MUSCLE-TARGETING COMPLEXES AND USES THEREOF
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 activity of a disease allele associated with muscle disease. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or RNAi oligonucleotide.
1 .- 91 . (canceled)
92 . A method of delivering an oligonucleotide to a muscle cell in a subject, the method comprising administering to the subject a muscle-targeting complex that comprise an anti-transferrin receptor antibody covalently linked to an oligonucleotide;
wherein the oligonucleotide is covalently linked at the 5′ end to a lysine residue of the anti-transferrin receptor antibody via a cleavable linker that comprises a valine-citrulline sequence, wherein the complexes are obtained by a cycloaddition reaction between an azide and an alkyne to form a triazole, wherein prior to the cycloaddition reaction, the azide is located on the cleavable linker that is covalently linked to the 5′ end of the oligonucleotide and the alkyne is provided in a bicyclononyne moiety that further covalently links to the anti-transferrin receptor antibody;
wherein the oligonucleotide i) comprises at least one modified nucleoside and/or at least one modified internucleoside linkage, ii) is 15-35 nucleotides in length, and iii) comprises a region of complementarity to an RNA encoded by a muscle disease gene in the muscle cell, wherein the region of complementarity is at least 12 nucleotides in length;
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; and
wherein the muscle-targeting complexes are administered to the subject by infusion and the oligonucleotide is released into the muscle cell following cleavage of the cleavable linker.
93 . The method of claim 92 , wherein the oligonucleotide comprises one or more 2′ modified nucleosides.
94 . The method of claim 93 , wherein the 2′ modified nucleoside is selected from the group consisting of: 2′-O-methyl, 2′-fluoro, 2′-O-methoxyethyl, and 2′, 4′-bridged nucleosides.
95 . The method of claim 92 , wherein the oligonucleotide comprises one or more modified internucleoside linkages.
96 . The method of claim 95 , wherein the modified internucleoside linkage is a phosphorothioate linkage.
97 . The method of claim 92 , wherein the oligonucleotide comprises one or more phosphorodiamidate morpholinos.
98 . The method of claim 92 , wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer.
99 . The method of claim 92 , wherein the oligonucleotide is 16-30 nucleotides in length.
100 . The method of claim 92 , wherein the region of complementarity is at least 16 nucleotides in length.
101 . The method of claim 92 , wherein the anti-transferrin receptor antibody binds to human TfR1 with a K D of less than 10 −8 M.
102 . The method of claim 92 , wherein the anti-transferrin receptor antibody is in the form of a ScFv, Fab fragment, Fab′ fragment, F(ab′)2 fragment, or Fv fragment.
103 . The method of claim 92 , wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.
104 . The method of claim 92 , wherein the cleavable linker further comprises one or more polyethylene glycol units.
105 . The method of claim 92 , wherein the muscle disease gene in the muscle cell is DMPK.
106 . The method of claim 92 , wherein the muscle disease gene in the muscle cell is DMD.
107 . The method of claim 92 , wherein the muscle disease gene in the muscle cell is DUX4.
108 . The method of claim 92 , wherein the muscle cell is a skeletal muscle cell, cardiac muscle cell, or smooth muscle cell.
109 . The method of claim 92 , wherein the subject is human.
110 . The method of claim 92 , wherein the subject is cynomolgus.
111 . The method of claim 92 , wherein the complexes are administered to the subject by intravenous infusion.