MUSCLE TARGETING COMPLEXES FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY
Aspects of the disclosure relate to oligonucleotides designed to target DUX4 RNAs and targeting complexes for delivering the oligonucleotides to cells (e.g., muscle cells) and uses thereof, particularly uses relating to treatment of disease (e.g., FSHD). Wherein a complex comprises an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured for reducing expression or activity of DUX4.
1 . A complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured for reducing expression or activity of DUX4, wherein the anti-TfR1 antibody comprises a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), a heavy chain complementarity determining region 3 (CDR-H3), a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), a light chain complementarity determining region 3 (CDR-L3) of any of the anti-TfR1 antibodies listed in Tables 2-7 and wherein the oligonucleotide comprises an antisense strand comprising a region of complementarity to a DUX4 sequence as set forth in SEQ ID NO: 160 or SEQ ID NO: 365.
2 . The complex of claim 1 , wherein the anti-TfR1 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL) of any of the anti-TfR1 antibodies listed in Table 3.
3 . The complex of any one of claim 1 or claim 2 , wherein the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76 and/or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75,
optionally wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 75.
4 . The complex of claim 1 or claim 2 , wherein the anti-TfR1 antibody is a Fab, optionally wherein the Fab comprises a heavy chain and a light chain of any of the anti-TfR1 Fabs listed in Table 5.
5 . The complex of claim 4 , wherein the Fab comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101 and/or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90,
optionally wherein the Fab 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.
6 . The complex of any one of claims 1-5 , wherein the oligonucleotide is 20-30 nucleotides in length.
7 . The complex of any one of claims 1-6 , wherein the oligonucleotide comprises a region of complementarity of at least 15 consecutive nucleotides to a DUX4 sequence as set forth in SEQ ID NO: 160 or SEQ ID NO: 365, optionally wherein the oligonucleotide comprises a region of complementarity of at least 15 consecutive nucleotides to a DUX4 sequence as set forth in any one of SEQ ID NOs: 161-168 or 213-288.
8 . The complex of any one of claims 1-7 , wherein the oligonucleotide comprises at least 15 consecutive nucleotides of any one of SEQ ID NOs: 169-176 or 289-364, wherein each thymine base (T) may independently and optionally be replaced with a uracil base (U), and each U may independently and optionally be replaced with a T, optionally wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs: 169-176 or 289-364.
9 . The complex of any one of claims 1-8 , wherein the oligonucleotide does not comprise the nucleotide sequence of SEQ ID NO: 151.
10 . The complex of any one of claims 1-9 , wherein the oligonucleotide further comprises a sense strand that hybridizes to the antisense strand to form a double stranded siRNA.
11 . The complex of any one of claims 1-10 , wherein the oligonucleotide comprises at least one modified internucleoside linkage.
12 . The complex of any one of claims 1-11 , wherein the oligonucleotide comprises one or more modified nucleosides, optionally wherein the one or more modified nucleosides are 2′-modified nucleosides.
13 . The complex of any one of claims 1-12 , wherein the antibody and the oligonucleotide are covalently linked via a linker, optionally wherein the linker is a cleavable linker, further optionally wherein the linker comprises a valine-citrulline sequence.
14 . A method of reducing DUX4 expression in a muscle cell, the method comprising contacting the muscle cell with an effective amount of the complex of any one of claims 1-13 for promoting internalization of the oligonucleotide to the muscle cell.
15 . The method of claim 14 , wherein the cell is in vitro.
16 . The method of claim 14 , wherein the cell is in a subject, optionally wherein the subject is human.
17 . A method of treating Facioscapulohumeral muscular dystrophy (FSHD), the method comprising administering to a subject in need thereof an effective amount of the complex of any one of claims 1-13 , wherein the subject has aberrant production of DUX4 protein, optionally wherein the subject is human.
18 . The method of any one of claims 16-17 , wherein the human subject has one or more deletions of a D4Z4 repeat in chromosome 4.
19 . The method of claim 18 , wherein the subject has 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or no D4Z4 repeats.
20 . An oligonucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 169-176 or 289-364.