Muscle targeting complexes and their use for treating facioscapulohumeral muscular dystrophy

Abstract

Aspects of the present disclosure relate to oligonucleotides (e.g., RNAi oligonucleotides such as siRNAs) designed to target DUX4 RNA, and targeting complexes for delivering the oligonucleotides to cells (e.g., muscle cells), and their uses, particularly for the treatment of diseases (e.g., FSHD).

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 133,156, filed Dec. 31, 2020, and U.S. Provisional Application No. 63 / 181,439, filed Apr. 29, 2021, both entitled "MUSCLE-TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY", under 35 U.S.C. § 119(e), the contents of each of which are incorporated herein by reference in their entirety.

[0002] Field of the Invention This application relates to targeting complexes for delivering oligonucleotides and molecular payloads (e.g., oligonucleotides) designed to target DUX4 RNA to cells, and their use, particularly for the treatment of diseases.

[0003] Reference to a Sequence Listing Submitted as a Text File via EFS-Web This application contains a Sequence Listing. This was submitted in ASCII format from EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy created on Dec. 30, 2021 is named D082470047WO00-SEQ-ZJG and is 708,513 bytes in size.

Background Art

[0004] Background Muscular dystrophy (MD) is a group of diseases characterized by progressive weakness and loss of muscle mass. These diseases are caused by mutations in genes that encode proteins necessary to form healthy muscle tissue. Facioscapulohumeral muscular dystrophy (FSHD) is a dominant genetic form of MD that primarily affects the muscles of the face, scapula, and upper arm. Other symptoms of FSHD include weakness of the abdominal muscles, retinal abnormalities, hearing loss, and joint pain and inflammation. FSHD is the most commonly seen of the nine types of MD that affect both adults and children, with an incidence rate worldwide of about 1 in 8,300 people. FSHD is caused by the abnormal production of a protein of unknown function, double homeobox 4 (DUX4). The DUX4 gene, which encodes the DUX4 protein, is located within the D4Z4 repeat region on chromosome 4 and is typically expressed only during fetal development, after which it is silenced by the hypermethylation of the D4Z4 repeats that surround the DUX4 gene. Two types of FSHD, type 1 and type 2, are described. Type 1, which accounts for about 95% of cases, is associated with a deletion of the D4Z4 repeats on chromosome 4. Unaffected individuals generally have more than 10 repeats arrayed in the subtelomeric region of chromosome 4, whereas the most common form of FSHD (FSHD1) is caused by a contraction to fewer than 10 repeats of the array and is associated with a decrease in the epigenetic silencing of DUX4 in skeletal muscle and a change-rich expression. Two variants of the 4q chromosome (4qA and 4qB) exist in the distal region of D4Z4. 4qA is in cis with a functional polyadenylation consensus site. Contractions on the 4qA allele are pathogenic because the DUX4 transcript is polyadenylated and translated into a stable protein. Type 2 FSHD, which accounts for about 5% of cases, is associated with mutations in the SMCHD1 gene on chromosome 18. Other than supportive therapies and treatments to address the symptoms of the disease, there is no effective treatment for FSHD. Summary of the Invention

[0005] Summary In several aspects, this disclosure provides oligonucleotides designed to target DUX4 RNA. In several embodiments, this disclosure provides oligonucleotides complementary to DUX4 RNA that are useful in reducing levels of DUX4 mRNA and / or protein associated with the pathological features of facioscapulohumeral muscular dystrophy (FSHD), including muscular atrophy, inflammation, and reduced differentiation potential and oxidative stress. In several embodiments, the oligonucleotides provided herein are designed to direct the RNAi-mediated degradation of DUX4 RNA. In several embodiments, the oligonucleotides are designed to efficiently involve the RNA-induced silencing complex (RISC) for DUX4 RNA degradation while also reducing off-target effects. In several embodiments, the oligonucleotides are designed to reduce levels of DUX4 RNA and / or protein. In several embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum stability. In several embodiments, the oligonucleotides are designed to have desired binding affinity. In several embodiments, the oligonucleotides are designed to have a desired toxicity and / or immunogenicity profile.

[0006] In some aspects, this disclosure provides complexes that target muscle cells (e.g., primary myoblasts) for the purpose of delivering a molecular payload (e.g., a DUX4-targeted oligonucleotide as described herein) to those cells. In some embodiments, the complexes provided herein are particularly useful, for example, for delivering a molecular payload that inhibits the expression or activity of DUX4 in subjects having or suspected of having facioscapulohumeral muscular dystrophy (FSHD). Accordingly, in some embodiments, the complexes provided herein include a muscle targeting agent (e.g., a muscle targeting antibody) that specifically binds to a receptor on the surface of muscle cells for the purpose of delivering the molecular payload to muscle cells. In some embodiments, the complex may be taken up into the cell via receptor-mediated internalization, and the molecular payload may be released into the cell to perform its function. For example, a complex modified to deliver an oligonucleotide may release the oligonucleotide so that the oligonucleotide can inhibit DUX4 gene expression in muscle cells. In some embodiments, the oligonucleotide is released by endosomal cleavage of a covalent linker connecting the oligonucleotide and the muscle targeting agent of the complex.

[0007] Some aspects of this disclosure provide a complex comprising a muscle targeting agent covalently linked to an oligonucleotide that targets double homeobox 4 (DUX4) mRNA, wherein the oligonucleotide comprises an antisense strand of 18-25 nucleotides in length, and includes SEQ ID NOs: 356, 501, 1398, 494, 509, 224, 1320, 561, 225, 226, 261, 265, 320, 341, 343, 388, 466, 4 The sequence includes complementary regions to the target sequence as represented by 83, 552, 560, 601, 921, 942, 953, 1294, 1296, 1301, 1321, 1322, 1323, 1324, 1325, 1373, 1394, 1395, 1523, 1531, 1548, 1558, and 1561, where the complementary regions are consecutive nucleosides of at least 16 in length.

[0008] In some embodiments, the muscle targeting agent is an anti-transferrin receptor (TfR) antibody.

[0009] In some embodiments, the oligonucleotide is an RNAi oligonucleotide.

[0010] In some aspects, the antisense strand contains one of the nucleotide sequences SEQ ID NOs: 3035, 3040, 3061, 3039, 3041, 3027, 3052, 3044, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3036, 3037, 3038, 3042, 3043, 3045, 3046, 3047, 3048, 3049, 3050, 3051, 3053, 3054, 3055, 3056, 3057, 3058, 3059, 3060, 3062, 3063, 3064, 3065, and 3066.

[0011] In some embodiments, the oligonucleotide further comprises a sense chain containing at least 18 consecutive nucleosides complementary to the antisense chain.

[0012] In some embodiments, the oligonucleotide comprises one or more modified nucleosides.

[0013] In some embodiments, one or more modified nucleosides are 2'-modified nucleotides, optionally selected from the following: 2'-fluoro(2'-F), 2'-O-methyl(2'-O-Me), 2'-O-methoxyethyl(2'-MOE), 2'-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'-O-DMAOE), 2'-O-dimethylaminopropyl(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O-DMAEOE), and 2'-ON-methylacetamide(2'-O-NMA).

[0014] In some embodiments, each 2'-modified nucleotide is 2'-O-methyl or 2'-fluoro(2'-F).

[0015] In some embodiments, the oligonucleotide comprises one or more phosphorothioate nucleoside linkages.

[0016] In some embodiments, one or more phosphorothioate nucleoside linkages are present on the antisense chain of the oligonucleotide.

[0017] In some embodiments, the ligation between two nucleosides at the 3' end of the antisense chain is a phosphorothioate nucleoside ligation.

[0018] In some embodiments, one or more cytidines of the alkyl group are 2'-modified 5-methylcytidines, optionally where 2'-modified 5-methylcytidines are 2'-O-Me-modified 5-methylcytidines or 2'-F-modified 5-methylcytidines.

[0019] In some aspects, the antisense chain is selected from modified versions of SEQ ID NOs. 3035, 3040, 3061, 3039, 3041, 3027, 3052, 3044, 3028, 3029, 3030, 3031, 3032, 3033, 3034, 3036, 3037, 3038, 3042, 3043, 3045, 3046, 3047, 3048, 3049, 3050, 3051, 3053, 3054, 3055, 3056, 3057, 3058, 3059, 3060, 3062, 3063, 3064, 3065, and 3066 listed in Table 8.

[0020] In some embodiments, the sense chain is selected from modified versions of sequence numbers 2995, 3000, 3021, 2999, 3001, 2987, 3012, 3004, 2988, 2989, 2990, 2991, 2992, 2993, 2994, 2996, 2997, 2998, 3002, 3003, 3005, 3006, 3007, 3008, 3009, 3010, 3011, 3013, 3014, 3015, 3016, 3017, 3018, 3019, 3020, 3022, 3023, 3024, 3025, and 3026 listed in Table 8.

[0021] In some embodiments, the oligonucleotide is an siRNA molecule selected from the siRNAs listed in Table 8.

[0022] In some embodiments, the antisense chain is selected from modified versions of sequence numbers 3040, 3061, 3027, 3037, 3039, 3041, 3044, and 3052 listed in Table 9.

[0023] In some embodiments, the sense chain is selected from modified versions of sequence numbers 3000, 3021, 2987, 2997, 2999, 3001, 3004, and 3012 listed in Table 9.

[0024] In some embodiments, the RNAi oligonucleotide is an siRNA molecule selected from the siRNAs listed in Table 9.

[0025] In some embodiments, the anti-TfR antibody comprises heavy chain complementarity-determining region 1 (CDR-H1), heavy chain complementarity-determining region 2 (CDR-H2), heavy chain complementarity-determining region 3 (CDR-H3), light chain complementarity-determining region 1 (CDR-L1), light chain complementarity-determining region 2 (CDR-L2), and light chain complementarity-determining region 3 (CDR-L3) of any of the anti-TfR antibodies listed in Table 2.

[0026] In some embodiments, the anti-TfR antibody comprises the heavy chain variable region (VH) and light chain variable region (VL) of any of the anti-TfR antibodies listed in Table 3.

[0027] In some embodiments, the anti-TfR antibody is Fab, where optionally Fab comprises the heavy and light chains of any of the anti-TfR Fabs listed in Table 5.

[0028] In some embodiments, anti-TfR antibodies are as follows: (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, CDR-H3 containing the amino acid sequence of SEQ ID NO: 29, CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32; (ii) CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32; or (ii) CDR-H1 containing the amino acid sequence of SEQ ID NO: 38, CDR-H2 containing the amino acid sequence of SEQ ID NO: 39, CDR-H3 containing the amino acid sequence of SEQ ID NO: 40, CDR-L1 containing the amino acid sequence of SEQ ID NO: 41, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 42 Includes.

[0029] In some embodiments, the anti-TfR antibody comprises VH, which contains the amino acid sequence of SEQ ID NO: 76, and VL, which contains the amino acid sequence of SEQ ID NO: 75.

[0030] In some embodiments, the anti-TfR antibody is Fab and comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 101 and a light chain containing the amino acid sequence of SEQ ID NO: 90.

[0031] In some embodiments, the muscle targeting agent and the antisense oligonucleotide are covalently linked via a linker, which optionally comprises a valine-citrulline sequence.

[0032] Furthermore, this specification provides a method for reducing DUX4 expression in muscle cells, the method comprising contacting the muscle cells with an amount of the complex described herein that is effective in promoting the internalization of oligonucleotides into the muscle cells.

[0033] Furthermore, provided herein are methods for treating facioscapulohumeral muscular dystrophy (FSHD), the methods comprising administering an effective amount of the complex described in any one of claims 1 to 18 to a subject in need thereof, wherein the subject has abnormal production of the DUX4 protein.

