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 composition comprising complexes comprising an anti-transferrin receptor (TfR) antibody covalently linked to at least one oligonucleotide, wherein the antibody is a Fab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90, and wherein each anti-TfR antibody of the complexes is on average covalently linked to 1 to 3 oligonucleotides, and wherein the oligonucleotide induces dystrophin exon 51 skipping, wherein the complexes of the composition comprise 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.
2. The composition of claim 1 , wherein the heavy chain of the antibody comprises an N-terminal pyroglutamate.
3. The composition of claim 1 , wherein the equilibrium dissociation constant (K D ) of binding of the antibody to the transferrin receptor is in a range from 10 −11 M to 10 −6 M.
4. The composition of claim 1 , wherein the oligonucleotide is 15-35 nucleotides in length.
5. The composition 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.
6. The composition 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.
7. The composition 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).
8. The composition of claim 1 , wherein the oligonucleotide comprises one or more modified nucleosides.
9. The composition of claim 1 , wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer.
10. The composition of claim 1 , wherein L1 comprises a structure of:
wherein the piperazine moiety links to the oligonucleotide, wherein L2 comprises
11. The composition 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 the composition of claim 1 .
13. The method of claim 12 , wherein the subject is human.
14. The method of claim 12 , wherein the subject is a cynomolgus.
15. The method of claim 12 , wherein the subject expresses a dystrophin pre-mRNA comprising a frameshift mutation.
16. The method of claim 12 , wherein the complex is intravenously administered to the subject.
17. The method of claim 12 , wherein the subject is amenable to dystrophin exon 51 skipping.
18. The method of claim 12 , wherein the subject has Duchenne muscular dystrophy.
19. The method of claim 12 , wherein the heavy chain of the antibody comprises an N-terminal pyroglutamate.