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 antibody covalently linked to an exon-skipping oligonucleotide that induces skipping of exon 53 of dystrophin (DMD),
wherein the anti-transferrin receptor antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region and the light chain variable region comprise human or humanized framework regions; 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;
wherein the oligonucleotide is 15 to 35 nucleotides in length and comprises a region of complementarity to exon 53 of a DMD transcript as set forth in SEQ ID NO: 295, wherein the region of complementarity is at least 12 nucleotides in length, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO);
wherein the oligonucleotide is covalently linked at the 5′ end to a lysine residue of the anti-transferrin receptor antibody via a cleavable linker comprising a valine-citrulline sequence; and
wherein the oligonucleotide is configured to induce skipping of exon 53 of the DMD pre-mRNA in a muscle cell.
2. The complex of claim 1 , wherein the region of complementarity is complementary to a target sequence of an oligonucleotide as set forth in any one of SEQ ID NOs: 222-242, 245, and 246.
3. The complex of claim 1 , wherein the oligonucleotide is 20-30 nucleotides in length.
4. The complex of claim 1 , wherein the region of complementarity is at least 16 nucleotides in length.
5. The complex of claim 1 , wherein the anti-transferrin receptor antibody binds to human TfR1 with a K D of less than 10 −8 M.
6. The complex of claim 1 , wherein the complex is obtained by a cycloaddition reaction between an azide and an alkyne to form a triazole.
7. The complex of claim 6 , 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.
8. The complex of claim 7 , wherein the cleavable linker further comprises one or more polyethylene glycol units.
9. The complex of claim 6 , 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.
10. The complex of claim 9 , wherein prior to the cycloaddition reaction the alkyne is provided in a bicyclononyne moiety.
11. The complex of claim 1 , wherein the muscle cell is a skeletal muscle cell, cardiac muscle cell, or smooth muscle cell.
12. A composition comprising the complex of claim 1 and a pharmaceutically acceptable carrier.
13. The composition of claim 12 , wherein the composition is formulated for intravenous administration.
14. A method of inducing skipping of exon 53 of a DMD pre-mRNA in a muscle cell of a subject expressing a DMD pre-mRNA comprising a frameshift mutation, the method comprising administering to the subject the complex of claim 1 .
15. The method of claim 14 , wherein the region of complementarity is complementary to a target sequence of an oligonucleotide as set forth in any one of SEQ ID NOs: 222-247 and 266.
16. The method of claim 14 , wherein the oligonucleotide promotes the expression or activity of a functional dystrophin protein in the muscle cell.
17. The method of claim 14 , wherein the muscle cell is a skeletal muscle cell, cardiac muscle cell, or smooth muscle cell.
18. The method of claim 14 , wherein the subject is human.
19. The method of claim 14 , wherein the subject is cynomolgus.
20. The method of claim 14 , wherein the complex is administered to the subject intravenously.