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 .- 72 . (canceled)
73 . A complex comprising an anti-transferrin receptor antibody covalently linked to an oligonucleotide that targets a dystrophin (DMD) pre-mRNA in a muscle cell,
wherein the oligonucleotide is 15 to 35 nucleotides in length and comprises a region of complementarity to the DMD pre-mRNA, wherein the region of complementarity is at least 12 nucleotides in length; and
wherein the anti-transferrin receptor antibody binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1, permits transferrin binding to TfR1, and cross-reacts with human TfR1 and cynomolgus TfR1.
74 . The complex of claim 73 , wherein the anti-transferrin receptor antibody is in the form of a ScFv, Fab fragment, Fab′ fragment, F(ab′)2 fragment, or Fv fragment.
75 . The complex of claim 73 , wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.
76 . The complex of claim 73 , wherein the anti-transferrin receptor antibody binds human TfR1 with a K D of 10 −11 M to 10 −6 M.
77 . The complex of claim 73 , wherein the anti-transferrin receptor antibody is a humanized antibody.
78 . The complex of claim 73 , wherein the oligonucleotide comprises one or more phosphorodiamidate morpholinos.
79 . The complex of claim 73 , wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).
80 . The complex of claim 73 , wherein the oligonucleotide is 16-30 nucleotides in length.
81 . The complex of claim 73 , wherein the region of complementarity is at least 16 nucleotides in length.
82 . The complex of claim 73 , wherein the oligonucleotide comprises a region of complementarity that is at least 12 nucleotides in length and is complementary to the target sequence of an oligonucleotide listed in Table 2.
83 . The complex of claim 73 , wherein the oligonucleotide comprises at least 16 consecutive nucleotides of an oligonucleotide listed in Table 2.
84 . The complex of claim 73 , wherein the anti-transferrin receptor antibody is covalently linked to the oligonucleotide via a cleavable linker comprising a valine-citrulline sequence.
85 . The complex of claim 73 , wherein the anti-transferrin receptor antibody is covalently linked to the oligonucleotide via conjugation to a lysine residue or a cysteine residue of the anti-transferrin receptor antibody.
86 . The complex of claim 73 , wherein the complex is configured to promote transferrin receptor mediated internalization of the oligonucleotide into a muscle cell.
87 . The complex of claim 73 , wherein the DMD pre-mRNA comprises one or more frameshift mutations.
88 . The complex of claim 87 , wherein the frameshift mutation is in exon 8, exon 23, exon 41, exon 44, exon 50, exon 51, exon 52, exon 53, or exon 55.
89 . The complex of claim 86 , wherein internalization of the complex in a muscle cell induces skipping of exon 44, exon 45, exon 51, exon 8, exon 23, exon 50, exon 52, exon 53, or exon 55 of DMD pre-mRNA in the muscle cell.
90 . The complex of claim 89 , wherein internalization of the complex in a muscle cell promotes the expression or activity of a functional dystrophin protein.
91 . A method of inducing skipping of an exon in a dystrophin (DMD) pre-mRNA in a muscle cell, the method comprising contacting the muscle cell with a complex comprising an anti-transferrin receptor antibody covalently linked to an oligonucleotide that targets the DMD pre-mRNA,
wherein the oligonucleotide is 15 to 35 nucleotides in length and comprises a region of complementarity to the DMD pre-mRNA, wherein the region of complementarity is at least 12 nucleotides in length; and
wherein the anti-transferrin receptor antibody binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1, permits transferrin binding to TfR1, and cross-reacts with human TfR1 and cynomolgus TfR1.
92 . A method of treating Duchenne Muscular Dystrophy (DMD) in a subject expressing a DMD pre-mRNA comprising one or more frameshift mutations, the method comprising administering to the subject a complex comprising an anti-transferrin receptor antibody covalently linked to an oligonucleotide that targets the DMD pre-mRNA in a muscle cell,
wherein the oligonucleotide is 15 to 35 nucleotides in length and comprises a region of complementarity to the DMD pre-mRNA, wherein the region of complementarity is at least 12 nucleotides in length; and
wherein the anti-transferrin receptor antibody binds in the range of C89 to F760 of human transferrin receptor protein 1 (TfR1) having an amino acid sequence as set forth in SEQ ID NO: 1, permits transferrin binding to TfR1, and cross-reacts with human TfR1 and cynomolgus TfR1.