MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING DYSTROPHINOPATHIES
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 promotes the expression or activity of a functional dystrophin protein. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide, e.g., an oligonucleotide that causes exon skipping in a mRNA expressed from a mutant DMD allele.
1 . A complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured for inducing skipping of exon 55 in a DMD pre-mRNA, wherein the oligonucleotide comprises a region of complementarity that is complementary with at least 8 consecutive nucleotides of any one of SEQ ID NOs: 160-779.
2 .- 4 . (canceled)
5 . The complex of claim 1 , wherein the anti-TfR1 antibody is a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, an scFv, an Fv, or a full-length IgG.
6 . The complex of claim 5 , wherein the anti-TfR1 antibody is a Fab fragment.
7 .- 8 . (canceled)
9 . The complex of claim 1 , wherein the anti-TfR1 antibody does not specifically bind to the transferrin binding site of the transferrin receptor 1 and/or wherein the anti-TfR1 antibody does not inhibit binding of transferrin to the transferrin receptor 1.
10 . The complex of claim 1 , wherein the oligonucleotide comprises a region of complementarity to at least 4 consecutive nucleotides of a splicing feature of the DMD pre-mRNA.
11 . The complex of claim 10 , wherein the splicing feature is an exonic splicing enhancer (ESE) in exon 55 of the DMD pre-mRNA, optionally wherein the ESE comprises a sequence of any one of SEQ ID NOs: 2031-2061.
12 . The complex of claim 10 , wherein the splicing feature is a branch point, a splice donor site, or a splice acceptor site, optionally wherein the splicing feature is across the junction of exon 54 and intron 54, in intron 54, across the junction of intron 54 and exon 55, across the junction of exon 55 and intron 55, in intron 55, or across the junction of intron 55 and exon 56 of the DMD pre-mRNA, and further optionally wherein the splicing feature comprises a sequence of any one of SEQ ID NOs: 2028-2030, 2062, and 2063.
13 . The complex of claim 1 , wherein the oligonucleotide comprises a sequence complementary to any one of SEQ ID NOs: 160-779 or comprises a sequence of any one of SEQ ID NOs: 780-2019, wherein each thymine base (T) may independently and optionally be replaced with a uracil base (U), and each U may independently and optionally be replaced with a T.
14 . The complex of claim 1 , wherein the oligonucleotide comprises a sequence of any one of SEQ ID NOs: 1400, 1402-1406, 1408, 1409, 1413, 1418-1420, 1483-1491, 1493, 1495, 1496, 1502-1506, 1508, 1510-1512, 1514, 1522-1524, 1529-1531, 1534, 1535, 1559, 1583, 1587, 1591, 1596, 1597, 1598, 1604, 1606, 1607, 1638, 1641, 1693-1695, 1702, 1703, 1766, 1813, 1988, and 1995, wherein each thymine base (T) may independently and optionally be replaced with a uracil base (U), and each U may independently and optionally be replaced with a T.
15 . The complex of claim 1 , wherein the oligonucleotide comprises one or more phosphorodiamidate morpholinos, optionally wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
16 . The complex of claim 1 , wherein the anti-TfR1 antibody is covalently linked to the oligonucleotide via a cleavable linker, optionally wherein the cleavable linker comprises a valine-citrulline sequence.
17 . The complex of claim 1 , wherein the anti-TfR1 antibody is covalently linked to the oligonucleotide via conjugation to a lysine residue or a cysteine residue of the antibody.
18 . An oligonucleotide that targets DMD, wherein the oligonucleotide comprises a region of complementarity to any one of SEQ ID NOs: 160-779, optionally wherein the region of complementarity comprises at least 15 consecutive nucleosides complementary to any one of SEQ ID NOs: 160-779.
19 . The oligonucleotide of claim 18 , wherein the oligonucleotide comprises at least 15 consecutive nucleosides of any one of SEQ ID NOs: 780-2019, optionally wherein the oligonucleotide comprises a sequence of any one of SEQ ID NOs: 780-2019, wherein each thymine base (T) may independently and optionally be replaced with a uracil base (U), and each U may independently and optionally be replaced with a T.
20 . A method of delivering an oligonucleotide to a cell, the method comprising contacting the cell with the complex of claim 1 .
21 . A method of promoting the expression or activity of a dystrophin protein in a cell, the method comprising contacting the cell with the complex of claim 1 in an amount effective for promoting internalization of the oligonucleotide to the cell, optionally wherein the cell is a muscle cell.