.5kDa. In some embodiments, the hybrid polymer is PolyArg12Cbp3.9kDa. In some embodiments, the hybrid polymer is PolyArg12Cbp16kDa. In some embodiments, the hybrid polymer is CPolyArg12Cbp1.5kDa. In some embodiments, the hybrid polymer is PolyArg12Cbp2kDa. In some embodiments, the hybrid polymer is PolyArg12bp2kDa. In some embodiments, the hybrid polymer is amide dextran. In some embodiments, the hybrid polymer is lysine dextran. In some embodiments, the hybrid polymer is PEG PEI 15kda. In some embodiments, the hybrid polymer is BPEI-G-PEG 550. In some embodiments, the hybrid polymer is BPEI-G-PEG 5000.

[0089] In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 100 to about 10,000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 100 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 200 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 300 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 400 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 500 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 600 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 700 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 800 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 900 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 1000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 2000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 3000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 4000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 5000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 6000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 7000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 8000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 9000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of about 10000 Daltons.

[0090] In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 100 to 10,000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 100 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 200 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 300 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 400 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 500 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 600 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 700 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 800 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 900 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 1000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 2000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 3000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 4000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 5000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 6000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 7000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 8000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 9000 Daltons. In some embodiments, the neutral portion of the hybrid polymer has a molecular weight of 10000 Daltons.

[0091] In some embodiments, the neutral portion of the hybrid polymer is a natural or synthetic polymer consisting of long chains of branched or unbranched monomers and / or crosslinked networks of monomers in two or three dimensions. In some embodiments, the neutral portion of the hybrid polymer comprises polysaccharides, lignin, rubber, or polyalkylene oxides (e.g., polyethylene glycol). Examples of the neutral portion of the hybrid polymer include, but are not limited to, α-, ω-dihydroxyl polyethylene glycol, biodegradable lactone-based polymers such as polyacrylic acid, polylactide acid (PLA), poly(glycolic acid) (PGA), polypropylene, polystyrene, polyolefins, polyamides, polycyanoacrylates, polyimides, polyethylene terephthalate (PET, PETG), polyethylene terephthalate (PETE), polytetramethylene glycol (PTG), or polyurethanes, and mixtures thereof. As used herein, mixtures refer to the use of different polymers within the same compound, as well as in reference to block copolymers. In some embodiments, a block copolymer is a polymer in which at least one section of the polymer is constructed from monomers of another polymer. In some embodiments, the neutral portion of the hybrid polymer comprises a polyalkylene oxide. In some embodiments, the neutral portion of the hybrid polymer comprises a poly(ethylene glycol) (PEG). In some embodiments, the neutral portion of the hybrid polymer comprises a branched poly(ethylene glycol) (PEG). In some embodiments, the neutral portion of the hybrid polymer comprises a linear poly(ethylene glycol) (PEG). In some embodiments, the hybrid polymer is a PEGylated cationic polypeptide. The hybrid polymer may comprise a PEG12-PEG24 polymer. Other suitable polymers as the neutral portion of the hybrid polymer are known in the art. See, for example, Thi TTH, et al., Polymers, 2020 vol. 12: 298; doi: 10.3390 / polym12020298, which is incorporated herein by reference in its entirety.

[0092] In some embodiments, the hybrid polymer and polynucleotide do not form aggregates or nanoparticles. In some embodiments, the charge ratio of the cationic polypeptide to the polynucleotide is about 0.25:1 to about 5:1. In some embodiments, the charge ratio of the cationic peptide to the polynucleotide is about 0.5:1 to about 5:1. In some embodiments, the charge ratio of the cationic peptide to the polynucleotide is about 1:1 to about 4:1. The charge ratio of the cationic polypeptide to the polynucleotide can be about 1:1 to about 2:1. In some embodiments, the charge ratio of the cationic polypeptide to the polynucleotide is about 1:1 or about 2:1.

[0093] In some embodiments, the hybrid polymer and polynucleotide do not form aggregates or nanoparticles. In some embodiments, the charge ratio of the cationic polymer to the polynucleotide is 0.25:1 to 5:1. In some embodiments, the charge ratio of the cationic polymer to the polynucleotide is 0.5:1 to 5:1. In some embodiments, the charge ratio of the cationic polymer to the polynucleotide is 1:1 to 4:1. The charge ratio of the cationic polymer to the polynucleotide may be 1:1 to 2:1. In some embodiments, the charge ratio of the cationic polymer to the polynucleotide is 1:1 or 2:1.

[0094] In some embodiments, at least 50% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 55% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 60% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 65% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 70% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 75% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 80% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 85% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 90% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 95% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 97% of the polynucleotides in the composition are present as monomeric species. In some embodiments, at least 99% of the polynucleotides in the composition are present as monomeric species. In some embodiments, all polynucleotides in the composition are present as monomeric species. In the context of non-conjugated polynucleotides in the composition, the term "monomeric species" refers to a complex that includes a single polynucleotide molecule and one or more hybrid polymers of the present disclosure. For example, in the case of non-conjugated polymers, a monomeric species includes a single polynucleotide and one or more hybrid polymers to which it is ionically bound. In the context of polynucleotides conjugated to a targeting molecule, since a targeting molecule can be conjugated to multiple polynucleotides (e.g., DAR is greater than 1), the term "monomeric species" refers to a complex that includes only polynucleotides conjugated to the same targeting molecule.For example, in the case of a polynucleotide conjugated to a targeting molecule, the monomeric species include a single targeting molecule, a polynucleotide conjugated to a targeting molecule (and only those polynucleotides), and one or more hybrid polymers to which the polynucleotide is ionically linked.

[0095] In some embodiments, the polynucleotide is designed to hybridize to or dimerize with another polynucleotide. In some embodiments, the targeting molecule conjugated to one or more polynucleotides is designed to bind to or dimerize with another target molecule. In some embodiments, at least 50% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 55% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 60% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 65% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 70% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 75% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 80% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 85% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 90% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 95% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 97% of the polynucleotides in the composition are present as dimeric species. In some embodiments, at least 99% of the polynucleotides in the composition are present as dimeric species. In some embodiments, all polynucleotides in the composition are present as dimeric species. In the context of non-conjugated polynucleotides in a composition, the term "dimeric species" refers to a complex comprising two polynucleotide molecules and one or more hybrid polymers of the present disclosure. For example, in the case of non-conjugated polymers, the dimeric species comprises two polynucleotides and one or more hybrid polymers to which they are ionically linked.In the context of a polynucleotide conjugated to a targeting molecule, since the targeting molecule can be conjugated to multiple polynucleotides (e.g., DAR is greater than 1), the term "dimeric species" refers to a complex that contains only a polynucleotide conjugated to two targeting molecules. For example, in the case of a polynucleotide conjugated to a targeting molecule, the dimeric species includes two targeting molecules, a polynucleotide conjugated to two targeting molecules (and only those polynucleotides), and one or more hybrid polymers to which the polynucleotides are ionically linked.

[0096] In some embodiments, the polynucleotide is designed to hybridize to two other polynucleotides or to trimerize with two other polynucleotides. In some embodiments, the targeting molecule conjugated to one or more polynucleotides is designed to bind to or trimerize with two targeting molecules. In some embodiments, at least 50% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 55% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 60% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 65% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 70% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 75% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 80% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 85% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 90% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 95% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 97% of the polynucleotides in the composition are present as trimer species. In some embodiments, at least 99% of the polynucleotides in the composition are present as trimer species. In some embodiments, all polynucleotides in the composition are present as trimer species. In the context of non-conjugated polynucleotides in the composition, the term "trimeric species" refers to a complex comprising three polynucleotide molecules and one or more hybrid polymers of the present disclosure. For example, in the case of non-conjugated polymers, the trimeric species comprises three polynucleotides and one or more hybrid polymers to which they are ionically linked.In the context of a polynucleotide conjugated to a targeting molecule, the targeting molecule may be conjugated to multiple polynucleotides (e.g., DAR is greater than 1), and therefore the term "trimeric species" refers to a complex that contains only a polynucleotide conjugated to three targeting molecules. For example, in the case of a polynucleotide conjugated to a targeting molecule, the trimeric species contains three targeting molecules, a polynucleotide conjugated to the three targeting molecules (and only those polynucleotides), and one or more hybrid polymers to which the polynucleotides are ionically linked.

[0097] In some embodiments, the polynucleotide is conjugated to a targeting molecule. In some embodiments, the targeting moiety includes amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and analogs or derivatives of all these classes of substances. Additional examples of targeting moieties also include steroids, such as cholesterol, phospholipids, di- and triacylglycerols, fatty acids, carbohydrates (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some embodiments, the targeting moiety is an antibody or a binding fragment thereof.

[0098] The targeting molecule can be an antibody or an antigen-binding fragment thereof, or a binding protein. In some embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof (e.g., a polynucleotide-antibody conjugate). In some embodiments, the antibody or binding fragment thereof is a human antibody or an antigen-binding fragment thereof, a humanized antibody or an antigen-binding fragment thereof, a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a monoclonal antibody or an antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv ...2 The antibody or antigen-binding fragment thereof is an immunoglobulin single variable domain (ISV) such as a diabody, a minibody, a Nanobody® molecule, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a vNAR, a centrin, a camelid antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, a Fab, a Fab-Fc, an Fv, a single chain Fv (scFv), a diabody, a minibody, and a Nanobody®. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody. Non-limiting examples of bispecific antibodies are in Bispecific T Cell Engagers (BiTEs) and Dual Affinity Retargeting Antibodies (DARTs). In some embodiments, the bispecific antibody is a trifunctional antibody or a bispecific miniantibody. In some embodiments, the bispecific antibody is a trifunctional antibody. In some embodiments, the trifunctional antibody is a full-length monoclonal antibody that comprises binding sites for two different antigens. In some embodiments, the bispecific antibody is a bispecific miniantibody. In some embodiments, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, a bis-scFv, (scFv)2, a diabody, a minibody, a triabody, a tetrabody, or a bispecific T cell engager (BiTE). In some embodiments, the bispecific T cell engager is a fusion protein comprising two single chain variable fragments (scFv), where the two scFvs target epitopes of two different antigens.

[0099] In some embodiments, the antibody or antigen-binding fragment thereof is a Fab. In some embodiments, the antibody or antigen-binding fragment thereof is a Fab-Fc. In some embodiments, the antibody or antigen-binding fragment thereof is an Fv. In some embodiments, the antibody or antigen-binding fragment thereof is a single chain Fv (scFv). In some embodiments, when the antibody or antigen-binding moiety is an scFv, the polynucleotide does not comprise bridging residues. In some embodiments, when the antibody or antigen-binding moiety is an scFv, the polynucleotide does not comprise cysteines. In some embodiments, the antibody or antigen-binding fragment thereof is a diabody. In some embodiments, the antibody or antigen-binding fragment thereof is a minibody. In some embodiments, the antibody or antigen-binding fragment thereof is an immunoglobulin single variable domain (ISV), such as a Nanobody® molecule. The Nanobody® can be Nanobody-HSA®.

[0100] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG molecule or is derived from an IgG molecule. The IgG molecule may be an IgG1 or IgG4 molecule. The antibody or antigen-binding fragment thereof may be an IgG1 molecule or may be derived therefrom. The antibody or antigen-binding fragment thereof may be an IgG2 molecule or may be derived therefrom. The antibody or antigen-binding fragment thereof may be an IgG3 molecule or may be derived therefrom. The antibody or antigen-binding fragment thereof may be an IgG4 molecule or may be derived therefrom.

[0101] Non-limiting examples of antibodies and antigen-binding fragments that can be used as targeting molecules in the present disclosure include FV55scFv, Fv55 diabody, 3TF12, and cetuximab. FV55 scFv is a monospecific scFv that binds to the human transferrin receptor (TfR1). The CDRs of FV55 scFv are identical to those of HB21, and FV55 scFv has V-terminal domains connected by (G4S)*3. H -V Loriented toward the 5'-terminal end and has a c-terminal cysteine ​​for conjugation. The molecular weight of FV55 scFv is about 26.5 kDa. See, for example, Haynes BF, Hemler M, Cotner T, Mann DL, Eisenbarth GS, Strominger JL, Fauci AS. Characterization of a monoclonal antibody (5E9) that defines a human cell surface antigen of cell activation. J Immunol. 1981; 127: 347-351. [PubMed: 6787129]. Fv55 diabodies contain two copies of FV55 scFv, except that the linker is (G4S) * N, where N is 1 or 2. The molecular weight of Fv55 diabodies is about 53 kDa. 3TF12 is a monospecific scFv that binds to the human transferrin receptor (TfR1). 3TF12 is a V connected by (G4S)*N H -V L When N is 3, 3tf12 is a monomeric scFv, and when N is 1, 3tf12 dimerizes to form a diabody. See, e.g., Crepin R. et al. Development of Human Single-Chain Antibodies to the Transferrin Receptor that Effectively Antagonize the Growth of Leukemias and Lymphomas. Cancer Research 2010, 70(13):5497-506. Cetuximab is a chimeric (mouse / human) monoclonal antibody and epidermal growth factor receptor (EGFR) inhibitor used to treat metastatic colorectal cancer and head and neck cancer. Cetuximab has a molecular weight of 145,781.92 g / mol.

[0102] In some embodiments, the targeting molecule is a binding protein. The binding protein can be a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor comprises the extracellular domain of the receptor. In some embodiments, the soluble receptor is an Fc fusion protein.

[0103] In some embodiments, the targeting molecule is a plasma protein. In some embodiments, the plasma protein comprises albumin. In some embodiments, the albumin is conjugated to the polynucleotide by one or more of the conjugation chemistries disclosed herein. In some cases, the albumin is conjugated to the polynucleotide by native ligation chemistry. In some cases, the albumin is conjugated to the polynucleotide by lysine conjugation.

[0104] In some cases, the targeting molecule is a steroid. Non-limiting exemplary steroids include cholesterol, phospholipids, di- and triacylglycerols, fatty acids, saturated, unsaturated, substituted, or other hydrocarbons, or combinations thereof. In some embodiments, the steroid is cholesterol or a cholesterol derivative. In some embodiments, the targeting molecule is cholesterol. In some embodiments, the steroid is conjugated to the polynucleotide by one or more of the conjugation chemistries disclosed herein. In some embodiments, the steroid is conjugated to the polynucleotide by native ligation chemistry.

[0105] In some embodiments, the targeting molecule is a polymer, including but not limited to a polynucleotide aptamer that binds to a specific surface marker on a cell.In some embodiments, the targeting molecule is a polynucleotide that does not hybridize to the target gene or mRNA, but instead can selectively bind to the cell surface marker, similar to an antibody that binds to its specific epitope on the cell surface marker.

[0106] In some embodiments, the targeting molecule is a polypeptide. In some embodiments, the polypeptide has a size of about 1 to about 3 kDa. In some embodiments, the polypeptide has a size of about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some embodiments, the targeting molecule is a polypeptide. In some embodiments, the polypeptide has a size of 1 to 3 kDa. In some embodiments, the polypeptide has a size of 1.2 to 2.8 kDa, 1.5 to 2.5 kDa, or 1.5 to 2 kDa. In some embodiments, the polypeptide is a bicyclic polypeptide. In some embodiments, the bicyclic polypeptide is a constrained bicyclic polypeptide. In some embodiments, the targeting molecule is a bicyclic polypeptide (e.g., Bicycle from Bicycle Therapeutics).

[0107] In additional embodiments, the targeting molecule is a small molecule. In some embodiments, the small molecule is an antibody-recruiting small molecule. In some embodiments, the antibody-recruiting small molecule comprises a target-binding end and an antibody-binding end, where the target-binding end is capable of recognizing and interacting with a cell surface receptor.

[0108] In some embodiments, the targeting molecule is a therapeutically or biologically active molecule.

[0109] In some embodiments, the polynucleotide is about 5 to about 100 nucleotides in length. In some embodiments, the polynucleotide is about 5 to about 50 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 30, about 15 to about 30, about 18 to about 25, about 18 to about 24, about 19 to about 23, or about 20 to about 22 nucleotides in length. In some embodiments, the polynucleotide is about 50 nucleotides in length. In some embodiments, the polynucleotide is about 49 nucleotides in length. In some embodiments, the polynucleotide is about 48 nucleotides in length. In some embodiments, the polynucleotide is about 47 nucleotides in length. In some embodiments, the polynucleotide is about 46 nucleotides in length. In some embodiments, the polynucleotide is about 45 nucleotides in length. In some embodiments, the polynucleotide is about 44 nucleotides in length. In some embodiments, the polynucleotide is about 43 nucleotides in length. In some embodiments, the polynucleotide is about 42 nucleotides in length. In some embodiments, the polynucleotide is about 41 nucleotides in length. In some embodiments, the polynucleotide is about 40 nucleotides in length. In some embodiments, the polynucleotide is about 39 nucleotides in length. In some embodiments, the polynucleotide is about 38 nucleotides in length. In some embodiments, the polynucleotide is about 37 nucleotides in length. In some embodiments, the polynucleotide is about 36 nucleotides in length. In some embodiments, the polynucleotide is about 35 nucleotides in length. In some embodiments, the polynucleotide is about 34 nucleotides in length. In some embodiments, the polynucleotide is about 33 nucleotides in length. In some embodiments, the polynucleotide is about 32 nucleotides in length. In some embodiments, the polynucleotide is about 31 nucleotides in length. In some embodiments, the polynucleotide is about 30 nucleotides in length. In some embodiments, the polynucleotide is about 29 nucleotides in length. In some embodiments, the polynucleotide is about 28 nucleotides in length.In some embodiments, the polynucleotide is about 27 nucleotides in length. In some embodiments, the polynucleotide is about 26 nucleotides in length. In some embodiments, the polynucleotide is about 25 nucleotides in length. In some embodiments, the polynucleotide is about 24 nucleotides in length. In some embodiments, the polynucleotide is about 23 nucleotides in length. In some embodiments, the polynucleotide is about 22 nucleotides in length. In some embodiments, the polynucleotide is about 21 nucleotides in length. In some embodiments, the polynucleotide is about 20 nucleotides in length. In some embodiments, the polynucleotide is about 19 nucleotides in length. In some embodiments, the polynucleotide is about 18 nucleotides in length. In some embodiments, the polynucleotide is about 17 nucleotides in length. In some embodiments, the polynucleotide is about 16 nucleotides in length. In some embodiments, the polynucleotide is about 15 nucleotides in length. In some embodiments, the polynucleotide is about 14 nucleotides in length. In some embodiments, the polynucleotide is about 13 nucleotides in length. In some embodiments, the polynucleotide is about 12 nucleotides in length. In some embodiments, the polynucleotide is about 11 nucleotides in length. In some embodiments, the polynucleotide is about 10 nucleotides in length. In some embodiments, the polynucleotide is about 9 nucleotides in length. In some embodiments, the polynucleotide is about 8 nucleotides in length. In some embodiments, the polynucleotide is about 7 nucleotides in length. In some embodiments, the polynucleotide is about 6 nucleotides in length. In some embodiments, the polynucleotide is about 5 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 50 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 45 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 40 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 35 nucleotides in length.In some embodiments, the polynucleotide is about 10 to about 30 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 25 nucleotides in length. In some embodiments, the polynucleotide is about 10 to about 20 nucleotides in length. In some embodiments, the polynucleotide is about 15 to about 25 nucleotides in length. In some embodiments, the polynucleotide is about 15 to about 30 nucleotides in length. In some embodiments, the polynucleotide is about 12 to about 30 nucleotides in length.

[0110] In some embodiments, the polynucleotide is 5-100 nucleotides in length. In some embodiments, the polynucleotide is 5-50 nucleotides in length. In some embodiments, the polynucleotide is 10-30, 15-30, 18-25, 18-24, 19-23, or 20-22 nucleotides in length. In some embodiments, the polynucleotide is 50 nucleotides in length. In some embodiments, the polynucleotide is 49 nucleotides in length. In some embodiments, the polynucleotide is 48 nucleotides in length. In some embodiments, the polynucleotide is 47 nucleotides in length. In some embodiments, the polynucleotide is 46 nucleotides in length. In some embodiments, the polynucleotide is 45 nucleotides in length. In some embodiments, the polynucleotide is 44 nucleotides in length. In some embodiments, the polynucleotide is 43 nucleotides in length. In some embodiments, the polynucleotide is 42 nucleotides in length. In some embodiments, the polynucleotide is 41 nucleotides in length. In some embodiments, the polynucleotide is 40 nucleotides in length. In some embodiments, the polynucleotide is 39 nucleotides in length. In some embodiments, the polynucleotide is 38 nucleotides in length. In some embodiments, the polynucleotide is 37 nucleotides in length. In some embodiments, the polynucleotide is 36 nucleotides in length. In some embodiments, the polynucleotide is 35 nucleotides in length. In some embodiments, the polynucleotide is 34 nucleotides in length. In some embodiments, the polynucleotide is 33 nucleotides in length. In some embodiments, the polynucleotide is 32 nucleotides in length. In some embodiments, the polynucleotide is 31 nucleotides in length. In some embodiments, the polynucleotide is 30 nucleotides in length. In some embodiments, the polynucleotide is 29 nucleotides in length. In some embodiments, the polynucleotide is 28 nucleotides in length. In some embodiments, the polynucleotide is 27 nucleotides in length.In some embodiments, the polynucleotide is 26 nucleotides in length. In some embodiments, the polynucleotide is 25 nucleotides in length. In some embodiments, the polynucleotide is 24 nucleotides in length. In some embodiments, the polynucleotide is 23 nucleotides in length. In some embodiments, the polynucleotide is 22 nucleotides in length. In some embodiments, the polynucleotide is 21 nucleotides in length. In some embodiments, the polynucleotide is 20 nucleotides in length. In some embodiments, the polynucleotide is 19 nucleotides in length. In some embodiments, the polynucleotide is 18 nucleotides in length. In some embodiments, the polynucleotide is 17 nucleotides in length. In some embodiments, the polynucleotide is 16 nucleotides in length. In some embodiments, the polynucleotide is 15 nucleotides in length. In some embodiments, the polynucleotide is 14 nucleotides in length. In some embodiments, the polynucleotide is 13 nucleotides in length. In some embodiments, the polynucleotide is 12 nucleotides in length. In some embodiments, the polynucleotide is 11 nucleotides in length. In some embodiments, the polynucleotide is 10 nucleotides in length. In some embodiments, the polynucleotide is 9 nucleotides in length. In some embodiments, the polynucleotide is 8 nucleotides in length. In some embodiments, the polynucleotide is 7 nucleotides in length. In some embodiments, the polynucleotide is 6 nucleotides in length. In some embodiments, the polynucleotide is 5 nucleotides in length. In some embodiments, the polynucleotide is 10-50 nucleotides in length. In some embodiments, the polynucleotide is 10-45 nucleotides in length. In some embodiments, the polynucleotide is 10-40 nucleotides in length. In some embodiments, the polynucleotide is 10-35 nucleotides in length. In some embodiments, the polynucleotide is 10-30 nucleotides in length. In some embodiments, the polynucleotide is 10-25 nucleotides in length.In some embodiments, the polynucleotide is 10-20 nucleotides in length. In some embodiments, the polynucleotide is 15-25 nucleotides in length. In some embodiments, the polynucleotide is 15-30 nucleotides in length. In some embodiments, the polynucleotide is 12-30 nucleotides in length.