[0034] Other aspects of this disclosure are as follows: Antisense chain: 5'-fCfUmCfUmCfAmUfUmCfUmGfAmAfAmCfCmAfAmAfUmC*fU*mG-3' (Sequence ID 3035) Sense chain: 5'-mGmAfUmUfUmGfGmUfUmUfCmAfGmAfAmUfGmAfGmAfG-3'(Sequence ID 2995); Antisense chain: 5'-fUfGmGfAmAfAmGfCmGfAmUfCmCfUmUfCmUfCmAfAmA*fG*mG-3'(Sequence ID 3040) Sense chain: 5'-mUmUfUmGfAmGfAmAfGmGfAmUfCmGfCmUfUmUfCmCfA-3'(Sequence ID 3000); Antisense chain: 5'-fCfCmGfGmUfAmUfUmCfUmUfCmCfUmCfGmCfUmGfAmG*fG*mG-3' (Sequence ID 3061) Sense chain: 5'-mCmUfCmAfGmCfGmAfGmGfAmAfGmAfAmUfAmCfCmGfG-3' (Sequence ID 3021); Antisense chain: 5'-fCfGmAfUmCfCmUfUmCfUmCfAmAfAmGfGmCfUmCfGmG*fA*mG-3'(Sequence ID 3039) Sense chain: 5'-mCmCfGmAfGmCfCmUfUmUfGmAfGmAfAmGfGmAfUmCfG-3' (Sequence ID 2999); Antisense chain: 5'-fGfCmGfAmUfGmCfCmUfGmGfAmAfAmGfCmGfAmUfCmC*fU*mU-3'(Sequence ID 3041) Sense chain: 5'-mGmGfAmUfCmGfCmUfUmUfCmCfAmGfGmCfAmUfCmGfC-3'(Sequence ID 3001); Antisense chain: 5'-fGfUmCfCmAfAmAfCmGfAmGfUmCfUmCfCmGfUmCfGmC*fC*mG-3' (Sequence ID 3027) Sense chain: 5'-mGmCfGmAfCmGfGmAfGmAfCmUfCmGfUmUfUmGfGmAfC-3' (Sequence ID 2987); Antisense chain: 5'-fUfUmCfUmAfGmGfAmGfAmGfGmUfUmGfCmGfCmCfUmG*fC*mU-3'(Sequence ID 3052) Sense chain: 5'-mCmAfGmGfCmGfCmAfAmCfCmUfCmUfCmCfUmAfGmAfA-3'(Sequence ID 3012); Antisense chain: 5'-fCfUmGfAmAfUmCfCmUfGmGfAmCfUmCfCmGfGmGfAmG*fG*mC-3'(Sequence ID 3044) Sense chain: 5'-mCmUfCmCfCmGfGmAfGmUfCmCfAmGfGmAfUmUfCmAfG-3' (Sequence ID 3004); Antisense chain: 5'-fUfCmCfGmCfUmCfAmAfAmGfCmAfGmGfCmUfCmGfCmA*fG*mG-3'(Sequence ID 3031) Sense chain: 5'-mUmGfCmGfAmGfCmCfUmGfCmUfUmUfGmAfGmCfGmGfA-3'(Sequence ID 2991); Antisense chain: 5'-fAfCmCfAmAfAmUfCmUfGmGfAmCfCmCfUmGfGmGfCmU*fC*mC-3'(Sequence ID 3034) Sense chain: 5'-mAmGfCmCfCmAfGmGfGmUfCmCfAmGfAmUfUmUfGmGfU-3' (Sequence ID 2994); Antisense chain: 5'-fGfGmAfAmUfGmCfCmGfAmUfGmGfCmCfUmGfGmGfCmC*fA*mG-3'(Sequence ID 3032) Sense chain: 5'-mGmGfCmCfCmAfGmGfCmCfAmUfCmGfGmCfAmUfUmCfC-3'(Sequence ID 2992); Antisense chain: 5'-fCfAmAfAmUfCmUfGmGfAmCfCmCfUmGfGmGfCmUfCmC*fG*mG-3'(Sequence ID 3033) Sense chain: 5'-mGmGfAmGfCmCfCmAfGmGfGmUfCmCfAmGfAmUfUmUfG-3'(Sequence ID 2993); Antisense chain: 5'-fGfGmAfCmUfCmCfGmGfGmAfGmGfCmCfCmGfUmCfUmC*fU*mC-3'(Sequence ID 3042) Sense chain: 5'-mGmAfGmAfCmGfGmGfCmCfUmCfCmCfGmGfAmGfUmCfC-3'(Sequence ID 3002); Antisense chain: 5'-fCfUmCfAmAfAmGfCmAfGmGfCmUfCmGfCmAfGmGfGmC*fC*mU-3'(Sequence ID 3030) Sense chain: 5'-mGmCfCmCfUmGfCmGfAmGfCmCfUmGfCmUfUmUfGmAfG-3'(Sequence ID 2990); Antisense chain: 5'-fAfUmUfCmCfCmGfCmCfGmGfUmGfCmUfGmCfCmUfCmA*fG*mC-3'(Sequence ID 3036) Sense chain: 5'-mUmGfAmGfGmCfAmGfCmAfCmCfGmGfCmGfGmGfAmAfU-3'(Sequence ID 2996); Antisense chain: 5'-fAfUmGfCmCfCmAfGmGfAmAfAmGfAmAfUmGfGmCfAmG*fU*mU-3'(Sequence ID 3065) Sense chain: 5'-mCmUfGmCfCmAfUmUfCmUfUmUfCmCfUmGfGmGfCmAfU-3' (Sequence ID 3025); Antisense chain: 5'-fGfUmUfUmCfUmAfGmGfAmGfAmGfGmUfUmGfCmGfCmC*fU*mG-3'(Sequence ID 3054) Sense chain: 5'-mGmGfCmGfCmAfAmCfCmUfCmUfCmCfUmAfGmAfAmAfC-3' (Sequence ID 3014); Antisense chain: 5'-fUfCmCfGmUfUmUfCmUfAmGfGmAfGmAfGmGfUmUfGmC*fG*mC-3'(Sequence ID 3057) Sense chain: 5'-mGmCfAmAfCmCfUmCfUmCfCmUfAmGfAmAfAmCfGmGfA-3' (Sequence ID 3017); Antisense chain: 5'-fGfAmAfAmCfUmCfCmGfGmGfCmUfCmGfCmCfAmGfGmA*fG*mC-3'(Sequence ID 3049) Sense chain: 5'-mUmCfCmUfGmGfCmGfAmGfCmCfCmGfGmAfGmUfUmUfC-3'(Sequence ID 3009); Antisense chain: 5'-fAfAmGfAmAfUmGfGmCfAmGfUmUfCmUfCmCfGmCfGmG*fU*mG-3'(Sequence ID 3064) Sense chain: 5'-mCmCfGmCfGmGfAmGfAmAfCmUfGmCfCmAfUmUfCmUfU-3' (Sequence ID 3024); Antisense chain: 5'-fCfGmUfUmUfCmUfAmGfGmAfGmAfGmGfUmUfGmCfGmC*fC*mU-3'(Sequence ID 3055) Sense chain: 5'-mGmCfGmCfAmAfCmCfUmCfUmCfCmUfAmGfAmAfAmCfG-3' (Sequence ID 3015); Antisense chain: 5'-fGfGmUfCmCfAmAfAmCfGmAfGmUfCmUfCmCfGmUfCmG*fC*mC-3'(Sequence ID 3028) Sense chain: 5'-mCmGfAmCfGmGfAmGfAmCfUmCfGmUfUmUfGmGfAmCfC-3'(Sequence ID 2988); Antisense chain: 5'-fGfCmGfGmUfGmUfGmGfAmGfUmCfUmCfUmCfAmCfCmG*fG*mG-3'(Sequence ID 3063) Sense chain: 5'-mCmGfGmUfGmAfGmAfGmAfCmUfCmCfAmCfAmCfCmGfC-3' (Sequence ID 3023); Antisense chain: 5'-fUfAmUfUmCfUmUfCmCfUmCfGmCfUmGfAmGfGmGfGmU*fG*mC-3'(Sequence ID 3059) Sense chain: 5'-mAmCfCmCfCmUfCmAfGmCfGmAfGmGfAmAfGmAfAmUfA-3' (Sequence ID 3019); Antisense chain: 5'-fGfGmGfUmCfCmAfAmAfCmGfAmGfUmCfUmCfCmGfUmC*fG*mC-3' (Sequence ID 3029) Sense chain: 5'-mGmAfCmGfGmAfGmAfCmUfCmGfUmUfUmGfGmAfCmCfC-3' (Sequence ID 2989); Antisense chain: 5'-fUfUmUfCmUfAmGfGmAfGmAfGmGfUmUfGmCfGmCfCmU*fG*mC-3'(Sequence ID 3053) Sense chain: 5'-mAmGfGmCfGmCfAmAfCmCfUmCfUmCfCmUfAmGfAmAfA-3' (Sequence ID 3013); Antisense chain: 5'-fCfAmGfAmAfAmCfUmCfCmGfGmGfCmUfCmGfCmCfAmG*fG*mA-3'(Sequence ID 3050) Sense chain: 5'-mCmUfGmGfCmGfAmGfCmCfCmGfGmAfGmUfUmUfCmUfG-3'(Sequence ID 3010); Antisense chain: 5'-fAfAmAfGmGfCmUfCmGfGmAfGmGfAmGfCmAfGmGfGmC*fG*mG-3'(Sequence ID 3038) Sense chain: 5'-mGmCfCmCfUmGfCmUfCmCfUmCfCmGfAmGfCmCfUmUfU-3' (Sequence ID 2998); Antisense chain: 5'-fGfCmUfUmUfUmGfCmCfCmGfGmGfUmGfCmGfGmAfGmG*fC*mC-3'(Sequence ID 3047) Sense chain: 5'-mCmCfUmCfCmGfCmAfCmCfCmGfGmGfCmAfAmAfAmGfC-3' (Sequence ID 3007); Antisense chain: 5'-fCfCmUfGmUfCmCfCmGfGmGfUmGfCmCfUmGfGmCfCmC*fU*mU-3'(Sequence ID 3045) Sense chain: 5'-mGmGfGmCfCmAfGmGfCmAfCmCfCmGfGmGfAmCfAmGfG-3' (Sequence ID 3005); Antisense chain: 5'-fUfGmAfAmUfCmCfUmGfGmAfCmUfCmCfGmGfGmAfGmG*fC*mC-3'(Sequence ID 3043) Sense chain: 5'-mCmCfUmCfCmCfGmGfAmGfUmCfCmAfGmGfAmUfUmCfA-3'(Sequence ID 3003); Antisense chain: 5'-fGfGmGfAmUfGmCfCmCfAmGfGmAfAmAfGmAfAmUfGmG*fC*mA-3'(Sequence ID 3066) Sense chain: 5'-mCmCfAmUfUmCfUmUfUmCfCmUfGmGfGmCfAmUfCmCfC-3' (Sequence ID 3026); Antisense chain: 5'-fGfAmGfUmCfUmCfUmCfAmCfCmGfGmGfCmCfUmAfGmA*fC*mC-3'(Sequence ID 3062) Sense chain: 5'-mUmCfUmAfGmGfCmCfCmGfGmUfGmAfGmAfGmAfCmUfC-3'(Sequence ID 3022); Antisense chain: 5'-fCfCmGfUmUfUmCfUmAfGmGfAmGfAmGfGmUfUmGfCmG*fC*mC-3'(Sequence ID 3056) Sense chain: 5'-mCmGfCmAfAmCfCmUfCmUfCmCfUmAfGmAfAmAfCmGfG-3' (Sequence ID 3016); Antisense chain: 5'-fCfGmGfUmCfCmUfCmCfCmGfGmCfUmUfUmUfGmCfCmC*fG*mG-3'(Sequence ID 3048) Sense chain: 5'-mGmGfGmCfAmAfAmAfGmCfCmGfGmGfAmGfGmAfCmCfG-3' (Sequence ID 3008); Antisense chain: 5'-fCfCmAfGmCfGmAfGmGfAmGfCmCfUmGfAmGfGmGfUmG*fG*mG-3' (Sequence ID 3046) Sense chain: 5'-mCmAfCmCfCmUfCmAfGmGfCmUfCmCfUmCfGmCfUmGfG-3' (Sequence ID 3006); Antisense chain: 5'-fGfCmUfUmCfCmAfGmCfGmAfGmGfCmGfGmCfCmUfCmU*fU*mC-3'(Sequence ID 3058) Sense chain: 5'-mAmGfAmGfGmCfCmGfCmCfUmCfGmCfUmGfGmAfAmGfC-3' (Sequence ID 3018); Antisense chain: 5'-fGfGmGfCmGfGmUfCmUfGmGfGmAfUmCfCmGfGmUfGmA*fC*mG-3'(Sequence ID 3037) Sense chain: 5'-mUmCfAmCfCmGfGmAfUmCfCmCfAmGfAmCfCmGfCmCfC-3' (Sequence ID 2997); Antisense chain: 5'-fGfUmAfUmUfCmUfUmCfCmUfCmGfCmUfGmAfGmGfGmG*fU*mG-3'(Sequence ID 3060) Sense chain: 5'-mCmCfCmCfUmCfAmGfCmGfAmGfGmAfAmGfAmAfUmAfC-3'(Sequence ID 3020); and Antisense chain: 5'-fUfGmCfUmGfCmAfGmAfAmAfCmUfCmCfGmGfGmCfUmC*fG*mC-3'(Sequence ID 3051) Sense chain: 5'-mGmAfGmCfCmCfGmGfAmGfUmUfUmCfUmGfCmAfGmCfA-3' (Sequence ID 3011) Provides oligonucleotides containing siRNA oligonucleotides selected from; Here, "m" refers to a 2'-O-methyl (2'-O-Me) modified nucleoside; "f" refers to a 2'-fluoro (2'-F) modified nucleoside; "*" refers to a phosphorothioate nucleoside linkage; and the absence of "*" between two nucleosides refers to a phosphodiester nucleoside linkage.

[0035] Other aspects of the present invention are as follows: Antisense chain: 5'-fUfGmGfAmAfAmGfxCmGfAmUfCmCfUmUfCmUfCmAfAmA*fG*mG-3'(Sequence ID 3040) Sense chain: 5'-mUmUfUmGfAmGfAmAfGmGfAmUfxCmGfCmUfUmUfCmCfA-3'(Sequence ID 3000); Antisense chain: 5'-fCfxCmGfGmUfAmUfUmCfUmUfCmCfUmxCfGmCfUmGfAmG*fG*mG-3' (Sequence ID 3061) Sense chain: 5'-mCmUfCmAfGmxCfGmAfGmGfAmAfGmAfAmUfAmCfxCmGfG-3' (Sequence ID 3021); Antisense chain: 5'-fGfUmCfCmAfAmAfxCmGfAmGfUmCfUmCfxCmGfUmxCfGmC*fxC*mG-3' (Sequence ID 3027) Sense chain: 5'-mGmxCfGmAfxCmGfGmAfGmAfCmUfxCmGfUmUfUmGfGmAfC-3'(Sequence ID 2987); Antisense chain: 5'-fGfGmGfxCmGfGmUfCmUfGmGfGmAfUmCfxCmGfGmUfGmA*fxC*mG-3'(Sequence ID 3037) Sense chain: 5'-mUmCfAmCfxCmGfGmAfUmCfCmCfAmGfAmCfxCmGfCmCfC-3'(Sequence ID 2997); Antisense chain: 5'-fxCfGmAfUmCfCmUfUmCfUmCfAmAfAmGfGmCfUmxCfGmG*fA*mG-3'(Sequence ID 3039) Sense chain: 5'-mCmxCfGmAfGmCfCmUfUmUfGmAfGmAfAmGfGmAfUmxCfG-3'(Sequence ID 2999); Antisense chain: 5'-fGfxCmGfAmUfGmCfCmUfGmGfAmAfAmGfxCmGfAmUfCmC*fU*mU-3'(Sequence ID 3041) Sense chain: 5'-mGmGfAmUfxCmGfCmUfUmUfCmCfAmGfGmCfAmUfxCmGfC-3'(Sequence ID 3001); Antisense chain: 5'-fCfUmGfAmAfUmCfCmUfGmGfAmCfUmCfxCmGfGmGfAmG*fG*mC-3'(Sequence ID 3044) Sense chain: 5'-mCmUfCmCfxCmGfGmAfGmUfCmCfAmGfGmAfUmUfCmAfG-3'(Sequence ID 3004); and Antisense chain: 5'-fUfUmCfUmAfGmGfAmGfAmGfGmUfUmGfxCmGfCmCfUmG*fC*mU-3'(Sequence ID 3052) Sense chain: 5'-mCmAfGmGfxCmGfCmAfAmCfCmUfCmUfCmCfUmAfGmAfA-3' (Sequence ID 3012) Provides oligonucleotides containing siRNA oligonucleotides selected from; Here, "m" refers to a 2'-O-methyl (2'-O-Me) modified nucleoside; "f" refers to a 2'-fluoro (2'-F) modified nucleoside; "mxC" refers to a 2'-O-Me modified 5-methylcytidine; "fxC" refers to a 2'-F modified 5-methylcytidine; "*" refers to a phosphorothioate nucleoside linkage; and the absence of "*" between two nucleosides refers to a phosphodiester nucleoside linkage.

