Muscle targeting complexes and uses thereof for treating myotonic dystrophy
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 expression or activity of a DMPK allele comprising a disease-associated-repeat. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or RNAi oligonucleotide.
1. A complex comprising an anti-transferrin receptor antibody covalently linked, via a protease-sensitive linker, to an oligonucleotide that targets a DMPK RNA,
wherein the complex is configured for delivering the oligonucleotide into muscle cells, wherein the oligonucleotide is 15, 16, 17, or 18 nucleotides in length and comprises a region of complementarity that is fully complementary, along a length of at least 14 contiguous nucleotides, to a coding region of a DMPK sequence as set forth in SEQ ID NO: 15,
wherein the oligonucleotide is configured to bring about degradation of the DMPK RNA via RNase H mediated degradation, wherein the oligonucleotide comprises a 5′-X-Y-Z-3′ formula, wherein X and Z are flanking regions comprising one or more 2′-modified nucleosides selected from the group consisting of: 2′-O-methyl, 2′-fluoro, 2′-O-methoxyethyl, and 2′,4′-bridged nucleosides, wherein Y is a gap region and each nucleoside in Y is a 2′-deoxyribonucleoside, and wherein the oligonucleotide comprises one or more phosphorothioate internucleoside linkages; and
wherein the anti-transferrin receptor antibody binds an extracellular domain of a transferrin receptor protein 1 (TfR1).
2. The complex of claim 1 , wherein the 5′ end of the oligonucleotide is covalently linked to a lysine in the anti-transferrin receptor antibody via the protease-sensitive linker.
3. The complex of claim 1 , wherein the protease-sensitive linker comprises a cleavage site of a lysosomal and/or endosomal protease.
4. The complex of claim 3 , wherein the lysosomal and/or endosomal protease is a cathepsin protease.
5. The complex of claim 1 , wherein the protease-sensitive linker comprises a valine-citrulline sequence.
6. The complex of claim 1 , wherein the protease sensitive linker comprises a triazole obtained by a cycloaddition reaction between an azide and an alkyne.
7. The complex of claim 6 , wherein prior to the cycloaddition reaction, the azide is located on the protease sensitive linker that is covalently linked to the 5′ end of the oligonucleotide and the alkyne is provided in a bicyclononyne moiety, and wherein the protease-sensitive linker further covalently links to the anti-transferrin receptor antibody.
8. The complex of claim 7 , wherein the protease-sensitive linker further comprises one or more polyethylene glycol units.
9. The complex of claim 1 , wherein each nucleotide in X and Z is a 2′-modified nucleoside.
10. The complex of claim 1 , wherein each internucleoside linkage in the oligonucleotide is a phosphorothioate linkage.
11. The complex of claim 1 , wherein the oligonucleotide is 16, 17 or 18 nucleotides in length.
12. The complex of claim 1 , wherein the region of complementarity is fully complementary, along a length of at least 16 contiguous nucleotides, to the coding region of the DMPK sequence.
13. The complex of claim 1 , wherein the anti-transferrin receptor antibody is in the form of a ScFv, Fab fragment, Fab′ fragment, F(ab′)2 fragment, or Fv fragment.
14. The complex of claim 13 , wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.
15. The complex of claim 1 , wherein the DMPK RNA comprises 38 to 200 repeating CUG units.
16. The complex of claim 1 , wherein administration of the complex to a subject results in reduction of DMPK RNA level by at least 50% in muscle cells of the subject.
17. The complex of claim 1 , wherein administration of the complex to a subject rescues splicing defects in muscle cells of the subject.
18. The complex of claim 1 , wherein the muscle cells are skeletal muscle cells, cardiac muscle cells, or smooth muscle cells.
19. The complex of claim 17 , wherein the subject is human.
20. The complex of claim 17 , wherein the subject is cynomolgus.
21. The complex of claim 1 , wherein the DMPK RNA contains a disease-associated repeat sequence in the 3′-UTR that is associated with myotonic dystrophy type 1 (DM1).