[0111] In some embodiments, the polynucleotide comprises RNA, DNA, or a combination thereof. In some embodiments, the polynucleotide comprises RNA. In some embodiments, the polynucleotide comprises DNA. In some embodiments, the polynucleotide comprises RNA and DNA. In some embodiments, the polynucleotide comprises a combination of DNA, RNA, and / or artificial nucleotide analogs. In some embodiments, the polynucleotide is a regulatory non-coding RNA (ncRNA). In some embodiments, the ncRNA comprises short non-coding RNA sequences expressed in the genome that control the expression or function of other biomolecules in mammalian cells. ncRNAs are generally less than 200 nucleotides in length, can be single-stranded or double-stranded, and can form non-linear secondary or tertiary structures. The ncRNA may include exogenous small interfering RNA (siRNA), microRNA (miRNA), small nuclear RNA (U-RNA), small nucleolar RNA (snoRNA), Piwi-binding RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small RNA derived from rDNA (srRNA), small RNA derived from transfer RNA (tsRNA), small RNA derived from ribosomal RNA (rsRNA), small RNA derived from large non-coding RNA (lncsRNA), or small RNA derived from messenger RNA (msRNA). In some embodiments, the polynucleotide is an engineered polynucleotide. The engineered polynucleotide may include DNA or RNA. In some embodiments, the engineered polynucleotide includes multiple nucleotides. In some embodiments, the engineered polynucleotide includes artificial nucleotide analogs. In some embodiments, the engineered polynucleotide includes DNA. In some embodiments, the DNA is genomic DNA, cell-free DNA, cDNA, fetal DNA, viral DNA, or maternal DNA. In some embodiments, the engineered polynucleotide includes RNA.In some embodiments, the RNA is a non-coding RNA element that regulates ribosomal RNA transcription by interacting with siRNA, ncRNA mimics, short hairpin RNA (shRNA), dicer-dependent siRNA (di-siRNA), antisense oligonucleotide (ASO), gapmer, mixmer, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), RNA-activating oligonucleotide (RNAa), transfer RNA (tRNA), ribosomal RNA (rRNA), heterogeneous nuclear RNA (hnRNA), promoter-associated RNA (pRNA), TIP5 (NoRC RNA), ribozymes, anti-microRNA (antimiR), aptamers, or exon-skipping oligonucleotides. In some embodiments, the engineered polynucleotide comprises a fully synthetic miRNA. A fully synthetic miRNA is one that is not derived from or based on an ncRNA. Alternatively, a fully synthetic miRNA may be based on an analysis of multiple potential target sequences or may be based on an isolated natural non-coding sequence that is not an ncRNA. In some embodiments, the polynucleotide is selected from the group consisting of siRNA, miRNA, miRNA mimic, antisense oligonucleotide (ASO), mRNA, and guide RNA. The polynucleotide may be an siRNA. In some embodiments, the polynucleotide is an miRNA. In some embodiments, the polynucleotide is an miRNA mimic. The polynucleotide may be miR-30 or a mimic of miR-30. Non-limiting examples of miR-30 mimics are shown in Table 3 below. Each miR-30 mimic in Table 3 is considered a separate embodiment. TIFF2025503504000004.tif80159

[0112] In some embodiments, the miR-30 mimic is selected from the group consisting of M30m1, M30m2, M30m3, and M30m4. In some embodiments, the miR-30 mimic is M30m1. In some embodiments, the miR-30 mimic is M30m2. In some embodiments, the miR-30 mimic is M30m3. In some embodiments, the miR-30 mimic is M30m4.

[0113] In some embodiments, it is an ASO. In some embodiments, the ASO is a DMPK ASO. In some embodiments, the ASO is a CAPN3 ASO. The ASO can be a dystrophy-targeting exon skipping ASO. The ASO can be a DUX4-targeting ASO. DUX4-targeting ASOs are known in the art. See, for example, WO2021 / 203043 and U.S. Provisional Patent Application No. 63 / 221,568, each of which is incorporated herein by reference in its entirety. Additional non-limiting examples of DUX4-targeting ASOs are shown in Table 4 below. Each DUX4-targeting ASO in Table 4 is considered a separate embodiment. In some embodiments, the DUX4-targeting ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32. In some embodiments, the DUX4-targeting ASO is ASDX2. In some embodiments, the DUX4-targeting ASO is ASDX4. In some embodiments, the DUX4-targeted ASO is ASDX23. In some embodiments, the DUX4-targeted ASO is ASDX26. In some embodiments, the DUX4-targeted ASO is ASDX32. TIFF2025503504000005.tif227159TIFF2025503504000006.tif218159

[0114] In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA targeting DUX4, DMPK, or CAPN3. In some embodiments, the polynucleotide comprises an siRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DUX4. In some embodiments, the polynucleotide comprises an ASO targeting DUX4. In some embodiments, the polynucleotide comprises a guide RNA targeting DUX4. In some embodiments, the polynucleotide comprises an siRNA targeting DMPK. In some embodiments, the polynucleotide comprises an miRNA targeting DMPK. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DMPK. In some embodiments, the polynucleotide comprises an ASO targeting DMPK. In some embodiments, the polynucleotide comprises an siRNA targeting CAPN3. In some embodiments, the polynucleotide comprises an miRNA targeting CAPN3. In some embodiments, the polynucleotide comprises an miRNA mimic targeting CAPN3. In some embodiments, the polynucleotide comprises an ASO that targets CAPN3.

[0115] In some embodiments, the polynucleotide is a coding RNA. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is a non-coding RNA. In some embodiments, the polynucleotide is a long non-coding RNA. In some embodiments, the polynucleotide is a guide RNA.

[0116] In some embodiments, a polynucleotide comprises one or more artificial nucleotide analogs. In some cases, the artificial nucleotide analog comprises a modification at one or more of the ribose moiety, the phosphate moiety, the nucleoside moiety, or a combination thereof. In some embodiments, one or more of the artificial nucleotide analogs is resistant to nucleases, for example ribonucleases such as RNase, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases, when compared to a natural polynucleotide. In some embodiments, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamide (2'-O-NMA) modified, LNA, EN Artificial nucleotide analogs, including A, PNA, HNA, BNA, 2'-O-ethyl (cEt), morpholino, methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof, are resistant to nucleases, e.g., ribonucleases such as RNase, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some embodiments, the 2'-O-methyl modified polynucleotide is nuclease-resistant (e.g., RNase-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some embodiments, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleotides are nuclease resistant (eg, RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant).In some embodiments, 2'-O-aminopropyl modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-deoxy modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, T-deoxy-2'-fluoro modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-O-aminopropyl (2'-O-AP) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant).In some embodiments, the LNA-modified polynucleotide is nuclease-resistant (e.g., RNase-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some embodiments, the ENA-modified polynucleotide is nuclease-resistant (e.g., RNase-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some embodiments, the HNA-modified polynucleotide is nuclease-resistant (e.g., RNase-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). The morpholino can be nuclease-resistant (e.g., RNase-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some embodiments, PNA-modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, methylphosphonate nucleotide-modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, thiol phosphonate nucleotide-modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, polynucleotides comprising 2'-fluoro N3-P5'-phosphoramidites are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, the 5' conjugates described herein inhibit 5'-3' exonucleolytic cleavage. In some embodiments, the 3' conjugates described herein inhibit 3'-5' exonucleolytic cleavage.

[0117] In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity to their mRNA targets compared to the equivalent natural polynucleotide. In some embodiments, the artificial nucleotide analogs include nucleic acids having modifications at the 2' hydroxyl group of the ribose moiety. In some embodiments, the modifications include H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some embodiments, the alkyl moiety further comprises a modification. In some embodiments, the modification includes an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some embodiments, the alkyl moiety further comprises heterosubstitution. In some embodiments, a carbon of a heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In some embodiments, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino. One or more of the artificial nucleotide analogues, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidites, may have increased binding affinity for their mRNA target compared to the equivalent naturally occurring polynucleotide.In some embodiments, 2'-O-methyl modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-O-aminopropyl modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-deoxy modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, T-deoxy-2'-fluoro modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-O-aminopropyl (2'-O-AP) modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides. In some embodiments, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides. In some embodiments, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides. In some embodiments, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides. In some embodiments, LNA-modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides.In some embodiments, ENA-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, PNA-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, HNA-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, morpholino-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, methylphosphonate nucleotide-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, thiolphosphonate nucleotide-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, polynucleotides comprising 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, increased affinity is indicated by a lower Kd, an increased melting temperature (Tm), or a combination thereof.

[0118] In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, 2'-O-ethyl (cEt), morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, phosphorodithioate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof.

[0119] In some embodiments, the artificial nucleotide analogues are 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 with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-haulidine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (e.g., 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queuosine, alkaosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, or pyridin-2-one), phenyl and modified phenyl groups, such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that function as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety and nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moiety, in some embodiments, is or is based on mannose, arabinose, glucopyranose, galactopyranose, 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.

[0120] In some embodiments, the polynucleotide comprises one or more phosphorothioate internucleotide linkages. In some embodiments, the polynucleotide comprises 2'-5' internucleotide linkages. In some embodiments, the 2'-5' internucleotide linkage(s) are at the 3' end, the 5' end, or both the 3' and 5' ends of one or both strands of the sequence. In some embodiments, the 2'-5' internucleotide linkage(s) are present at various other locations within one or both strands of the sequence. In some embodiments, the polynucleotide comprises a terminal cap molecule at the 3' end, the 5' end, or both the 3' and 5' ends.

[0121] In some embodiments, the targeting molecule and the polynucleotide are combined to provide a synergistic therapeutic or biological effect.

[0122] In some embodiments, the polynucleotide is directly conjugated to the targeting molecule. The polynucleotide may be conjugated to the targeting molecule via a linker. Suitable linkers for conjugating the polynucleotide to the targeting molecule are known in the art. See, for example, WO2017 / 173408, which is incorporated herein by reference in its entirety. In some embodiments, the linker is a hydrophobic linker. The linker may be a peptide linker. In some embodiments, the linker is a chemical linker. The chemical linker may be a polymer linker. In some embodiments, the chemical linker is linear. In some embodiments, the chemical linker is cyclic.

[0123] In some embodiments, the polymer linker comprises PEG, a sugar, a fatty acid, a phosphate, pyrophosphate, or polysarcosine. In some embodiments, the polymer linker comprises PEG. In some embodiments, the polymer linker comprises a sugar. In some embodiments, the polymer linker comprises a fatty acid. In some embodiments, the polymer linker comprises a phosphate. In some embodiments, the polymer linker comprises pyrophosphate. In some embodiments, the polymer linker comprises polysarcosine. The linker can be a high molecular weight PEG linker. In some embodiments, the high molecular weight PEG linker comprises 1,000 to 5,000 PEG monomers (i.e., PEG1k to PEG5k). In some embodiments, the high molecular weight PEG linker is PEG1k. In some embodiments, the high molecular weight PEG linker is PEG1.5k. In some embodiments, the high molecular weight PEG linker is PEG2k. In some embodiments, the high molecular weight PEG linker is PEG3k. In some embodiments, the high molecular weight PEG linker is PEG4k. In some embodiments, the high molecular weight PEG linker is PEG5k.

[0124] In some embodiments, the linker is a low molecular weight PEG linker. In some embodiments, the low molecular weight PEG linker comprises 4 to 100 PEG monomers (i.e., PEG4 to PEG100). In some embodiments, the low molecular weight PEG linker is PEG12 to PEG48. In some embodiments, the low molecular weight PEG linker is PEG12 to PEG24. In some embodiments, the low molecular weight PEG linker is PEG12 to PEG18. In some embodiments, the low molecular weight PEG linker is PEG6 to PEG18. In some embodiments, the low molecular weight PEG linker is PEG4. In some embodiments, the low molecular weight PEG linker is PEG5. In some embodiments, the low molecular weight PEG linker is PEG6. In some embodiments, the low molecular weight PEG linker is PEG7. In some embodiments, the low molecular weight PEG linker is PEG8. In some embodiments, the low molecular weight PEG linker is PEG9. In some embodiments, the low molecular weight PEG linker is PEG10. In some embodiments, the low molecular weight PEG linker is PEG11. In some embodiments, the low molecular weight PEG linker is PEG12. In some embodiments, the low molecular weight PEG linker is PEG13. In some embodiments, the low molecular weight PEG linker is PEG14. In some embodiments, the low molecular weight PEG linker is PEG15. In some embodiments, the low molecular weight PEG linker is PEG16. In some embodiments, the low molecular weight PEG linker is PEG17. In some embodiments, the low molecular weight PEG linker is PEG18. In some embodiments, the low molecular weight PEG linker is PEG19. In some embodiments, the low molecular weight PEG linker is PEG20. In some embodiments, the low molecular weight PEG linker is PEG21. In some embodiments, the low molecular weight PEG linker is PEG22. In some embodiments, the low molecular weight PEG linker is PEG23. In some embodiments, the low molecular weight PEG linker is PEG24. In some embodiments, the low molecular weight PEG linker is PEG25.In some embodiments, the low molecular weight PEG linker is PEG26. In some embodiments, the low molecular weight PEG linker is PEG27. In some embodiments, the low molecular weight PEG linker is PEG28. In some embodiments, the low molecular weight PEG linker is PEG29. In some embodiments, the low molecular weight PEG linker is PEG30. In some embodiments, the low molecular weight PEG linker is PEG31. In some embodiments, the low molecular weight PEG linker is PEG32. In some embodiments, the low molecular weight PEG linker is PEG33. In some embodiments, the low molecular weight PEG linker is PEG34. In some embodiments, the low molecular weight PEG linker is PEG35. In some embodiments, the low molecular weight PEG linker is PEG36. In some embodiments, the low molecular weight PEG linker is PEG37. In some embodiments, the low molecular weight PEG linker is PEG38. In some embodiments, the low molecular weight PEG linker is PEG39. In some embodiments, the low molecular weight PEG linker is PEG40. In some embodiments, the low molecular weight PEG linker is PEG41. In some embodiments, the low molecular weight PEG linker is PEG42. In some embodiments, the low molecular weight PEG linker is PEG43. In some embodiments, the low molecular weight PEG linker is PEG44. In some embodiments, the low molecular weight PEG linker is PEG45. In some embodiments, the low molecular weight PEG linker is PEG46. In some embodiments, the low molecular weight PEG linker is PEG47. In some embodiments, the low molecular weight PEG linker is PEG48. In some embodiments, the low molecular weight PEG linker is PEG49. In some embodiments, the low molecular weight PEG linker is PEG50. In some embodiments, the low molecular weight PEG linker is PEG51. In some embodiments, the low molecular weight PEG linker is PEG52. In some embodiments, the low molecular weight PEG linker is PEG53. In some embodiments, the low molecular weight PEG linker is PEG54.In some embodiments, the low molecular weight PEG linker is PEG55. In some embodiments, the low molecular weight PEG linker is PEG56. In some embodiments, the low molecular weight PEG linker is PEG57. In some embodiments, the low molecular weight PEG linker is PEG58. In some embodiments, the low molecular weight PEG linker is PEG59. In some embodiments, the low molecular weight PEG linker is PEG60. In some embodiments, the low molecular weight PEG linker is PEG61. In some embodiments, the low molecular weight PEG linker is PEG62. In some embodiments, the low molecular weight PEG linker is PEG63. In some embodiments, the low molecular weight PEG linker is PEG64. In some embodiments, the low molecular weight PEG linker is PEG65. In some embodiments, the low molecular weight PEG linker is PEG66. In some embodiments, the low molecular weight PEG linker is PEG67. In some embodiments, the low molecular weight PEG linker is PEG68. In some embodiments, the low molecular weight PEG linker is PEG69. In some embodiments, the low molecular weight PEG linker is PEG70. In some embodiments, the low molecular weight PEG linker is PEG71. In some embodiments, the low molecular weight PEG linker is PEG72. In some embodiments, the low molecular weight PEG linker is PEG73. In some embodiments, the low molecular weight PEG linker is PEG74. In some embodiments, the low molecular weight PEG linker is PEG75. In some embodiments, the low molecular weight PEG linker is PEG76. In some embodiments, the low molecular weight PEG linker is PEG77. In some embodiments, the low molecular weight PEG linker is PEG78. In some embodiments, the low molecular weight PEG linker is PEG79. In some embodiments, the low molecular weight PEG linker is PEG80. In some embodiments, the low molecular weight PEG linker is PEG81. In some embodiments, the low molecular weight PEG linker is PEG82. In some embodiments, the low molecular weight PEG linker is PEG83.In some embodiments, the low molecular weight PEG linker is PEG84. In some embodiments, the low molecular weight PEG linker is PEG85. In some embodiments, the low molecular weight PEG linker is PEG86. In some embodiments, the low molecular weight PEG linker is PEG87. In some embodiments, the low molecular weight PEG linker is PEG88. In some embodiments, the low molecular weight PEG linker is PEG89. In some embodiments, the low molecular weight PEG linker is PEG90. In some embodiments, the low molecular weight PEG linker is PEG91. In some embodiments, the low molecular weight PEG linker is PEG92. In some embodiments, the low molecular weight PEG linker is PEG93. In some embodiments, the low molecular weight PEG linker is PEG94. In some embodiments, the low molecular weight PEG linker is PEG95. In some embodiments, the low molecular weight PEG linker is PEG96. In some embodiments, the low molecular weight PEG linker is PEG97. In some embodiments, the low molecular weight PEG linker is PEG98. In some embodiments, the low molecular weight PEG linker is PEG99. In some embodiments, the low molecular weight PEG linker is PEG100.

[0125] In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable. The linker may be cleavable in vivo. In some embodiments, the cleavable linker is selected from the group consisting of a disulfide linker, a self-immolative peptide polymer hybrid, and a sulfatase-promoted aryl sulfate linker. In some embodiments, the cleavable linker is a disulfide linker. The cleavable linker may be a self-immolative peptide polymer hybrid. In some embodiments, the cleavable linker is a sulfatase-promoted aryl sulfate linker. In some embodiments, the self-immolative peptide polymer hybrid comprises glucuronic acid, para-amino-benzoyloxy (PAB), 7-amino-3-hydroxyethyl-coumarin (7-AHC), or a Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX). In some embodiments, the self-immolative peptide polymer hybrid comprises glucuronic acid. In some embodiments, the self-immolative peptide polymer hybrid comprises para-amino-benzoyloxy (PAB). In some embodiments, the self-immolative peptide polymer hybrid comprises 7-amino-3-hydroxyethyl-coumarin (7-AHC). In some embodiments, the self-immolative peptide polymer hybrid comprises an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX).

[0126] In some embodiments, the cleavable linker is cleaved via reduction, hydrolysis, proteolysis, photocleavage, chemical cleavage, enzymatic cleavage, or bioorthogonal cleavage. In some embodiments, the cleavable linker is cleaved by reduction. In some embodiments, the cleavable linker is cleaved by hydrolysis. In some embodiments, the cleavable linker is cleaved by proteolysis. In some embodiments, the cleavable linker is cleaved by photocleavage. In some embodiments, the cleavable linker is cleaved by chemical cleavage. The chemical cleavage may be by Fe II-mediated β-elimination of TRX. In some embodiments, the cleavable linker is cleaved by enzymatic cleavage. The enzymatic cleavage may be by non-proteolytic sulfatase, β-galactosidase / glucuronidase, or pyrophosphate phosphatase. In some embodiments, the enzymatic cleavage is by non-proteolytic sulfatase. In some embodiments, the enzymatic cleavage is by β-galactosidase / glucuronidase. In some embodiments, the enzymatic cleavage is by pyrophosphate phosphatase. In some embodiments, the cleavable linker is cleaved by bioorthogonal cleavage. The bioorthogonal cleavage can be by Cu I-BTTAA or free copper ion mediated cleavage. In some embodiments, the linker is an acid cleavable linker.

[0127] In some embodiments, the linker comprises a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group). In some embodiments, the linker includes a homobifunctional cross-linker, a heterobifunctional cross-linker, etc. In some embodiments, the linker is a traceless linker (or a zero-length linker). In some embodiments, the linker is a non-polymeric linker. In some embodiments, the linker is a non-peptide linker, or a linker that does not include an amino acid residue.

[0128] In some embodiments, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3,3'-dithiobis(sulfosuccinimidyl propionate (DTSSP)), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDST), ethylene glycobis (Ethylene glycol) succinimidyl succinate (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DP Examples of suitable aryl ethers include, but are not limited to, bis-[2-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-ρ-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0129] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include amine-reactive and sulfhydryl crosslinkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (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)toluamide]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-iodophenyl)succinimidyl (4-iodoactyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoactyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y-maleimidobutyryloxy)succinimide esters (GMBs), N-(gamma-maleimidobutyloxy)sulfosuccinimide esters (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino )hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers,For example, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive crosslinkers, for example, N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate, Sulfosuccinimidyl-2-(p-azido-salicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-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-1,3'-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 Succinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive crosslinkers, such as 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB),These include, but are not limited to, N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl-reactive and photoreactive crosslinkers, such as p-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers, such as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers, such as p-azidophenylglyoxal (APG).

[0130] In some embodiments, the linker comprises a reactive functional group. In some embodiments, the reactive functional group comprises a nucleophilic group that reacts with an electrophilic group present on the binding moiety. Exemplary electrophilic groups include carbonyl groups, such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0131] In some embodiments, the linker comprises a maleimide group. In some embodiments, the maleimide group is also referred to as a maleimide spacer. In some embodiments, the maleimide group further includes caproic acid to form maleimidocaproyl (mc). In some embodiments, the linker comprises maleimidocaproyl (mc). In some embodiments, the linker is maleimidocaproyl (mc). In other cases, the maleimide group comprises a maleimidomethyl group, e.g., succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), as described above.

[0132] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some embodiments, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) and incorporates a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby precluding the maleimide from undergoing elimination via a retro-Michael reaction. In some embodiments, 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 embodiments, the linker comprises a self-stabilizing maleimide. In some embodiments, the linker is a self-stabilizing maleimide.

[0133] In some embodiments, the linker comprises a peptide moiety. In some embodiments, the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some embodiments, the peptide moiety is a cleavable peptide moiety (e.g., either enzymatically or chemically). In some embodiments, the peptide moiety is a non-cleavable peptide moiety. In some embodiments, 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, Τφ-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some embodiments, the linker comprises a peptide moiety, e.g., 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 embodiments, the linker comprises Val-Cit. In some embodiments, the linker is Val-Cit.

[0134] In some embodiments, the linker comprises a benzoic acid group, or a derivative thereof. In some embodiments, the benzoic acid group, or a derivative thereof, comprises para-aminobenzoic acid (PABA). In some embodiments, the benzoic acid group, or a derivative thereof, comprises gamma-aminobutyric acid (GABA).

[0135] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some embodiments, the maleimide group is maleimidocaproyl (mc). In some embodiments, the peptide group is val-cit. In some embodiments, the benzoic acid group is PABA. In some embodiments, the linker comprises a mc-val-cit group. In some embodiments, the linker comprises a val-cit-PABA group. In additional cases, the linker comprises a mc-val-cit-PABA group.

[0136] In some embodiments, the linker is a self-immolative linker or a self-eliminating linker. In some embodiments, the linker is a self-immolative linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some embodiments, the linker comprises the linker described in U.S. Pat. No. 9,089,614 or PCT Publication No. WO2015038426 (each of which is incorporated herein by reference in its entirety).

[0137] In some embodiments, the linker is a dendritic linker. In some embodiments, the dendritic linker comprises a branched multifunctional linker moiety. In some embodiments, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some embodiments, the dendritic linker comprises a PAMAM dendrimer.

[0138] In some embodiments, the linker is a traceless linker, or a linker that does not leave a linker moiety (e.g., atom or linker group) on polynucleotide or targeting molecule after cleavage.Exemplary traceless linkers include, but are not limited to, germanium linker, silicon linker, sulfur linker, selenium linker, nitrogen linker, phosphorus linker, boron linker, chromium linker, or phenylhydrazide linker.In some embodiments, the linker is the traceless aryl-triazene linker described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15):2493-2497 (2013). In some embodiments, the linker is a traceless linker as described in Blaney, et al., 'Traceless solid-phase organic synthesis,' Chem. Rev. 102:2607-2024 (2002). In some embodiments, the linker is a traceless linker as described in U.S. Pat. No. 6,821,783, which is incorporated herein by reference in its entirety.

