Polynucleotide conjugates comprising
Disclosed herein are polynucleic acid molecules, pharmaceutical compositions, and methods of use for antibody-PLN targeting oligonucleotide conjugates (AOC).
1 . A polynucleotide conjugate comprising an anti-transferrin receptor antibody or antigen-binding fragment thereof conjugated to a polynucleotide that mediates the downregulation of PLN mRNA expression levels in a cardiac muscle cell, wherein the polynucleotide hybridizes to a nucleic acid sequence at positions selected from 216-244 of a PLN mRNA comprising an Arg14del (R14del) mutation, wherein the polynucleotide is a double-stranded RNA molecule.
2 . The polynucleotide conjugate of claim 1 , wherein the polynucleotide is a double-stranded small interfering RNA (siRNA) comprising a guide strand and a passenger strand.
3 . The polynucleotide conjugate of claim 2 , wherein the passenger strand comprises a nucleic acid sequence having at least 16, 17, 18, 19, 20, or 21 consecutive nucleotides from a sequence selected from SEQ ID NOs: 316-324 and 346-354.
4 . The polynucleotide conjugate of claim 2 , wherein the guide strand comprises a nucleic acid sequence having at least 16, 17, 18, 19, 20, or 21 consecutive nucleotides from a sequence selected from SEQ ID NOs: 302-310 and 330-338.
5 . The polynucleotide conjugate of claim 3 , wherein the passenger strand comprises a nucleic acid sequence selected from SEQ ID NOs: 316-324 and 346-354.
6 . The polynucleotide conjugate of claim 4 , wherein the guide strand comprises a nucleic acid sequence selected from SEQ ID NOs: 302-310 and 330-338.
7 . The polynucleotide conjugate of claim 1 , wherein the polynucleotide comprises at least one 2′-modified nucleotide, at least one modified internucleotide linkage, or at least one inverted abasic moiety.
8 . The polynucleotide conjugate of claim 7 , wherein the at least one 2′-modified nucleotide comprises 2′-O-methyl, 2′-O-methoxyethyl(2′-O-MOE), 2′-O-aminopropyl, 2′-deoxy, 2′-deoxy-2′-fluoro, 2′-O-aminopropyl(2′-O-AP), 2′-O-dimethylaminoethyl(2′-O-DMAOE), 2′-O-dimethylaminopropyl(2′-O-DMAP), 2′-O-dimethylaminoethyloxyethyl(2′-O-DMAEOE), 2′—O—N-methylacetamido (2′-O-NMA) modified nucleotide, locked nucleic acid (LNA), ethylene nucleic acid (ENA), or a combination thereof.
9 . The polynucleotide conjugate of claim 7 , wherein the at least one modified internucleotide linkage comprises a phosphorothioate linkage or a phosphorodithioate linkage.
10 . The polynucleotide conjugate of claim 1 , wherein the polynucleotide comprises a 5′-terminal vinylphosphonate-modified nucleotide.
11 . The polynucleotide conjugate of claim 1 , wherein the anti-transferrin receptor antibody or antigen binding fragment thereof comprises a non-human antibody or antigen binding fragment thereof, a human antibody or antigen binding fragment thereof, a humanized antibody or antigen binding fragment thereof, a chimeric antibody or antigen binding fragment thereof, a monoclonal antibody or antigen binding fragment thereof, a monovalent Fab′, a divalent Fab2, a single-chain variable fragment (scFv), a diabody, a minibody, a nanobody, a single-domain antibody (sdAb), or a camelid antibody or antigen binding fragment thereof.
12 . The polynucleotide conjugate of claim 1 , wherein the polynucleotide conjugate has a polynucleotide to antibody ratio of from about 1 to about 4.
13 . The polynucleotide conjugate of claim 1 , wherein polynucleotide conjugate comprises a linker connecting the anti-transferrin receptor antibody or antigen-binding fragment thereof to the polynucleotide.
14 . A polynucleotide molecule for modulating PLN mRNA expression levels, wherein the polynucleotide molecule comprises a nucleic acid sequence selected from SEQ ID NOs: 302-310 and 330-338, and wherein the polynucleotide molecule is a double-stranded RNA molecule.
15 . The polynucleotide molecule of claim 14 , wherein the polynucleotide molecule preferentially downregulates an expression level of a mutated PLN mRNA, relative to a wild-type PLN mRNA.
16 . A conjugate comprising an anti-transferrin receptor antibody or antigen-binding fragment thereof conjugated to a polynucleotide molecule that decreases PLN mRNA expression levels in a cardiac muscle cell, wherein the anti-transferrin receptor antibody or antigen-binding fragment thereof comprises a VH sequence of SEQ ID NO: 376, 377, 378, 379, or 380, and a VL sequence of SEQ ID NOs: 381, 382, 383, 384, or 385, and wherein the polynucleotide molecule comprises a guide strand having a nucleic acid sequence selected from SEQ ID NOs: 302-310 and 330-338 and a passenger strand having a nucleic acid sequence selected from SEQ ID NOs: 316-324 and 346-354.
17 . The conjugate of claim 16 , wherein the passenger strand or the guide strand comprises a 5′-terminal vinylphosphonate-modified nucleotide.
18 . The conjugate of claim 16 , wherein the conjugate comprises a linker connecting the anti-transferrin receptor antibody or antigen-binding fragment thereof to the polynucleotide molecule.
19 . The conjugate of claim 18 , wherein the linker comprises a maleimide group.
20 . The conjugate of claim 16 , wherein the polynucleotide molecule is selected from the group consisting of the guide strand sequence of SEQ ID NO: 302 and the passenger strand sequence of SEQ ID NO: 316; the guide strand sequence of SEQ ID NO: 303 and the passenger strand sequence of SEQ ID NO: 317; the guide strand sequence of SEQ ID NO: 304 and the passenger strand sequence of SEQ ID NO: 318; the guide strand sequence of SEQ ID NO: 331 and the passenger sequence of SEQ ID NO: 347; and the guide strand sequence of SEQ ID NO: 332 and the passenger strand sequence of SEQ ID NO: 348.
21 . A method of treating a cardiomyopathy associated with PLN mutation in a subject in need thereof, comprising administering to said subject a polynucleotide conjugate comprising an anti-transferrin receptor antibody or antigen-binding fragment thereof conjugated to a polynucleotide that mediates the downregulation of PLN mRNA expression levels in a cardiac muscle cell, wherein the polynucleotide hybridizes to a nucleic acid sequence at positions selected from 216-244 of a PLN mRNA, thereby treating cardiomyopathy in said subject, wherein the PLN mutation is an Arg14del (R14del) mutation, and wherein the polynucleotide molecule is a double-stranded RNA molecule.
22 . The method of claim 21 , wherein the cardiomyopathy associated with PLN mutation is a dilated cardiomyopathy or a hypertrophic cardiomyopathy.
23 . The method of claim 22 , wherein the dilated cardiomyopathy is a genetic dilated cardiomyopathy associated with one or more mutations selected from TTN, LMNA, RI3M20, SCN5A, MYH7, TNNT2, and TPMI mutations.
24 . The method of claim 22 , wherein the hypertrophic cardiomyopathy is associated with one or more mutations selected from MYH7, MYBPC3, TNNT2, TNNC, and TPM1 mutations.