[0036] Another aspect of the present invention provides a complex comprising a muscle targeting agent covalently linked to an oligonucleotide targeting double homeobox 4 (DUX4) mRNA, wherein the oligonucleotide comprises an antisense chain of 18 to 25 nucleotides in length and a region of complementarity to the target sequence as represented by SEQ ID NOs. 163 to 1574, wherein the complementarity region is a sequence of at least 16 consecutive nucleosides. In some embodiments, the muscle targeting agent is an anti-transferrin receptor (TfR) antibody. In some embodiments, the oligonucleotide is an RNAi oligonucleotide. In some embodiments, the antisense chain comprises one nucleotide sequence from SEQ ID NOs. 1575 to 2986. [Brief explanation of the drawing]

[0037] Simple description of the drawing [Figure 1] Figure 1 shows a non-limiting schematic diagram illustrating the effect of transfecting cells with siRNA.

[0038] [Figure 2] Figure 2 shows a non-restrictive schematic diagram illustrating the activity of a muscle targeting complex containing siRNA.

[0039] [Figure 3]Figures 3A-3B show non-restrictive schematic diagrams illustrating the activity of siRNA-containing muscle targeting complexes in in vivo mouse muscle tissue (gastrocnemius and heart) compared to vehicle-treated controls. (N=4 C57BL / 6 WT mice)

[0040] [Figure 4A-B] Figures 4A-4E illustrate non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing siRNA. [Figure 4C-D] Figures 4A-4E illustrate non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing siRNA. [Figure 4E] Figures 4A-4E illustrate non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing siRNA.

[0041] [Figure 5A] Figures 5A–5B show the activity of DUX4-targeted siRNAs listed in Table 8 in knockdown of DUX4 mRNA expression in Hepa1–6 cells. Figure 5A shows the activity of siRNAs in knockdown of DUX4 mRNA when Hepa1–6 cells were treated with 2 nM or 10 nM of each indicated siRNA. [Figure 5B] Figure 5B shows the dose-response curve for siRNA9 that produces an IC50 value of 176 pM.

[0042] [Figure 6A-D] Figures 6A–6H are dose-response curves showing the reduction of MBD3L2 mRNA after transfection of AB1080 immortalized FSHD patient-derived myotubes with specific DUX4-targeted siRNAs listed in Table 8 at various concentrations. The siRNAs tested were siRNA9 (Figure 6A); siRNA14 (Figure 6B); siRNA35 (Figure 6C), siRNA13 (Figure 6D), siRNA15 (Figure 6E), siRNA1 (Figure 6F), siRNA26 (Figure 6G), and siRNA18 (Figure 6H). [Figure 6E-H]Figures 6A–6H are dose-response curves showing the reduction of MBD3L2 mRNA after transfection of AB1080 immortalized FSHD patient-derived myotubes with specific DUX4-targeted siRNAs listed in Table 8 at various concentrations. The siRNAs tested were siRNA9 (Figure 6A); siRNA14 (Figure 6B); siRNA35 (Figure 6C), siRNA13 (Figure 6D), siRNA15 (Figure 6E), siRNA1 (Figure 6F), siRNA26 (Figure 6G), and siRNA18 (Figure 6H). [Figure 7] Figure 7 shows the composite mRNA levels of three DUX4 transcriptome markers (MBD3L2, TRIM43, and ZSCAN4) in myotubes from AB1080 immortalized FSHD patients after incubation with siRNA conjugates containing siRNA9, siRNA14, or siRNA35 (corresponding to siRNA9, siRNA14, and siRNA35 in Table 8) covalently linked with anti-TfR Fab 3M12 VH4 / Vκ3. The anti-TfR Fab was covalently linked to the 3' end of the sense strand of each siRNA via a linker, and the corresponding antisense strand was annealed to the sense strand. [Modes for carrying out the invention]

[0043] Detailed description Several aspects of this disclosure provide oligonucleotides designed to target DUX4 RNA. In some embodiments, this disclosure provides oligonucleotides complementary to DUX4 RNA that are useful in reducing levels of DUX4 mRNA and / or protein associated with the pathological conditions of facioscapulohumeral muscular dystrophy (FSHD), including muscular atrophy, inflammation, and reduced differentiation ability and oxidative stress. In some embodiments, the oligonucleotides provided herein are designed to direct RNAi-mediated degradation of DUX4 RNA. In some embodiments, the oligonucleotides are designed to efficiently involve the RNA-induced silencing complex (RISC) for DUX4 RNA degradation while also reducing off-target effects. In some embodiments, the oligonucleotides are designed to reduce levels of DUX4 RNA and / or protein. In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum stability. In some embodiments, the oligonucleotides are designed to have desired binding affinity. In some embodiments, the oligonucleotides are designed to have a desired toxicity and / or immunogenicity profile.

[0044] In several aspects, this disclosure provides a complex comprising a muscle targeting agent covalently linked to a DUX4-targeted oligonucleotide for effective delivery of the oligonucleotide to muscle cells. In some embodiments, the complex is particularly useful for delivering a molecular payload that inhibits the expression or activity of a target gene in muscle cells, for example, in subjects having or suspected of having a rare muscle disease. For example, in some embodiments, the complex is provided to target DUX4 to treat subjects having FSHD. In some embodiments, the complex provided herein comprises an oligonucleotide that inhibits the expression of DUX4 in subjects having one or more D4Z4 repeat deletions on chromosome 4.

[0045] Further aspects of this disclosure, including the definition of terms, are provided below.

[0046] I. Definition To administer (give): As used herein, the term “administer” or “give” means to provide a complex to a subject in a physiologically and / or pharmacologically useful manner (for example, to treat a disease in the subject).

[0047] about: When used herein, the terms “approximately” or “about” refer to a value similar to the given reference value when applied to one or more values ​​of interest. In some embodiments, unless otherwise stated or evident from the context (except where such a figure exceeds 100% of a feasible value), the terms “approximately” or “about” refer to a broad range of values ​​that fall within plus or minus (greater than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than these values.

[0048] antibody: As used herein, the term “antibody” refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant, for example, a paratope that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from an antibody of a camelid animal or a nanobody derived from a shark antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody comprises a framework having a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant region selected from the group consisting of constant regions of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. In some embodiments, the antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or (for example, and), a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody comprises a constant region, for example, an Fc region. The immunoglobulin constant region refers to the heavy chain or light chain constant region. The amino acid sequences of the human IgG heavy chain and light chain constant region and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of the antibody described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, the antibody described herein comprises a human gamma-1 CH1 domain, a CH2 domain, and / or (for example, and), a CH3 domain. In some embodiments, the amino acid sequence of the VH domain includes the amino acid sequence of the human gamma (γ) heavy chain constant region, for example, any sequence known in the art.Non-limiting examples of human constant region sequences are described in the art; see, for example, U.S. Patent No. 5,693,780 and Kabat EA et al. (1991) above. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, SUMOylation, and / or methylation (e.g., and)). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glyciaeation (GPI anchor attachment), and / or phosphoglycosylation (e.g., and). In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide containing one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues linked by a peptide bond and is used to link one or more antigen-binding sites. Examples of linker polypeptides have been reported (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Furthermore, antibodies may also be part of larger immunoadhesion molecules formed by covalent or noncovalent bonds between the antibody or antibody moiety and one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to construct tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to construct divalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol.Immunol. 31:1047-1058).

[0049] CDR: As used herein, the term “CDR” refers to the complementarity-determining region within an antibody variable sequence. A typical antibody molecule contains a heavy chain variable region (VH) and a light chain variable region (VL), which are typically involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as “complementarity-determining regions” (“CDR”), which are interspersed with more conserved regions known as “framework regions” (“FR”). Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methodologies known in the art, for example, by Kabat definitions, IMGT definitions, Chothia definitions, AbM definitions, and / or (as an example, and) contact definitions, all of which are well known in the art.For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark) imgt.org, Lefranc, M.-P. et al., Nucleic Acids. Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[Epub],5:45-60(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,33:D593-597(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,37:D1006-1012(2009);Lefranc,M.-P.et al.,Nucleic Acids Res.,43:D413-422(2015);Chothia et al.,(1989)Nature 342:877;Chothia,C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. See al(1997)J.Molec.Biol.273:927-948; and Almagro,J.Mol.Recognit.17:132-143(2004). Also see hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDR may mean CDR as defined by any method known in the art.Two antibodies having the same CDR mean that, if determined in the same way, for example by the IMGT definition, the two antibodies have the same amino acid sequence of their CDR.

[0050] There are three CDRs in each of the variable regions of the heavy and light chains, designated as CDR1, CDR2, and CDR3 for each variable region. The term “CDR set,” as used herein, refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The precise boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al., Sequence of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and (1991)) not only provides a unique residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Subportions of the CDRs may be designated as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol This is described in 262(5):732-45(1996). Further CDR boundary definitions do not have to strictly adhere to one of the systems above, but may still overlap with Kabat CDRs, and may be shortened or lengthened based on predictions or experimental findings that specific residues, groups of residues, or even the entire CDR do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are shown in Table 1. Table 1. CDR definition [Table 1] 1 IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark), imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999) 2 Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 3 Chothia et al., J.Mol.Biol.196:901-917(1987))

[0051] CDR-grafted antibodies: The term "CDR-conjugated antibody" refers to an antibody that contains heavy chain and light chain variable region sequences from one species, but in which one or more sequences of the VH and / or VL CDR regions are replaced with CDR sequences from another species. This includes antibodies that have mouse heavy chain and light chain variable regions, but in which one or more of the mouse CDR (e.g., CDR3) are replaced with human CDR sequences.

[0052] Chimeric antibodies: The term "chimeric antibody" refers to an antibody that contains heavy chain and light chain variable region sequences from one species and a constant region sequence from another species, such as an antibody that has mouse heavy chain and light chain variable regions linked to a human constant region.

[0053] Complementary: As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleotides or two sets of nucleotides. In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in a bond between two nucleotides or two sets of nucleotides. The term “complementary” may also refer to the capacity for precise pairing between two nucleosides or two sets of nucleosides. In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in a bond between two nucleosides or two sets of nucleosides. For example, if a base of an oligonucleotide at a certain position can hydrogen bond with a base of a target nucleic acid (e.g., mRNA) at a corresponding position, then the bases are considered complementary at that position. Base pairings may include both standard Watson-Crick base pairings and non-Watson-Crick base pairings (e.g., Wobble base pairings and Hoogsteen base pairings). For example, in some embodiments, as complementary base pairings, an adenosine base (A) is complementary to a thymidine base (T) or a uracil base (U), a cytosine base (C) is complementary to a guanosine base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U, or T and is considered complementary to them. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U, or T.

[0054] Conserved amino acid substitutions: As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the amino acid substitution is made. Variants may be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, references summarizing such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made to amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0055] Covalently linked: As used herein, the term “covalently linked” refers to the characteristic of two or more molecules linked together by at least one covalent bond. In some embodiments, two molecules may be covalently linked together by a single bond (e.g., a disulfide bond or disulfide bridge) acting as an intermolecular linker. However, in some embodiments, two or more molecules may be covalently linked together by a molecule acting as a linker, which binds two or more molecules together through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be an incleavable linker.

[0056] Cross-reaction: As used herein, and in the context of targeting agents (e.g., antibodies), the term “cross-reacting” refers to the property of an agent to be able to specifically bind to one or more antigens of a similar type or class (e.g., multiple homologous, paralogous, or orthologous antigens) with similar affinity or binding activity. For example, in some embodiments, an antibody that cross-reacts to antigens of a similar type or class between humans and non-human primates (e.g., human transferrin receptors and non-human primate transferrin receptors) is capable of binding to human antigens and non-human primate antigens with similar affinity or binding activity. In some embodiments, an antibody cross-reacts to human antigens and rodent antigens of a similar type or class. In some embodiments, an antibody cross-reacts to rodent antigens of a similar type or class and non-human primate antigens. In some embodiments, an antibody cross-reacts to human antigens, non-human primate antigens, and rodent antigens of a similar type or class.

[0057] DUX4: As used herein, the term “DUX4” refers to the gene encoding double homeobox 4, a protein commonly expressed during fetal development and in the testes of adult males. In some embodiments, DUX4 may be a human gene (Gene ID: 100288687), a non-human animal of the non-human primate order (e.g., Gene ID: 750891, Gene ID: 100405864), or a rodent gene (e.g., Gene ID: 306226). In humans, DUX4 gene expression outside of fetal development and the testes is associated with facioscapulohumeral muscular dystrophy. In addition, several human transcript variants encoding various protein isoforms have been characterized (e.g., as annotated with GenBank RefSeq accessions: NM_001293798.2, NM_001306068.2, NM_001363820.1).

[0058] Facioscapulohumeral Muscular Dystrophy (FSHD): As used herein, the term “facioscapulohumeral muscular dystrophy (FSHD)” refers to a genetic disorder caused by mutations in the DUX4 or SMCHD1 gene, characterized primarily by decreased muscle mass and atrophy in the muscles of the face, scapula, and upper arm. Two types of the disorder, type 1 and type 2, are described. Type 1 is associated with a deletion in the D4Z4 repeat region on chromosome 4 containing the DUX4 gene. In some embodiments, type 1 is associated with a deletion in the D4Z4 repeat region on variant 4qA of the chromosome 4 allele containing the DUX4 gene. Type 2 is associated with a mutation in the SMCHD1 gene. Both type 1 and type 2 FSHD are characterized by abnormal production of the DUX4 protein after fetal development and outside the testes. Facioscapulohumeral muscular dystrophy, the genetic basis of the disorder, and associated symptoms are described in the art. (For example, see Campbell, AE, et al., "Facioscapulohumeral dystrophy: Activating an early embryonic transcriptional program in human skeletal muscle," Human Mol Genet. (2018); and Tawil, R. "Facioscapulohumeral muscular dystrophy," Handbook Clin. Neurol. (2018), 148:541-548.) FSHD type 1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 158900. FSHD type 2 is associated with OMIM Entry # 158901.

