MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR MODULATION OF GENES ASSOCIATED WITH MUSCLE HEALTH
Aspects of the disclosure relate to molecular payloads that modulate the expression or activity of genes involved in muscle growth and maintenance (e.g., MSTN, INHBA, ACVR1B, MLCK1, ACVR1, FBXO32, TRIM63, MEF2D, KLF15, MED1, MED13, and/or PPP1R3A), and complexes comprising a muscle-targeting agent covalently linked to such molecular payloads. In some embodiments, the muscle-targeting agent specifically binds to an internalizing cell surface receptor on a muscle cell (e.g., a cardiac muscle cell, a smooth muscle cell, a skeletal muscle cell). 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 1 antibody covalently linked to a molecular payload that modulates the expression or activity of myostatin (MSTN), inhibin beta A (INHBA), activin receptor type-1B (ACVR1B), myosin light chain kinase (MLCK1), activin A receptor type-1 (ACVR1), atrogin-1 (FBXO32), tripartite motif containing 63 (TRIM63), myocyte-specific enhancer factor 2D (MEF2D), Krüppel-like factor 15 (KLF15), Mediator complex subunit 1 (MED1), Mediator complex subunit 13 (MED13), and/or protein phosphatase 1 regulatory subunit 3A (PPP1R3A), wherein the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) as set forth in SEQ ID NO: 51, a heavy chain complementarity determining region 2 (CDR-H2) as set forth in SEQ ID NO: 52, a heavy chain complementarity determining region 3 (CDR-H3) as set forth in SEQ ID NO: 53, a light chain complementarity determining region 1 (CDR-L1) as set forth in SEQ ID NO: 54, a light chain complementarity determining region 2 (CDR-L2) as set forth in SEQ ID NO: 55, and a light chain complementarity determining region 3 (CDR-L3) as set forth in SEQ ID NO: 50.
2 . The antibody of claim 1 , wherein the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 61; and/or a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 62.
3 . The complex of claim 2 , wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 61 and a VL comprising the amino acid sequence of SEQ ID NO: 62.
4 . The complex of claim 1 , wherein the antibody is selected from the group consisting of a full-length IgG, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFv, and a Fv.
5 . The complex of claim 4 , wherein the antibody is a full-length IgG.
6 . (canceled)
7 . The complex of claim 4 , wherein the antibody is a Fab.
8 .- 9 . (canceled)
10 . The complex 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.
11 .- 12 . (canceled)
13 . The complex of claim 1 , wherein the molecular payload comprises an oligonucleotide comprising an antisense strand comprising a region of complementarity to an MSTN target sequence, an INHBA target sequence, an ACVR1B target sequence, a MLCK1 target sequence, an ACVR1 target sequence, a FBXO32 target sequence, a TRIM63 target sequence, a MEF2D target sequence, a KLF15 target sequence, a MED1 target sequence, a MED13 target sequence, or a PPP1R3A target sequence.
14 .- 35 . (canceled)
36 . The complex of claim 13 , wherein the oligonucleotide further comprises a sense strand that hybridizes to the antisense strand to form a double stranded siRNA.
37 . The complex of claim 13 , wherein the oligonucleotide comprises one or more modified nucleosides and/or one or more phosphorothioate internucleoside linkages.
38 . The complex of claim 37 , wherein the one or more modified nucleosides are 2′ modified nucleotides.
39 . (canceled)
40 . The complex of claim 36 , wherein the oligonucleotide is an siRNA listed in Table 10, Table 13, Table 16, Table 19, Table 22, Table 25, Table 28, Table 31, Table 34, Table 37, or Table 40.
41 . The complex of claim 1 , wherein the antibody is covalently linked to the molecular payload via
(i) a cleavable linker, optionally wherein the cleavable linker comprises a valine-citrulline sequence; or
(ii) a non-cleavable linker, optionally wherein the non-cleavable linker is an alkane linker.
42 . A method of reducing MSTN, INHBA, ACVR1B, MLCK1, ACVR1, FBXO32, TRIM63, MEF2D, KLF15, MED1, MED13, and/or PPP1R3A expression in a muscle cell, the method comprising contacting the muscle cell with an effective amount of the complex of claim 1 for promoting internalization of the molecular payload to the muscle cell.
43 . A method of treating muscle atrophy the method comprising administering to a subject in need thereof an effective amount of the complex of claim 1 , wherein the subject has elevated expression or activity of MSTN, INHBA, and/or ACVR1B, and the complex comprises a molecular payload that modulates the expression or activity of MSTN, INHBA, and/or ACVR1B.
44 . A method of treating irritable bowel syndrome (IBS) or irritable bowel disease (IBD) the method comprising administering to a subject in need thereof an effective amount of the complex claim 1 , wherein the subject has elevated levels of MLCK1 protein and the complex comprises a molecular payload that modulates the expression or activity of MLCK1.
45 . A method of treating a subject having a disease associated with elevated level of ACVR1, the method comprising administering to the subject an effective amount of the complex of claim 1 , wherein the complex comprises a molecular payload that modulates the expression or activity of ACVR1.
46 . A method of treating muscle atrophy the method comprising administering to a subject in need thereof an effective amount of the complex of claim 1 , wherein the subject has elevated expression or activity of FBXO32 or TRIM63, and the complex comprises a molecular payload that modulates the expression or activity of FBXO32 or TRIM63.
47 . A method of treating a heart disease, the method comprising administering to a subject in need thereof an effective amount of the complex of claim 1 , wherein the subject has elevated expression or activity of MEF2D, KLF15, MED1, MED13, and/or PPP1R3A, and the complex comprises a molecular payload that modulates the expression or activity of MEF2D, KLF15, MED1, MED13, and/or PPP1R3A.
48 .- 49 . (canceled)
50 . An oligonucleotide comprising an siRNA listed in Tables 9, 10, 12, 13, 15, 16, 18, 19, 21, 22, 24, 25, 27, 28, 30, 21, 33, 34, 36, 37, 39, and 40.