[0139] In some embodiments, the linker comprises a functional group that provides steric hindrance at the binding site between the linker and the conjugate moiety (e.g., a polynucleotide or targeting molecule disclosed herein). In some embodiments, the steric hindrance is around a disulfide bond. Exemplary linkers that exhibit steric hindrance include heterobifunctional linkers, such as the heterobifunctional linkers described above. In some embodiments, linkers that exhibit steric hindrance include SMCC and SPDB.

[0140] In some embodiments, the linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker comprises a hydrazone bond that is susceptible to hydrolytic cleavage. In some embodiments, the acid-cleavable linker comprises a thiomaleamic acid linker. In some embodiments, 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).

[0141] In some embodiments, the linker is a cyclic amine or cyclic amine as described in U.S. Pat. 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 / 0309257, 2016 / 0309260, 2015 / 0309271, 2015 / 0309272, 2015 / 0309273, 2015 / 0309274, 2015 / 0309275, 2015 / 0309276, 2015 / 0309277, 2015 / 0309278, 2015 / 0309279, 2015 / 0309279, 2015 / 0309277, 2015 / 0309279 ... 037360 or 2014 / 0294851, or PCT Publication Nos. WO2015057699, WO2014080251, WO2014197854, WO2014145090 or WO2014177042, each of which is incorporated by reference in its entirety.

[0142] In some embodiments, the linker is conjugated to a lysine residue, a cysteine ​​residue, a histidine residue, or a non-natural amino acid residue in the targeting molecule. In some embodiments, the linker is conjugated to a lysine residue in the targeting molecule. In some embodiments, the linker is conjugated to a cysteine ​​residue in the targeting molecule. In some embodiments, the linker is conjugated to a histidine residue in the targeting molecule. In some embodiments, the linker is conjugated to a non-natural amino acid residue in the targeting molecule.

[0143] In some embodiments, the linker is conjugated to the targeting molecule by chemical conjugation or enzymatic conjugation. In some embodiments, the linker is conjugated to the targeting molecule by chemical conjugation. Chemical conjugation can include acylation and click chemistry. In some embodiments, the linker is conjugated to the targeting molecule by enzymatic conjugation. Enzymatic conjugation can be via a sortase or transferase enzyme.

[0144] In some embodiments, the polynucleotide is conjugated to the targeting molecule by a chemical ligation process. In some embodiments, the polynucleotide is conjugated to the targeting molecule by native ligation. In some embodiments, 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 embodiments, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, the polynucleotide is conjugated to the targeting molecule by native ligation chemistry, either site-specifically or non-specifically.

[0145] In some embodiments, the polynucleotide is conjugated to the targeting molecule by a site-specific method using the "traceless" coupling technology (Philochem). In some embodiments, the "traceless" coupling technology utilizes the N-terminal 1,2-aminothiol group on the targeting molecule, which is then conjugated to a polynucleotide containing an aldehyde group (see Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS134(13):5887-5892(2012)).

[0146] In some embodiments, polynucleotides are conjugated to targeting molecules by site-specific methods that utilize unnatural amino acids incorporated into the targeting molecule. In some embodiments, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some embodiments, the keto group of pAcPhe is selectively coupled to an alkoxy-amine derived conjugate moiety to form an oxime bond (see Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS109(40):16101-16106 (2012)).

[0147] In some embodiments, polynucleotides are conjugated to targeting molecules by a site-specific method that utilizes an enzyme-catalyzed process. In some embodiments, the site-specific method utilizes SMARTag™ technology (Redwood). In some embodiments, SMARTag™ technology involves the creation of a formylglycine (FGly) residue from a cysteine ​​by a formylglycine-generating enzyme (FGE) through an oxidative process in the presence of an aldehyde tag, followed by conjugation of FGly to an alkylhydrene-functionalized polynucleotide via hydrazino-Pictet-Spengler (HIPS) ligation (see Wu et al., "Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag," PNAS106(9):3000-3005(2009); Agarwal, et al., "A Pictet-Spengler ligation for protein chemical modification," PNAS110(1):46-51(2013)).

[0148] In some embodiments, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some embodiments, polynucleotides are conjugated to targeting molecules utilizing a microbial transglutaminase-catalyzed process. In some embodiments, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine in a recognition sequence and a primary amine of a functionalized polynucleotide. In some embodiments, 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)).

[0149] In some embodiments, the polynucleotide is conjugated to the targeting molecule by the methods described in PCT Publication No. WO2014 / 140317 (incorporated herein by reference in its entirety), which utilizes a sequence-specific transpeptidase. In some embodiments, the polynucleotide is conjugated to the targeting molecule by the methods described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540 (each of which is incorporated herein by reference in its entirety).

[0150] In some embodiments, each targeting molecule is conjugated to 1-8 polynucleotide molecules (i.e., a drug:antibody ratio (DAR) of 1-8). In some embodiments, each targeting molecule is conjugated to 1 polynucleotide molecule (DAR of 1). In some embodiments, each targeting molecule is conjugated to 2 polynucleotide molecules (DAR of 2). In some embodiments, each targeting molecule is conjugated to 3 polynucleotide molecules (DAR of 3). In some embodiments, each targeting molecule is conjugated to 4 polynucleotide molecules (DAR of 4). In some embodiments, each targeting molecule is conjugated to 5 polynucleotide molecules (DAR of 5). In some embodiments, each targeting molecule is conjugated to 6 polynucleotide molecules (DAR of 6). In some embodiments, each targeting molecule is conjugated to 7 polynucleotide molecules (DAR of 7). In some embodiments, each targeting molecule is conjugated to eight polynucleotide molecules (a DAR of 8).

[0151] In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than about 30 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than about 40 kDa. The polynucleotide conjugate targeting molecule can have a molecular weight of more than about 50 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than about 60 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of about 7,500 kDa or less.

[0152] In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 30 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 40 kDa. The polynucleotide conjugate targeting molecule can have a molecular weight of more than 50 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 60 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of 7,500 kDa or less.

[0153] In some embodiments, the polynucleotide conjugate is selected from the group consisting of cetuximab-DBCO-C9-M30m3 (DAR of 3), cetuximab-DBCO-C4 / P5-M30m3 (DAR of 3), cetuximab-DBCO-PEG9-M30m3 (DAR of 3), cetuximab-DBCO-PEG9-M30m3 (DAR of 2), cetuximab-DBCO-PEG9-M30m3 (DAR of 4), cetuximab-DBCO-PEG In some embodiments, the polynucleotide conjugate is selected from the group consisting of cetuximab-DBCO-C9-M30m3 (DAR of 3). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-C4 / P5-M30m3 (DAR of 3). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-PEG9-M30m3 (DAR of 3). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-PEG9-M30m3 (DAR of 2). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-PEG9-M30m3 (DAR of 4). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-PEG9-M30m3 (DAR of 6). In some embodiments, the polynucleotide conjugate is cetuximab-linear-PEG13-M30m3 (DAR of 4). In some embodiments, the polynucleotide conjugate is 3tf12-DBCO-PEG8-NCD5 (DAR of 1). In some embodiments, the polynucleotide conjugate is 3tf12-DBCO-PEG8-M30m3 (DAR of 1). In some embodiments, the polynucleotide conjugate is Fv55-SMCC-M30m3 (DAR of 1).In some embodiments, the polynucleotide conjugate is Fv55-PEG8-DBCO-M30m3 (DAR of 1). In some embodiments, the polynucleotide conjugate is Fv55-PEG8-DBCO-M30m3 (DAR of 2). In some embodiments, the polynucleotide conjugate is selected from the antibody-polynucleotide conjugates listed in Table 5 below. Each antibody-polynucleotide conjugate in Table 5 is considered a separate embodiment. In some embodiments, the polynucleotide conjugate is selected from the antibody-polynucleotide conjugates listed in Table 6 below. Each antibody-polynucleotide conjugate in Table 6 is considered a separate embodiment. TIFF2025503504000007.tif224159TIFF2025503504000008.tif242156TIFF2025503504000009.t if121159TIFF2025503504000010.tif239159TIFF2025503504000011.tif242157TIFF20255035040 00012.tif242153TIFF2025503504000013.tif242150TIFF2025503504000014.tif242153TIFF202 5503504000015.tif242155TIFF2025503504000016.tif242157TIFF2025503504000017.tif242154

[0154] In some embodiments, the composition comprises: (a) cetuximab-DBCO-C9-M30m3(DAR3) and PEG12-poly-(D-Arg)12; (b) cetuximab-DBCO-C4 / P5-M30m3(DAR3) and PEG12-poly-(D-Arg)12; (c) cetuximab-DBCO-PEG9-M30m3(DAR3) and PEG12-poly-(D-Arg)12; (d) cetuximab-DBCO-PEG9 (e) cetuximab-DBCO-PEG9-M30m3 (DAR4) and PEG12-poly-(D-Arg)12, (f) cetuximab-DBCO-PEG9-M30m3 (DAR6) and PEG12-poly-(D-Arg)12, (g) cetuximab-linear-PEG13-M30m3 (DAR4) and PEG12-poly-(D-Arg)12, (h) 3 (i) tf12-DBCO-PEG8-NCD5 and poly(L-Arg)9, (i) 3tf12-DBCO-PEG8-M30m3 and poly(L-Arg)9, (j) Fv55-SMCC-M30m3 and PEG12-poly(L-Arg)12, (k) Fv55-PEG30-M30m3 and PEG12-poly(L-Arg)12, (l) cetuximab-PEG4-azide-DBCO-PEG5-M30m3(DAR2) and PEG 12PolyArg12{d}, (m) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2) and PolyArg12Cbp3.9kDa, (n) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PEG12PolyArg12{d}, (o) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12Cbp3.9kDa, (p) cetuximab-PEG4-azide-DBCO-PEG5-M30m3(DAR4) and PolyArg12C-PEG2000Da, (q) cetuximab-PEG4-azide-DBCO-PEG5-M30m3(DAR4) and PolyArg12C-PEG5000Da, (r) cetuximab-PEG4-azide-DBCO-PEG5-M30m3(DAR4) and PolyArg12C-dextran5000Da, (s) cetuximab-SMCC-M30m3(DAR4) and PEG12PolyArg12{d}, (t) cetuximab-MCVCPABcPNP-M30m3(DAR4) and PEG12PolyArg12{d}, (u) cetuximab-MCPEG4VCPABcPNP-M30m3 (DAR 4) and PEG12PolyArg12{d}, (v) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR 2.5) and PEG12PolyArg12{d}, (w) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR 4.5) and PEG12PolyArg12{d}, (x) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR 6.5) and PEG12PolyArg12{d}, or (y) cetuximab-C4(azide-DBCO)C5-M30m3 and PEG12PolyArg12. In some embodiments, the composition comprises cetuximab-DBCO-C9-M30m3 (DAR3) and PEG12-poly-(D-Arg). 12 In some embodiments, the composition comprises cetuximab-DBCO-C4 / P5-M30m3 (DAR3) and PEG12-poly-(D-Arg) 12 In some embodiments, the composition comprises cetuximab-DBCO-PEG9-M30m3 (DAR3) and PEG12-poly-(D-Arg) 12 In some embodiments, the composition comprises cetuximab-DBCO-PEG9-M30m3 (DAR2) and PEG12-poly-(D-Arg) 12 In some embodiments, the composition comprises cetuximab-DBCO-PEG9-M30m3(DAR4) and PEG12-poly-(D-Arg) 12In some embodiments, the composition comprises cetuximab-DBCO-PEG9-M30m3(DAR6) and PEG12-poly-(D-Arg) 12 In some embodiments, the composition comprises cetuximab-linear-PEG13-M30m3(DAR4) and PEG12-poly-(D-Arg) 12 In some embodiments, the composition comprises 3tf12-DBCO-PEG8-NCD5 and Poly(L-Arg) 9 In some embodiments, the composition comprises 3tf12-DBCO-PEG8-M30m3 and Poly(L-Arg) 9 In some embodiments, the composition comprises Fv55-SMCC-M30m3 and PEG12-Poly(L-Arg) 12 In some embodiments, the composition comprises Fv55-PEG30-M30m3 and PEG12-Poly(L-Arg) 12In some embodiments, the composition comprises cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2) and PEG12PolyArg12{d}. In some embodiments, the composition comprises cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2) and PolyArg12Cbp 3.9 kDa. In some embodiments, the composition comprises cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PEG12PolyArg12{d}. In some embodiments, the composition comprises cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12Cbp 3.9 kDa. In some embodiments, the composition comprises cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-PEG2000Da. In some embodiments, the composition comprises cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-PEG5000Da. In some embodiments, the composition comprises cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-Dextran5000Da. In some embodiments, the composition comprises cetuximab-SMCC-M30m3 (DAR4) and PEG12PolyArg12{d}. In some embodiments, the composition comprises cetuximab-MCVCPABcPNP-M30m3 (DAR4) and PEG12PolyArg12{d}. In some embodiments, the composition comprises cetuximab-MCPEG4VCPABcPNP-M30m3 (DAR4) and PEG12PolyArg12{d}. In some embodiments, the composition comprises cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2.5) and PEG12PolyArg12{d}. In some embodiments, the composition comprises cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4.5) and PEG12PolyArg12{d}.In some embodiments, the composition comprises Cetuximab-PEG4-Azide-DBCO-PEG5-M30m3(DAR6.5) and PEG12PolyArg12{d}. In some embodiments, the composition comprises Cetuximab-C4(Azide-DBCO)C5-M30m3 and PEG12PolyArg12. In some embodiments, the composition comprises any of the antibody-polynucleotide conjugates and related hybrid polymers disclosed in Table 5 above. Each of the antibody-polynucleotide conjugates and related hybrid polymers disclosed in Table 5 is considered a separate embodiment.

[0155] Polynucleotide Conjugates A second aspect of the present disclosure provides a polynucleotide conjugate. In some embodiments, the polynucleotide conjugate comprises a polynucleotide conjugated to a targeting molecule.

[0156] In some embodiments, targeting moieties include amino acids, peptides, polypeptides, proteins, antibodies, antigens, toxins, hormones, lipids, nucleotides, nucleosides, sugars, carbohydrates, polymers such as polyethylene glycol and polypropylene glycol, and analogs or derivatives of all these classes of substances. Additional examples of targeting moieties also include steroids, such as cholesterol, phospholipids, di- and triacylglycerols, fatty acids, carbohydrates (e.g., saturated, unsaturated, or substituted), enzyme substrates, biotin, digoxigenin, and polysaccharides. In some embodiments, the targeting moiety is an antibody or a binding fragment thereof.

[0157] The targeting molecule can be an antibody or an antigen-binding fragment thereof, or a binding protein. In some embodiments, the targeting molecule is an antibody or an antigen-binding fragment thereof (e.g., a polynucleotide-antibody conjugate). In some embodiments, the antibody or binding fragment thereof is a human antibody or an antigen-binding fragment thereof, a humanized antibody or an antigen-binding fragment thereof, a murine antibody or an antigen-binding fragment thereof, a chimeric antibody or an antigen-binding fragment thereof, a monoclonal antibody or an antigen-binding fragment thereof, a monovalent Fab', a bivalent Fab2, a F(ab)'3 fragment, a single chain variable fragment (scFv), a bis-scFv ... 2, an immunoglobulin single variable domain (ISV) such as a diabody, a minibody, a Nanobody® molecule, a triabody, a tetrabody, a disulfide stabilized Fv protein (dsFv), a single domain antibody (sdAb), an Ig NAR, a vNAR, a centrin, a camelid antibody or antigen-binding fragment thereof, a bispecific antibody or antigen-binding fragment thereof, or a chemically modified derivative thereof. In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, a Fab-Fc, an Fv, a single chain Fv (scFv), a diabody, a minibody, and a Nanobody® molecule. In some embodiments, the antibody or antigen-binding fragment thereof is a monoclonal antibody. In some embodiments, the antibody or antigen-binding fragment thereof is a bispecific antibody. Non-limiting examples of bispecific antibodies are in Bispecific T Cell Engagers (BiTEs) and Dual Affinity Retargeting Antibodies (DARTs). In some embodiments, the bispecific antibody is a trifunctional antibody or a bispecific miniantibody. In some embodiments, the bispecific antibody is a trifunctional antibody. In some embodiments, the trifunctional antibody is a full-length monoclonal antibody that comprises binding sites for two different antigens. In some embodiments, the bispecific antibody is a bispecific miniantibody. In some embodiments, the bispecific miniantibody comprises a bivalent Fab2, F(ab)'3 fragment, a bis-scFv, (scFv)2, a diabody, a minibody, a triabody, a tetrabody, or a bispecific T cell engager (BiTE). In some embodiments, the bispecific T cell engager is a fusion protein comprising two single chain variable fragments (scFv), where the two scFvs target epitopes of two different antigens.

[0158] In some embodiments, the antibody or antigen-binding fragment thereof is a Fab. In some embodiments, the antibody or antigen-binding fragment thereof is a Fab-Fc. In some embodiments, the antibody or antigen-binding fragment thereof is an Fv. In some embodiments, the antibody or antigen-binding fragment thereof is a single chain Fv (scFv). In some embodiments, when the antibody or antigen-binding moiety is an scFv, the polynucleotide does not comprise bridging residues. In some embodiments, when the antibody or antigen-binding moiety is an scFv, the polynucleotide does not comprise cysteines. In some embodiments, the antibody or antigen-binding fragment thereof is a diabody. In some embodiments, the antibody or antigen-binding fragment thereof is a minibody. In some embodiments, the antibody or antigen-binding fragment thereof is an immunoglobulin single variable domain (ISV), such as a Nanobody® molecule. The Nanobody® can be Nanobody-HSA®.

[0159] In some embodiments, the antibody or antigen-binding fragment thereof is an IgG molecule or is derived from an IgG molecule. The IgG molecule may be an IgG1 or IgG4 molecule. The antibody or antigen-binding fragment thereof may be an IgG1 molecule or may be derived therefrom. The antibody or antigen-binding fragment thereof may be an IgG2 molecule or may be derived therefrom. The antibody or antigen-binding fragment thereof may be an IgG3 molecule or may be derived therefrom. The antibody or antigen-binding fragment thereof may be an IgG4 molecule or may be derived therefrom.

[0160] Non-limiting examples of antibodies and antigen-binding fragments that can be used as targeting molecules in the present disclosure include FV55scFv, Fv55 diabody, 3TF12, and cetuximab. FV55 scFv is a monospecific scFv that binds to the human transferrin receptor (TfR1). The CDRs of FV55 scFv are identical to those of HB21, and FV55 scFv has V-terminal domains connected by (G4S)*3. H -V Loriented toward the 5'-terminal end and has a c-terminal cysteine ​​for conjugation. The molecular weight of FV55 scFv is about 26.5 kDa. See, for example, Haynes BF, Hemler M, Cotner T, Mann DL, Eisenbarth GS, Strominger JL, Fauci AS. Characterization of a monoclonal antibody (5E9) that defines a human cell surface antigen of cell activation. J Immunol. 1981; 127: 347-351. [PubMed: 6787129]. Fv55 diabodies contain two copies of FV55 scFv, except that the linker is (G4S) * N, where N is 1 or 2. The molecular weight of Fv55 diabodies is about 53 kDa. 3TF12 is a monospecific scFv that binds to the human transferrin receptor (TfR1). 3TF12 is a V connected by (G4S)*N H -V L When N is 3, 3tf12 is a monomeric scFv, and when N is 1, 3tf12 dimerizes to form a diabody. See, e.g., Crepin R. et al. Development of Human Single-Chain Antibodies to the Transferrin Receptor that Effectively Antagonize the Growth of Leukemias and Lymphomas. Cancer Research 2010, 70(13):5497-506. Cetuximab is a chimeric (mouse / human) monoclonal antibody and epidermal growth factor receptor (EGFR) inhibitor used to treat metastatic colorectal cancer and head and neck cancer. Cetuximab has a molecular weight of 145,781.92 g / mol.

[0161] In some embodiments, the targeting molecule is a binding protein. The binding protein can be a soluble receptor or a soluble ligand. In some embodiments, the soluble receptor comprises the extracellular domain of the receptor. In some embodiments, the soluble receptor is an Fc fusion protein.

[0162] In some embodiments, the targeting molecule is a plasma protein. In some embodiments, the plasma protein comprises albumin. In some embodiments, the albumin is conjugated to the polynucleotide by one or more of the conjugation chemistries disclosed herein. In some cases, the albumin is conjugated to the polynucleotide by native ligation chemistry. In some cases, the albumin is conjugated to the polynucleotide by lysine conjugation.

[0163] In some cases, the targeting molecule is a steroid. Non-limiting exemplary steroids include cholesterol, phospholipids, di- and triacylglycerols, fatty acids, saturated, unsaturated, substituted, or other hydrocarbons, or combinations thereof. In some embodiments, the steroid is cholesterol or a cholesterol derivative. In some embodiments, the targeting molecule is cholesterol. In some embodiments, the steroid is conjugated to the polynucleotide by one or more of the conjugation chemistries disclosed herein. In some embodiments, the steroid is conjugated to the polynucleotide by native ligation chemistry.

[0164] In some embodiments, the targeting molecule is a polymer, including but not limited to a polynucleotide aptamer that binds to a specific surface marker on a cell.In some embodiments, the targeting molecule is a polynucleotide that does not hybridize to the target gene or mRNA, but instead can selectively bind to the cell surface marker, similar to an antibody that binds to its specific epitope on the cell surface marker.

[0165] In some embodiments, the targeting molecule is a polypeptide. In some embodiments, the polypeptide has a size of about 1 to about 3 kDa. In some embodiments, the polypeptide has a size of about 1.2 to about 2.8 kDa, about 1.5 to about 2.5 kDa, or about 1.5 to about 2 kDa. In some embodiments, the polypeptide is a bicyclic polypeptide. In some embodiments, the bicyclic polypeptide is a constrained bicyclic polypeptide. In some embodiments, the targeting molecule is a bicyclic polypeptide (e.g., Bicycle from Bicycle Therapeutics).

[0166] In additional embodiments, the targeting molecule is a small molecule. In some embodiments, the small molecule is an antibody-recruiting small molecule. In some embodiments, the antibody-recruiting small molecule comprises a target-binding end and an antibody-binding end, where the target-binding end is capable of recognizing and interacting with a cell surface receptor.

[0167] In some embodiments, the targeting molecule is a therapeutically or biologically active molecule.