[0059] Framework: As used herein, the term “framework” or “framework sequence” refers to the sequence remaining in the variable region after subtracting the CDRs. Since the precise definition of a CDR sequence can be determined by various systems, the meaning of a framework sequence depends on correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 on the light chain, and CDR-H1, CDR-H2, and CDR-H3 on the heavy chain) also divide the framework regions on the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. When referred to elsewhere, framework regions that do not specify a particular sub-region as FR1, FR2, FR3, or FR4 represent the combined FRs within the variable region of a naturally occurring single immunoglobulin chain. When used herein, FR represents one of four subregions, and FR(plural) represents two or more of the four subregions containing the framework region. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0060] Human antibodies: When used herein, the term “human antibody” is intended to encompass antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of this disclosure may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences (mutations introduced, for example, by random mutagenesis or site-directed mutagenesis in vitro, or by somatic mutation in vivo), particularly in CDRs, especially CDR3. However, when used herein, the term “human antibody” is not intended to encompass antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, are conjugated onto a human framework sequence.

[0061] Humanized antibodies: The term “humanized antibody” refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH sequence and / or (e.g., and) VL sequence has been modified to be more “human-like,” i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-conjugated antibody in which a human CDR sequence is introduced onto non-human VH and VL sequences and replaced with the corresponding non-human CDR sequence. In one embodiment, a humanized anti-transferrin receptor antibody and antigen-binding moiety are provided. Such an antibody may be produced by obtaining a mouse anti-transferrin receptor monoclonal antibody using existing hybridoma technology followed by in vitro genetic engineering for humanization (e.g., disclosed in PCT publication WO 2005 / 123126 A2 by Kasaian et al.).

[0062] Internalized cell surface receptors: As used herein, the term “internalizing cell surface receptor” refers, for example, to a cell surface receptor that is internalized by a cell in response to an external stimulus (e.g., a ligand that binds to the receptor). In some embodiments, the internalizing cell surface receptor is internalized by endocytosis. In some embodiments, the internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalizing cell surface receptor is internalized by clathrin-independent pathways, such as phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or (e.g., and), an extracellular domain, which optionally further comprises a ligand-binding domain. In some embodiments, the cell surface receptor becomes internalized by the cell after ligand binding. In some embodiments, the ligand may be a muscle targeting agent or a muscle targeting antibody. In some embodiments, the internalizing cell surface receptor is a transferrin receptor.

[0063] Isolated antibodies: When used herein, "isolated antibody" is intended to refer to an antibody for which there are substantially no other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to the transferrin receptor has substantially no other antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to the transferrin receptor complex may have cross-reactivity to other antigens, such as transferrin receptor molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or (for example, and) chemicals.

[0064] Kabat numbering: The terms “Kabat numbering,” “Kabat definition,” and “Kabat labeling” are used interchangeably herein. These terms refer to a system of numbering amino acid residues that are recognized in the art but are more variable (i.e., highly variable) than other amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding moiety (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable region extends from amino acid positions 31-35 for CDR1, from amino acid positions 50-65 for CDR2, and from amino acid positions 95-102 for CDR3. In the light chain variable region, the hypervariable region extends to amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

[0065] Molecular payload: As used herein, the term “molecular payload” refers to a molecule or species that functions to modulate a biological outcome. In some embodiments, the molecular payload is ligated to or otherwise linked to a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide containing a chain having a region complementary to a target gene.

[0066] Muscle targeting agents: As used herein, the term “muscle targeting agent” refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on the muscle cell may be a membrane protein, e.g., an endogenous membrane protein or a superficial membrane protein. Typically, a muscle targeting agent specifically binds to an antigen on the muscle cell that facilitates the internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent can specifically bind to an internalizing cell surface receptor on the muscle and be internalized into the muscle cell through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, protein, peptide, nucleic acid (e.g., an aptamer), or antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0067] Muscle targeting antibodies: As used herein, the term “muscle-targeting antibody” refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, a muscle-targeting antibody specifically binds to an antigen on a muscle cell that facilitates the internalization of the muscle-targeting antibody (and any associated molecular payload) into the muscle cell. In some embodiments, a muscle-targeting antibody specifically binds to an internalized cell surface receptor present on the muscle cell. In some embodiments, a muscle-targeting antibody is an antibody that specifically binds to a transferrin receptor.

[0068] Oligonucleotides: As used herein, the term “oligonucleotide” refers to an oligomeric nucleic acid compound with a length of up to 200 nucleotides. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNAs, gapmers, mixmers, phosphorodiamidite morpholino, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), etc. Oligonucleotides may be single-stranded or double-stranded. In some embodiments, oligonucleotides may contain one or more modified nucleotides (e.g., 2'-O-methylglycosyrup, purine, or pyrimidine modifications). In some embodiments, oligonucleotides may contain one or more modified nucleosides (e.g., 2'-O-methylglycosyrup, purine, or pyrimidine modifications). In some embodiments, oligonucleotides may contain one or more modified nucleotide linkages. In some embodiments, oligonucleotides may contain one or more modified nucleoside linkages. In some embodiments, oligonucleotides may contain one or more phosphorothioate linkages, which may be in the stereochemical configuration of Rp or Sp.

[0069] Recombinant antibodies: When used herein, the term “recombinant human antibody” refers to all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (as described in detail herein), antibodies isolated from recombinant combinatorial human antibody libraries (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from human immunoglobulin gene transgenic animals (e.g., mice) (e.g., Taylor, LD, et al.) It is intended to include antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences, as described by al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370). Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, in vivo somatic mutagenesis), and therefore, although the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to the VH and VL sequences of the human germline, they may not be naturally present in the germline repertoire of human antibodies in vivo.One aspect of the present disclosure provides fully human antibodies capable of binding to human transferrin receptors, which may be produced using techniques well known in the art, for example, but not limited to, techniques using a human Ig phage library (e.g., disclosed in PCT publication WO 2005 / 007699 A2 by Jermutus et al.).

[0070] Domain of complementarity: As used herein, the term “complementary region” refers to a nucleotide sequence (e.g., a nucleotide sequence of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., a nucleotide sequence of a target nucleic acid) such that the two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary to the cognate nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95%, or 99% complementary). In some embodiments, the complementary region contains one, two, three, or four mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0071] Binds specifically: As used herein, the term “specifically binds” refers to the ability of a molecule to bind to a binding partner to a degree of affinity or binding activity that the molecule can be used to distinguish the binding partner from a suitable control in a binding assay or other binding context. With respect to antibodies, the term “specifically binds” refers to the ability of an antibody to bind to a specific antigen to a degree of affinity or binding activity (for example, to the extent that it allows preferential targeting to a cell (e.g., muscle cells) through binding to the antigen, as described herein,) compared to a suitable reference antigen, or an antigen that the antibody can be used to distinguish a particular antigen from other antigens. In some embodiments, when the antibody binds to the target, at least about 10 -4 M, 10-5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or K less than this D When having, the antibody binds specifically to the target. In some embodiments, the antibody binds specifically to an epitope of the tip domain of the transferrin receptor, for example, the transferrin receptor.

[0072] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient suspected of having or having a disease, for example, a human patient. In some embodiments, the subject is a human patient suspected of having or having FSHD.

[0073] Transferrin receptor: As used herein, the term “transferrin receptor” (also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalized cell surface receptor that binds to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may originate from humans (NCBI Gene ID 7037), non-human primates (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodents (e.g., NCBI Gene ID 22042). In addition, several human transcript variants encoding various isoforms of the receptor have been characterized (e.g., those annotated with GenBank RefSeq accessions: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0074] 2' Modified nucleoside: As used herein, the terms “2'-modified nucleoside” and “2'-modified ribonucleoside” are used interchangeably and refer to nucleosides having a sugar moiety modified at the 2' position. In some embodiments, a 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, where the 2' and 4' positions of the sugar are cross-linked (e.g., by methylene, ethylene, or (S)-restricted ethyl cross-linking). In some embodiments, a 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, where, for example, the 2' position of the sugar moiety is substituted. Non-limiting examples of 2'-modified nucleosides include: 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acids (LNA, methylene-bridged nucleic acids), ethylene-bridged nucleic acids (ENA), and (S)-restricted ethyl-bridged nucleic acids (cEt). In some embodiments, the 2'-modified nucleosides described herein are high-affinity modified nucleosides, and oligonucleotides containing 2'-modified nucleosides have increased affinity for target sequences compared to unmodified oligonucleotides. An example of the structure of a 2'-modified nucleoside is provided below: [ka] Although these examples are shown with phosphate groups, all nucleoside linkages are intended between 2'-modified nucleosides.

[0075] II. Complexes Provided herein are targeting agents, for example, complexes comprising an antibody covalently linked to a molecular payload. In some embodiments, the complex comprises a muscle-targeting antibody covalently linked to an oligonucleotide. The complex may include an antibody that specifically binds to a single antigen site, or an antibody that binds to at least two antigen sites, which may be present on the same antigen or on different antigens.

[0076] The complex may be used to modulate the activity or function of at least one gene, protein, and / or (for example, and) nucleic acid. In some embodiments, the molecular payload present with the complex is responsible for the modulation of the gene, protein, and / or (for example, and) nucleic acid. The molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any other molecular entity capable of modulating the activity or function of the gene, protein, and / or (for example, and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide targeting DUX4 in muscle cells.

[0077] In some embodiments, the complex comprises a muscle targeting agent (e.g., an anti-transferrin receptor antibody) covalently linked to a molecular payload (e.g., an antisense oligonucleotide targeting DUX4).

[0078] A. Muscle targeting agents Several aspects of this disclosure provide muscle targeting agents, for example, muscle targeting agents for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents can bind to muscle cells, for example, via specific binding to antigens on muscle cells, and deliver the bound molecular payload to the muscle cells. In some embodiments, the molecular payload is bound to the muscle targeting agent (for example, covalently) and, upon binding of the muscle targeting agent to an antigen on a muscle cell, is internalized into the muscle cell, for example, via endocytosis. It should be understood that various types of muscle targeting agents may be used in accordance with this disclosure. It should also be understood that any muscle target (for example, a muscle surface protein) can be targeted by any type of muscle targeting agent described herein. For example, muscle targeting agents may contain, or consist of, nucleic acids (for example, DNA or RNA), peptides (for example, antibodies), lipids (for example, microvesicles), or sugar moieties (for example, polysaccharides). In some embodiments, muscle targeting agents may include or consist of small molecules. Exemplary muscle targeting agents are described in more detail herein, however, it should be understood that the exemplary muscle targeting agents provided herein are not intended to be limiting.

[0079] Several aspects of this disclosure provide muscle targeting agents that specifically bind to antigens on muscles, such as skeletal muscle, smooth muscle, or cardiac muscle. In some embodiments, any of the muscle targeting agents provided herein bind (specifically, for example) to antigens on skeletal muscle cells, smooth muscle cells, and / or (for example, and) cardiac muscle cells.

[0080] Interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins) can achieve both tissue localization and selective uptake into muscle cells. In some embodiments, molecules that are substrates of muscle uptake transporters are useful for delivering molecular payloads into muscle tissue. Binding to muscle surface recognition elements, followed by endocytosis, can allow even macromolecules such as antibodies to enter muscle cells. As another example, a molecular payload conjugated to transferrin or an anti-transferrin receptor antibody may be taken up by muscle cells via binding to the transferrin receptor and then endocytosed, for example, via clathrin-mediated endocytosis.

[0081] The use of muscle-targeting agents can also be useful for enriching molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with the effect in other tissues. In some embodiments, muscle-targeting agents enrich bound molecular payloads in muscle cells compared to other cell types within the subject. In some embodiments, muscle-targeting agents enrich bound molecular payloads in muscle cells (e.g., skeletal muscle cells, smooth muscle cells, or cardiomyocytes) to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more than in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or adipocytes). In some embodiments, the toxicity of the molecular payload in a target when conjugated to a muscle targeting agent is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when it is delivered to the target.

[0082] In some embodiments, muscle recognition elements (e.g., muscle cell antigens) may be required to achieve muscle selectivity. For example, the muscle targeter may be a small molecule that is a substrate of a muscle-specific uptake transporter. Another example is that the muscle targeter may be an antibody that enters muscle cells via transporter-mediated endocytosis. Yet another example is that the muscle targeter may be a ligand that binds to cell surface receptors on muscle cells. It should be understood that while transporter-based approaches provide a direct pathway to cell entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0083] i. Muscle targeting antibody In some embodiments, muscle targeting agents are antibodies. Generally, the high specificity of these antibodies against their target antigens provides the potential to selectively target muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and / or cardiomyocytes). This specificity may also limit off-target toxicity. Examples of antibodies capable of targeting surface antigens of muscle cells have been reported and are within the scope of this disclosure. For example, antibodies that target the surface of muscle cells are described in Arahata K., et al. "Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide" Nature 1988;333:861-3; Song KS, et al. "Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins" J Biol Chem 1996;271:15160-5; and Weisbart RH et al. "Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb" Mol Immunol. 2003 Mar,39(13):783-9; the entire contents of each of these are incorporated herein by reference.

