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 an oligonucleotide that induces dystrophin (DMD) exon skipping, wherein the antibody comprises a heavy chain variable region (VH) comprising SEQ ID NO: 79 and a light chain variable region (VL) comprising SEQ ID NO: 80.
2 . 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.
3 . The complex of claim 2 , wherein the antibody is a Fab fragment.
4 . The complex of claim 3 , wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103; and a light chain comprising the amino acid sequence of SEQ ID NO: 95.
5 . The complex of claim 1 , wherein the oligonucleotide induces skipping of exon 8, exon 23, exon 35, exon 43, exon 44, exon 45, exon 46, exon 50, exon 51, exon 52, exon 53, and/or exon 55 of DMD.
6 . The complex of claim 1 , wherein the heavy chain of the antibody comprises an N-terminal pyroglutamate.
7 . The complex of claim 1 , wherein the oligonucleotide is 15-35 nucleotides in length.
8 . The complex of claim 1 , wherein the oligonucleotide comprises a region of complementarity to a dystrophin RNA, wherein the region of complementarity is 12-35 nucleotides in length.
9 . The complex of claim 1 , wherein the oligonucleotide comprises a region of complementarity to the target sequence of an oligonucleotide as set forth in any one of SEQ ID NOs: 131, and 151-401, wherein the region of complementarity is 12-35 nucleotides in length.
10 . The complex of claim 1 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 131, and 151-401, wherein any one or more of the uracil bases (U's) in the oligonucleotide may optionally be a thymine base (T).
11 . The complex of claim 1 , wherein the oligonucleotide comprises at least one modified internucleoside linkage.
12 . The complex of claim 11 , wherein the at least one modified internucleoside linkage is a phosphorothioate linkage.
13 . The complex of claim 1 , wherein the oligonucleotide comprises one or more modified nucleosides.
14 . The complex of claim 13 , wherein the one or more modified nucleosides are 2′-modified nucleosides.
15 . The complex of claim 1 , wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer.
16 . The complex of claim 1 , wherein the antibody is covalently linked to the oligonucleotide via a cleavable linker comprising a valine-citrulline sequence.
17 . The complex of claim 1 , wherein the antibody is covalently linked to the oligonucleotide via conjugation to a lysine residue or a cysteine residue of the antibody.
18 . 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 oligonucleotide to the cell.
19 . 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 .