[0168] In some embodiments, the polynucleotide comprises RNA, DNA, or a combination thereof. In some embodiments, the polynucleotide comprises RNA. In some embodiments, the polynucleotide comprises DNA. In some embodiments, the polynucleotide comprises RNA and DNA. In some embodiments, the polynucleotide comprises a combination of DNA, RNA, and / or artificial nucleotide analogs. In some embodiments, the polynucleotide is a regulatory non-coding RNA (ncRNA). In some embodiments, the ncRNA comprises short non-coding RNA sequences expressed in the genome that control the expression or function of other biomolecules in mammalian cells. ncRNAs are generally less than 200 nucleotides in length, can be single-stranded or double-stranded, and can form non-linear secondary or tertiary structures. The ncRNA may include exogenous small interfering RNA (siRNA), microRNA (miRNA), small nuclear RNA (U-RNA), small nucleolar RNA (snoRNA), Piwi-binding RNA (piRNA), repeat-associated small interfering RNA (rasiRNA), small RNA derived from rDNA (srRNA), small RNA derived from transfer RNA (tsRNA), small RNA derived from ribosomal RNA (rsRNA), small RNA derived from large non-coding RNA (lncsRNA), or small RNA derived from messenger RNA (msRNA). In some embodiments, the polynucleotide is an engineered polynucleotide. The engineered polynucleotide may include DNA or RNA. In some embodiments, the engineered polynucleotide includes multiple nucleotides. In some embodiments, the engineered polynucleotide includes artificial nucleotide analogs. In some embodiments, the engineered polynucleotide includes DNA. In some embodiments, the DNA is genomic DNA, cell-free DNA, cDNA, fetal DNA, viral DNA, or maternal DNA. In some embodiments, the engineered polynucleotide includes RNA.In some embodiments, the RNA is an siRNA, an ncRNA mimic, a short hairpin RNA (shRNA), a dicer-dependent siRNA (di-siRNA), an antisense oligonucleotide (ASO), a gapmer, a mixer, a double-stranded RNA (dsRNA), a single-stranded RNAi (ssRNAi), a DNA-directed RNA interference (ddRNAi), an RNA-activating oligonucleotide (RNAa), an aptamer, or an exon-skipping oligonucleotide. In some embodiments, the engineered polynucleotide comprises a fully synthetic miRNA. A fully synthetic miRNA is one that is not derived from or based on an ncRNA. Alternatively, a fully synthetic miRNA may be based on an analysis of multiple potential target sequences or may be based on an isolated naturally occurring non-coding sequence that is not an ncRNA. In some embodiments, the polynucleotide is selected from the group consisting of an siRNA, an miRNA, an miRNA mimic, an antisense oligonucleotide (ASO), an mRNA, and a guide RNA. The polynucleotide may be an siRNA. In some embodiments, the polynucleotide is an miRNA. In some embodiments, the polynucleotide is an miRNA mimic. The polynucleotide can be miR-30 or a mimic of miR-30. Non-limiting examples of miR-30 mimics are shown in Table 3 above.

[0169] In some embodiments, the miR-30 mimic is selected from the group consisting of M30m1, M30m2, M30m3, and M30m4. In some embodiments, the miR-30 mimic is M30m1. In some embodiments, the miR-30 mimic is M30m2. In some embodiments, the miR-30 mimic is M30m3. In some embodiments, the miR-30 mimic is M30m4.

[0170] In some embodiments, it is an ASO. In some embodiments, the ASO can target and inhibit multiple genes associated with the disorder. In some embodiments, the ASO targets an autosomal dominantly mutated gene that causes a genetic disorder. In some embodiments, the ASO targets DMPK. In some embodiments, the ASO targets CAPN3. The ASO can target DUX4. DUX4-targeted ASOs are known in the art. See, for example, WO2021 / 203043 and U.S. Provisional Patent Application No. 63 / 221,568, each of which is incorporated herein by reference in its entirety. Additional non-limiting examples of DUX4-targeted ASOs are shown in Table 4 above. In some embodiments, the DUX4-targeted ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32. In some embodiments, the DUX4-targeted ASO is ASDX2. In some embodiments, the DUX4-targeted ASO is ASDX4. In some embodiments, the DUX4-targeted ASO is ASDX23. In some embodiments, the DUX4-targeted ASO is ASDX26. In some embodiments, the DUX4-targeted ASO is ASDX32.

[0171] In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA targeting DUX4, DMPK, or CAPN3. In some embodiments, the polynucleotide comprises an siRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DUX4. In some embodiments, the polynucleotide comprises an ASO targeting DUX4. In some embodiments, the polynucleotide comprises a guide RNA targeting DUX4. In some embodiments, the polynucleotide comprises an siRNA targeting DMPK. In some embodiments, the polynucleotide comprises an miRNA targeting DMPK. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DMPK. In some embodiments, the polynucleotide comprises an ASO targeting DMPK. In some embodiments, the polynucleotide comprises an siRNA targeting CAPN3. In some embodiments, the polynucleotide comprises an miRNA targeting CAPN3. In some embodiments, the polynucleotide comprises an miRNA mimic targeting CAPN3. In some embodiments, the polynucleotide comprises an ASO that targets CAPN3.

[0172] In some embodiments, the polynucleotide is a coding RNA. In some embodiments, the polynucleotide is an mRNA. In some embodiments, the polynucleotide is a non-coding RNA. In some embodiments, the polynucleotide is a long non-coding RNA. In some embodiments, the polynucleotide is a guide RNA.

[0173] In some embodiments, a polynucleotide comprises one or more artificial nucleotide analogs. In some embodiments, one or more of the artificial nucleotide analogs described herein are resistant to nucleases, e.g., ribonucleases such as RNases, deoxyribonucleases such as DNases, or exonucleases such as 5'-3' and 3'-5' exonucleases, when compared to naturally occurring polynucleotides. In some embodiments, the artificial nucleotide analogs are resistant to nucleases, e.g., ribonucleases such as RNases, deoxyribonucleases such as DNases, or exonucleases such as 5'-3' and 3'-5' exonucleases. In some embodiments, the artificial nucleotide analogs are resistant to nucleases, e.g., 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). Modifications, including LNA, ENA, PNA, HNA, morpholino, methyl phosphonate nucleotides, thiol phosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof, are resistant to nucleases, e.g., ribonucleases such as RNase, deoxyribonucleases such as DNase, or exonucleases such as 5'-3' exonucleases and 3'-5' exonucleases. In some embodiments, the 2'-O-methyl modified polynucleotide is nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-O-aminopropyl modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant).In some embodiments, 2'-deoxy modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, T-deoxy-2'-fluoro modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-O-aminopropyl (2'-O-AP) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, LNA-modified polynucleotides are nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant).In some embodiments, ENA-modified polynucleotides are nuclease-resistant (e.g., RNase-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some embodiments, HNA-modified polynucleotides are nuclease-resistant (e.g., RNase-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). Morpholinos can be nuclease-resistant (e.g., RNase-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some embodiments, PNA-modified polynucleotides are nuclease-resistant (e.g., RNase-resistant, DNase-resistant, 5'-3' exonuclease-resistant, or 3'-5' exonuclease-resistant). In some embodiments, the methylphosphonate nucleotide-modified polynucleotide is nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, the thiol phosphonate nucleotide-modified polynucleotide is nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, the polynucleotide comprising a 2'-fluoro N3-P5'-phosphoramidite is nuclease resistant (e.g., RNase resistant, DNase resistant, 5'-3' exonuclease resistant, or 3'-5' exonuclease resistant). In some embodiments, the 5' conjugates described herein inhibit 5'-3' exonuclease cleavage. In some embodiments, the 3' conjugates described herein inhibit 3'-5' exonuclease cleavage.

[0174] In some embodiments, one or more of the artificial nucleotide analogs described herein have increased binding affinity to their mRNA targets compared to the equivalent natural polynucleotide. In some embodiments, the artificial nucleotide analogs include nucleic acids having modifications at the 2' hydroxyl group of the ribose moiety. In some embodiments, the modifications include H, OR, R, halo, SH, SR, NH2, NHR, NR2, or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogen, sulfur, thiol, thioether, thioester, amine (primary, secondary, or tertiary), amide, ether, ester, alcohol, and oxygen. In some embodiments, the alkyl moiety further comprises a modification. In some embodiments, the modification includes an azo group, a keto group, an aldehyde group, a carboxyl group, a nitro group, a nitroso group, a nitrile group, a heterocyclic (e.g., imidazole, hydrazino, or hydroxylamino) group, an isocyanate or cyanate group, or a sulfur-containing group (e.g., sulfoxide, sulfone, sulfide, or disulfide). In some embodiments, the alkyl moiety further comprises heterosubstitution. In some embodiments, a carbon of a heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In some embodiments, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino. One or more of the artificial nucleotide analogues, including 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, or 2'-fluoro N3-P5'-phosphoramidites, may have increased binding affinity for their mRNA target compared to the equivalent naturally occurring polynucleotide.In some embodiments, 2'-O-methyl modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-O-methoxyethyl (2'-O-MOE) modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-O-aminopropyl modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-deoxy modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, T-deoxy-2'-fluoro modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-O-aminopropyl (2'-O-AP) modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, 2'-O-dimethylaminoethyl (2'-O-DMAOE) modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides. In some embodiments, 2'-O-dimethylaminopropyl (2'-O-DMAP) modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides. In some embodiments, TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides. In some embodiments, 2'-ON-methylacetamide (2'-O-NMA) modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides. In some embodiments, LNA-modified polynucleotides have increased binding affinity to their mRNA target compared to comparable native polynucleotides.In some embodiments, ENA-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, PNA-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, HNA-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, morpholino-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, methylphosphonate nucleotide-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, thiolphosphonate nucleotide-modified polynucleotides have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, polynucleotides comprising 2'-fluoro N3-P5'-phosphoramidites have increased binding affinity to their mRNA targets compared to comparable native polynucleotides. In some embodiments, increased affinity is indicated by a lower Kd, an increased melting temperature (Tm), or a combination thereof.

[0175] In some embodiments, the artificial nucleotide analogs include 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl, 2'-deoxy, T-deoxy-2'-fluoro, 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), TO-dimethylaminoethyloxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamide (2'-O-NMA) modifications, LNA, ENA, PNA, HNA, morpholino, methylphosphonate nucleotides, thiolphosphonate nucleotides, 2'-fluoro N3-P5'-phosphoramidites, or combinations thereof.

[0176] In some embodiments, the artificial nucleotide analogues are 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 with modifications at the 5-position, 5-(2-amino)propyluridine, 5-halocytidine, 5-haulidine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine, 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (e.g., 7-deaza-adenosine, 6-azouridine, 6-azocytidine, or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (such as 2-thiouridine, 4-thiouridine, and 2-thiocytidine), dihydrouridine, pseudouridine, queuosine, alkaosine, naphthyl and substituted naphthyl groups, any O- and N-alkylated purines and pyrimidines (such as N6-methyladenosine, 5-methylcarbonylmethyluridine, uridine 5-oxyacetic acid, pyridin-4-one, or pyridin-2-one), phenyl and modified phenyl groups, such as aminophenol or 2,4,6-trimethoxybenzene, modified cytosines that function as G-clamp nucleotides, 8-substituted adenines and guanines, 5-substituted uracils and thymines, azapyrimidines, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonyl alkylated nucleotides. Modified nucleotides also include nucleotides modified with respect to the sugar moiety and nucleotides having sugars or analogs thereof that are not ribosyl. For example, the sugar moiety, in some embodiments, is or is based on mannose, arabinose, glucopyranose, galactopyranose, 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.

[0177] In some embodiments, the polynucleotide comprises one or more phosphorothioate internucleotide linkages. In some embodiments, the polynucleotide comprises 2'-5' internucleotide linkages. In some embodiments, the 2'-5' internucleotide linkage(s) are at the 3' end, the 5' end, or both the 3' and 5' ends of one or both strands of the sequence. In some embodiments, the 2'-5' internucleotide linkage(s) are present at various other locations within one or both strands of the sequence. In some embodiments, the polynucleotide comprises a terminal cap molecule at the 3' end, the 5' end, or both the 3' and 5' ends.

[0178] In some embodiments, the targeting molecule and the polynucleotide are combined to provide a synergistic therapeutic or biological effect.

[0179] In some embodiments, the polynucleotide is directly conjugated to the targeting molecule. The polynucleotide may be conjugated to the targeting molecule via a linker. Suitable linkers for conjugating the polynucleotide to the targeting molecule are known in the art. See, for example, WO2017 / 173408, which is incorporated herein by reference in its entirety. In some embodiments, the linker is a hydrophobic linker. The linker may be a peptide linker. In some embodiments, the linker is a chemical linker. The chemical linker may be a polymer linker. In some embodiments, the chemical linker is linear. In some embodiments, the chemical linker is cyclic.

[0180] In some embodiments, the polymer linker comprises PEG, a sugar, a fatty acid, a phosphate, pyrophosphate, or polysarcosine. In some embodiments, the polymer linker comprises PEG. In some embodiments, the polymer linker comprises a sugar. In some embodiments, the polymer linker comprises a fatty acid. In some embodiments, the polymer linker comprises a phosphate. In some embodiments, the polymer linker comprises pyrophosphate. In some embodiments, the polymer linker comprises polysarcosine. The linker can be a high molecular weight PEG linker. In some embodiments, the high molecular weight PEG linker comprises 1,000 to 5,000 PEG monomers (i.e., PEG1k to PEG5k). In some embodiments, the high molecular weight PEG linker is PEG1k. In some embodiments, the high molecular weight PEG linker is PEG1.5k. In some embodiments, the high molecular weight PEG linker is PEG2k. In some embodiments, the high molecular weight PEG linker is PEG3k. In some embodiments, the high molecular weight PEG linker is PEG4k. In some embodiments, the high molecular weight PEG linker is PEG5k.

[0181] In some embodiments, the linker is a low molecular weight PEG linker. In some embodiments, the low molecular weight PEG linker comprises 4 to 100 PEG monomers (i.e., PEG4 to PEG100). In some embodiments, the low molecular weight PEG linker is PEG12 to PEG48. In some embodiments, the low molecular weight PEG linker is PEG12 to PEG24. In some embodiments, the low molecular weight PEG linker is PEG12 to PEG18. In some embodiments, the low molecular weight PEG linker is PEG6 to PEG18. In some embodiments, the low molecular weight PEG linker is PEG4. In some embodiments, the low molecular weight PEG linker is PEG5. In some embodiments, the low molecular weight PEG linker is PEG6. In some embodiments, the low molecular weight PEG linker is PEG7. In some embodiments, the low molecular weight PEG linker is PEG8. In some embodiments, the low molecular weight PEG linker is PEG9. In some embodiments, the low molecular weight PEG linker is PEG10. In some embodiments, the low molecular weight PEG linker is PEG11. In some embodiments, the low molecular weight PEG linker is PEG12. In some embodiments, the low molecular weight PEG linker is PEG13. In some embodiments, the low molecular weight PEG linker is PEG14. In some embodiments, the low molecular weight PEG linker is PEG15. In some embodiments, the low molecular weight PEG linker is PEG16. In some embodiments, the low molecular weight PEG linker is PEG17. In some embodiments, the low molecular weight PEG linker is PEG18. In some embodiments, the low molecular weight PEG linker is PEG19. In some embodiments, the low molecular weight PEG linker is PEG20. In some embodiments, the low molecular weight PEG linker is PEG21. In some embodiments, the low molecular weight PEG linker is PEG22. In some embodiments, the low molecular weight PEG linker is PEG23. In some embodiments, the low molecular weight PEG linker is PEG24. In some embodiments, the low molecular weight PEG linker is PEG25.In some embodiments, the low molecular weight PEG linker is PEG26. In some embodiments, the low molecular weight PEG linker is PEG27. In some embodiments, the low molecular weight PEG linker is PEG28. In some embodiments, the low molecular weight PEG linker is PEG29. In some embodiments, the low molecular weight PEG linker is PEG30. In some embodiments, the low molecular weight PEG linker is PEG31. In some embodiments, the low molecular weight PEG linker is PEG32. In some embodiments, the low molecular weight PEG linker is PEG33. In some embodiments, the low molecular weight PEG linker is PEG34. In some embodiments, the low molecular weight PEG linker is PEG35. In some embodiments, the low molecular weight PEG linker is PEG36. In some embodiments, the low molecular weight PEG linker is PEG37. In some embodiments, the low molecular weight PEG linker is PEG38. In some embodiments, the low molecular weight PEG linker is PEG39. In some embodiments, the low molecular weight PEG linker is PEG40. In some embodiments, the low molecular weight PEG linker is PEG41. In some embodiments, the low molecular weight PEG linker is PEG42. In some embodiments, the low molecular weight PEG linker is PEG43. In some embodiments, the low molecular weight PEG linker is PEG44. In some embodiments, the low molecular weight PEG linker is PEG45. In some embodiments, the low molecular weight PEG linker is PEG46. In some embodiments, the low molecular weight PEG linker is PEG47. In some embodiments, the low molecular weight PEG linker is PEG48. In some embodiments, the low molecular weight PEG linker is PEG49. In some embodiments, the low molecular weight PEG linker is PEG50. In some embodiments, the low molecular weight PEG linker is PEG51. In some embodiments, the low molecular weight PEG linker is PEG52. In some embodiments, the low molecular weight PEG linker is PEG53. In some embodiments, the low molecular weight PEG linker is PEG54.In some embodiments, the low molecular weight PEG linker is PEG55. In some embodiments, the low molecular weight PEG linker is PEG56. In some embodiments, the low molecular weight PEG linker is PEG57. In some embodiments, the low molecular weight PEG linker is PEG58. In some embodiments, the low molecular weight PEG linker is PEG59. In some embodiments, the low molecular weight PEG linker is PEG60. In some embodiments, the low molecular weight PEG linker is PEG61. In some embodiments, the low molecular weight PEG linker is PEG62. In some embodiments, the low molecular weight PEG linker is PEG63. In some embodiments, the low molecular weight PEG linker is PEG64. In some embodiments, the low molecular weight PEG linker is PEG65. In some embodiments, the low molecular weight PEG linker is PEG66. In some embodiments, the low molecular weight PEG linker is PEG67. In some embodiments, the low molecular weight PEG linker is PEG68. In some embodiments, the low molecular weight PEG linker is PEG69. In some embodiments, the low molecular weight PEG linker is PEG70. In some embodiments, the low molecular weight PEG linker is PEG71. In some embodiments, the low molecular weight PEG linker is PEG72. In some embodiments, the low molecular weight PEG linker is PEG73. In some embodiments, the low molecular weight PEG linker is PEG74. In some embodiments, the low molecular weight PEG linker is PEG75. In some embodiments, the low molecular weight PEG linker is PEG76. In some embodiments, the low molecular weight PEG linker is PEG77. In some embodiments, the low molecular weight PEG linker is PEG78. In some embodiments, the low molecular weight PEG linker is PEG79. In some embodiments, the low molecular weight PEG linker is PEG80. In some embodiments, the low molecular weight PEG linker is PEG81. In some embodiments, the low molecular weight PEG linker is PEG82. In some embodiments, the low molecular weight PEG linker is PEG83.In some embodiments, the low molecular weight PEG linker is PEG84. In some embodiments, the low molecular weight PEG linker is PEG85. In some embodiments, the low molecular weight PEG linker is PEG86. In some embodiments, the low molecular weight PEG linker is PEG87. In some embodiments, the low molecular weight PEG linker is PEG88. In some embodiments, the low molecular weight PEG linker is PEG89. In some embodiments, the low molecular weight PEG linker is PEG90. In some embodiments, the low molecular weight PEG linker is PEG91. In some embodiments, the low molecular weight PEG linker is PEG92. In some embodiments, the low molecular weight PEG linker is PEG93. In some embodiments, the low molecular weight PEG linker is PEG94. In some embodiments, the low molecular weight PEG linker is PEG95. In some embodiments, the low molecular weight PEG linker is PEG96. In some embodiments, the low molecular weight PEG linker is PEG97. In some embodiments, the low molecular weight PEG linker is PEG98. In some embodiments, the low molecular weight PEG linker is PEG99. In some embodiments, the low molecular weight PEG linker is PEG100.

[0182] In some embodiments, the linker is non-cleavable. In some embodiments, the linker is cleavable. The linker may be cleavable in vivo. In some embodiments, the cleavable linker is selected from the group consisting of a disulfide linker, a self-immolative peptide polymer hybrid, and a sulfatase-promoted aryl sulfate linker. In some embodiments, the cleavable linker is a disulfide linker. The cleavable linker may be a self-immolative peptide polymer hybrid. In some embodiments, the cleavable linker is a sulfatase-promoted aryl sulfate linker. In some embodiments, the self-immolative peptide polymer hybrid comprises glucuronic acid, para-amino-benzoyloxy (PAB), 7-amino-3-hydroxyethyl-coumarin (7-AHC), or a Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX). In some embodiments, the self-immolative peptide polymer hybrid comprises glucuronic acid. In some embodiments, the self-immolative peptide polymer hybrid comprises para-amino-benzoyloxy (PAB). In some embodiments, the self-immolative peptide polymer hybrid comprises 7-amino-3-hydroxyethyl-coumarin (7-AHC). In some embodiments, the self-immolative peptide polymer hybrid comprises an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX).

[0183] In some embodiments, the cleavable linker is cleaved via reduction, hydrolysis, proteolysis, photocleavage, chemical cleavage, enzymatic cleavage, or bioorthogonal cleavage. In some embodiments, the cleavable linker is cleaved by reduction. In some embodiments, the cleavable linker is cleaved by hydrolysis. In some embodiments, the cleavable linker is cleaved by proteolysis. In some embodiments, the cleavable linker is cleaved by photocleavage. In some embodiments, the cleavable linker is cleaved by chemical cleavage. The chemical cleavage may be by Fe II-mediated β-elimination of TRX. In some embodiments, the cleavable linker is cleaved by enzymatic cleavage. The enzymatic cleavage may be by non-proteolytic sulfatase, β-galactosidase / glucuronidase, or pyrophosphate phosphatase. In some embodiments, the enzymatic cleavage is by non-proteolytic sulfatase. In some embodiments, the enzymatic cleavage is by β-galactosidase / glucuronidase. In some embodiments, the enzymatic cleavage is by pyrophosphate phosphatase. In some embodiments, the cleavable linker is cleaved by bioorthogonal cleavage. The bioorthogonal cleavage can be by Cu I-BTTAA or free copper ion mediated cleavage. In some embodiments, the linker is an acid cleavable linker.

[0184] In some embodiments, the linker comprises a C1-C6 alkyl group (e.g., a C5, C4, C3, C2, or C1 alkyl group). In some embodiments, the linker includes a homobifunctional cross-linker, a heterobifunctional cross-linker, etc. In some embodiments, the linker is a traceless linker (or a zero-length linker). In some embodiments, the linker is a non-polymeric linker. In some embodiments, the linker is a non-peptide linker, or a linker that does not include an amino acid residue.

[0185] In some embodiments, the linker comprises a homobifunctional linker. Exemplary homobifunctional linkers include Lomant's reagent dithiobis(succinimidyl propionate) DSP, 3,3'-dithiobis(sulfosuccinimidyl propionate (DTSSP)), disuccinimidyl suberate (DSS), bis(sulfosuccinimidyl)suberate (BS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfoDST), ethylene glycobis (Ethylene glycol) succinimidyl succinate (EGS), disuccinimidyl glutarate (DSG), N,N'-disuccinimidyl carbonate (DSC), dimethyl adipimidate (DMA), dimethyl pimelimidate (DMP), dimethyl suberimidate (DMS), dimethyl-3,3'-dithiobispropionimidate (DTBP), 1,4-di-3'-(2'-pyridyldithio)propionamido)butane (DP Examples of suitable aryl ethers include, but are not limited to, bis-[2-(4-azidosalicylamido)ethyl]disulfide (BASED), formaldehyde, glutaraldehyde, 1,4-butanediol diglycidyl ether, adipic acid dihydrazide, carbohydrazide, o-toluidine, 3,3'-dimethylbenzidine, benzidine, α,α'-ρ-diaminodiphenyl, diiodo-p-xylenesulfonic acid, N,N'-ethylene-bis(iodoacetamide), or N,N'-hexamethylene-bis(iodoacetamide).