[0084] a. Anti-transferrin receptor (TfR) antibody Several aspects of this disclosure are based on the recognition that agents that bind to transferrin receptors, such as anti-transferrin receptor antibodies, can target muscle cells. Transferrin receptors are internalized cell surface receptors that transport transferrin across the cell membrane and contribute to the regulation of intracellular iron levels and homeostasis. Several aspects of this disclosure provide transferrin receptor-binding proteins capable of binding to transferrin receptors. Consequently, aspects of this disclosure provide binding proteins (e.g., antibodies) that bind to transferrin receptors. In some embodiments, binding proteins that bind to transferrin receptors are internalized into muscle cells along with any bound molecular payload. As used herein, antibodies that bind to transferrin receptors may also be interchangeably referred to as transferrin receptor antibodies, anti-transferrin receptor antibodies, or anti-TfR antibodies. Antibodies that bind to transferrin receptors, such as specifically binding antibodies, may be internalized into cells upon binding to the transferrin receptor, for example, through receptor-mediated endocytosis.

[0085] It should be understood that anti-TfR antibodies may be produced, synthesized, and / or (for example, and) derivatized using several known methodologies, e.g., library design using phage display. Exemplary methodologies are characterized in the art and incorporated by reference (Diez, P. et al. "High-throughput phage-display screening in array format", Enzyme and microbial technology, 2015, 79, 34-41.; Christoph MH and Stanley, JR "Antibody Phage Display: Technique and Applications", J Invest Dermatol. 2014, 134:2.; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies". 1985, Springer). In other embodiments, anti-TfR antibodies have been characterized or disclosed to date.Antibodies that specifically bind to transferrin receptors are known in the art (for example, U.S. Patent No. 4,364,934, filed 12 / 4 / 1979, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing the same"; U.S. Patent No. 8,409,573, filed 6 / 14 / 2006, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Patent No. 9,708,406, filed 5 / 20 / 2014, "Anti-transferrin receptor antibodies and methods of use"; U.S. Patent No. 9,611,323, filed 12 / 19 / 2014, "Low affinity blood brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed 12 / 24 / 2014, "Novel anti-Transferrin receptor antibody that passes through blood-brain barrier"; Schneider C. et al. "Structural features"). of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9."J Biol Chem.1982,257:14,8516-8522.;Lee et al."Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse"2000,J See Pharmacol. Exp. Ther., 292:1048-1052).

[0086] In some embodiments, the anti-TfR antibodies described herein bind to the transferrin receptor with high specificity and affinity. In some embodiments, the anti-TfR antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor or to an epitope that becomes exposed to the antibody. In some embodiments, the anti-TfR antibodies provided herein specifically bind to transferrin receptors from humans, non-human primate animals, mice, rats, etc. In some embodiments, the anti-TfR antibodies provided herein bind to the human transferrin receptor. In some embodiments, the anti-TfR antibodies described herein bind to the amino acid segments of human or non-human primate animal transferrin receptors provided in SEQ ID NOs. 105-108. In some embodiments, the anti-TfR antibodies described herein bind to the amino acid segments corresponding to amino acids 90-96 of the human transferrin receptor as represented by SEQ ID NO. 105, which are not in the apex domain of the transferrin receptor.

[0087] In some embodiments, the anti-TfR1 antibody described herein (for example, anti-TfR clone 8 in Table 2 below) binds to an epitope in TfR1, where the epitope comprises residues at amino acids 214-241 and / or 354-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibody described herein binds to an epitope comprising residues at amino acids 214-241 and 354-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibody described herein binds to an epitope comprising one or more residues of human TfR1 as represented by SEQ ID NO: 105, Y222, T227, K231, H234, T367, S368, S370, T376, and S378. In some embodiments, the anti-TfR1 antibodies described herein bind to epitopes comprising human TfR1 residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 as represented by SEQ ID NO: 105.

[0088] In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and its variants in Table 2 below) bind to an epitope in TfR1, where the epitope comprises residues at amino acids 258-291 and / or 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and its variants in Table 2 below) bind to an epitope comprising residues at amino acids 258-291 and 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and its variants in Table 2 below) bind to an epitope comprising one or more residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as represented by SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and its variants in Table 2 below) bind to epitopes comprising human TfR1 residues K261, S273, Y282, T362, S368, S370, and K371 as represented by SEQ ID NO: 105.

[0089] An example of a human transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows: (Sequence ID 105).

[0090] Examples of non-human primate transferrin receptor amino acid sequences corresponding to the NCBI sequence NP_001244232.1 (transferrin receptor protein 1, Macaca mulatta) are as follows: (Sequence ID 106).

[0091] An example of a non-human primate transferrin receptor amino acid sequence corresponding to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: (Sequence ID 107).

[0092] An example of a mouse transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_001344227.1 (transferrin receptor protein 1, Mus musculus) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYVKIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKNVLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTRLDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF (SEQ ID NO: 108).

[0093] In some embodiments, anti-TfR antibodies bind to the following amino acid segment of the receptor: FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and do not inhibit the binding interaction between the transferrin receptor and transferrin and / or (for example, and) human hemochromatosis protein (also known as HFE). In some embodiments, the anti-TfR antibodies described herein do not bind to the epitope of SEQ ID NO: 109.

[0094] Appropriate methodologies may be used, for example, through the use of recombinant DNA protocols to obtain and / or (for example, and) produce antibodies, antibody fragments, or antigen conjugates. In some embodiments, antibodies may also be produced through the generation of hybridomas (see, for example, Kohler, G and Milstein, C. "Continuous cultures of fused cells secreting antibody of predefined specificity" Nature, 1975, 256:495-497). The antigen of interest may be used as an immunogen of any type or entity, for example, recombinant or naturally occurring type or entity. Hybridomas are screened using standard methods, for example, ELISA screening, to find at least one hybridoma that produces an antibody targeting a specific antigen. Antibodies may also be produced through screening of protein expression libraries expressing the antibody (for example, phage display libraries). Phage display library designs may also be used in several embodiments (see, for example, U.S. Patent No. 5,223,409, 3 / 1 / 1991, "Directed evolution of novel binding proteins"; WO 1992 / 18619, 4 / 10 / 1992, "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, 5 / 1 / 1991, "Recombinant library screening methods"; WO 1992 / 20791, 5 / 15 / 1992, "Methods for producing members of specific binding pairs"; and WO 1992 / 15679, 2 / 28 / 1992, "Improved epitope displaying phage"). In some embodiments, antigens of interest may be used to immunize non-human animals, such as rodents or goats.In some embodiments, once antibodies have been obtained from non-human animals, they may be modified using a number of methodologies, including, for example, recombinant DNA techniques. Examples of antibody production and methodological additions are also known in the art (see, for example, Harlow et al., "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988).

[0095] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, SUMOylation, and / or methylation (e.g., and)). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glycialysis (GPI anchor attachment), and / or phosphoglycosylation (e.g., and). In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the sugar molecules are present in numbers of approximately 1–10, 1–5, 5–10, 1–4, 1–3, or 2. In some embodiments, the glycosylated antibody is glycosylated whole or partially. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell (which may optionally be deficient in enzymes in the N- or O-glycosylation pathway, e.g., glycosyltransferase). In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules as described in the international patent application publication WO2014065661, published on 1 May 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0096] In some embodiments, the anti-TfR antibodies of this disclosure comprise the VL domain and / or (for example, and) the VH domain of any one anti-TfR antibody selected from any one of Tables 2-7, and comprise a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (for example, IgG2a and IgG2b) of an immunoglobulin molecule. Non-limiting examples of human constant regions have been described in the art; see, for example, Kabat EA et al. (1991) above.

[0097] In some embodiments, drugs that bind to transferrin receptors, such as anti-TfR antibodies, can target muscle cells and / or mediate the transport of drugs across the blood-brain barrier (for example, and). Transferrin receptors are internalized cell surface receptors that transport transferrin across the cell membrane and contribute to the regulation of intracellular iron levels and homeostasis. Some aspects of this disclosure provide transferrin receptor-binding proteins capable of binding to transferrin receptors. Antibodies that bind to transferrin receptors, such as specifically binding antibodies, may be internalized into cells upon binding to the transferrin receptor, for example, through receptor-mediated endocytosis.

[0098] In some aspects, antibodies that bind to transferrin receptors with high specificity and affinity are provided herein. In some aspects, the anti-TfR antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor or to an epitope that becomes exposed to the antibody. In some aspects, the anti-TfR antibodies provided herein specifically bind to transferrin receptors from humans, non-human primate animals, mice, rats, etc. In some aspects, the anti-TfR antibodies provided herein bind to human transferrin receptors. In some aspects, the anti-TfR antibodies described herein bind to amino acid segments of human or non-human primate animal transferrin receptors provided in SEQ ID NOs. 105-108. In some aspects, the anti-TfR antibodies described herein bind to amino acid segments corresponding to amino acids 90-96 of the human transferrin receptor as represented by SEQ ID NO. 105, which are not in the apex domain of the transferrin receptor. In some aspects, the anti-TfR antibodies described herein bind to TfR1 but not to TfR2.

[0099] In some embodiments, the anti-TFR antibody is at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13The antibodies specifically bind to TfR1 (e.g., human or non-human primate TfR1) by a binding affinity of M, or a smaller binding affinity (e.g., indicated by Kd). In some embodiments, the anti-TfR antibodies described herein bind to TfR1 by a KD in the sub-nanomole range. In some embodiments, the anti-TfR antibodies described herein selectively bind to transferrin receptor 1 (TfR1) but not to transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR antibodies described herein bind to human TfR1 and cynoporin TfR1 (e.g., 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 The antibodies do not bind to mouse TfR1 (by M or a smaller Kd). The affinity and binding kinetics of anti-TfR antibodies can be tested using any preferred method, including but not limited to biosensor technologies (e.g., OCTET or BIACORE). In some embodiments, the binding of any one of the anti-TfR antibodies described herein does not compete with or inhibit transferrin binding to TfR1. In some embodiments, the binding of any one of the anti-TfR antibodies described herein does not compete with or inhibit HFE-beta-2-microglobulin binding to TfR1.

[0100] Table 2 provides a non-limiting list of anti-TfR antibodies. Table 2. Examples of anti-TfR antibodies [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] *The location of the mutation follows the Kabat numbering of each VH sequence containing the mutation.

[0101] In some embodiments, the anti-TfR antibody of this disclosure is a variant of any one of the anti-TfR antibodies provided in Table 2. In some embodiments, the anti-TfR antibody of this disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, which are the same as CDR-H1, CDR-H2, and CDR-H3 in any one of the anti-TfR antibodies provided in Table 2, and comprises a humanized heavy chain variable region and / or (for example, and) a light chain variable region.

[0102] Examples of amino acid sequences of humanized anti-TfR antibodies described herein are provided in Table 3. Table 3. Variable regions of anti-TfR antibodies [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] * The location of the mutation follows the Kabat numbering of each VH sequence containing the mutation. ** CDRs following the Kabat numbering system are shown in bold.

[0103] In some embodiments, the anti-TfR antibody of this disclosure comprises a VH containing any one of the anti-TfR antibodies provided in Table 3, CDR-H1, CDR-H2, and CDR-H3, and includes one or more amino acid variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) in the framework region when compared to each VH provided in Table 3. Alternatively or in addition (e.g., in addition), the anti-TfR antibody of this disclosure comprises a VL containing any one of the anti-TfR antibodies provided in Table 3, CDR-L1, CDR-L2, and CDR-L3, and includes one or more amino acid variations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) in the framework region when compared to each VL provided in Table 3.

[0104] In some embodiments, the anti-TfR antibody of this disclosure comprises a VH containing any one of the anti-TfR antibodies provided in Table 3, CDR-H1, CDR-H2, and CDR-H3, and contains at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) of the same amino acid sequence in the framework region when compared to each VH provided in Table 3. Alternatively or in addition (e.g., in addition), the anti-TfR antibody of this disclosure comprises a VL containing any one of the anti-TfR antibodies provided in Table 3, CDR-L1, CDR-L2, and CDR-L3, and contains at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) of the same amino acid sequence in the framework region when compared to each VL provided in Table 3.

[0105] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 69, and VL, which contains the amino acid sequence of SEQ ID NO: 70.

[0106] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 71, and VL, which contains the amino acid sequence of SEQ ID NO: 70.

[0107] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 72, and VL, which contains the amino acid sequence of SEQ ID NO: 70.

[0108] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 73, and VL, which contains the amino acid sequence of SEQ ID NO: 74.

[0109] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 73, and VL, which contains the amino acid sequence of SEQ ID NO: 75.

[0110] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 76, and VL, which contains the amino acid sequence of SEQ ID NO: 74.

[0111] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 76, and VL, which contains the amino acid sequence of SEQ ID NO: 75.

[0112] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 77, and VL, which contains the amino acid sequence of SEQ ID NO: 78.

[0113] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 79, and VL, which contains the amino acid sequence of SEQ ID NO: 80.

[0114] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 77, and VL, which contains the amino acid sequence of SEQ ID NO: 80.

[0115] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH containing the amino acid sequence of SEQ ID NO: 154 and VL containing the amino acid sequence of SEQ ID NO: 155.

[0116] In some embodiments, the anti-TfR antibodies described herein are full-length IgG, which may comprise the heavy chain constant region and light chain constant region from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR antibodies described herein may comprise the heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may belong to any preferred origin, e.g., human, mouse, rat, or rabbit. In a particular example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of the human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 81).

[0117] In some embodiments, the heavy chain of any of the anti-TfR antibodies described herein comprises a mutant human IgG1 constant region. For example, the introduction of an LALA mutation on the CH2 domain of human IgG1 (a mutant derived from mAb b12 mutated to replace the lower hinge residues Leu234 Leu235 with Ala234 and Ala235) is known to reduce Fcγ receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). Mutant human IgG1 constant regions are provided below (mutations are in bold and underlined): [ka]

[0118] In some embodiments, any light chain of any of the anti-TfR antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 83).

[0119] The heavy and light chain constant regions of other antibodies are well known in the art and are provided, for example, in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0120] In some embodiments, the anti-TfR antibodies described herein include a heavy chain comprising a VH or any of its variants as listed in Table 3, and a heavy chain comprising a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR antibodies described herein include a heavy chain comprising a heavy chain comprising a heavy chain constant region containing only 25 amino acid variations (for example, only 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) as listed in Table 3. In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising VH or any of its variants as listed in Table 3, and a heavy chain comprising a heavy chain constant region as represented by SEQ ID NO: 81. In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising VH or any of its variants as listed in Table 3, and a heavy chain comprising a heavy chain constant region as represented by SEQ ID NO: 82.

