MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY
Aspects of the disclosure relate to complexes comprising a muscle-targeting agent covalently linked to a molecular payload. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload inhibits expression or activity of a DMPK allele comprising a disease-associated-repeat. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or RNAi oligonucleotide.
1 . A complex comprising an anti-transferrin receptor (TfR) antibody covalently linked to a molecular payload configured for reducing expression or activity of DMPK,
wherein the anti-TfR antibody comprises:
a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 77; and/or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 78.
2 . The complex of claim 1 , wherein the antibody comprises:
a VH comprising an amino acid sequence of SEQ ID NO: 77 and a VL comprising an amino acid sequence of SEQ ID NO: 78.
3 . The complex of claim 1 , wherein the antibody is selected from the group consisting of a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, an scFv, an Fv, and a full-length IgG.
4 . The complex of claim 3 , wherein the antibody is a Fab fragment.
5 . The complex of claim 4 , wherein the antibody comprises:
a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 93.
6 . The complex of claim 4 , wherein the antibody comprises:
a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 93.
7 . The complex of claim 1 , wherein the antibody does not specifically bind to the transferrin binding site of the transferrin receptor and/or wherein the antibody does not inhibit binding of transferrin to the transferrin receptor.
8 . The complex of claim 1 , wherein the antibody is cross-reactive with extracellular epitopes of two or more of a human, non-human primate and rodent transferrin receptor.
9 . The complex of claim 1 , wherein the complex is configured to promote transferrin receptor mediated internalization of the molecular payload into a muscle cell.
10 . The complex of claim 1 , wherein the molecular payload is an oligonucleotide.
11 . The complex of claim 10 , wherein the oligonucleotide comprises at least 15 consecutive nucleotides of SEQ ID NOs: 148-383 and 621-638, wherein any one or more of the thymidine bases (T's) in the oligonucleotide may optionally be a uridine base (U) and/or any one or more of the U's may optionally be a T.
12 . The complex of claim 11 , wherein the oligonucleotide comprises a sequence comprising any one of SEQ ID NOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212, 215, 218, 222, 248, and 264, wherein any one or more of the U's in the oligonucleotide may optionally be a T.
13 . The complex of claim 10 , wherein the oligonucleotide comprises a region of complementarity to at least 15 consecutive nucleotides of any one of SEQ ID NO: 384-619.
14 . The complex of claim 10 , wherein the oligonucleotide mediates RNAse H-mediated cleavage of a DMPK mRNA transcript.
15 . The complex of claim 10 , wherein the oligonucleotide comprises a 5′-X-Y-Z-3′ formula, wherein X and Z are flanking regions comprising one or more 2′-modified nucleosides selected from the group consisting of: 2′-O-methyl, 2′-fluoro, 2′-O-methoxyethyl, and 2′,4′-bridged nucleosides, and wherein Y is a gap region and each nucleoside in Y is a 2′-deoxyribonucleoside.
16 . The complex of claim 10 , wherein the oligonucleotide comprises one or more phosphorothioate internucleoside linkages.
17 . The complex of claim 1 , wherein the antibody is covalently linked to the molecular payload via a cleavable linker; optionally wherein the cleavable linker comprises a valine-citrulline sequence; and optionally wherein the antibody is covalently linked to the molecular payload via conjugation to lysine residue or cysteine residue of the antibody.
18 .- 19 . (canceled)
20 . The complex of claim 1 , wherein reducing expression comprises reducing RNA levels of DMPK, optionally wherein the reduced RNA levels are in the nucleus of a cell, optionally wherein the cell is a muscle cell, and optionally wherein the DMPK is encoded from an allele comprising a disease-associated repeat.
21 . (canceled)
22 . A method of reducing DMPK expression in a cell, the method comprising contacting the cell with the complex of claim 1 in an effective amount for promoting internalization of the molecular payload in the cell, optionally wherein the cell is a muscle cell.
23 . A method of treating a subject having an expansion of a disease-associated-repeat of a DMPK allele that is associated with myotonic dystrophy, the method comprising administering to the subject an effective amount of the complex of claim 1 .
24 .- 25 . (canceled)