[0186] In some embodiments, the linker comprises a heterobifunctional linker. Exemplary heterobifunctional linkers include amine-reactive and sulfhydryl crosslinkers, such as N-succinimidyl 3-(2-pyridyldithio)propionate (sPDP), long-chain N-succinimidyl 3-(2-pyridyldithio)propionate (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)toluamide]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-iodophenyl)succinimidyl (4-iodoactyl)aminobenzoate (sIAB), sulfosuccinimidyl (4-iodoactyl)aminobenzoate (sulfo-sIAB), succinimidyl-4-(p-maleimidophenyl)butyrate (sMPB), sulfosuccinimidyl-4-(p-maleimidophenyl)butyrate (sulfo-sMPB), N-(y-maleimidobutyryloxy)succinimide esters (GMBs), N-(gamma-maleimidobutyloxy)sulfosuccinimide esters (sulfo-GMBs), succinimidyl 6-((iodoacetyl)amino )hexanoate (sIAX), succinimidyl 6-[6-(((iodoacetyl)amino)hexanoyl)amino]hexanoate (sIAXX), succinimidyl 4-(((iodoacetyl)amino)methyl)cyclohexane-1-carboxylate (sIAC), succinimidyl 6-((((4-iodoacetyl)amino)methyl)cyclohexane-1-carbonyl)amino)hexanoate (sIACX), p-nitrophenyl iodoacetate (NPIA), carbonyl-reactive and sulfhydryl-reactive crosslinkers,For example, 4-(4-N-maleimidophenyl)butyric acid hydrazide (MPBH), 4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide-8 (M2C2H), 3-(2-pyridyldithio)propionyl hydrazide (PDPH), amine-reactive and photoreactive crosslinkers, for example, N-hydroxysuccinimidyl-4-azidosalicylate (NHs-AsA), N-hydroxysulfosuccinimidyl-4-azidosalicylate (sulfo-NHs-AsA), sulfosuccinimidyl-(4-azidosalicylamido)hexanoate, Sulfosuccinimidyl-2-(p-azido-salicylamido)ethyl-1,3'-dithiopropionate (sAsD), N-hydroxysuccinimidyl-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-1,3'-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 Succinimidyl 2-(7-azido-4-methylcoumarin-3-acetamido)ethyl-1,3'-dithiopropionate (sAED), sulfosuccinimidyl 7-azido-4-methylcoumarin-3-acetate (sulfo-sAMCA), p-nitrophenyl diazopyruvate (pNPDP), p-nitrophenyl-2-diazo-3,3,3-trifluoropropionate (PNP-DTP), sulfhydryl-reactive and photoreactive crosslinkers, such as 1-(p-azidosalicylamido)-4-(iodoacetamido)butane (AsIB),These include, but are not limited to, N-[4-(p-azidosalicylamido)butyl]-3'-(2'-pyridyldithio)propionamide (APDP), benzophenone-4-iodoacetamide, benzophenone-4-maleimidocarbonyl-reactive and photoreactive crosslinkers, such as p-azidobenzoylhydrazide (ABH), carboxylate-reactive and photoreactive crosslinkers, such as 4-(p-azidosalicylamido)butylamine (AsBA), and arginine-reactive and photoreactive crosslinkers, such as p-azidophenylglyoxal (APG).

[0187] In some embodiments, the linker comprises a reactive functional group. In some embodiments, the reactive functional group comprises a nucleophilic group that reacts with an electrophilic group present on the binding moiety. Exemplary electrophilic groups include carbonyl groups, such as aldehydes, ketones, carboxylic acids, esters, amides, enones, acyl halides, or acid anhydrides. In some embodiments, the reactive functional group is an aldehyde. Exemplary nucleophilic groups include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides.

[0188] In some embodiments, the linker comprises a maleimide group. In some embodiments, the maleimide group is also referred to as a maleimide spacer. In some embodiments, the maleimide group further includes caproic acid to form maleimidocaproyl (mc). In some embodiments, the linker comprises maleimidocaproyl (mc). In some embodiments, the linker is maleimidocaproyl (mc). In other cases, the maleimide group comprises a maleimidomethyl group, e.g., succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sMCC), or sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-sMCC), as described above.

[0189] In some embodiments, the maleimide group is a self-stabilizing maleimide. In some embodiments, the self-stabilizing maleimide utilizes diaminopropionic acid (DPR) and incorporates a basic amino group adjacent to the maleimide to provide intramolecular catalysis of thiosuccinimide ring hydrolysis, thereby precluding the maleimide from undergoing elimination via a retro-Michael reaction. In some embodiments, 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 embodiments, the linker comprises a self-stabilizing maleimide. In some embodiments, the linker is a self-stabilizing maleimide.

[0190] In some embodiments, the linker comprises a peptide moiety. In some embodiments, the peptide moiety comprises at least 2, 3, 4, 5, 6, 7, 8, or more amino acid residues. In some embodiments, the peptide moiety is a cleavable peptide moiety (e.g., either enzymatically or chemically). In some embodiments, the peptide moiety is a non-cleavable peptide moiety. In some embodiments, 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, Τφ-Cit, Phe-Ala, Ala-Leu-Ala-Leu, or Gly-Phe-Leu-Gly. In some embodiments, the linker comprises a peptide moiety, e.g., 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 embodiments, the linker comprises Val-Cit. In some embodiments, the linker is Val-Cit.

[0191] In some embodiments, the linker comprises a benzoic acid group, or a derivative thereof. In some embodiments, the benzoic acid group, or a derivative thereof, comprises para-aminobenzoic acid (PABA). In some embodiments, the benzoic acid group, or a derivative thereof, comprises gamma-aminobutyric acid (GABA).

[0192] In some embodiments, the linker comprises one or more of a maleimide group, a peptide moiety, and / or a benzoic acid group, in any combination. In some embodiments, the linker comprises a combination of a maleimide group, a peptide moiety, and / or a benzoic acid group. In some embodiments, the maleimide group is maleimidocaproyl (mc). In some embodiments, the peptide group is val-cit. In some embodiments, the benzoic acid group is PABA. In some embodiments, the linker comprises a mc-val-cit group. In some embodiments, the linker comprises a val-cit-PABA group. In additional cases, the linker comprises a mc-val-cit-PABA group.

[0193] In some embodiments, the linker is a self-immolative linker or a self-eliminating linker. In some embodiments, the linker is a self-immolative linker. In other cases, the linker is a self-eliminating linker (e.g., a cyclized self-eliminating linker). In some embodiments, the linker comprises the linker described in U.S. Pat. No. 9,089,614 or PCT Publication No. WO2015038426 (each of which is incorporated herein by reference in its entirety).

[0194] In some embodiments, the linker is a dendritic linker. In some embodiments, the dendritic linker comprises a branched multifunctional linker moiety. In some embodiments, the dendritic linker is used to increase the molar ratio of polynucleotide B to binding moiety A. In some embodiments, the dendritic linker comprises a PAMAM dendrimer.

[0195] In some embodiments, the linker is a traceless linker, or a linker that does not leave a linker moiety (e.g., atom or linker group) on polynucleotide or targeting molecule after cleavage.Exemplary traceless linkers include, but are not limited to, germanium linker, silicon linker, sulfur linker, selenium linker, nitrogen linker, phosphorus linker, boron linker, chromium linker, or phenylhydrazide linker.In some embodiments, the linker is the traceless aryl-triazene linker described in Hejesen, et al., "A traceless aryl-triazene linker for DNA-directed chemistry," Org Biomol Chem 11(15):2493-2497 (2013). In some embodiments, the linker is a traceless linker as described in Blaney, et al., 'Traceless solid-phase organic synthesis,' Chem. Rev. 102:2607-2024 (2002). In some embodiments, the linker is a traceless linker as described in U.S. Pat. No. 6,821,783, which is incorporated herein by reference in its entirety.

[0196] In some embodiments, the linker comprises a functional group that provides steric hindrance at the binding site between the linker and the conjugate moiety (e.g., a polynucleotide or targeting molecule disclosed herein). In some embodiments, the steric hindrance is around a disulfide bond. Exemplary linkers that exhibit steric hindrance include heterobifunctional linkers, such as the heterobifunctional linkers described above. In some embodiments, linkers that exhibit steric hindrance include SMCC and SPDB.

[0197] In some embodiments, the linker is an acid-cleavable linker. In some embodiments, the acid-cleavable linker comprises a hydrazone bond that is susceptible to hydrolytic cleavage. In some embodiments, the acid-cleavable linker comprises a thiomaleamic acid linker. In some embodiments, 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).

[0198] In some embodiments, the linker is a cyclic amine or cyclic amine as described in U.S. Pat. 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 / 0309257, 2016 / 0309260, 2015 / 0309271, 2015 / 0309272, 2015 / 0309273, 2015 / 0309274, 2015 / 0309275, 2015 / 0309276, 2015 / 0309277, 2015 / 0309278, 2015 / 0309279, 2015 / 0309279, 2015 / 0309277, 2015 / 0309279 ... 037360 or 2014 / 0294851, or PCT Publication Nos. WO2015057699, WO2014080251, WO2014197854, WO2014145090 or WO2014177042, each of which is incorporated by reference in its entirety.

[0199] In some embodiments, the linker is conjugated to a lysine residue, a cysteine ​​residue, a histidine residue, or a non-natural amino acid residue in the targeting molecule. In some embodiments, the linker is conjugated to a lysine residue in the targeting molecule. In some embodiments, the linker is conjugated to a cysteine ​​residue in the targeting molecule. In some embodiments, the linker is conjugated to a histidine residue in the targeting molecule. In some embodiments, the linker is conjugated to a non-natural amino acid residue in the targeting molecule.

[0200] In some embodiments, the linker is conjugated to the targeting molecule by chemical conjugation or enzymatic conjugation. In some embodiments, the linker is conjugated to the targeting molecule by chemical conjugation. Chemical conjugation can include acylation and click chemistry. In some embodiments, the linker is conjugated to the targeting molecule by enzymatic conjugation. Enzymatic conjugation can be via a sortase or transferase enzyme.

[0201] In some embodiments, the polynucleotide is conjugated to the targeting molecule by a chemical ligation process. In some embodiments, the polynucleotide is conjugated to the targeting molecule by native ligation. In some embodiments, 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 embodiments, the conjugation is as described in U.S. Patent No. 8,936,910. In some embodiments, the polynucleotide is conjugated to the targeting molecule by native ligation chemistry, either site-specifically or non-specifically.

[0202] In some embodiments, the polynucleotide is conjugated to the targeting molecule by a site-specific method using the "traceless" coupling technology (Philochem). In some embodiments, the "traceless" coupling technology utilizes the N-terminal 1,2-aminothiol group on the targeting molecule, which is then conjugated to a polynucleotide containing an aldehyde group (see Casi et al., "Site-specific traceless coupling of potent cytotoxic drugs to recombinant antibodies for pharmacodelivery," JACS134(13):5887-5892(2012)).

[0203] In some embodiments, polynucleotides are conjugated to targeting molecules by site-specific methods that utilize unnatural amino acids incorporated into the targeting molecule. In some embodiments, the unnatural amino acid comprises p-acetylphenylalanine (pAcPhe). In some embodiments, the keto group of pAcPhe is selectively coupled to an alkoxy-amine derived conjugate moiety to form an oxime bond (see Axup et al., "Synthesis of site-specific antibody-drug conjugates using unnatural amino acids," PNAS 109(40):16101-16106 (2012)).

[0204] In some embodiments, polynucleotides are conjugated to targeting molecules by a site-specific method that utilizes an enzyme-catalyzed process. In some embodiments, the site-specific method utilizes SMARTag™ technology (Redwood). In some embodiments, SMARTag™ technology involves the creation of a formylglycine (FGly) residue from a cysteine ​​by a formylglycine-generating enzyme (FGE) through an oxidative process in the presence of an aldehyde tag, followed by conjugation of FGly to an alkylhydrene-functionalized polynucleotide via hydrazino-Pictet-Spengler (HIPS) ligation (see Wu et al., "Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag," PNAS106(9):3000-3005(2009); Agarwal, et al., "A Pictet-Spengler ligation for protein chemical modification," PNAS110(1):46-51(2013)).

[0205] In some embodiments, the enzyme-catalyzed process includes microbial transglutaminase (mTG). In some embodiments, polynucleotides are conjugated to targeting molecules utilizing a microbial transglutaminase-catalyzed process. In some embodiments, mTG catalyzes the formation of a covalent bond between the amide side chain of a glutamine in a recognition sequence and a primary amine of a functionalized polynucleotide. In some embodiments, 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)).

[0206] In some embodiments, the polynucleotide is conjugated to the targeting molecule by the methods described in PCT Publication No. WO2014 / 140317 (incorporated herein by reference in its entirety), which utilizes a sequence-specific transpeptidase. In some embodiments, the polynucleotide is conjugated to the targeting molecule by the methods described in U.S. Patent Publication Nos. 2015 / 0105539 and 2015 / 0105540 (each of which is incorporated herein by reference in its entirety).

[0207] In some embodiments, each targeting molecule is conjugated to 1-8 polynucleotide molecules (i.e., a drug:antibody ratio (DAR) of 1-8). In some embodiments, each targeting molecule is conjugated to 1 polynucleotide molecule (DAR of 1). In some embodiments, each targeting molecule is conjugated to 2 polynucleotide molecules (DAR of 2). In some embodiments, each targeting molecule is conjugated to 3 polynucleotide molecules (DAR of 3). In some embodiments, each targeting molecule is conjugated to 4 polynucleotide molecules (DAR of 4). In some embodiments, each targeting molecule is conjugated to 5 polynucleotide molecules (DAR of 5). In some embodiments, each targeting molecule is conjugated to 6 polynucleotide molecules (DAR of 6). In some embodiments, each targeting molecule is conjugated to 7 polynucleotide molecules (DAR of 7). In some embodiments, each targeting molecule is conjugated to eight polynucleotide molecules (a DAR of 8).

[0208] In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than about 30 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than about 40 kDa. The polynucleotide conjugate targeting molecule can have a molecular weight of more than about 50 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than about 60 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of about 7,500 kDa or less.

[0209] In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 30 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 40 kDa. The polynucleotide conjugate targeting molecule can have a molecular weight of more than 50 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of more than 60 kDa. In some embodiments, the polynucleotide conjugate targeting molecule has a molecular weight of 7,500 kDa or less.

[0210] In some embodiments, the polynucleotide conjugate is selected from the group consisting of cetuximab-DBCO-C9-M30m3 (DAR of 3), cetuximab-DBCO-C4 / P5-M30m3 (DAR of 3), cetuximab-DBCO-PEG9-M30m3 (DAR of 3), cetuximab-DBCO-PEG9-M30m3 (DAR of 2), cetuximab-DBCO-PEG9-M30m3 (DAR of 4), cetuximab-DBCO-PEG In some embodiments, the polynucleotide conjugate is selected from the group consisting of cetuximab-DBCO-C9-M30m3 (DAR of 3). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-C4 / P5-M30m3 (DAR of 3). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-PEG9-M30m3 (DAR of 3). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-PEG9-M30m3 (DAR of 2). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-PEG9-M30m3 (DAR of 4). In some embodiments, the polynucleotide conjugate is cetuximab-DBCO-PEG9-M30m3 (DAR of 6). In some embodiments, the polynucleotide conjugate is cetuximab-linear-PEG13-M30m3 (DAR of 4). In some embodiments, the polynucleotide conjugate is 3tf12-DBCO-PEG8-NCD5 (DAR of 1). In some embodiments, the polynucleotide conjugate is 3tf12-DBCO-PEG8-M30m3 (DAR of 1). In some embodiments, the polynucleotide conjugate is Fv55-SMCC-M30m3 (DAR of 1).In some embodiments, the polynucleotide conjugate is Fv55-PEG8-DBCO-M30m3 (DAR of 1). In some embodiments, the polynucleotide conjugate is Fv55-PEG8-DBCO-M30m3 (DAR of 2). In some embodiments, the polynucleotide conjugate is selected from the antibody-polynucleotide conjugates listed in Table 5 above. In some embodiments, the polynucleotide conjugate is selected from the antibody-polynucleotide conjugates listed in Table 6 above. Each of the APCs disclosed in Table 5 or Table 6 is considered a separate embodiment.

[0211] Treatment of genetic diseases The third aspect of the present disclosure provides a method for treating genetic disease in a subject in need of such treatment.In some embodiments, the method comprises administering to the subject a therapeutically effective amount of any of the compositions for delivering polynucleotides disclosed herein.In some embodiments, the method comprises administering to the subject a therapeutically effective amount of any of the polynucleotide conjugates disclosed herein.

[0212] The genetic disease disclosed herein can be a cancer, a neurological disorder, a fibrotic disease, a scarring disease, an autoimmune disease, or an inherited genetic disorder.

[0213] In some embodiments, the genetic disease is a neurological disorder. In some embodiments, the neurological disorder is acquired epileptiform aphasia, acute disseminated encephalomyelitis, adrenoleukodystrophy, agenesis of the corpus callosum, agnosia, Aicardi syndrome, Alexander disease, Alpers disease, alternating hemiplegia, Alzheimer's disease, amyotrophic lateral sclerosis (see motor neuron diseases), anencephaly, Angeman syndrome, hemangiomatosis, anoxia, aphasia, apraxia, arachnoid cyst, arachnoiditis, Arnold-Chiari malformation, arteriovenous malformation, Asperger's syndrome, ataxia telangiectasia, attention deficit hyperactivity disorder, autism, auditory processing disorder, autonomic neuropathy. , back pain, Batten disease, Behçet's disease, Bell's palsy, benign essential blepharospasm, benign focal muscular atrophy, benign intracranial hypertension, bilateral frontal-parietal gyri, Binswanger's disease, blepharospasm, Bloch-Sulzberger syndrome, brachial plexus injury, brain abscess, brain injury, brain tumor, Brown-Séquard syndrome, Canavan disease, carpal tunnel syndrome (CTS), burning pain, central pain syndrome, central pontine myelinolysis, centronuclear myopathy, head disorder, cerebral aneurysm, cerebral arteriosclerosis, cerebral atrophy, cerebral gigantism, cerebral palsy, Charcot-Marie-Tooth disease, Chiari malformation, chorea, chronic inflammation chronic demyelinating polyneuropathy (CIDP), chronic pain, chronic regional pain syndrome, Coffin-Lowry syndrome, coma including persistent vegetative state, congenital bilateral facial palsy, corticobasal degeneration, cranial arteritis, craniosynostosis, Creutzfeldt-Jakob disease, cumulative trauma disorder, Cushing's syndrome, cytomegalovirus inclusion body disease (CIBD), cytomegalovirus infection, Dandy-Walker syndrome, Dawson's disease, Domorsia syndrome, Dejerine-Klumpke palsy, Dejerine-Sottas disease, delayed sleep phase syndrome, dementia, dermatomyositis, neurointegrative disorders Movement disorder, diabetic neuropathy, diffuse sclerosis, autonomic neuropathy, dyscalculia, dysgraphia, dyslexia, dystonia, early infantile epileptic encephalopathy, empty sella syndrome, encephalitis, cerebral herniation, trigeminal nerve region hemangiomatosis, encopresis, epilepsy, Erb's palsy, erythromelalgia, essential tremor, Fabry's disease, Fahr's syndrome, syncope, familial spastic paralysis, febrile convulsions, Fisher's syndrome, Friedreich's ataxia, FART syndrome, Gaucher's disease, Gerstmann's syndrome, giant cell arteritis, giant cell inclusion disease, globoid cell leukodystrophy, ectopic gray matter, Guillain-Barre syndrome,HTLV-1 associated myelopathy, Hallervorden-Spatz disease, head injury, headache, hemifacial spasm, hereditary spastic paraplegia, hereditary polyneuropathic ataxia, otic varicella, herpes zoster, Hirayama syndrome, holoprosencephaly, Huntington's disease, hydranencephaly, hydrocephalus, hypercortisolism, hypertrophic cardiomyopathy, hypoxia, immune-mediated encephalomyelitis, inclusion body myositis, incontinentia pigmenti, infantile phytanic acid storage disease, infantile Refsum disease, infantile spasms, inflammatory myopathy, intracranial cysts, increased intracranial pressure, Joubert syndrome, Kearns-Sayre syndrome, Kennedy disease, Kinsbone syndrome, Klippel-Feil syndrome, Krabbe syndrome disease, Kugelberg-Welander disease, Kuru, Lafora disease, Lambert-Eaton myasthenic syndrome, Landau-Kleffner syndrome, lateral myelocorticoid (Wallenberg) syndrome, learning disabilities, Leigh disease, Lennox-Gastaut syndrome, Lesch-Nyhan syndrome, leukodystrophy, dementia with Lewy bodies, cerebrospinal fluid defects, locked-in syndrome, Lou Gehrig's disease, lumbar disc disease, Lyme disease - neurological sequelae, Machado-Joseph disease (spinocerebellar ataxia type 3), megalencephaly, maple syrup urine disease, Marfan syndrome, megalencephaly, Melkerson-Rossenthal syndrome, Ménière's disease, meningeal inflammation, Menkes disease, metachromatic leukodystrophy, microcephaly, migraine, Miller Fisher syndrome, mild stroke, mitochondrial myopathy, Moebius syndrome, juvenile unilateral upper limb muscular atrophy, motor neuron disease, motor skills disorder, Moyamoya disease, mucopolysaccharidosis, multiinfarct dementia, multifocal motor neuropathy, multiple sclerosis, multiple system atrophy, muscular dystrophy, myalgic encephalomyelitis, myasthenia gravis, myelinating diffuse sclerosis, infantile myoclonic encephalopathy, myoclonus, myopathy, myotubular myopathy, congenital myotonia, narcolepsy, neurofibromatosis, neuroleptic relaxation Drug-induced malignant syndrome, Neurological symptoms of AIDS, Neurological sequelae of lupus, Neuromyotonia, Neuronal ceroid lipofuscinosis, Neuronal migration disorder, Niemann-Pick disease, Non-24-hour sleep-wake syndrome, Nonverbal learning disorder, O'Sullivan-McLeod syndrome, Occipital neuralgia, Latent spinal dysraphism, Ohtahara syndrome, Olivopontocerebellar atrophy, Opsoclonus-myoclonus syndrome, Optic neuritis, Orthostatic hypotension, Overuse syndrome, Recurrent vision, Parkinson's disease, Paramyotonia congenita, Paraneoplastic disorders, Seizures, Parry-Romberg syndrome, Romberg syndrome,Pelizaeus-Merzbach disease, periodic paralysis, peripheral neuropathy, persistent vegetative state, diffuse neuropathy, photophotic sneeze reflex, phytanic acid storage disease, Pick's disease, nerve compression, pituitary tumors, PMG, polio, polymicrogyria, polymyositis, porencephaly, post-polio syndrome, post-herpetic neuralgia (PHN), post-infectious encephalomyelitis, postural hypotension, Prader-Willi syndrome, primary lateral sclerosis, prion disease, progressive hemifacial atrophy also known as Romberg syndrome, progressive multifocal leukoencephalopathy, progressive sclerosing polydystrophy, progressive supranuclear paralysis paralysis, pseudotumor cerebri, Ramsay Hunt syndrome (types I and II), Rasmussen's encephalitis, reflex sympathetic dystrophy syndrome, Refsum's disease, repetitive movement disorder, repetitive stress injury, restless legs syndrome, retroviral-associated myelopathy, Rett syndrome, Reye's syndrome, Romberg's syndrome, rabies, chorea, Sandoff's disease, schizophrenia, Schilder's disease, schizencephaly, sensory integration disorder, septo-optic dysplasia, shaken baby syndrome, shingles, Shy-Drager syndrome, Sjogren's syndrome, sleep apnea, sleep disease, gastric sneeze reflex, Sotos syndrome, convulsions, spinal cord injury, spinal tumor, spinal muscular atrophy, spinal stenosis, Steele-Richardson-Olszewski syndrome, see progressive supranuclear palsy, spinocerebellar ataxia, stiff-body syndrome, stroke, Sturge-Weber syndrome, subacute sclerosing panencephalitis, subcortical arteriosclerotic encephalopathy, superficial hemosiderosis, Syndenham chorea, syncope, synesthesia, syringomyelia, tardive dyskinesia, Tay-Sachs disease, temporal arteritis, tethered spinal cord syndrome, Thomsen's disease, thoracic outlet syndrome, trigeminal nerve In some embodiments, the neurological disorder is selected from the group consisting of: chronic pain, Todd's palsy, Tourette's syndrome, transient ischemic attacks, transmissible spongiform encephalopathy, transverse myelitis, traumatic brain injury, tremors, trigeminal neuralgia, tropical spastic paraparesis, trypanosomiasis, tuberous sclerosis, vasculitis including temporal arteritis, von Hippel-Lindau disease (VHL), Biliusk encephalomyelitis (VE), Wallenberg syndrome, Werdnig-Hoffmann disease, West syndrome, whiplash injury, Williams syndrome, Wilson's disease, X-linked spinal and bulbar muscular atrophy, and Zellweger syndrome. In some embodiments, the neurological disorder is a movement disorder, e.g., multiple system atrophy (MSA).