[0121] In some embodiments, the anti-TfR antibody described herein comprises a light chain comprising a VL or any of its variants as listed in Table 3, and a light chain comprising a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR antibody described herein comprises a light chain comprising a VL or any of its variants as listed in Table 3, and a light chain comprising a light chain constant region containing 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR antibody described herein comprises a light chain comprising a VL or any of its variants as listed in Table 3, and a light chain comprising a light chain constant region as represented by SEQ ID NO: 83.

[0122] Examples of IgG heavy and light chain amino acid sequences of the described anti-TfR antibodies are provided in Table 4 below. Table 4. Heavy and light chain sequences of examples of anti-TfR Fabs [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] * The location of the mutation follows the Kabat numbering of each VH sequence containing the mutation. ** CDRs following the Kabat numbering system are shown in bold; VH / VL sequences are underlined.

[0123] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) when compared to the heavy chain shown in any one of SEQ ID NOs. Alternatively or in addition (for example, in addition), the anti-TfR antibody of the present disclosure comprises a light chain having 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the light chain shown in any one of SEQ ID NOs.

[0124] In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs. 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or in addition (e.g., in addition), the anti-TfR antibody described herein comprises a light chain containing an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs. 85, 89, 90, 93, 95, and 157. In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs. 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or in addition (for example, in addition), the anti-TfR antibodies described herein include a light chain comprising any one amino acid sequence of SEQ ID NOs. 85, 89, 90, 93, 95, and 157.

[0125] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 84 and a light chain containing the amino acid sequence of SEQ ID NO: 85.

[0126] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 86 and a light chain containing the amino acid sequence of SEQ ID NO: 85.

[0127] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 87 and a light chain containing the amino acid sequence of SEQ ID NO: 85.

[0128] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 88 and a light chain containing the amino acid sequence of SEQ ID NO: 89.

[0129] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 88 and a light chain containing the amino acid sequence of SEQ ID NO: 90.

[0130] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 91 and a light chain containing the amino acid sequence of SEQ ID NO: 89.

[0131] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 91 and a light chain containing the amino acid sequence of SEQ ID NO: 90.

[0132] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 92 and a light chain containing the amino acid sequence of SEQ ID NO: 93.

[0133] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 94 and a light chain containing the amino acid sequence of SEQ ID NO: 95.

[0134] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 92 and a light chain containing the amino acid sequence of SEQ ID NO: 95.

[0135] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 156 and a light chain containing the amino acid sequence of SEQ ID NO: 157.

[0136] In some embodiments, the anti-TfR antibody is a Fab fragment, Fab' fragment, or F(ab')2 fragment of an intact antibody (full-length antibody). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by standard methods (e.g., by recombination or by digesting the heavy chain constant region of full-length IgG using an enzyme such as papain). For example, the F(ab')2 fragment can be produced by pepsin or papain digestion of the antibody molecule, and the Fab fragment can be produced by reducing the disulfide crosslinks of the F(ab')2 fragment. In some embodiments, the heavy chain constant region on the Fab fragment of the anti-TfR1 antibody described herein comprises the amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96).

[0137] In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 96. In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing a heavy chain constant region that is at least 25 amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to SEQ ID NO: 96. In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing a heavy chain constant region that is at least 25 amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to SEQ ID NO: 96.

[0138] In some embodiments, the anti-TfR antibody described herein comprises a light chain containing a VL or any of its variants as listed in Table 3, and a light chain containing a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR antibody described herein comprises a light chain containing a VL or any of its variants as listed in Table 3, and a light chain containing a light chain constant region with 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR antibody described herein comprises a light chain containing a VL or any of its variants as listed in Table 3, and a light chain containing a light chain constant region represented by SEQ ID NO: 83.

[0139] Examples of Fab heavy and light chain amino acid sequences of the described anti-TfR antibodies are provided in Table 5 below. Table 5. Heavy and light chain sequences of examples of anti-TfR Fabs. [Table 5-1] [Table 5-2] [Table 5-3] * The location of the mutation follows the Kabat numbering of each VH sequence containing the mutation. ** CDRs following the Kabat numbering system are shown in bold; VH / VL sequences are underlined.

[0140] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the heavy chain as represented by any one of SEQ ID NOs. Alternatively or in addition (for example, in addition), the anti-TfR antibody of the present disclosure comprises a light chain having 25 or fewer amino acid variations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the light chain as represented by any one of SEQ ID NOs.

[0141] In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs. Alternatively or in addition (e.g., in addition), the anti-TfR antibody described herein comprises a light chain containing an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs. In some embodiments, the anti-TfR antibody described herein comprises a heavy chain containing an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs. 97-103, 158, and 159. Alternatively or in addition (for example, in addition), the anti-TfR antibodies described herein include a light chain comprising any one amino acid sequence of SEQ ID NOs. 85, 89, 90, 93, 95, and 157.

[0142] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 97 and a light chain containing the amino acid sequence of SEQ ID NO: 85.

[0143] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 98 and a light chain containing the amino acid sequence of SEQ ID NO: 85.

[0144] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 99 and a light chain containing the amino acid sequence of SEQ ID NO: 85.

[0145] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 100 and a light chain containing the amino acid sequence of SEQ ID NO: 89.

[0146] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 100 and a light chain containing the amino acid sequence of SEQ ID NO: 90.

[0147] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 101 and a light chain containing the amino acid sequence of SEQ ID NO: 89.

[0148] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 101 and a light chain containing the amino acid sequence of SEQ ID NO: 90.

[0149] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 102 and a light chain containing the amino acid sequence of SEQ ID NO: 93.

[0150] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 103 and a light chain containing the amino acid sequence of SEQ ID NO: 95.

[0151] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 102 and a light chain containing the amino acid sequence of SEQ ID NO: 95.

[0152] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 158 and a light chain containing the amino acid sequence of SEQ ID NO: 157.

[0153] In some embodiments, the anti-TfR antibody of the present disclosure comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 159 and a light chain containing the amino acid sequence of SEQ ID NO: 157.

[0154] Other known anti-TfR antibodies Any other suitable anti-TfR antibody known in the art may be used as a muscle targeting agent in the conjugate disclosed herein. Examples of known anti-TfR antibodies (including relevant references and binding epitopes) are listed in Table 6. In some embodiments, the anti-TfR antibody comprises a complementation determining region (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any of the anti-TfR antibodies provided herein, for example, those listed in Table 6.

[0155] Table 6 - List of anti-TfR antibody clones, including relevant references and binding epitope information. [Table 6-1] [Table 6-2] [Table 6-3]

[0156] In some aspects, the anti-TfR antibody of this disclosure comprises one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-TfR antibodies selected from Table 6. In some aspects, the anti-TfR antibody comprises CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-TfR antibodies selected from Table 6. In some aspects, the anti-transferrin antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-TfR antibodies selected from Table 6.

[0157] In some embodiments, the anti-TfR antibody of this disclosure comprises any antibody that includes the heavy chain variable domain and / or (for example, and) the light chain variable domain of any anti-TfR antibody, such as any one of the anti-TfR antibodies selected from Table 6. In some embodiments, the anti-TfR antibody of this disclosure comprises any antibody that includes the heavy chain variable and light chain variable pair of any anti-TfR antibody, such as any one of the anti-TfR antibodies selected from Table 6.

[0158] Aspects of this disclosure provide anti-TfR antibodies having heavy-chain variable (VH) and / or (e.g., and) light-chain variable (VL) domain amino acid sequences homologous to any of those described herein. In some embodiments, the anti-TfR antibody includes a heavy-chain variable sequence or a light-chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy-chain variable sequence and / or any light-chain variable sequence of any anti-TfR antibody, such as any one of the anti-TfR antibodies selected from Table 6. In some embodiments, the homologous heavy-chain variable and / or (e.g., and) light-chain variable amino acid sequences are not varied in any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur within the heavy-chain variable and / or (e.g., and) light-chain variable sequences that exclude any of the CDR sequences provided herein. In some embodiments, any of the anti-TfR antibodies provided herein include a heavy chain variable sequence and a light chain variable sequence that include a framework sequence which is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequence of any anti-TfR antibody, such as any one of the anti-TfR antibodies selected from Table 6.

[0159] Examples of transferrin receptor antibodies that may be used in accordance with this disclosure are described in the international patent publication WO 2016 / 081643 (incorporated herein by reference). The amino acid sequences of these antibodies are provided in Table 7. Table 7. Heavy and light chain CDRs of known anti-TfR examples. [Table 7-1] [Table 7-2]

[0160] In some aspects, the anti-TfR antibodies of this disclosure include CDR-H1, CDR-H2, and CDR-H3, which are the same as CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. Alternatively or in addition (for example, in addition), the anti-TfR antibodies of this disclosure include CDR-L1, CDR-L2, and CDR-L3, which are the same as CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

[0161] In some embodiments, the anti-TfR antibody of this disclosure contains CDR-L3, which has three or fewer amino acid variations (e.g., three, two, or one or fewer amino acid variations) compared to CDR-L3 as shown in Table 7. In some embodiments, the anti-TfR antibody of this disclosure contains CDR-L3 having one amino acid variation compared to CDR-L3 as shown in Table 7. In some embodiments, the anti-TfR antibody of this disclosure contains CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system). In some embodiments, the anti-TfR antibodies of this disclosure include CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2, which are the same as CDR-H1, CDR-H2, and CDR-H3 shown in Table 7, and include CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system).

[0162] In some embodiments, the anti-TfR antibody of this disclosure comprises a heavy chain CDR as shown in Table 7, and a heavy chain CDR that is identical to it by at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) in combination. Alternatively or in addition (e.g., in addition), the anti-TfR antibody of this disclosure comprises a light chain CDR as shown in Table 7, and a light chain CDR that is identical to it by at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) in combination.

[0163] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 124. Alternatively or in addition (for example, in addition), the anti-TfR antibody of the present disclosure comprises VL, which contains the amino acid sequence of SEQ ID NO: 125.

[0164] In some embodiments, the anti-TfR antibody of the present disclosure comprises VH, which contains the amino acid sequence of SEQ ID NO: 128. Alternatively or in addition (for example, in addition), the anti-TfR antibody of the present disclosure comprises VL, which contains the amino acid sequence of SEQ ID NO: 129.

[0165] In some embodiments, the anti-TfR antibody of the present disclosure comprises a VH having 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to a VH as represented by SEQ ID NO: 128. Alternatively or in addition (for example, in addition), the anti-TfR antibody of the present disclosure comprises a VL having 15 or fewer amino acid variations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to a VL as represented by SEQ ID NO: 129.

[0166] In some embodiments, the anti-TfR antibody of this disclosure is a full-length IgG1 antibody that may contain heavy chain constant regions and light chain constant regions from a human antibody. In some embodiments, any heavy chain of the anti-TfR antibody as described herein may contain a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may belong to any preferred source, e.g., human, mouse, rat, or rabbit. In a particular example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (Sequence ID 81).

[0167] In some embodiments, any light chain of the anti-TfR antibody described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (Sequence ID 83).

[0168] In some embodiments, the anti-TfR antibody described herein is a chimeric antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 132. Alternatively or in addition (for example, in addition), the anti-TfR antibody described herein comprises a light chain containing the amino acid sequence of SEQ ID NO: 133.

[0169] In some embodiments, the anti-TfR antibody described herein is a fully human antibody comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 134. Alternatively or in addition (for example, in addition), the anti-TfR antibody described herein comprises a light chain containing the amino acid sequence of SEQ ID NO: 135.

[0170] In some embodiments, the anti-TfR antibody is an antigen-binding fragment (Fab) of an intact antibody (full-length antibody). In some embodiments, the anti-TfR Fab described herein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 136. Alternatively or in addition (for example, in addition), the anti-TfR Fab described herein comprises a light chain containing the amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-TfR Fab described herein comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 137. Alternatively or in addition (for example, in addition), the anti-TfR Fab described herein comprises a light chain containing the amino acid sequence of SEQ ID NO: 135.

[0171] The anti-TfR antibodies described herein may be in any form, but are not limited to, intact (i.e., full-length) antibodies, their antigen-binding fragments (such as Fab, Fab', F(ab')2, Fv, etc.), single-chain antibodies, bispecific antibodies, or antibodies comprising nanobodies. In some embodiments, the anti-TfR antibodies described herein are scFv. In some embodiments, the anti-TfR antibodies described herein are scFv-Fab (e.g., scFv condensed with a portion of the constant region). In some embodiments, the anti-TfR antibodies described herein are scFv condensed with a constant region (e.g., the human IgG1 constant region as represented by SEQ ID NO: 81).

[0172] In some embodiments, conservative mutations can be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where, when determined, for example based on the crystal structure, the residue is unlikely to be involved in interaction with the target antigen (e.g., a transferrin receptor). In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1) and / or (e.g., and) in the hinge region) to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or (e.g., and).

[0173] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased) as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, for example, to facilitate the assembly of the light and heavy chains, or to change the stability of the antibody (e.g., increased or decreased), or to facilitate linker conjugation.

[0174] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeting antibodies described herein (e.g., numbered according to the Kabat numbering system (e.g., Kabat's EU index), in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region) to increase or decrease the affinity of the antibody for Fc receptors on effector cells (e.g., activated Fc receptors). Mutations in the Fc region of an antibody that increase or decrease its affinity for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof, are known to those of skill in the art. Examples of mutations in Fc receptors of an antibody that can be made to alter its affinity for Fc receptors are described, e.g., in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publications No. WO 02 / 060919; No. WO 98 / 23289; and No. WO 97 / 34631 (which are incorporated herein by reference).

[0175] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably, the Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the in vivo half-life of the antibody. See, e.g., International Publications No. WO 02 / 060919; No. WO 98 / 23289; and No. WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that would alter (e.g., increase or decrease) the in vivo half-life of an antibody.

