Muscle targeting complexes and uses thereof for treating dystrophinopathies

Abstract

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.