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 (TfR) antibody covalently linked to a molecular payload configured for promoting the expression or activity of a DMD gene, wherein the 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 the amino acid sequence of SEQ ID NO: 77 and a VL comprising the 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, a scFv, a 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: 101; and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89.
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 muscle-targeting antibody does not inhibit binding of transferrin to the transferrin receptor.
8 . The complex claim 1 , wherein the molecular payload is an oligonucleotide.
9 . The complex of claim 8 , wherein the oligonucleotide promotes exon skipping in a DMD RNA.
10 . The complex of claim 9 , wherein the oligonucleotide promotes skipping of an exon of DMD in the range of exon 8 to exon 55.
11 . (canceled)
12 . The complex of claim 1 , wherein the oligonucleotide comprises a region of complementarity to one or more full or partial exonic splicing enhancers (ESE) of a DMD transcript.
13 . The complex of claim 8 , wherein the oligonucleotide comprises a region of complementarity to a target sequence comprising one or more full or partial ESEs as set forth in SEQ ID NOs: 402-436 and 2043-2238.
14 . The complex of claim 8 , wherein the oligonucleotide promotes skipping of exon 51.
15 . The complex of claim 8 , wherein the oligonucleotide is 20-30 nucleotides in length and comprises a region of complementarity to a target sequence comprising at least 4 consecutive nucleotides of an ESE as set forth in any one of SEQ ID NOs: 402-436.
16 . The complex of claim 8 , wherein the oligonucleotide comprises any one of SEQ ID NOs: 437-1241, or comprises a region of complementarity to any one of SEQ ID NOs: 1242-2046.
17 . The complex of claim 8 , wherein the oligonucleotide comprises a region of complementarity to a target sequence of an oligonucleotide listed in Table 14, optionally wherein the oligonucleotide comprises a sequence listed in Table 14, wherein any one or more of the uracil bases (U's) in the oligonucleotide may optionally be a thymine base (T).
18 .- 22 . (canceled)
23 . The complex of claim 8 , wherein the oligonucleotide comprises one or more phosphorodiamidate morpholinos, optionally wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
24 . The complex of claim 1 , wherein the antibody is covalently linked to the molecular payload via a cleavable linker, wherein the cleavable linker comprises a valine-citrulline sequence.
25 .- 26 . (canceled)
27 . A method of promoting the expression or activity of a DMD 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 molecular payload to the cell, optionally wherein the cell is a muscle cell.
28 . A method of treating a subject having a mutated DMD allele that is associated with a dystrophinopathy, the method comprising administering to the subject an effective amount of the complex of claim 1 .