Muscle-targeting complexes comprising an anti-transferrin receptor antibody linked to an oligonucleotide and methods of delivering oligonucleotide to a subject
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 DUX4. 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 to a 5′ end or a 3′ end of an oligonucleotide,
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 and wherein the anti-transferrin receptor antibody does not specifically bind to the transferrin binding site of TfR1;
wherein the oligonucleotide comprises one or more modifications and comprises a region of complementarity of at least 15 nucleotides in length to the nucleotide sequence as set forth in SEQ ID NO: 199, wherein the oligonucleotide is in the range of 15-30 nucleotides in length;
wherein the one or more modifications comprise a 2′-modified nucleoside selected from the group consisting of: a 2′-O-methyl nucleoside, a 2′-fluoro nucleoside, a 2′-O-methoxyethyl nucleoside, 2′,4′-bridged nucleosides, and combinations thereof, and/or comprise a modified backbone comprising one or more phosphorothioate linkages.
2. The complex of claim 1 , wherein the oligonucleotide is 15-23 nucleotides in length.
3. The complex of claim 1 , wherein the region of complementarity is at least 16 nucleotides in length.
4. The complex of claim 1 , wherein the region of complementarity is at least 17 nucleotides in length.
5. The complex of claim 1 , wherein the region of complementarity is at least 18 nucleotides in length.
6. The complex of claim 1 , wherein the region of complementarity is at least 19 nucleotides in length.
7. The complex of claim 1 , wherein the oligonucleotide comprises a modified backbone comprising one or more phosphorothioate linkages.
8. The complex of claim 1 , wherein the oligonucleotide comprises a gap region flanked both 5′ and 3′ by flanking regions, wherein the flanking regions comprises one or more 2′-modified nucleosides selected from the group consisting of: 2′-O-methyl, 2′-fluoro, 2′-O-methoxyethyl, 2′,4′-bridged nucleosides, and combinations thereof, and wherein each nucleoside of the gap region is a 2′-deoxyribonucleoside.
9. The complex of claim 8 , wherein each nucleotide of the flanking regions is a 2′-modified nucleoside.
10. The complex of claim 9 , wherein each internucleoside linkage in the oligonucleotide is a phosphorothioate linkage.
11. The complex of claim 1 , wherein the oligonucleotide is a double-stranded molecule comprising the single strand hybridized to a complementary strand.
12. The complex of claim 1 , wherein the anti-transferrin receptor antibody further comprises one or more sugar or carbohydrate molecules.
13. The complex of claim 12 , wherein the one or more sugar or carbohydrate molecules comprise a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, a phospholipid unit, or combinations thereof.
14. The complex of claim 1 , wherein the oligonucleotide is covalently linked to a lysine in the anti-transferrin receptor antibody via a cleavable linker.
15. The complex of claim 14 , wherein the cleavable linker comprises a valine-citrulline sequence.
16. The complex of claim 15 , wherein the complex is obtained by a cycloaddition reaction between an azide and an alkyne to form a triazole.
17. The complex of claim 16 , wherein, prior to the cycloaddition reaction, the azide is covalently linked to the valine-citrulline sequence of the cleavable linker that is covalently linked to the oligonucleotide and the alkyne is provided in a bicyclononyne moiety that further covalently links to the anti-transferrin receptor antibody.
18. The complex of claim 14 , wherein the cleavable linker further comprises one or more polyethylene glycol units.
19. 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.
20. The complex of claim 1 , wherein the anti-transferrin receptor antibody is in the form of a Fab fragment.
21. A method of delivering an oligonucleotide to a subject, the method comprising intravenously administering to the subject the complex of claim 1 .
22. The method of claim 21 , wherein the subject has a muscular dystrophy.
23. The method of claim 21 , wherein the subject has myotonic dystrophy type I.
24. The method of claim 21 , wherein the oligonucleotide is delivered to a muscle of the subject.
25. The method of claim 24 , wherein the muscle cell is a skeletal muscle cell, a cardiac muscle cell, or a smooth muscle cell.
26. The method of claim 21 , wherein the subject is human.
27. The method of claim 21 , wherein the subject is cynomolgus.