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 at least one oligonucleotide, wherein the antibody comprises:
(i) a heavy chain comprising a heavy chain variable region (VH) and a human IgG CH1 domain and lacking a human IgG CH2 domain and CH3 domain, wherein the heavy chain comprises the amino acid sequence of SEQ ID NO: 101, and
(ii) a light chain comprising a light chain variable region (VL) and a human kappa light chain constant region, wherein the light chain comprises the amino acid sequence of SEQ ID NO: 90;
wherein each oligonucleotide is covalently linked to a lysine residue of the anti-TfR antibody, and wherein the oligonucleotide induces dystrophin exon 51 skipping.
2. The complex of claim 1 , wherein the heavy chain of the antibody comprises an N-terminal pyroglutamate.
3. The complex of claim 1 , wherein the oligonucleotide is 15-35 nucleotides in length.
4. 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.
5. 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: 333-357, 745, and 2254, wherein the region of complementarity is 12-35 nucleotides in length.
6. The complex of claim 1 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 333-357, 745, and 2254, wherein any one or more of the thymine bases (T's) in the oligonucleotide may optionally be a uracil base (U), and wherein any one or more of the uracil bases (U's) in the oligonucleotide may optionally be a thymine base (T).
7. The complex of claim 1 , wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer.
8. The complex of claim 1 , wherein the antibody is covalently linked to each oligonucleotide via a cleavable linker comprising a valine-citrulline sequence.
9. The complex of claim 8 , wherein the complex comprises a structure of:
wherein n is 3 and m is 4, wherein L1 comprises a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, —O—, —N(R A )—, —S—, —C(═O)—, —C(═O)O—, —C(═O)NR A —, —NR A C(═O)—, —NR A C(═O) R A —, —C(═O) R A —, —NR A C(═O)O—, —NR A C(═O)N (R A )—, —OC(═O)—, —OC(═O)O—, —OC(—O)N (R A )—, —S(O) 2 NR A —, —NR A S(O) 2 —, or a combination thereof, wherein each R A is independently hydrogen or substituted or unsubstituted alkyl.
10. The complex of claim 9 , wherein L1 comprises a structure of:
wherein the piperazine moiety links to the oligonucleotide, wherein L2 comprises
11. The complex of claim 10 , wherein L2 comprises
12. A method of inducing dystrophin exon 51 skipping in muscle cells of a subject, the method comprising administering to the subject a composition comprising the complex of claim 1 .
13. The method of claim 12 , wherein the subject is human.
14. The method of claim 12 , wherein the subject has Duchenne muscular dystrophy.
15. The method of claim 12 , wherein the heavy chain of the antibody comprises an N-terminal pyroglutamate.
16. A complex comprising an anti-transferrin receptor (TfR) antibody covalently linked to at least one oligonucleotide, wherein the antibody comprises:
(i) a heavy chain comprising a heavy chain variable region (VH) and a portion of a heavy chain constant region, wherein the VH comprises the amino acid sequence of SEQ ID NO: 76 and the portion of the heavy chain constant region consists of the amino acid sequence of SEQ ID NO: 96, and
(ii) a light chain comprising the amino acid sequence of SEQ ID NO: 90;
wherein each oligonucleotide is covalently linked to a lysine residue of the anti-TfR antibody, and wherein the oligonucleotide induces dystrophin exon 51 skipping.
17. The complex of claim 16 , wherein the heavy chain of the antibody comprises an N-terminal pyroglutamate.
18. The complex of claim 16 , wherein the portion of the heavy chain constant region consists of a CH1 domain and a portion of a hinge region.
19. The complex of claim 16 , wherein the oligonucleotide is 15-35 nucleotides in length and comprises a region of complementarity to a dystrophin RNA, wherein the region of complementarity is 12-35 nucleotides in length.
20. The complex of claim 16 , 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: 333-357, 745, and 2254, wherein the region of complementarity is 12-35 nucleotides in length.
21. The complex of claim 16 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 333-357, 745, and 2254, wherein any one or more of the thymine bases (T's) in the oligonucleotide may optionally be a uracil base (U), and wherein any one or more of the uracil bases (U's) in the oligonucleotide may optionally be a thymine base (T).
22. The complex of claim 16 , wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer.
23. The complex of claim 16 , wherein the antibody is covalently linked to each oligonucleotide via a cleavable linker comprising a valine-citrulline sequence.
24. The complex of claim 23 , wherein the complex comprises a structure of:
wherein n is 3 and m is 4, wherein L1 comprises a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, —O—, —N(R A )—, —S—, —C(═O)—, —C(═O)O—, —C(═O)NR A —, —NR A C(═O)—, —NR A C(═O) R A —, —C(═O) R A —, —NR A C(═O)O—, —NR A C(═O)N (R A )—, —OC(═O)—, —OC(═O)O—, —OC(═O)N (R A )—, —S(O) 2 NR A , —NR A S(O) 2 —, or a combination thereof, wherein each R A is independently hydrogen or substituted or unsubstituted alkyl.
25. The complex of claim 24 , wherein L1 comprises a structure of:
wherein the piperazine moiety links to the oligonucleotide, wherein L2 comprises
26. The complex of claim 25 , wherein L2 comprises
27. A method of inducing dystrophin exon 51 skipping in muscle cells of a subject, the method comprising administering to the subject a composition comprising the complex of claim 16 .
28. The method of claim 27 , wherein the subject is human.
29. The method of claim 27 , wherein the subject has Duchenne muscular dystrophy.
30. The method of claim 27 , wherein the heavy chain of the antibody comprises an N-terminal pyroglutamate.