[0176] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably Fc or hinge-Fc domain fragment) to reduce the half-life of the anti-TfR antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341-447 of human IgG1) in numbering according to the Kabat EU index (Kabat EA et al. (1991) above). In some embodiments, the IgG1 constant region of the antibodies described herein includes a methionine (M) to tyrosine (Y) substitution at position 252, numbered according to the EU index as found in Kabat, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256. See U.S. No. 7,658,921 (which is incorporated herein by reference). This type of mutant IgG, referred to as the “YTE mutant,” has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody includes an IgG constant region containing one, two, or three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as found in Kabat.

[0177] In some embodiments, one or more amino acid substitutions are introduced into the Fc region of the IgG constant region to alter one or more effector functions of the anti-TfR antibody. The effector ligand with altered affinity for itself can be, for example, an Fc receptor or a C1 component of complement. This approach is described in more detail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation (through point mutation or other means) of a constant region domain can reduce binding of the circulating antibody to Fc receptors, thereby increasing tumor localization. For a description of mutations that delete or inactivate the constant region and thereby increase tumor localization, see, by way of example, U.S. Pat. Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region (which may also reduce binding to Fc receptors) (see, by way of example, Shields R L et al., (2001) J Biol Chem 276:6591-604).

[0178] In some embodiments, one or more amino acid residues in the constant region of the muscle-targeting antibody described herein may be replaced with different amino acid residues so that the antibody may have modified Clq binding and / or (for example, and) reduced or absent complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are modified to alter the complement-binding ability of the antibody. This approach is described in further international publication WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) to cells and / or (for example, and) to increase the antibody's affinity for the Fcγ receptor. This approach is described in further international publication WO 00 / 42072.

[0179] In some embodiments, the heavy chain and / or (for example, and) light chain variable domain(s) sequences(s) of the antibodies provided herein may be used, as described elsewhere herein, to generate, for example, CDR-conjugated antibodies, chimeric antibodies, humanized antibodies, or compound human antibodies, or antigen-binding fragments. As will be understood by those skilled in the art, any variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and the variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody will maintain its specific binding ability to the transferrin receptor such that it may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% binding to the transferrin receptor compared to the original antibody from which it is derived.

[0180] In some embodiments, the antibodies provided herein include mutations that confer desired properties to the antibody. For example, to avoid potential complications resulting from Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may contain a stabilizing "Adair" mutation.

[0181] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, SUMOylation, and / or methylation (e.g., and)). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glycialysis (GPI anchor attachment), and / or phosphoglycosylation (e.g., and). In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the sugar molecules are present in numbers of approximately 1–10, 1–5, 5–10, 1–4, 1–3, or 2. In some embodiments, the glycosylated antibody is glycosylated whole or partially. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell (which may optionally be deficient in enzymes in the N- or O-glycosylation pathway, e.g., glycosyltransferase). In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules as described in the international patent application publication WO2014065661, published on 1 May 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0182] In some embodiments, any one of the anti-TfR antibodies described herein may comprise a signal peptide (e.g., an N-terminal signal peptide) on the heavy chain and / or (e.g., and) light chain sequences. In some embodiments, the anti-TfR1 antibody described herein comprises either one of the VH and VL sequences, either one of the IgG heavy chain and light chain sequences, or either one of the Fab heavy chain and light chain sequences described herein, and further comprises a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide comprises the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0183] In some embodiments, antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also called pyroglutamate formation (pyroGlu), may occur in the antibody at N-terminal glutamate (Glu) and / or glutamine (Gln) residues during production. Therefore, antibodies identified as having a sequence containing an N-terminal glutamate or glutamine residue should be understood to include antibodies that have undergone pyroglutamate formation due to post-translational modification. In some embodiments, pyroglutamate formation occurs in the heavy chain sequence. In some embodiments, pyroglutamate formation occurs in the light chain sequence.

[0184] b. Other muscle-targeting antibodies In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin IIb, or CD63. In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to myogenic precursor proteins. Exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha-7, integrin alpha-7 beta-1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to skeletal muscle proteins. The exemplified skeletal muscle proteins include, but are not limited to, alpha-sarcoglycans, beta-sarcoglycans, calpain inhibitors, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycans, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha-7, integrin alpha-7 beta-1, integrin beta-1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to smooth muscle proteins. The exemplary smooth muscle proteins include, but are not limited to, alpha-smooth muscle actin, VE-cadherin, cardesmon / CALD1, carponin 1, desmin, histamine H2 R, motilin R / GPR38, transgelin / TAGLN, and vimentin. However, it should be understood that antibodies against additional targets are within the scope of this disclosure, and that the list of exemplary targets provided herein is not intended to be limiting.

[0185] c. Characteristics / Changes of Antibodies In some embodiments, conservative mutations can be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where, when determined, for example based on the crystal structure, the residue is unlikely to be involved in interaction with the target antigen (e.g., a transferrin receptor). In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1) and / or (e.g., and) in the hinge region) to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or (e.g., and).

[0186] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased) as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, for example, to facilitate the assembly of the light and heavy chains, or to change the stability of the antibody (e.g., increased or decreased), or to facilitate linker conjugation.

[0187] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1) and / or (e.g., and) in the hinge region) to increase or decrease the antibody's affinity for Fc receptors on the surface of effector cells. Mutations in the Fc region of antibodies that increase or decrease the antibody's affinity for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof are known to those skilled in the art. Examples of mutations in the Fc receptor of an antibody that may be made to alter the antibody's affinity for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and international publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631 (these are incorporated herein by reference).

[0188] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably an Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. For example, see international publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that would alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0189] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the Kabat EU index (Kabat EA et al. (1991) above). In some embodiments, the IgG1 constant region of the antibodies described herein includes a methionine (M) to tyrosine (Y) substitution at position 252, numbered according to the EU index as found in Kabat, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256. See U.S. No. 7,658,921 (which is incorporated herein by reference). This type of mutant IgG, referred to as the “YTE mutant,” has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody includes an IgG constant region containing one, two, or three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as found in Kabat.

[0190] In some embodiments, one or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function (one or more) of an anti-transferrin receptor antibody. The effector ligand with altered affinity to itself may be, for example, the Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patents 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) may reduce the binding of the circulating antibody to the Fc receptor, thereby increasing tumor localization. For descriptions of mutations that delete or inactivate the constant region and thereby increase tumor localization, see, for example, U.S. Patents 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region (which may reduce binding to the Fc receptor) (see, for example, Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0191] In some embodiments, one or more amino acid residues in the constant region of the muscle-targeting antibody described herein may be replaced with different amino acid residues so that the antibody may have modified Clq binding and / or (for example, and) reduced or absent complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are modified to alter the complement-binding ability of the antibody. This approach is described in further international publication WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) to cells and / or (for example, and) to increase the antibody's affinity for the Fcγ receptor. This approach is described in further international publication WO 00 / 42072.

[0192] In some embodiments, the heavy chain and / or (for example, and) light chain variable domain(s) sequences(s) of the antibodies provided herein may be used, as described elsewhere herein, to generate, for example, CDR-conjugated antibodies, chimeric antibodies, humanized antibodies, or compound human antibodies, or antigen-binding fragments. As will be understood by those skilled in the art, any variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and the variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody will maintain its specific binding ability to the transferrin receptor such that it may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% binding to the transferrin receptor compared to the original antibody from which it is derived.

[0193] In some embodiments, the antibodies provided herein include mutations that confer desired properties to the antibody. For example, to avoid potential complications resulting from Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may contain a stabilizing "Adair" mutation.

[0194] As provided herein, the antibodies of this disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be attached at its C-terminus to a light chain constant region-like Cκ or Cλ. Similarly, the VH domain or a portion thereof may be attached to all or some heavy chain-like IgA, IgD, IgE, IgG, and IgM and any isotype subclass. The antibody may also encompass a preferred constant region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of this disclosure may be combined with any preferred constant region to encompass the VH and VL domains, or their antigen-binding regions.

[0195] ii. Muscle-targeting peptides Several aspects of this disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences that bind to specific cell types (for example, peptide sequences with a length of 5 to 20 amino acids) are described. For example, cell-targeting peptides are mentioned in Vines e., et al., A. "Cell-penetrating and cell-targeting peptides in drug delivery" Biochim Biophys Acta 2008, 1786:126-38; Jarver P., et al., "In vivo biodistribution and efficacy of peptide mediated delivery" Trends Pharmacol Sci 2010; 31:528-35; Samoylova TI, et al., "Elucidation of muscle-binding peptides by phage display screening" Muscle Nerve 1999; 22:460-6; ​​U.S. Patent No. 6,329,501, issued December 11, 2001, titled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE"; and Samoylov AM, et al., "Recognition of cell-specific binding of phage display derived peptides using an acoustic wave sensor." Biomol This is described in Eng 2002;18:269-72; the entire contents of each of these are incorporated herein by reference. Selectivity to desired tissues, e.g., muscle, can be achieved by designing peptides to interact with specific cell surface antigens (e.g., receptors). Skeletal muscle targeting has been investigated, and a wide range of molecular payloads can be delivered. These approaches, which do not have many of the practical disadvantages of large antibodies or viral particles, may have high selectivity to muscle tissue. Consequently, in some embodiments, muscle targeting agents are muscle-targeting peptides ranging in length from 4 to 50 amino acids.In some embodiments, muscle-targeting peptides have a length of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids. Muscle-targeting peptides can be generated using one of several methods, such as phage display.

[0196] In some embodiments, muscle-targeting peptides may bind to internalized cell surface receptors (e.g., transferrin receptors) that are overexpressed or relatively highly expressed in muscle cells compared to other cells. In some embodiments, muscle-targeting peptides may target transferrin receptors (e.g., bind to transferrin receptors). In some embodiments, transferrin receptor-targeting peptides may contain naturally occurring ligands, e.g., segments of transferrin. In some embodiments, transferrin receptor-targeting peptides are described in U.S. Patent No. 6,743,893, 11 / 30 / 2000, “RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR”. In some embodiments, the transferrin receptor-targeting peptide is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells." BMC Cancer. 2011 Aug 18;11:359. In some embodiments, the transferrin receptor-targeting peptide is as described in U.S. Patent No. 8,399,653, 5 / 20 / 2011, "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY."

[0197] As described above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides were identified using a phage display library that presents peptapeptides on the surface. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 3071) bound to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. As a result, in some embodiments, the muscle targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 3071). This peptide exhibited improved specificity for binding to myocardial and skeletal muscle tissues after intravenous injection into mice, with reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have been identified using phage display. For example, in the context of treatment of DMD, a 12-amino acid peptide was identified by a phage display library for muscle targeting. See Yoshida D., et al., "Targeting of salicylate to skin and muscle following topical injections in rats." Int J Pharm 2002;231:177-84; the entire content of which is hereby incorporated by reference. Here, a 12-amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO: 3072) was identified, and this muscle-targeting peptide showed improved binding to C2C12 cells compared to the ASSLNIA (SEQ ID NO: 3071) peptide.

[0198] Any additional method for identifying peptides selective to muscle (e.g., skeletal muscle) more than other cell types involves in vitro selection, which is described in Ghosh D., et al., "Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting" J Virol 2005;79:13667-72; the entire content of which is incorporated herein by reference. Nonspecific cell binders were selected by pre-incubating random 12-mer peptide phage display libraries with a mixture of non-muscle cell types. Following rounds of selection, the 12-amino acid peptide TARGEHKEEELI (SEQ ID NO: 3073) appeared most frequently. Consequently, in some embodiments, muscle targeting agents contain the amino acid sequence TARGEHKEEELI (SEQ ID NO: 3073).

[0199] Muscle targeting agents may be amino acid-containing molecules or peptides. Muscle targeting peptides may correspond to sequences of proteins that preferentially bind to protein receptors found in muscle cells. In some embodiments, muscle targeting peptides contain high propensity of hydrophobic amino acids (e.g., valine) so that the peptides can preferentially target muscle cells. In some embodiments, muscle targeting peptides have not been characterized or disclosed to date. These peptides may be recalled, produced, synthesized, and / or (e.g., and) derivatized using one of several methodologies, e.g., a phage-dispenseed peptide library, a one-bead-one-compound peptide library, or a positional scanning synthetic peptide combinatorial library. The example methodologies are characterized in the relevant technical field and are incorporated by reference (Gray, BP and Brown, KC "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides" Chem Rev. 2014, 114:2, 1020-1081.; Samoylova, TI and Smith, BF "Elucidation of muscle-binding peptides by phage display screening." Muscle Nerve, 1999, 22:4, 460-6).In several embodiments, muscle-targeting peptides have been disclosed to date (see, for example, Writer MJet al. "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display." J. Drug Targeting. 2004; 12: 185; Cai, D. "BDNF-mediated enhancement of inflammation and injury in the aging heart." Physiol Genomics. 2006, 24: 3, 191-7.; Zhang, L. "Molecular profiling of heart endothelial cells." Circulation, 2005, 112: 11, 1601-11.; McGuire, MJet al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004, 342: 1, 171-82). The exemplary muscle-targeting peptides include the following amino acid sequences: CQAQGQLVC (SEQ ID NO: 3074), CSERSMNFC (SEQ ID NO: 3075), CPKTRRVPC (SEQ ID NO: 130), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 3076), ASSLNIA (SEQ ID NO: 3071), CMQHSMRVC (SEQ ID NO: 3077), and DDTRHWG (SEQ ID NO: 131). In some embodiments, the muscle-targeting peptides may contain approximately 2–25 amino acids, approximately 2–20 amino acids, approximately 2–15 amino acids, approximately 2–10 amino acids, or approximately 2–5 amino acids. The muscle-targeting peptides may contain naturally occurring amino acids, such as cysteine, alanine, or amino acids that are not naturally occurring, or modified amino acids.Amino acids that do not exist in nature include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the muscle-targeting peptide may be linear; in other embodiments, the muscle-targeting peptide may be cyclic (e.g., bicyclic) (see, for example, Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

[0200] iii. Muscle-targeting receptor ligands The muscle targeting agent may be a ligand, for example, a ligand that binds to a receptor protein. The muscle targeting ligand may be a protein that binds to an internalized cell surface receptor expressed by muscle cells, for example, transferrin. Consequently, in some embodiments, the muscle targeting agent is transferrin, or a derivative thereof that binds to a transferrin receptor. Alternatively, the muscle targeting ligand may be a small molecule, for example, a lipophilic small molecule that preferentially targets muscle cells compared to other cell types. Exemplary lipophilic small molecules that may target muscle cells include compounds containing cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolenic acid, linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and alkoxy acids.