[0214] In some embodiments, the genetic disease is an autoimmune disease. In some embodiments, the genetic disease is an autoimmune disease. In some embodiments, the genetic disease is an autoimmune disease, such as acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, allergic asthma, allergic rhinitis, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune aplastic anemia, autoimmune autonomic dysfunction, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune immunodeficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune pulmonary fibrosis, ... Autoimmune thyroid disease, Axonal and neuropathic neuropathies, Baro's disease, Behçet's disease, Bullous pemphigoid, Cardiomyopathy, Castlemen's disease, Celiac sprue (non-tropical), Chagas disease, Chronic fatigue syndrome, Chronic inflammatory demyelinating polyneuropathy (CIDP), Chronic relapsing multifocal osteomyelitis (CRMO), Churg-Strauss syndrome, Cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, Cold agglutinins, Congenital heart block, Coxsackie myocarditis, Crest's disease, Essential mixed cryoglobulinemia, Demyelinating neuropathy, Dermatomyositis, David's disease Neuromyelitis optica, discoid lupus, Dressler's syndrome, endometriosis, eosinophilic fasciitis, erythema nodosum, experimental allergic encephalomyelitis, Evan's syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture's syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, immunomodulatory lipoproteins, inclusion body myositis, insulin-dependent diabetes mellitus (type 1) , interstitial cystitis, juvenile arthritis, juvenile diabetes mellitus, Kawasaki disease, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignified conjunctivitis, linear immunoglobulin A disease (LAD), lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic), neutropenia, ocular cicatricial pemphigoid, optic neuritis, palindromic rheumatoid arthritis, PANDAS (Streptococcal pediatric autoimmune neuropsychiatric disorders),Paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Parry-Romberg syndrome, Parsonage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS syndrome, polyarteritis nodosa, autoimmune polyglandular syndrome types I, II and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, anti-inflammatory Sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, scleroderma, Slogren's syndrome, sperm-testicular autoimmunity, stiff-body syndrome, subacute bacterial endocarditis (SBE), sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteriopathy, thrombocytopenic purpura (TPP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular dermatitis, vitiligo or Wegener's granulomatosis or chronic active hepatitis, primary biliary cirrhosis, cadilated cardiomyopathy, myocarditis, autoimmune polyendocrine adenoma syndrome type I (APS-I), cystic fibrosis vasculitis, acquired hypoparathyroidism, coronary artery disease, pemphigus foliaceus, pemphigus vulgaris, Rasmussen's encephalitis, autoimmune gastritis, insulin hypoglycemic syndrome (Hirata's disease), type B insulin resistance, acanthosis, systemic lupus erythematosus (SLE), pernicious anemia, resistant Lyme arthritis, polyneuropathy, demyelinating diseases, atopic dermatitis, autoimmune hypothyroidism, vitiligo, thyroid function ophthalmopathy, autoimmune celiac disease, ACTH deficiency, dermatomyositis, Sjögren's syndrome, systemic sclerosis, progressive systemic sclerosis, morphea, primary antiphospholipid syndrome, chronic idiopathic urticaria, connective tissue syndrome, necrotizing crescentic glomerulonephritis (NCGN), systemic vasculitis, Raynaud's syndrome, chronic liver disease, visceral leishmaniasis, autoimmune C1 deficiency, membranoproliferative glomerulonephritis (MPGN), prolonged clotting time, immunodeficiency, atherosclerosis, neuronal disorders, paraneoplastic pemphigus, paraneoplastic systemic stiffening syndrome, paraneoplastic encephalomyelitis, subacute autonomic neuropathy, cancer-associated retinopathy,Selected from the group consisting of paraneoplastic opsoclonus-myoclonus-ataxia, lower motor neuron syndrome, and Lambert-Eaton myasthenic syndrome.

[0215] In some embodiments, the genetic disease is AIDS, anthrax, botulism, brucellosis, chancroid, chlamydia infection, cholera, coccidioidomycosis, cryptosporidiosis, cyclosporiasis, diphtheria, ehrlichiosis, arboviral encephalitis, enterohemorrhagic E. coli, giardiasis, gonorrhea, dengue fever, Haemophilus influenzae, leprosy, hantavirus pulmonary syndrome, hemolytic uremic syndrome, hepatitis A, hepatitis B, hepatitis C, human immunodeficiency virus, legionnaires' disease, listeriosis, Lyme disease, malaria, measles meningococcal disease, mumps, pertussis (whooping cough), cough), plague, paralytic poliomyelitis, psittacosis, Q fever, rabies, Rocky Mountain spotted fever, rubella, congenital rubella syndrome, dysentery, smallpox, streptococcal disease (invasive group A), streptococcal toxic shock syndrome, pneumococcus, syphilis, tetanus, toxic shock syndrome, trichinosis, tuberculosis, tularemia, typhoid fever, vancomycin-intermediate resistant Staphylococcus aureus, chickenpox, yellow fever, variant Creutzfeldt-Jakob disease (vCJD), Ebola hemorrhagic fever, echinococcus, Hendra virus infection, human monkeypox, influenza A, influenza B, H5N1, Lassa fever, Marburg The virus may be selected from the group consisting of hemorrhagic fever, Nipah virus, Onyong fever, Rift Valley fever, herpes, HIV, HCV genotype 1, HCV genotype 2, HCV genotype 3, HCV genotype 4, HCV genotype 5, HCV genotype 6, SARS-CoV-2 (COVID-19), SARS-CoV (SARS), MERS-CoV (MERS), 229E coronavirus, NL63 coronavirus, OC43 coronavirus, CoV-HKU1 (HKU1), alpha coronavirus, beta coronnavirus, Venezuelan equine encephalitis, and West Nile virus.

[0216] In some embodiments, the genetic disease is a fibrotic disease, a scarring disease, or both. In some embodiments, the fibrotic or scarring disease is selected from the group consisting of pulmonary fibrosis, cystic fibrosis, idiopathic pulmonary fibrosis, radiation-induced fibrosis, myocardial fibrosis, bridging fibrosis, liver cirrhosis, gliosis, arteriosclerosis, arthrofibrosis, Crohn's disease, Dupuytren's contracture, keloids, mediastinal fibrosis, myelofibrosis, Peyronie's disease, nephrogenic systemic fibrosis, progressive massive fibrosis, retroperitoneal fibrosis, scleroderma / systemic sclerosis, and adhesive capsulitis.

[0217] In some embodiments, the genetic disease is cancer. In some embodiments, the cancer is thyroid cancer, adrenal cortical carcinoma, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis, central nervous system (CNS) cancer, peripheral nervous system (PNS) cancer, breast cancer, Castleman's disease, cervical cancer, childhood non-Hodgkin's lymphoma, lymphoma, colon and rectal cancer, endometrial cancer, esophageal cancer, Ewing family tumors (e.g., Ewing's sarcoma), eye cancer, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, hairy cell leukemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, childhood leukemia, The cancer is selected from the group consisting of chronic lymphocytic leukemia, chronic myelogenous leukemia, liver cancer, lung cancer, pulmonary carcinoid tumor, non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, myeloproliferative disease, nasal and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (adult soft tissue cancer), melanoma skin cancer, non-melanoma skin cancer, gastric cancer, testicular cancer, thymic cancer, uterine cancer (e.g., uterine sarcoma), vaginal cancer, vulvar cancer, or Waldenstrom's macroglobulinemia. In some cases, the cancer may be selected from the list maintained by the National Cancer Institute (https: / / www.cancer.gov / types).

[0218] The conditions or diseases disclosed herein may include hyperproliferative disorders. Malignant hyperproliferative disorders may be stratified into risk groups, such as low risk and medium-high risk. Hyperproliferative disorders may include, but may not be limited to, cancer, hyperplasia, or tumor. In some cases, the hyperproliferative cancer may be breast cancer, such as ductal carcinoma, medullary carcinoma, colloid carcinoma, tubular carcinoma, and inflammatory breast cancer of the breast ductal tissue; ovarian cancer, including epithelial ovarian tumors, such as ovarian adenocarcinoma, and adenocarcinoma that has metastasized from the ovary into the peritoneal cavity; uterine cancer; cervical cancer, such as epithelial cervical adenocarcinoma, including squamous cell carcinoma and adenocarcinoma; prostate cancer, for example, selected from the following: adenocarcinoma or adenocarcinoma metastasized to bone; pancreatic cancer, such as epithelioid cell carcinoma of the pancreatic ductal tissue and adenocarcinoma of the pancreatic duct; transitional cell carcinoma in the bladder, urothelial carcinoma (transitional cell carcinoma). bladder cancer, such as bladder cancer, squamous cell carcinoma, adenocarcinoma, and small cell carcinoma; leukemia, such as acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, myelodysplasia, myeloproliferative disorders, acute myeloid leukemia (AML), chronic myelogenous leukemia (CML), mastocytosis, chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and myelodysplastic syndromes (MDS); bone cancer; squamous cell carcinoma, adenocarcinoma, and lung cancer, such as non-small cell lung cancer (NSCLC), which can be classified as large cell anaplastic carcinoma, non-small cell lung cancer, and small cell lung cancer; skin cancer, such as basal cell carcinoma, melanoma, squamous cell carcinoma, and actinic keratosis (which can develop into squamous cell carcinoma); ocular retinoblastoma; cutaneous or intraocular (eye) melanoma; primary liver cancer (cancer that begins in the liver); kidney cancer; autoimmune deficiency syndrome (AIDS)-related lymphomas, such as diffuse large B-cell lymphoma, B-cell immunoblastic lymphoma, and small non-cleaving lymphoma. cell lymphoma; Kaposi's sarcoma; virally induced cancers including Hepatitis B virus (HBV), Hepatitis C virus (HCV), and hepatocellular carcinoma; human lymphotropic virus type 1 (HTLV-1) and adult T-cell leukemia / lymphoma; and human papilloma virus (HPV) and cervical cancer; central nervous system (CNS) cancers, such as primary brain tumors (including gliomas (astrocytoma, anaplastic astrocytoma, glioblastoma multiforme), oligodendroglioma, ependymoma, meningioma, lymphoma, schwannoma, medulloblastoma);The cancer may be a peripheral nervous system (PNS) cancer, such as malignant peripheral nerve sheath tumors (MPNST), including acoustic neuroma, neurofibroma, and schwannoma, malignant fibrous cell tumor, malignant fibrous histiocytoma, malignant meningioma, malignant mesothelioma, and malignant mixed Mullerian tumor; oral cavity and oropharyngeal cancer, such as hypopharyngeal carcinoma, laryngeal carcinoma, nasopharyngeal carcinoma, and oropharyngeal carcinoma; gastric cancer, such as lymphoma, gastric stromal tumor, and carcinoid tumor; testicular cancer, such as germ cell tumors (GCT) (including seminoma and non-seminoma), and gonadal stromal tumor (including Leydig cell tumor and Sertoli cell tumor); thymic carcinoma, such as thymoma, thymic carcinoma, Hodgkin's disease, non-Hodgkin's lymphoma carcinoid, or carcinoid tumor; rectal cancer; and colon cancer. In some cases, the diseases that are stratified, classified, characterized, or diagnosed by the methods of the present disclosure include, but are not limited to, thyroid diseases, such as, for example, but not limited to, benign thyroid diseases, including, for example, but not limited to, follicular adenoma, Hurthle cell adenoma, lymphocytic thyroiditis, and thyroid hyperplasia. In some cases, the diseases that are stratified, classified, characterized, or diagnosed by the methods of the present disclosure include, but are not limited to, malignant thyroid diseases, such as, for example, but not limited to, follicular carcinoma, follicular variant of papillary thyroid carcinoma, medullary carcinoma, and papillary carcinoma.

[0219] In some embodiments, the genetic disease is a hereditary genetic disorder resulting from an abnormality in a gene or chromosome. Hereditary genetic disorders can be divided into two categories: single gene disorders, and multifactorial and polygenic (complex) disorders. Single gene disorders can be the result of a single mutated gene. Inheriting single gene disorders can include, but are not limited to, autosomal dominant, autosomal recessive, X-chromosome dominant, X-chromosome recessive, Y-chromosome and mitochondrial inheritance. In some embodiments, one mutated copy of a gene is required for a person to be affected by an autosomal dominant disorder. Examples of autosomal dominant disorders can include, but are not limited to, Huntington's disease, neurofibromatosis type 1, Marfan syndrome, hereditary nonpolyposis colorectal cancer, or hereditary multiple exostoses. In an autosomal recessive disorder, two copies of a gene must be mutated for a subject to be affected by an autosomal recessive disorder. Examples of this type of disorder may include, but are not limited to, cystic fibrosis, sickle cell disease (also partial sickle cell disease), Tay-Sachs disease, Niemann-Pick disease, or spinal muscular atrophy. X-dominant disorders are caused by mutations in genes on the X chromosome, such as X-dominant hypophosphatemic rickets. Some X-dominant conditions, such as Rett syndrome, incontinentia pigmenti type 2, and Aicardi syndrome, can be fatal. X-recessive disorders are also caused by mutations in genes on the X chromosome. Examples of this type of disorder may include, but are not limited to, hemophilia A, Duchenne muscular dystrophy, red-green color blindness, muscular dystrophy, and male pattern baldness. Y-linked disorders are caused by mutations in the Y chromosome. Examples may include, but are not limited to, male infertility and hirsutism. Genetic disorders of mitochondrial inheritance, also known as maternal inheritance, may apply to genes within the mitochondrial DNA, such as Lieber's hereditary optic neuropathy.

[0220] Inherited genetic disorders may also be complex, multifactorial, or polygenic. Polygenic inherited genetic disorders may involve multiple genes acting in combination with lifestyle and environmental factors. Complex genetic disorders may cluster within families, but do not have a clear inheritance pattern. Multifactorial or polygenic disorders include, but are not limited to, heart disease, diabetes, asthma, autism, autoimmune diseases such as multiple sclerosis, cancer, ciliopathies, cleft palate, hypertension, inflammatory bowel disease, mental retardation, or obesity.

[0221] Other exemplary inherited genetic disorders include 1p36 deletion syndrome, 21-hydroxylase deficiency, 22q11.2 deletion syndrome, aceruloplasminemia, achondroplasia type II, achondroplasia, acute intermittent porphyria, adenylsuccinate lyase deficiency, adrenoleukodystrophy, Alexander disease, alkaptonuria, alpha-1 antitrypsin deficiency, Alström syndrome, Alzheimer's disease (types 1, 2, 3, and 4), hereditary amelogenesis imperfecta, amyotrophic lateral sclerosis, amyotrophic lateral sclerosis type 2, amyotrophic lateral sclerosis type 4, amyotrophic lateral sclerosis type 5, amyotrophic lateral sclerosis type 6, amyotrophic lateral sclerosis type 7, amyotrophic lateral sclerosis type 8, amyotrophic lateral sclerosis type 9, amyotrophic lateral sclerosis type 10, amyotrophic lateral sclerosis type 11, amyotrophic lateral sclerosis type 12, amyotrophic lateral sclerosis type 13, amyotrophic lateral sclerosis type 14, amyotrophic lateral sclerosis type 15, amyotrophic lateral sclerosis type 16, amyotrophic lateral sclerosis type 17, amyotrophic lateral sclerosis type 18, amyotrophic lateral sclerosis type 19, amyotrophic lateral sclerosis type 20, amyotrophic lateral sclerosis type 21, amyotrophic lateral sclerosis type 22, amyotrophic lateral sclerosis type 23, amyotrophic lateral sclerosis type 24, amyotrophic lateral sclerosis type 25, amyotrophic lateral sclerosis type 26, amyotrophic lateral sclerosis type 27, am type 4, androgen insensitivity syndrome, anemia, Angelman syndrome, Apert syndrome, ataxia telangiectasia, Bear-Stevenson craniosynostosis syndrome, Benjamin syndrome, beta thalassemia, biotinidase deficiency, Birt-Hogg-Dube syndrome, bladder cancer, Bloom syndrome, bone disease, breast cancer, kyphotic dysplasia, Canavan disease, cancer, celiac disease, chronic granulomatous disease (CGD), Charcot-Marie-Tooth disease, Charcot-Marie-Tooth disease type 1, Charcot-Marie-Tooth disease type 4, Charcot-Marie-Tooth disease type 2, Charcot-Marie-Tooth disease Leigh-Tooth disease type 4, Cockayne syndrome, Coffin-Lowry syndrome, collagen diseases types II and XI, colon cancer, congenital absence of the vas deferens, congenital bilateral absence of the vas deferens, congenital diabetes mellitus, congenital erythropoietic porphyria, congenital heart disease, congenital hypothyroidism, connective tissue disease, Cowden syndrome, cri-a-cat syndrome, Crohn's disease, fibrostenosis, Crouzon syndrome, Crouzon cutaneous skeletal syndrome, cystic fibrosis, de Grouchy syndrome, degenerative neurological diseases, Dent's disease, developmental disorders, Di George syndrome, distal spinal muscular atrophy type V, Down syndrome, dwarfism, Ehlers-Danlos syndrome group, Ehlers-Danlos syndrome multiple joint laxity type, Ehlers-Danlos syndrome classic type, Ehlers-Danlos syndrome skin fragility type, Ehlers-Danlos syndrome kyphoscoliosis type, vascular type, erythropoietic protoporphyria, Fabry disease, facial injuries and disorders, factor V Leiden thrombosis, familial adenomatous polyposis, familial dysautonomia, Fanconi anemia, FG syndrome, fragile X syndrome, Friedreich ataxia, Friedreich ataxia, G6PD deficiency, galactosemia, Gaucher disease (types 1, 2 and 3), hereditary brain diseases, glycine encephalopathy, hemochromatosis type 2,Haemochromatosis type 4, Harlequin ichthyosis, Head and brain malformations, Hearing impairment and deafness, Hearing impairment in children, Haemochromatosis (neonatal, type 2 and type 3), Haemophilia, Myelohepatic porphyria, Hereditary coproporphyria, Hereditary multiple exostoses, Hereditary neuropathy with pressure palsies, Hereditary nonpolyposis colorectal cancer, Homocystinuria, Huntington's disease, Hutchinson-Gillfold-Progeria syndrome, Hyperoxaluria, Primary hyperphenylalaninemia, Hypochondrogenesis, Hypochondroplasia, IDISC15, Incontinentia pigmenti, Gaucher disease in childhood, Hereditary ascending spastic paralysis of childhood, Infertility, Jackson-Weiss syndrome , Joubert syndrome, juvenile primary lateral sclerosis, Kennedy disease, Klinefelter syndrome, Kniest dysplasia, Krabbe disease, learning disabilities, Lesch-Nyhan syndrome, leukodystrophy, Li-Fraumeni syndrome, lipoprotein lipase deficiency, familial male reproductive disorders, Marfan syndrome, McCune-Albright syndrome, McLeod syndrome, Mediterranean fever, familial Menkes disease, Menkes syndrome, metabolic disorders, methemoglobinemia beta globin type, methemoglobinemia, hereditary methemoglobinemia, methylmalonic acidemia, Micro syndrome, microcephaly, movement disorders, Mowat-Wilson syndrome, mucopolysaccharidoses (MPS) I), Muenke syndrome, muscular dystrophy, Duchenne and Becker muscular dystrophy, myotonic dystrophy, myotonic dystrophy type 1 and type 2, limb-girdle muscular dystrophy, Pompe disease, neonatal hemochromatosis, neurofibromatosis, neurofibromatosis type 1, neurofibromatosis type 2, neurofibromatosis type I, neurofibromatosis type II, neurological diseases, neuromuscular diseases, Niemann-Pick disease, nonketotic hyperglycinemia, nonsyndromic hearing loss, nonsyndromic hearing loss autosomal recessive, Noonan syndrome, osteogenesis imperfecta (types I and III) , Otospondylomegalyepiphyseal dysplasia, Pantothenate kinase-associated neurodegeneration, Patau syndrome (trisomy 13), Pendred syndrome, Peutz-Jeghers syndrome, Pfeiffer syndrome, Phenylketonuria, Porphyria, Porphyria cutanea tarda, Prader-Willi syndrome, Primary pulmonary hypertension, Prion diseases, Progeria, Propionic acidemia, Protein C deficiency, Protein S deficiency, Pseudo-Gaucher disease, Pseudoxanthoma elasticum, Retinopathy, Ret syndrome, Retinoblastoma FA-Friedreich ataxia,Rubinstein-Taybi syndrome, Sandoff disease, Sensory and Autonomic Neuropathy Type III, Sickle Cell Anemia, Skeletal Muscle Regeneration, Cutaneous Pigmentation, Smith-Lemli-Opitz Syndrome, Speech and Communication Disorders, Spinal Muscular Atrophy, Spinobulbar Muscular Atrophy, Spinocerebellar Ataxia, Spondyloepiphyseal Dysplasia, Strudwick Type, Spondyloepiphyseal Dysplasia Congenita, Stickler Syndrome, Stickler Syndrome COL2A1, Tay-Sachs Disease, Tetrahydrobiopterin Deficiency, Thanatophoric Dysplasia, Diabetes and Sensorineural Hearing Loss These conditions may include, but are not limited to, thiamine-responsive megaloblastic anemia, thyroid disease, Tourette's syndrome, Treacher-Collins syndrome, triple X syndrome, tuberous sclerosis, Turner syndrome, Usher syndrome, variegate porphyria, von Hippel-Lindau disease, Waardenburg syndrome, Weissenbach-Zweimüller syndrome, Wilson's disease, Wolf-Hirschhorn syndrome, xeroderma pigmentosum, X-linked severe combined immunodeficiency, X-linked sideroblastic anemia, or X-linked spinobulbar muscular atrophy.

