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 (TfR) antibody covalently linked to an oligonucleotide,
wherein the anti-TfR antibody comprises
a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 76; and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 75, and wherein the oligonucleotide targets a Dystrophia Myotonica Protein Kinase (DMPK) RNA.
2 . The complex of claim 1 , wherein the VH of the antibody comprises an N-terminal pyroglutamate.
3 . The complex of claim 1 , wherein the oligonucleotide is 15-25 nucleotides in length.
4 . The complex of claim 3 , wherein the oligonucleotide comprises a region of complementarity to SEQ ID NO: 131, wherein the region of complementarity is 12-25 nucleotides in length.
5 . The complex of claim 3 , wherein the oligonucleotide comprises a region of complementarity to any one of SEQ ID NO: 384-619, wherein the region of complementarity is 12-25 nucleotides in length.
6 . The complex of claim 5 , wherein the region of complementarity is to a sequence as set forth in any one of SEQ ID NOs: 395, 398, 408, 410, 416, 418, 424, 426, 431, 432, 437, 439, 448, 451, 454, 458, 484, 500.
7 . The complex of claim 5 , wherein the region of complementarity is to a sequence as set forth in SEQ ID NO: 458.
8 . The complex of claim 5 , wherein the region of complementarity is to a sequence as set forth in SEQ ID NO: 464.
9 . The complex of claim 5 , wherein the region of complementarity is to a sequence as set forth in SEQ ID NO: 468.
10 . The complex of claim 3 , wherein the oligonucleotide comprises at least 15 consecutive nucleotides of any one of SEQ ID NOs: 148-383.
11 . The complex, of claim 10 , wherein any one or more of the thymine bases (T's) in the oligonucleotide is a uracil base (U).
12 . The complex of claim 3 , wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 148-383.
13 . The complex of claim 12 , wherein any one or more of the thymine bases (T's) in the oligonucleotide is a uracil base (U).
14 . The complex of claim 3 , wherein the oligonucleotide comprises at least 15 consecutive nucleotides of a sequence set forth in any one of SEQ ID NOs: 159, 162, 172, 174, 180, 182, 188, 190, 195, 196, 201, 203, 212, 215, 218, 222, 248, and 264.
15 . The complex of claim 14 , wherein any one or more of the thymine bases (T's) in the oligonucleotide is a uracil base (U).
16 . The complex of claim 3 , wherein the oligonucleotide comprises at least 15 consecutive nucleotides of SEQ ID NO: 222.
17 . The complex of claim 16 , wherein any one or more of the thymine bases (T's) in the oligonucleotide is a uracil base (U).
18 . The complex of claim 3 , wherein the oligonucleotide comprises at least 15 consecutive nucleotides of SEQ ID NO: 228.
19 . The complex of claim 18 , wherein any one or more of the thymine bases (T's) in the oligonucleotide is a uracil base (U).
20 . The complex of claim 3 , wherein the oligonucleotide comprises at least 15 consecutive nucleotides of SEQ ID NO: 232.
21 . The complex of claim 20 , wherein any one or more of the thymine bases (T's) in the oligonucleotide is a uracil base (U).
22 . The complex of claim 1 , wherein the oligonucleotide 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.
23 . The complex of claim 1 , wherein the oligonucleotide comprises one or more internucleoside phosphorothioate linkages.
24 . The complex of claim 1 , 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, 2′,4′-bridged nucleosides, and a combination thereof, and wherein Y is a gap region and each nucleoside in Y is a 2′-deoxyribonucleoside.
25 . The complex of claim 24 , wherein the oligonucleotide comprises one or more internucleoside phosphorothioate linkages.
26 . The complex of claim 1 , wherein the antibody is covalently linked to the oligonucleotide via a linker.
27 . The complex of claim 26 , wherein the linker comprises a cleavable linker.
28 . The complex of claim 26 , wherein the linker comprises a valine-citrulline sequence.
29 . The complex of claim 1 , 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.
30 . A composition comprising complexes, wherein each complex comprises an anti-transferrin receptor (TfR) antibody covalently linked to at least one oligonucleotide, wherein the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 76 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 75, wherein each anti-TfR antibody of the complexes is on average covalently linked to 1 to 3 oligonucleotides, and wherein the oligonucleotide targets a Dystrophia Myotonica Protein Kinase (DMPK) RNA.
31 . The composition of claim 30 , wherein the VH of the antibody comprises an N-terminal pyroglutamate.
32 . A method of reducing DMPK expression in muscle cells of a subject, the method comprising administering to the subject a composition comprising the complex of claim 1 .
33 . The method of claim 32 , wherein the subject is human.
34 . The method of claim 32 , wherein the subject is a cynomolgus.
35 . The method of claim 32 , wherein the subject has myotonic dystrophy, type 1 (DM1).
36 . The method of claim 32 , wherein the method reduces DMPK RNA and/or protein levels in muscle cells of the subject.
37 . The method of claim 32 , wherein the subject comprises a mutant DMPK allele comprising disease-associated CTG repeats.
38 . The method of claim 32 , wherein the composition is intravenously administered to the subject.
39 . The method of claim 32 , wherein the VH of the antibody comprises an N-terminal pyroglutamate.
40 . A method of treating myotonic dystrophy 1 (DM1) in a subject, the method comprising administering to the subject a composition comprising the complex of claim 1 .
41 . The method of claim 40 , wherein the subject is human.
42 . The method of claim 40 , wherein the composition is intravenously administered to the subject.