[0222] In some embodiments, the genetic disease is a viral infection. The viral infection may be caused by a virus selected from the group consisting of adenovirus, anellovirus, arenavirus, astrovirus, bunyavirus, calicivirus, coronavirus, filovirus, flavivirus, hepadnavirus, herpesvirus, orthomyxovirus, papillomavirus, paramyxovirus, parvovirus, picornavirus, pneumovirus, polyomavirus, poxvirus, reovirus, retrovirus, rhabdovirus, and togavirus. In some embodiments, the virus is an adeno-associated virus, Aichi virus, Australian bat lyssavirus, BK polyomavirus, Banna virus, Barmah Forest virus, Bunyamwera virus, Lacrosse bunyavirus, Snowshoe hare bunyavirus, Cercopithecine herpesvirus, Chandipura virus, Chikungunya virus, Cosavirus A, Cowpox virus, Coxsackievirus, Crimean-Congo hemorrhagic fever virus, Dengue virus, Dhoori virus, Dugbe virus, virus), Dubenhague virus, Eastern equine encephalitis virus, Ebola virus, Echovirus, Encephalomyocarditis virus, Epstein-Barr virus, European bat lyssavirus, GB virus C / G hepatitis virus, Hantavirus, Hendra virus, Hepatitis A virus, Hepatitis B virus, Hepatitis C virus, Hepatitis E virus, Hepatitis D virus, Horsepox virus, Human adenovirus, Human astrovirus, Human coronavirus, Human cytomegalovirus, Human enterovirus 68, Human enterovirus 70, Human hepatitis human herpesvirus 1, human herpesvirus 2, human herpesvirus 6, human herpesvirus 7, human herpesvirus 8, human immunodeficiency virus, human papillomavirus 1, human papillomavirus 2, human papillomavirus 16, human papillomavirus 18, human parainfluenza, human parvovirus B19, human respiratory syncytial virus, human rhinovirus, human SARS coronavirus, human spumaretrovirus, human T-lymphotropic virus, human torovirus, influenza A virus, influenza B virus,Influenza C virus, Isfahan virus, JC polyomavirus, Japanese encephalitis virus, Junin arenavirus, KI polyomavirus, Kunjin virus, Lagos bat virus, Lake Victoria Marburg virus, Langat virus, Lassa virus, Rosedale virus, Louping ill virus, Lymphocytic choriomeningitis virus, Machupo virus, Mayaro virus, MERS coronavirus, Measles virus, Mengo encephalomyocarditis virus, Merkel cell polyomavirus, Mokola virus, Molluscum contagiosum virus, Monkeypox virus, Mumps virus, Murray Valley encephalitis virus, New York virus, Nipah virus, Norwalk virus, O'nyong-nyong virus, Orf virus, Oropouche virus, Pichinde virus, Polio virus, Punta Toro phlebovirus, Puuma lavirus, Rabies virus The virus is selected from the group consisting of rabies virus, Rift Valley fever virus, Rosavirus A, Ross River virus, Rotavirus A, Rotavirus B, Rotavirus C, Rubella virus, Sagiyama virus, Sarivirus A, Sicilian sandfly fever virus, Sapporo virus, SARS coronavirus 2, Semliki forest virus, Seoul virus, Simian foamy virus, Simian virus 5, Sindbis virus, Southampton virus, St. Louis encephalitis virus, Tick-borne Powassan virus, Torque teno virus, Tuscany virus, Ukuniemi virus, Vaccinia virus, Varicella zoster virus, Smallpox virus, Venezuelan equine encephalitis virus, Vesicular stomatitis virus, Western equine encephalitis virus, WU polyomavirus, West Nile virus, Yaba monkey tumor virus, Yaba-like disease virus, Yellow fever virus, and Zika virus.

[0223] In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA that targets a viral gene. In some embodiments, the polynucleotide comprises an siRNA that targets a viral gene. In some embodiments, the polynucleotide comprises an miRNA that targets a viral gene. In some embodiments, the polynucleotide comprises an miRNA mimic that targets a viral gene. In some embodiments, the polynucleotide comprises an ASO that targets a viral gene. In some embodiments, the polynucleotide comprises a guide RNA that targets a viral gene. The polynucleotide may be conjugated to a targeting molecule that specifically binds to a viral protein or a protein on the surface of a host cell for the virus. In some embodiments, the polynucleotide and the targeting molecule act synergistically in treating a viral infection.

[0224] In some embodiments, the genetic disease is cancer. In some embodiments, the cancer is characterized by overexpression of a cancer gene. In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA that targets the cancer gene. In some embodiments, the polynucleotide comprises an siRNA that targets the cancer gene. In some embodiments, the polynucleotide comprises an miRNA that targets the cancer gene. In some embodiments, the polynucleotide comprises an miRNA mimic that targets the cancer gene. In some embodiments, the polynucleotide comprises an ASO that targets the cancer gene. In some embodiments, the polynucleotide comprises a guide RNA that targets the cancer gene.

[0225] In some embodiments, the cancer is characterized by a reduction in the expression of a tumor suppressor gene. The polynucleotide may comprise an mRNA molecule that encodes the tumor suppressor gene. In some embodiments, the polynucleotide comprises a guide RNA that restores the expression of the tumor suppressor gene.

[0226] In some embodiments, the polynucleotide is conjugated to a targeting molecule that specifically binds to tumor cells of the cancer. In some embodiments, the targeting molecule specifically binds to epidermal growth factor receptor, and the polynucleotide is a miR-30 miRNA or a mimic thereof. In some embodiments, the targeting molecule specifically binds to ACVR1, and the polynucleotide is a miR-30 miRNA or a mimic thereof. In some embodiments, the targeting molecule specifically binds to ACVR1, and the polynucleotide is a miR-26 miRNA or a mimic thereof. In some embodiments, the targeting molecule specifically binds to TFR. In some embodiments, the targeting molecule specifically binds to TFR is selected from the group consisting of FV55 scFv, Fv55 diabody, and 3TF12. In some embodiments, the targeting molecule specifically binds to TFR is FV55 scFv. In some embodiments, the targeting molecule specifically binds to TFR is FV55 diabody. In some embodiments, the targeting molecule specifically binds to TFR is 3TF12. Polynucleotides and targeting molecules may act synergistically in the treatment of cancer.

[0227] In some embodiments, the genetic disease is a neuromuscular disease. The neuromuscular disease may be a muscular dystrophy. In some embodiments, the muscular dystrophy is facioscapulohumeral muscular dystrophy (FSHD). In some embodiments, the polynucleotide comprises an siRNA, miRNA, miRNA mimic, ASO, or guide RNA targeting DUX4, DMPK, or CAPN3. In some embodiments, the polynucleotide comprises an siRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA targeting DUX4. In some embodiments, the polynucleotide comprises an miRNA mimic targeting DUX4. In some embodiments, the polynucleotide comprises an ASO targeting DUX4. In some embodiments, the polynucleotide comprises a guide RNA targeting DUX4. In some embodiments, the ASO targeting DUX is selected from the group consisting of DUX4-targeting ASOs disclosed in Table 4. In some embodiments, the DUX4-targeting ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32. In some embodiments, the DUX4-targeted ASO is ASDX2. In some embodiments, the DUX4-targeted ASO is ASDX4. In some embodiments, the DUX4-targeted ASO is ASDX23. In some embodiments, the DUX4-targeted ASO is ASDX26. In some embodiments, the DUX4-targeted ASO is ASDX32. In some embodiments, the polynucleotide comprises an siRNA that targets DMPK. In some embodiments, the polynucleotide comprises an miRNA that targets DMPK. In some embodiments, the polynucleotide comprises an miRNA mimic that targets DMPK. In some embodiments, the polynucleotide comprises an ASO that targets DMPK. In some embodiments, the polynucleotide comprises an siRNA that targets CAPN3. In some embodiments, the polynucleotide comprises an miRNA that targets CAPN3. In some embodiments, the polynucleotide comprises an miRNA mimic that targets CAPN3.In some embodiments, the polynucleotide comprises an ASO that targets CAPN3.

[0228] In some embodiments, the muscular dystrophy is Duchenne muscular dystrophy.In some embodiments, the polynucleotide is the mRNA that encodes dystrophin or utrophin.In some embodiments, the polynucleotide is a guide RNA that restores the expression of dystrophin or utrophin.

[0229] In some embodiments, the polynucleotide is conjugated to a targeting molecule that specifically binds to the marker on the surface of skeletal muscle cells of the subject.The targeting molecule can specifically bind to ACVR1.In some embodiments, the targeting molecule specifically binds to ACVR1, and the polynucleotide is DUX4 targeting ASO.

[0230] In some embodiments, the polynucleotide and the targeting molecule exhibit synergistic effects in the treatment of muscular dystrophy.

[0231] Anti-transferrin receptor antibody A fourth aspect of the present disclosure provides an antibody or antigen-binding fragment thereof that specifically binds to human transferrin receptor (TfR1). In some embodiments, the antibody or antigen-binding fragment thereof has the amino acid sequence (underlined CDRs): The heavy chain variable region (VH) containing TIFF2025503504000018.tif28159 and the amino acid sequence (underlined CDRs): and a light chain variable region (VL) comprising TIFF2025503504000019.tif16159.

[0232] In some embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a full length antibody, an immunoglobulin single variable domain (ISV) such as a Fab, Fab-Fc, Fv, single chain Fv (scFv), a diabody, a minibody, and a Nanobody® molecule. In some embodiments, the antibody or antigen-binding fragment thereof is an scFv. In some embodiments, the antibody or antigen-binding fragment thereof is a diabody.

[0233] In some embodiments, the VH and VL are connected by a linker. In some embodiments, the linker comprises the amino acid sequence GGGGS. In some embodiments, the linker comprises the amino acid sequence (GGGGS) N and N is 1 to 3.

[0234] In some embodiments, the antibody or antigen-binding fragment thereof is an scFv, and the VH and VL are connected by a linker, and the linker has the amino acid sequence (GGGGS). 3 In some embodiments, the antibody or antigen-binding fragment thereof is a diabody, wherein the VH and VL are connected by a linker, and the linker comprises the amino acid sequence (GGGGS). N where N is 1 or 2.

[0235] Exemplary embodiments Particular embodiments of the present disclosure are described in the following numbered paragraphs.

[0236] 1. A composition comprising a hybrid polymer and a polynucleotide, said hybrid polymer comprising a cationic portion and a neutral portion.

[0237] 2. The composition of embodiment 1, wherein the cationic portion of the hybrid polymer interacts with the polynucleotide non-covalently, e.g., via ionic interactions.

[0238] 3. The composition of embodiment 1 or 2, wherein the cationic portion of the hybrid polymer is a cationic polypeptide.

[0239] 4. The composition of embodiment 3, wherein the cationic polypeptide is a poly-arginine polypeptide.

[0240] 5. The composition of embodiment 3, wherein the cationic polypeptide is a poly-lysine polypeptide.

[0241] 6. The composition of embodiment 3, wherein the cationic polypeptide comprises arginine and lysine residues.

[0242] 7. The composition of embodiment 3, wherein the cationic polypeptide comprises protamine.

[0243] 8. The composition of any one of embodiments 3 to 7, wherein said cationic polypeptide comprises an L-amino acid residue.

[0244] 9. The composition of any one of embodiments 3 to 7, wherein the cationic polypeptide comprises D-amino acid residues.

[0245] 10. The composition of any one of embodiments 3 to 7, wherein said cationic polypeptide comprises L-amino acid residues and D-amino acid residues.

[0246] 11. The composition according to any one of embodiments 3 to 10, wherein said cationic polypeptide comprises 9 to 18 amino acid residues.

[0247] 12. The composition of embodiment 11, wherein the cationic polypeptide comprises 12 amino acid residues.

[0248] 13. The composition of embodiment 3, wherein said cationic polypeptide is selected from the group of cationic polypeptides disclosed in Table 1.

[0249] 14. The composition of embodiment 1 or 2, wherein the cationic portion of the hybrid polymer comprises a cationic polymer having a size of about 600 to about 2000 daltons.

[0250] 15. The composition of embodiment 14, wherein the cationic polymer is a linear polymer.

[0251] 16. The composition of embodiment 14, wherein the cationic polymer is a branched polymer.

[0252] 17. The composition according to any one of embodiments 14 to 16, wherein the cationic polymer is selected from the group consisting of gelatin, glucosamine, N-acetylglucosamine, chitosan, cationic dextran, cationic cyclodextrin, cationic cellulose, polyethyleneimine (PEI), polyamidoamine (PAA), poly(amino-co-ester) (PAE), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), or cationic lipids, such as DOTAP (N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium) chloride, poly[N,N-diethylaminoethyl methacrylate] (PDEAEMA), cationic mucic acid polymer (cMAP), and DOPE (dioleylphosphatidylethanolamine).

[0253] 18. The composition of any one of the preceding embodiments, wherein the neutral portion of the hybrid polymer comprises a polymer having a size of about 100 to about 1000 daltons.

[0254] 19. The composition of embodiment 18, wherein the neutral portion of the hybrid polymer comprises poly(ethylene glycol) (PEG).

[0255] 20. The composition of any one of embodiments 1-13 and 18-19, wherein the hybrid polymer is a PEGylated cationic polypeptide.

[0256] 21. The composition of embodiment 19 or 20, wherein the hybrid polymer comprises a PEG12-PEG24 polymer.

[0257] 22. The composition of any one of the preceding embodiments, wherein the hybrid polymer and the polynucleotide do not form aggregates or nanoparticles.

[0258] 23. The composition according to any one of the preceding embodiments, wherein the charge ratio of the cationic polypeptide to the polynucleotide is between 0.25:1 and 5:1.

[0259] 24. The composition of embodiment 23, wherein the charge ratio of the cationic polypeptide to the polynucleotide is 0.5:1 to 5:1.

[0260] 25. The composition of embodiment 23, wherein the charge ratio of the cationic polypeptide to the polynucleotide is 1:1 to 4:1.

[0261] 26. The composition of embodiment 23, wherein the charge ratio of the cationic polypeptide to the polynucleotide is 1:1 to 2:1.

[0262] 27. The composition of embodiment 23, wherein the charge ratio of the cationic polypeptide to the polynucleotide is 1:1 or 2:1.

[0263] 28. The hybrid polymer is PEG12PolyArg12{d}, PEG12PolyArg6, PEG12PolyArg6C, PEG24PolyArg12C, PEG24PolyArg12, PEG24PolyArg9, PolyArg12C-PEG2000Da, PolyArg12C-PEG5000Da, PolyArg12C-Dextran5000Da, PEG12PolyArg12, PEG12P 3. The composition of claim 1 or 2, wherein the polyArg9d, PEG1000DaPolyArg12, PEG2000DaPolyArg12, PEG5000DaPolyArg12, PolyArg12Cbp1.5kDa, PolyArg12Cbp3.9kDa, PolyArg12Cbp16kDa, CPolyArg12Cbp1.5kDa, PolyArg12Cbp2kDa, PolyArg12bp2kDa, amide dextran, lysine dextran, PEG PEI 15kda, BPEI-G-PEG550, and BPEI-G-PEG5000.

[0264] 29. The composition of any one of embodiments 1 to 28, wherein the polynucleotide is conjugated to a targeting molecule.

[0265] 30. The composition of embodiment 29, wherein the targeting molecule is an antibody or an antigen-binding fragment thereof, or a binding protein.

[0266] 31. The composition of embodiment 30, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, a Fab-Fc, an Fv, a single-chain Fv (scFv), a diabody, a minibody, and a Nanobody® molecule.

[0267] 32. The composition of embodiment 31, wherein the bispecific antibody is a bispecific T cell engager (BiTE) or a dual affinity retargeting antibody (DART).

[0268] 33. The composition of embodiment 31, wherein the Nanobody® is Nanobody-HSA®.

[0269] 34. The composition of any one of embodiments 30 to 33, wherein the antibody or antigen-binding fragment thereof is an IgG molecule or is derived from an IgG molecule.

[0270] 35. The composition of embodiment 34, wherein the IgG molecule is an IgG1 or IgG4 molecule.

[0271] 36. The composition of embodiment 30, wherein the binding protein is a soluble receptor or a soluble ligand.

[0272] 37. The composition of embodiment 36, wherein the soluble receptor comprises the extracellular domain of the receptor.

[0273] 38. The composition of embodiment 36 or 37, wherein the soluble receptor is an Fc fusion protein.

[0274] 39. The composition according to any one of embodiments 29 to 38, wherein the targeting molecule is a therapeutically or biologically active molecule.

[0275] 40. The composition of any one of embodiments 1 to 39, wherein the polynucleotide is selected from the group consisting of siRNA, ncRNA mimic, short hairpin RNA (shRNA), dicer-dependent siRNA (di-siRNA), antisense oligonucleotide (ASO), gapmer, mixmer, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), RNA activating oligonucleotide (RNAa), aptamer, exon skipping oligonucleotide, miRNA, miRNA mimic, mRNA, and guide RNA.

[0276] 41. The composition of embodiment 40, wherein the polynucleotide is an miRNA mimic.

[0277] 42. The composition of embodiment 41, wherein the miRNA mimic mimics miR-30.

[0278] 43. The composition of embodiment 42, wherein the polynucleotide is an miRNA mimic selected from the group consisting of M30m1, M30m2, M30m3, and M30m4.

[0279] 44. The composition of embodiment 43, wherein the polynucleotide is M30m3.

[0280] 45. The composition of embodiment 40, wherein the polynucleotide is an ASO.

[0281] 46. ​​The composition of embodiment 45, wherein the ASO is a DUX4-targeting ASO.

[0282] 47. The composition of embodiment 46, wherein the DUX4-targeting ASO is selected from the group consisting of DUX4-targeting ASOs disclosed in Table 4.

[0283] 48. The composition of embodiment 47, wherein the DUX4-targeting ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32.

[0284] 49. A composition described in any one of embodiments 40 to 48, wherein the targeting molecule and the polynucleotide provide a synergistic therapeutic or biological effect.

[0285] 50. The composition of any one of embodiments 29 to 49, wherein the polynucleotide is directly conjugated to the targeting molecule.

[0286] 51. The composition of any one of embodiments 29 to 49, wherein the polynucleotide is conjugated to the targeting molecule via a linker.

[0287] 52. The composition of embodiment 51, wherein the linker is a hydrophobic linker.

[0288] 53. The composition of embodiment 51, wherein the linker is a peptide linker.

[0289] 54. The composition of embodiment 51, wherein the linker is a chemical linker.

[0290] 55. The composition of embodiment 54, wherein the chemical linker is a polymer linker.

[0291] 56. The composition of embodiment 54 or 55, wherein the chemical linker is linear.

[0292] 57. The composition of embodiment 54 or 55, wherein the chemical linker is cyclic.

[0293] 58. The composition of embodiment 55, wherein the polymer linker comprises PEG, a sugar, a fatty acid, a phosphate, pyrophosphate, or polysarcosine.

[0294] 59. The composition of embodiment 58, wherein the linker is a high molecular weight PEG linker.

[0295] 60. The composition of embodiment 58, wherein the linker is a low molecular weight PEG linker.

[0296] 61. The composition of any one of embodiments 51-60, wherein the linker is non-cleavable.

[0297] 62. The composition of any one of embodiments 51-60, wherein the linker is cleavable.

[0298] 63. The composition of embodiment 62, wherein the linker is cleavable in vivo.

[0299] 64. The composition of embodiment 62 or 63, wherein the cleavable linker is selected from the group consisting of a disulfide linker, a self-immolative peptide polymer hybrid, and a sulfatase-promoted aryl sulfate linker.

[0300] 65. The composition of embodiment 64, wherein the self-immolative peptide polymer hybrid comprises glucuronic acid, para-amino-benzoyloxy (PAB), 7-amino-3-hydroxyethyl-coumarin (7-AHC), or an Fe(II)-reactive 1,2,4-trioxolane scaffold (TRX).

[0301] 66. The composition of any one of embodiments 62 to 65, wherein the cleavable linker can be cleaved via reduction, hydrolysis, proteolysis, photocleavage, chemical cleavage, enzymatic cleavage, and bioorthogonal cleavage.

[0302] 67. The composition of embodiment 66, wherein the chemical cleavage is by Fe II-mediated beta-elimination of TRX.

[0303] 68. The composition of embodiment 66, wherein the enzymatic cleavage is by non-proteolytic sulfatase, β-galactosidase / glucuronidase, or pyrophosphate phosphatase.

[0304] 69. The composition of embodiment 66, wherein the bioorthogonal cleavage is by CuI-BTTAA or free copper ion mediated cleavage.

[0305] 70. The composition of any one of embodiments 51-69, wherein the linker is conjugated to a lysine residue, a cysteine ​​residue, a histidine residue, or a non-natural amino acid residue in the targeting molecule.

[0306] 71. The composition according to any one of embodiments 51 to 70, wherein the linker is conjugated to the targeting molecule by chemical or enzymatic conjugation.

[0307] 72. The composition of embodiment 71, wherein the chemical conjugation comprises acylation and click chemistry.

[0308] 73. The composition of embodiment 71, wherein the enzymatic conjugation is mediated by a sortase or transferase enzyme.

[0309] 74. The composition of any one of embodiments 29 to 73, wherein each targeting molecule is conjugated to 1 to 8 polynucleotide molecules (DAR of 1 to 8).

[0310] 75. The composition of embodiment 74, wherein each targeting molecule is conjugated to one polynucleotide molecule (DAR1).

[0311] 76. The composition of embodiment 74, wherein each targeting molecule is conjugated to two polynucleotide molecules (DAR2).

[0312] 77. The composition of embodiment 74, wherein each targeting molecule is conjugated to three polynucleotide molecules (DAR3).

[0313] 78. The composition of embodiment 74, wherein each targeting molecule is conjugated to four polynucleotide molecules (DAR4).

[0314] 79. The composition of embodiment 74, wherein each targeting molecule is conjugated to five polynucleotide molecules (DAR5).

[0315] 80. The composition of embodiment 74, wherein each targeting molecule is conjugated to six polynucleotide molecules (DAR6).

[0316] 81. The composition of embodiment 74, wherein each targeting molecule is conjugated to seven polynucleotide molecules (DAR7).

[0317] 82. The composition of embodiment 74, wherein each targeting molecule is conjugated to eight polynucleotide molecules (DAR8).

[0318] 83. The composition of any one of embodiments 29 to 82, wherein the polynucleotide conjugate targeting molecule has a molecular weight of more than 30 kDa.

[0319] 84. The composition of embodiment 83, wherein the polynucleotide conjugate targeting molecule has a molecular weight of more than 40 kDa.

[0320] 85. The composition of embodiment 84, wherein the polynucleotide conjugate targeting molecule has a molecular weight of more than 50 kDa.

[0321] 86. The composition described in embodiment 85, wherein the polynucleotide conjugate targeting molecule has a molecular weight of more than 60 kDa.

[0322] 87. A composition described in any one of embodiments 29 to 86, wherein the polynucleotide conjugate targeting molecule has a molecular weight of 7,500 kDa or less.

[0323] 88. The polynucleotide conjugate is selected from the group consisting of cetuximab-DBCO-C9-M30m3 (DAR3), cetuximab-DBCO-C4 / P5-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR2), cetuximab-DBCO-PEG9-M30m3 (DAR4), cetuximab-DBCO-PEG9-M30m3 (DAR6), cetuximab-linear-PEG13-M30m3 (DAR4 ... Mab-PEG4-azide-DBCO-PEG5-M30m3 (DAR1), Cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2), Cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4), Cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2.5), Cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4.5), Cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR6.5), cetuximab-SMCC-M30m3(DAR4)(SMCC), cetuximab-MCVCPABcPNP-M30m3(DAR4)(MCVCPABcPNP), cetuximab-MCPEG4VCPABcPNP-M30m3(DAR4)(MCPEG4VCPABcPNP), cetuximab-C4-azide-DBCO-C5-M30m3, cetuximab-PEG4-azide-DBCO-PEG4-m30m3(DAR4), cetuximab-MC-PEG4-ValCit-PABc-M30m3(DAR1), cetuximab-MC-PEG4-ValCit-PABc-M30m3(DAR2), cetuximab-MC-PEG4-ValCit-PABc-M30m3(DAR3), 30. The composition of embodiment 29, wherein the ASO-carbon 4-DBCO-carbon 5-3tf12 (DAR1), Cetuximab-MC-PEG4-ValCit-PABc-M30m3 (DAR4), 3tf12-DBCO-PEG8-NCD5 (DAR1), 3tf12-DBCO-PEG8-M30m3 (DAR1), Fv55-SMCC-M30m3 (DAR1), Fv55-PEG8-DBCO-M30m3 (DAR1), Fv55-PEG8-DBCO-M30m3 (DAR2), Fv55-Linker-M30m3 (DAR2), Fv55-DBCO-PEG8-M30m1 (DAR1), Fv55-DBCO-PEG8-M30m1 (DAR2), and ASO-carbon 4-DBCO-carbon 5-3tf12 (DAR1).

[0324] 89. The composition of embodiment 29, wherein the polynucleotide conjugate is selected from the group consisting of antibody-polynucleotide conjugates disclosed in Table 5 or Table 6.

[0325] 90. The composition comprises: (a) cetuximab-DBCO-C9-M30m3 (DAR3) and PEG12-poly-(D-Arg)12, (b) cetuximab-DBCO-C4 / P5-M30m3 (DAR3) and PEG12-poly-(D-Arg)12, (c) cetuximab-DBCO-PEG9-M30m3 (DAR3) and PEG12-poly-(D-Arg)12, (d) cetuximab-DBCO-PEG9-M30m3 (DAR2) and (e) cetuximab-DBCO-PEG9-M30m3 (DAR4) and PEG12-poly-(D-Arg)12, (f) cetuximab-DBCO-PEG9-M30m3 (DAR6) and PEG12-poly-(D-Arg)12, (g) cetuximab-linear-PEG13-M30m3 (DAR4) and PEG12-poly-(D-Arg)12, (h) 3tf12-DBC O-PEG8-NCD5 and poly(L-Arg)9, (i) 3tf12-DBCO-PEG8-M30m3 and poly(L-Arg)9, (j) Fv55-SMCC-M30m3 and PEG12-poly(L-Arg)12, (k) Fv55-PEG30-M30m3 and PEG12-poly(L-Arg)12, (l) Cetuximab-PEG4-azide-DBCO-PEG5-M30m3(DAR2) and PEG12Po lyArg12{d}, (m) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2) and PolyArg12Cbp3.9kDa, (n) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PEG12PolyArg12{d}, (o) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12Cbp3.9 kDa, (p) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-PEG2000Da, (q) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-PEG5000Da, (r) cetuximab-PEG4-azide-DBCO-PEG5-M30m 3 (DAR4) and PolyArg12C-dextran 5000Da, (s) cetuximab-SMCC-M30m3 (DAR4) and PEG12PolyArg12{d}, (t) cetuximab-MCVCPABcPNP-M30m3 (DAR4) and PEG12PolyArg12{d}, (u) cetuximab-MCPEG4VCPABcPNP-M30m3 (D 3. The composition of embodiment 1, comprising: (a) cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DAR4) and PEG12PolyArg12{d}, (b) cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DAR5.5) and PEG12PolyArg12{d}, (c) cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DAR6.5) and PEG12PolyArg12{d}, (d) cetuximab-C4(azido-DBCO)C5-M30m3 and PEG12PolyArg12, or (e) any of the antibody-polynucleotide conjugate and hybrid polymer combinations disclosed in Table 5.

[0326] 91. A polynucleotide conjugate comprising a polynucleotide conjugated to a targeting molecule.

[0327] 92. The polynucleotide conjugate of embodiment 91, wherein the targeting molecule is an antibody or an antigen-binding fragment thereof, or a binding protein.

[0328] 93. The polynucleotide conjugate of embodiment 92, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, Fab-Fc, Fv, single-chain Fv (scFv), a diabody, a minibody, a vNAR, a centrin, and a Nanobody® molecule.

[0329] 94. The polynucleotide conjugate of embodiment 93, wherein the bispecific antibody is a bispecific T cell engager (BiTE) or a dual affinity retargeting antibody (DART).

[0330] 95. The polynucleotide conjugate of embodiment 93, wherein the Nanobody® is Nanobody-HSA®.

[0331] 96. The polynucleotide conjugate of any one of embodiments 92 to 95, wherein the antibody or antigen-binding fragment thereof is an IgG molecule or is derived from an IgG molecule.

[0332] 97. The polynucleotide conjugate according to embodiment 96, wherein the IgG molecule is an IgG1 or IgG4 molecule.

[0333] 98. The polynucleotide conjugate according to embodiment 92, wherein the binding protein is a soluble receptor or a soluble ligand.

[0334] 99. The polynucleotide conjugate of embodiment 98, wherein the soluble receptor comprises the extracellular domain of a receptor.

[0335] 100. The polynucleotide conjugate of embodiment 98 or 99, wherein the soluble receptor is an Fc fusion protein.

[0336] 101. A polynucleotide conjugate according to any one of embodiments 91 to 100, wherein the targeting molecule is a therapeutically or biologically active molecule.

[0337] 102. The polynucleotide conjugate according to any one of embodiments 91 to 101, wherein the polynucleotide is selected from the group consisting of siRNA, ncRNA mimic, short hairpin RNA (shRNA), dicer-dependent siRNA (di-siRNA), antisense oligonucleotide (ASO), gapmer, mixmer, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), RNA activating oligonucleotide (RNAa), aptamer, exon skipping oligonucleotide, miRNA, miRNA mimic, mRNA, and guide RNA.

[0338] 103. The polynucleotide conjugate of embodiment 102, wherein the polynucleotide is an miRNA mimic.

[0339] 104. The polynucleotide conjugate of embodiment 103, wherein the miRNA mimic mimics miR-30.

[0340] 105. The polynucleotide conjugate of embodiment 104, wherein the polynucleotide is an miRNA mimic selected from the group consisting of M30m1, M30m2, M30m3, and M30m4.

[0341] 106. The polynucleotide conjugate of embodiment 105, wherein the polynucleotide is M30m3.

[0342] 107. The polynucleotide conjugate of embodiment 102, wherein the polynucleotide is an ASO.

[0343] 108. The polynucleotide conjugate of embodiment 107, wherein the ASO is a DUX4-targeting ASO.

[0344] 109. The polynucleotide conjugate of embodiment 108, wherein the DUX4-targeting ASO is selected from the group consisting of the DUX4-targeting ASOs disclosed in Table 4.

[0345] 110. The polynucleotide conjugate of embodiment 109, wherein the DUX4-targeting ASO is selected from the group consisting of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32.

[0346] 111. A polynucleotide conjugate according to any one of embodiments 101 to 110, wherein the targeting molecule and the polynucleotide provide a synergistic therapeutic or biological effect.

[0347] 112. The polynucleotide conjugate according to any one of embodiments 91 to 111, wherein the polynucleotide is directly conjugated to the targeting molecule.

[0348] 113. The composition of any one of embodiments 91 to 111, wherein the polynucleotide is conjugated to the targeting molecule via a linker.

[0349] 114. The polynucleotide conjugate according to embodiment 113, wherein the linker is a hydrophobic linker.

[0350] 115. The polynucleotide conjugate according to embodiment 113, wherein the linker is a peptide linker.

[0351] 116. The polynucleotide conjugate according to embodiment 113, wherein the linker is a chemical linker.

[0352] 117. The polynucleotide conjugate according to embodiment 116, wherein the chemical linker is a polymer linker.

[0353] 118. The polynucleotide conjugate according to embodiment 116 or 117, ...

Claims

1. A composition comprising a hybrid polymer and a polynucleotide, said hybrid polymer comprising a cationic portion and a neutral portion.

2. The composition of claim 1 , wherein the cationic portion of the hybrid polymer interacts with the polynucleotide via a non-covalent interaction (e.g., an ionic interaction).

3. The composition of claim 1 or 2, wherein the cationic portion of the hybrid polymer is a cationic polypeptide, such as a poly-arginine polypeptide or a poly-lysine polypeptide.

4. The composition of claim 3 , wherein the cationic polypeptide comprises L-amino acid residues, D-amino acid residues, or both.

5. The composition of claim 3 or 4, wherein the cationic polypeptide comprises 9 to 18 amino acid residues, for example 12 amino acid residues.

6. The composition of claim 3 , wherein the cationic polypeptide is selected from the group of cationic polypeptides disclosed in Table 1.

7. The composition of claim 1 or 2, wherein the cationic portion of the hybrid polymer comprises a cationic polymer having a size of about 600 to about 2000 Daltons.

8. 8. The composition of claim 7, wherein the cationic polymer is selected from the group consisting of gelatin, glucosamine, N-acetylglucosamine, chitosan, cationic dextran, cationic cyclodextrin, cationic cellulose, polyethyleneimine (PEI), polyamidoamine (PAA), poly(amino-co-ester) (PAE), poly[2-(N,N-dimethylamino)ethyl methacrylate] (PDMAEMA), or cationic lipids such as DOTAP (N-(1-(2,3-dioleyloxy)propyl)-N,N,N trimethylammonium) chloride, poly[N,N-diethylaminoethyl methacrylate] (PDEAEMA), cationic mucic acid polymer (cMAP), and DOPE (dioleylphosphatidylethanolamine).

9. The composition of any one of claims 1 to 8, wherein the neutral portion of the hybrid polymer comprises a polymer having a size of about 100 to about 1000 Daltons.

10. The composition of claim 9, wherein the neutral portion of the hybrid polymer comprises poly(ethylene glycol) (PEG), such as a PEG12-PEG24 polymer.

11. The composition of any one of claims 1 to 6 and 9 to 10, wherein the hybrid polymer is a PEGylated cationic polypeptide.

12. The composition according to any one of claims 1 to 11, wherein the hybrid polymer and the polynucleotide do not form aggregates or nanoparticles.

13. The composition of any one of claims 1 to 12, wherein the charge ratio of the cationic polypeptide to the polynucleotide is from 0.25:1 to 5:1, such as from 1:1 to 2:

1.

14. The hybrid polymers are PEG12PolyArg12{d}, PolyArg6, PEG12PolyArg6, PEG12PolyArg6C, PEG24PolyArg12C, PEG24PolyArg12, PEG24PolyArg9, PolyArg12C-PEG2000Da, PolyArg12C-PEG5000Da, PolyArg12C-Dextran5000Da, PEG12PolyArg12, PEG12PolyArg 9d, PEG1000DaPolyArg12, PEG2000DaPolyArg12, PEG5000DaPolyArg12, PolyArg12Cbp1.5kDa, PolyArg12Cbp3.9kDa, PolyArg12Cbp16kDa, CPolyArg12Cbp1.5kDa, PolyArg12Cbp2kDa, PolyArg12bp2kDa, amide dextran, lysine dextran, spermine, agmatine, arginine, PEG PEI 15kda, BPEI-G-PEG550, and BPEI-G-PEG5000.

15. The composition of any one of claims 1 to 14, wherein the polynucleotide is conjugated to a targeting molecule such as an antibody or antigen-binding fragment thereof, or a binding protein (e.g. a soluble receptor or soluble ligand).

16. 16. The composition of claim 15, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, a Fab-Fc, an Fv, a single chain Fv (scFv), a diabody, a minibody, and a Nanobody® molecule.

17. The composition of claim 15 or 16, wherein the targeting molecule is a therapeutically or biologically active molecule.

18. 18. The composition of any one of claims 1 to 17, wherein the polynucleotide is selected from the group consisting of siRNA, ncRNA mimics, short hairpin RNA (shRNA), dicer-dependent siRNA (di-siRNA), antisense oligonucleotides (ASO), gapmers, mixmers, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), RNA activating oligonucleotides (RNAa), aptamers, exon skipping oligonucleotides, miRNA, miRNA mimics, mRNA, and guide RNA.

19. The composition of claim 18, wherein the polynucleotide is a miRNA mimic such as a miR-30 mimic, e.g., M30m1, M30m2, M30m3 and M30m4.

20. The composition of claim 18, wherein the polynucleotide is a DUX4-targeting ASO, such as an ASO of ASDX2, ASDX4, ASDX23, ASDX26, or ASDX32.

21. The composition of any one of claims 17 to 20, wherein the targeting molecule and the polynucleotide provide a synergistic therapeutic or biological effect.

22. The composition of any one of claims 15 to 21, wherein the polynucleotide is directly conjugated to the targeting molecule.

23. 22. The composition of any one of claims 15 to 21, wherein the polynucleotide is conjugated to the targeting molecule via a linker, such as a hydrophobic linker, a peptide linker, or a chemical linker, including polymeric, linear, and cyclic chemical linkers.

24. 24. The composition of claim 23, wherein the polymer linker comprises PEG, a sugar, a fatty acid, a phosphate, pyrophosphate, or polysarcosine.

25. The composition of claim 23 or 24, wherein the linker is non-cleavable.

26. 25. The composition of claim 23 or 24, wherein the linker is cleavable, such as a disulfide linker, a self-immolative peptide polymer hybrid, or a sulfatase-promoted aryl sulfate linker.

27. The composition of any one of claims 23 to 26, wherein the linker is conjugated to a lysine residue, a cysteine ​​residue, a histidine residue, or a non-natural amino acid residue in the targeting molecule.

28. The composition of any one of claims 15 to 27, wherein each targeting molecule is conjugated to 1 to 8 polynucleotide molecules (1 to 8 DAR).

29. The polynucleotide conjugates include cetuximab-DBCO-C9-M30m3 (DAR3), cetuximab-DBCO-C4 / P5-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR2), cetuximab-DBCO-PEG9-M30m3 (DAR4), cetuximab-DBCO-PEG9-M30m3 (DAR6), cetuximab-linear-PEG13-M30m3 (DAR4), cetuximab-PEG4-azido-DBCO-PEG5 -M30m3 (DAR1), cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DAR2), cetuximab-PEG 4-azido-DBCO-PEG5-M30m3 (DAR4), cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DA R2.5), cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DAR4.5), cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DAR6.5), cetuximab-SMCC-M30m3 (DAR4) (SMCC), cetuximab- MCVCPABcPNP-M30m3 (DAR4) (MCVCPABcPNP), cetuximab-MCPEG4VCPABcPNP-M30m3 (DAR4) (MCPEG4VCPABcPNP), cetuximab-C4-azide-DBCO-C5-M30m3, cetuximab-PEG4-azide-DBCO-PEG4-m30m3 (DAR4), cetuximab-MC-PEG4-ValCit-PABc-M30m3 (DAR1), cetuximab-MC-PEG4-ValCit-PABc-M30m3 (DAR2), cetuximab-MC-PE G4-ValCit-PABc-M30m3 (DAR3), cetuximab-MC-PEG4-ValCit-PABc-M30m3 (DAR3) AR4), 3tf12-DBCO-PEG8-NCD5 (DAR1), 3tf12-DBCO-PEG8-M30m3 (DAR1), Fv 55-SMCC-M30m3 (DAR1), Fv55-PEG8-DBCO-M30m3 (DAR1), Fv55-PEG8-DBCO -M30m3 (DAR2), Fv55-linker-M30m3 (DAR2), Fv55-DBCO-PEG8-M30m1 (DAR1),The composition of claim 15, which is selected from the group consisting of Fv55-DBCO-PEG8-M30m1 (DAR2), and ASO-carbon4-DBCO-carbon5-3tf12 (DAR1).

30. The composition of claim 15, wherein the polynucleotide conjugate is selected from the group consisting of antibody-polynucleotide conjugates disclosed in Table 5 or Table 6.

31. The composition comprises: (a) cetuximab-DBCO-C9-M30m3 (DAR3) and PEG12-poly-(D-Arg)12, (b) cetuximab-DBCO-C4 / P5-M30m3 (DAR3) and PEG12-poly-(D-Arg)12, (c) cetuximab-DBCO-PEG9-M30m3 (DAR3) and PEG12-poly-(D-Arg)12, (d) cetuximab-DBCO-PEG9-M30m3 (DAR2) and PEG12-poly-(D-Arg)12, (e) cetuximab-DBCO-PEG9-M30m3 (DAR4) and PEG12-poly -(D-Arg)12, (f) cetuximab-DBCO-PEG9-M30m3 (DAR6) and PEG12-poly-(D-Arg)12, (g) cetuximab-linear-PEG13-M30m3 (DAR4) and PEG12-poly-(D-Arg)12, (h) 3tf12-DBCO-PEG8-NCD5 and poly(L-Arg)9, (i) 3tf12-DBCO-PEG8-M30m3 and poly(L-Arg)9, (j) Fv55-SMCC-M30m3 and PEG12-poly(L-Arg)12, (k) Fv55-PEG30-M30m3 and PE G12-Poly(L-Arg)12, (l) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2) and PEG12PolyArg12{d}, (m) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2) and PolyArg12Cbp3.9kDa, (n) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PEG12PolyArg12{d}, (o) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12 Cbp 3.9 kDa, (p) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-PEG2000 Da, (q) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-PEG5000 Da, (r) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR4) and PolyArg12C-dextran 5000 Da, (s) cetuximab-SMCC-M30m3 (DAR4) and PEG12PolyArg12{d},(t) cetuximab-MCVCPABcPNP-M30m3 (DAR4) and PEG12PolyArg12{d}, (u) cetuximab-MCPEG4VCPABcPNP-M30m3 (DAR4) and PEG12PolyArg12{d}, (v) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR2.5) and PEG12PolyArg12{d}, (w) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 ( 2. The composition of claim 1, comprising any of the following combinations of antibody-polynucleotide conjugates and hybrid polymers disclosed in Table 5: (i) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR 4.5) and PEG12PolyArg12{d}, (ii) cetuximab-PEG4-azide-DBCO-PEG5-M30m3 (DAR 6.5) and PEG12PolyArg12{d}, (iii) cetuximab-C4(azide-DBCO)C5-M30m3 and PEG12PolyArg12, or (iv) any of the combinations of antibody-polynucleotide conjugates and hybrid polymers disclosed in Table 5.

32. A polynucleotide conjugate comprising a polynucleotide conjugated to a targeting molecule.

33. 33. The polynucleotide conjugate of claim 32, wherein the targeting molecule is an antibody or an antigen-binding fragment thereof, or a binding protein (e.g., a soluble receptor or soluble ligand).

34. 34. The polynucleotide conjugate of claim 33, wherein the antibody or antigen-binding fragment thereof is selected from the group consisting of a monoclonal antibody, a bispecific antibody, an immunoglobulin single variable domain (ISV) such as a Fab, Fab-Fc, Fv, single chain Fv (scFv), a diabody, a minibody, a vNAR, a centirrin, and a Nanobody® molecule.

35. The polynucleotide conjugate of any one of claims 32 to 34, wherein the targeting molecule is a therapeutically or biologically active molecule.

36. 37. The polynucleotide conjugate of any one of claims 32 to 36, wherein the polynucleotide is selected from the group consisting of siRNA, ncRNA mimics, short hairpin RNA (shRNA), dicer-dependent siRNA (di-siRNA), antisense oligonucleotides (ASO), gapmers, mixmers, double-stranded RNA (dsRNA), single-stranded RNAi (ssRNAi), DNA-directed RNA interference (ddRNAi), RNA activating oligonucleotides (RNAa), aptamers, exon skipping oligonucleotides, miRNA, miRNA mimics, mRNA, and guide RNA.

37. 37. The polynucleotide conjugate of claim 36, wherein the polynucleotide is a miRNA mimic such as a miR-30 mimic, e.g., M30m1, M30m2, M30m3, and M30m4.

38. 37. The polynucleotide conjugate of claim 36, wherein the polynucleotide is a DUX4-targeting ASO, such as an ASO of ASDX2, ASDX4, ASDX23, ASDX26, and ASDX32.

39. The polynucleotide conjugate of any one of claims 32 to 38, wherein the targeting molecule and the polynucleotide provide a synergistic therapeutic or biological effect.

40. The polynucleotide conjugate of any one of claims 32 to 39, wherein the polynucleotide is directly conjugated to the targeting molecule.

41. The polynucleotide conjugate of any one of claims 32 to 39, wherein the polynucleotide is conjugated to the targeting molecule via a linker, such as a hydrophobic linker, a peptide linker, or a chemical linker, including polymeric, linear, and cyclic chemical linkers.

42. 42. The polynucleotide conjugate of claim 41, wherein the polymer linker comprises PEG, a sugar, a fatty acid, phosphate, pyrophosphate, or polysarcosine.

43. 43. The polynucleotide conjugate of claim 41 or 42, wherein the linker is non-cleavable.

44. 43. The polynucleotide conjugate of claim 41 or 42, wherein the linker is cleavable, such as a disulfide linker, a self-immolative peptide polymer hybrid, and a sulfatase-promoted aryl sulfate linker.

45. The polynucleotide conjugate of any one of claims 41 to 44, wherein the linker is conjugated to a lysine residue, a cysteine ​​residue, a histidine residue, or a non-natural amino acid residue in the targeting molecule.

46. The polynucleotide conjugate of any one of claims 32 to 45, wherein each targeting molecule is conjugated to one to eight polynucleotide molecules (DAR1-8).

47. The polynucleotide conjugates include cetuximab-DBCO-C9-M30m3 (DAR3), cetuximab-DBCO-C4 / P5-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR3), cetuximab-DBCO-PEG9-M30m3 (DAR2), cetuximab-DBCO-PEG9-M30m3 (DAR4), cetuximab-DBCO-PEG9-M30m3 (DAR6), cetuximab-linear-PEG13-M30m3 (DAR4), cetuximab-PEG4-azido-DBCO-PEG5 -M30m3 (DAR1), cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DAR2), cetuximab-PEG 4-azido-DBCO-PEG5-M30m3 (DAR4), cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DA R2.5), cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DAR4.5), cetuximab-PEG4-azido-DBCO-PEG5-M30m3 (DAR6.5), cetuximab-SMCC-M30m3 (DAR4) (SMCC), cetuximab- MCVCPABcPNP-M30m3 (DAR4) (MCVCPABcPNP), cetuximab-MCPEG4VCPABcPNP-M30m3 (DAR4) (MCPEG4VCPABcPNP), cetuximab-C4-azide-DBCO-C5-M30m3, cetuximab-PEG4-azide-DBCO-PEG4-m30m3 (DAR4), cetuximab-MC-PEG4-ValCit-PABc-M30m3 (DAR1), cetuximab-MC-PEG4-ValCit-PABc-M30m3 (DAR2), cetuximab-MC-PE G4-ValCit-PABc-M30m3 (DAR3), cetuximab-MC-PEG4-ValCit-PABc-M30m3 (DAR3) AR4), 3tf12-DBCO-PEG8-NCD5 (DAR1), 3tf12-DBCO-PEG8-M30m3 (DAR1), Fv 55-SMCC-M30m3 (DAR1), Fv55-PEG8-DBCO-M30m3 (DAR1), Fv55-PEG8-DBCO -M30m3 (DAR2), Fv55-linker-M30m3 (DAR2), Fv55-DBCO-PEG8-M30m1 (DAR1),The polynucleotide conjugate of claim 32, which is selected from the group consisting of Fv55-DBCO-PEG8-M30m1 (DAR2), and ASO-carbon4-DBCO-carbon5-3tf12 (DAR1).

48. The polynucleotide conjugate of claim 32, wherein the polynucleotide conjugate is selected from the group consisting of antibody-polynucleotide conjugates disclosed in Table 5 or Table 6.

49. 11. A method of treating a genetic disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of a composition of any one of claims 1 to 31 or a polynucleotide conjugate of any one of claims 32 to 48.

50. 50. The method of claim 49, wherein the genetic disease is selected from the group consisting of a viral infection, cancer, and a neuromuscular disease.

51. 51. The method of claim 50, wherein the neuromuscular disease is a muscular dystrophy, such as facioscapulohumeral muscular dystrophy (FSHD) or Duchenne muscular dystrophy.

52. 1. An antibody or antigen-binding fragment thereof that specifically binds to the human transferrin receptor (TfR1), the antibody or antigen-binding fragment thereof having the amino acid sequence: QVQVQDSGGELVQPGGSLRVSCKASGFNIKDSYMHWVRQAPGKGLEWVAFIDPETGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGSIYWYFDVWGK and a light chain variable region (VL) comprising the amino acid sequence DIQMTQSPSSLSASVGQRVTITCRASQSLLNSSNQKNSLGWYQQKPGKAPKLLIYFASTRQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHYSTPLTFGQGTKVDIKRC.

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