[0201] iv. Muscle-targeting aptamers Muscle-targeting agents may be aptamers that preferentially target muscle cells compared to other cell types, such as RNA aptamers. In some embodiments, muscle-targeting aptamers have not been characterized or disclosed to date. These aptamers may be recalled, produced, synthesized, and / or (for example, and) derivatized using one of several methodologies, e.g., systematic evolution of ligands by exponential enrichment. The exemplary methodologies are characterized in the art and incorporated by reference (Yan, AC and Levy, M. "Aptamers and aptamer targeted delivery" RNA biology, 2009, 6:3, 316-20.; Germer, K. et al. "RNA aptamers and their therapeutic and diagnostic applications." Int. J. Biochem. Mol. Biol. 2013; 4:27-40). In some embodiments, muscle-targeted aptamers have been disclosed to date (see, for example, Phillippou, S. et al. "Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers." Mol Ther Nucleic Acids. 2018, 10: 199-214.; Thiel, WH et al. "Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation." Mol Ther. 2016, 24: 4, 779-87). Exemplary muscle-targeted aptamers include the A01B RNA aptamer and RNA Apt 14. In some embodiments, the aptamer is a nucleic acid-based aptamer, an oligonucleotide aptamer, or a peptide aptamer. In some embodiments, the aptamer may be approximately 5–15 kDa, approximately 5–10 kDa, approximately 10–15 kDa, approximately 1–5 Daa, approximately 1–3 kDa, or smaller.

[0202] v. Other muscle targeting agents One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use substrates of muscle transporter proteins, such as transporter proteins expressed on the muscle fiber sheath. In some embodiments, muscle targeting agents are substrates of influx transporters specific to muscle tissue. In some embodiments, the influx transporters are specific to skeletal muscle tissue. The two major classes of transporters expressed on the muscle fiber sheath of skeletal muscle are (1) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue, and (2) the solute carrier (SLC) superfamily, which can facilitate the influx of substrates into skeletal muscle. In some embodiments, muscle targeting agents are substrates that bind to the ABC or SLC superfamily of transporters. In some embodiments, the substrates that bind to the ABC or SLC superfamily of transporters are naturally occurring substrates. In some embodiments, the substrates that bind to the ABC or SLC superfamily of transporters are naturally occurring substrates, such as synthetic derivatives that bind to the ABC or SLC superfamily of transporters.

[0203] In some embodiments, the muscle targeting agent is any of the muscle targeting agents described herein (e.g., antibodies, nucleic acids, small molecules, peptides, aptamers, lipids, sugar moieties) that target the SLC superfamily of transporters. In some embodiments, the muscle targeting agent is a substrate of the SLC superfamily of transporters. The SLC transporter is either in equilibrium or utilizes a proton or sodium ion gradient created across the membrane to propel the transport of the substrate. Exemplary SLC transporters with high expression in skeletal muscle include, but are not limited to, the SATT transporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7 transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), LAT3 transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-J transporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2 transporter (FLJ46769; SLC22A5), ENT transporter (ENT1; SLC29A1 and ENT2; SLC29A2), PAT2 transporter (SLC36A2), and SAT2 transporter (KIAA1382; SLC38A2). These transporters can provide an opportunity for muscle targeting by facilitating the influx of substrates into skeletal muscle.

[0204] In some embodiments, muscle targeting agents are substrates of the equilibrium nucleoside transporter 2 (ENT2) transporter. Compared to other transporters, ENT2 has one of the highest expression mRNAs in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs, including the brain, heart, placenta, thymus, pancreas, prostate, and kidneys, but is particularly abundant in skeletal muscle. Human ENT2 facilitates the uptake of its substrates according to their concentration gradients. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleic acid bases. The hENT2 transporter has low affinity for all nucleosides except inosine (adenosine, guanosine, uridine, thymidine, and cytidine). Consequently, in some embodiments, muscle targeting agents are ENT2 substrates. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and clopharabine. In some embodiments, any of the muscle targeting agents provided herein are related to a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle targeting agent is noncovalently linked to the molecular payload.

[0205] In some embodiments, the muscle targeting agent is a substrate of an organic cation / carnitine transporter (OCTN2), which is a sodium ion-dependent high-affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, mildronate, acetylcarnitine, or any derivative thereof that binds to OCTN2. In some embodiments, carnitine, mildronate, acetylcarnitine, or their derivatives are covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0206] The muscle targeting agent may be a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be hemoduvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, hemoduvelin may be full length or fragmentary, or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with a functional hemoduvelin protein. In some embodiments, the hemoduvelin mutant may be a soluble fragment, may lack N-terminal signaling, and / or (for example, and), may lack a C-terminal anchoring domain. In some embodiments, hemoduberin may be annotated with GenBank RefSeq accession numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It should be understood that hemoduberin may originate from humans, non-human primates, or rodents.

[0207] B. Molecular payload Several aspects of this disclosure provide oligonucleotides designed to target DUX4 RNA to modulate molecular payloads, such as DUX4 expression or activity. In some embodiments, this disclosure provides oligonucleotides complementary to DUX4 RNA that are useful in reducing levels of DUX4 mRNA and / or protein associated with the pathological features of facioscapulohumeral muscular dystrophy (FSHD), including muscular atrophy, inflammation, and reduced differentiation potential and oxidative stress. In some embodiments, the oligonucleotides provided herein are designed to direct RNAi-mediated degradation of DUX4 RNA. In some embodiments, the oligonucleotides are designed to efficiently involve the RNA-induced silencing complex (RISC) for DUX4 RNA degradation while also reducing off-target effects. In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum stability. In some embodiments, the oligonucleotides are designed to have desired binding affinity. In some embodiments, the oligonucleotides are designed to have a desired toxicity and / or immunogenicity profile.

[0208] In some embodiments, DUX4-targeted oligonucleotides include a chain having a region complementary to DUX4 RNA. Exemplary oligonucleotides are described in more detail herein, but it should be understood that the exemplary oligonucleotides provided herein are not intended to be limiting.

[0209] i. oligonucleotides In some embodiments, the DUX4-targeted oligonucleotides provided herein are designed to induce RNAi-mediated degradation of DUX4 mRNA. In some embodiments, the DUX4-targeted oligonucleotides provided herein include an antisense strand complementary to DUX4 mRNA. In some embodiments, the oligonucleotides provided herein further include a sense strand forming a double-stranded oligonucleotide (e.g., siRNA). In some embodiments, it should be understood that by incorporating a functional sequence (e.g., an antisense strand sequence) from one format into the other format, an oligonucleotide of one format (e.g., an antisense oligonucleotide) may be suitably adapted to another format (e.g., an siRNA oligonucleotide).

[0210] Any suitable oligonucleotide may be used as a molecular payload as described herein. Examples of oligonucleotides useful for targeting DUX4 include U.S. Patent No. 9,988,628, published February 2, 2017, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. Patent No. 9,469,851, published October 30, 2014, entitled "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4"; U.S. Patent Application Publication No. 20120225034, published September 6, 2012, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; and "MORPHOLINO TARGETING DUX4 FOR TREATING PCT patent application publication number WO 2013 / 120038, published on August 15, 2013, entitled "FSHD"; Chen et al., "Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics," Molecular Therapy, 2016, 24:8, 1405-1411; and Ansseau et al., "Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy (FSHD)," Genes, 2017, 8, 93. The entire contents of each of these are incorporated herein by reference. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA, or another oligonucleotide that hybridizes with the target DUX4 gene or mRNA.

[0211] In some embodiments, the oligonucleotides described herein have regions of complementarity to sequences as shown below: human DUX4 corresponding to NCBI sequence NM_001293798.2 (SEQ ID NO: 160) or NCBI sequence NM_001306068.3 (SEQ ID NO: 161) as shown below, and / or (as an example, and) mouse DUX4 corresponding to NCBI sequence NM_001081954.1 (SEQ ID NO: 162) as shown below. Other non-limiting examples of human DUX4 mRNA is NCBI sequence: NM_033178, GenBank accession number FJ439133, AF117653, HM101229, HM101230, HM101232, HM101233, HM101234, HM101235, HM101240, HM101241, HM101242, HM101243, HM1 01244, HM101245, HM101246, HM101247, HM101248, HM101249, HM101250, HM101251 and HM190160, HM190161, HM190162, HM190163, HM190164, HM190165, HM190166, HM190167, HM 190168, HM190169, HM190170, HM190171, HM190172, HM190173, HM190174, HM190175, HM 190176, HM190177, HM190178, HM190179, HM190180, HM190181, HM190182, HM190183, HM1 This includes 90184, HM190185, HM190186, HM190187, HM190188, HM190189, HM190190, HM190191, HM190192, HM190193, HM190194, HM190195, and HM190196, each of which is incorporated herein by reference.In some embodiments, oligonucleotides may have a region complementary to a hypomethylated and reduced D4Z4 repeat, as described in Daxinger, et al., "Genetic and Epigenetic Contributors to FSHD," Lim JW, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1;24(17):4817-4828 (the entire contents of each of these are incorporated herein by reference), published in Curr Opin Genet Dev in 2015.

[0212] In some embodiments, the oligonucleotide may have a region of complementarity to a sequence represented as follows, which is an example of a human DUX4 gene sequence (NM_001293798.2) (SEQ ID NO: 160):

[0213] In some embodiments, the oligonucleotide may have a region of complementarity to a sequence represented as follows, which is an example of a human DUX4 gene sequence (NM_001306068.3) (SEQ ID NO: 161):

[0214] In some embodiments, the oligonucleotide may have a region of complementarity to a sequence represented as follows, which is an example of a mouse DUX4 gene sequence (SEQ ID NO: 162) (NM_001081954.1):

[0215] In some embodiments, the oligonucleotide may have regions of complementarity to the DUX4 gene sequence of multiple species, selected from, for example, human, mouse, and non-human species. In some embodiments, the non-human species is cynomolgus monkey.

[0216] i. Size / arrangement of oligonucleotides Oligonucleotides may be of various different lengths, for example, depending on the format. In some embodiments, oligonucleotides are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or longer. In some embodiments, oligonucleotides are 8-50 nucleotides, 8-40 nucleotides, 8-32 nucleotides, 10-15 nucleotides, 10-20 nucleotides, 15-25 nucleotides, 21-23 nucleotides, and so on. In some embodiments, the oligonucleotides have lengths of 8–32 nucleotides, 15–29 nucleotides, 15–27 nucleotides, 15–20 nucleotides, 20–25 nucleotides, 21–27 nucleotides, 23–27 nucleotides, 25–30 nucleotides, or 25–32 nucleotides.

[0217] In some embodiments, for the purposes of this disclosure, a complementary nucleic acid sequence of an oligonucleotide is specifically hybridizable to or specific to a target nucleic acid when, under conditions where the binding of the sequence to a target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) causing loss of activity (e.g., inhibition of translation) or loss of expression (e.g., degradation of target mRNA), and where avoidance of nonspecific binding is desired, such as under physiological conditions in the case of in vivo assays or therapeutic treatments and in vitro assays, and under conditions where the assay is performed under favorable stringency conditions. Therefore, in some embodiments, the oligonucleotide may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the consecutive nucleotides of the target nucleic acid. In some embodiments, the complementary nucleotide sequence does not need to be specifically hybridizable to the target nucleic acid or 100% complementary to the target sequence that is specific to the target nucleic acid. In some embodiments, the oligonucleotide contains one or more mismatched nucleic acid bases compared to the target nucleic acid. In some embodiments, activity with respect to the target is reduced by such mismatch, but activity with respect to non-targets is reduced by a greater amount (i.e., selectivity for the target nucleic acid is increased and off-target effects are reduced). In some embodiments, the target nucleic acid is a pre-mRNA molecule or an mRNA molecule.

[0218] In some embodiments, the oligonucleotide includes a complementary region to the target nucleic acid having a length in the range of 8–15, 8–30, 8–40, or 10–50, or 5–50, or 5–40 nucleotides. In some embodiments, the oligonucleotide includes a complementary region to the target nucleic acid having a length in the range of 8–32, 15–29, 15–27, 21–27, or 23–27 nucleotides. In some embodiments, the oligonucleotide includes a complementary region to the target nucleic acid having a length in the range of 15–29, 15–27, 15–20, 20–25, 21–27, 23–27, 25–27, or 25–32 nucleotides. In some embodiments, the complementary region of the oligonucleotide to the target nucleic acid is of a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, the complementary region is complementary to at least 8 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain 1, 2, or 3 base mismatches compared to some consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

[0219] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a sequence containing any one of sequence numbers 1575-2986 and 3027-3066. In some embodiments, the oligonucleotide comprises a sequence containing any one of sequence numbers 1575-2986 and 3027-3066. In some embodiments, the oligonucleotide comprises a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity with at least 12 or at least 15 consecutive nucleotides of any one of sequence numbers 1575-2986 and 3027-3066. In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a sequence containing any one of sequence numbers 3027-3066. In some embodiments, the oligonucleotide comprises a sequence containing one of sequence numbers 3027-3066. In some embodiments, the oligonucleotide comprises a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity with at least 12 or at least 15 consecutive nucleotides of one of sequence numbers 3027-3066.

[0220] In some embodiments, the oligonucleotide includes a region of complementarity to the target sequence as represented by any one of SEQ ID NOs: 163-1574. In some embodiments, the oligonucleotide includes a region of complementarity to the target sequence as represented by any one of SEQ ID NOs: 2987-3026. In some embodiments, the oligonucleotide includes a region of complementarity that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementary to at least 12 or at least 15 consecutive nucleotides of the target sequence as represented by any one of SEQ ID NOs: 163-1574. In some embodiments, the oligonucleotide includes a region of complementarity that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementary to at least 12 or at least 15 consecutive nucleotides of the target sequence as represented by any one of SEQ ID NOs: 2987-3026. In some embodiments, the complementary region is a nucleotide with a length of at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 19, or at least 20 nucleotides. In some embodiments, the complementary region is a nucleotide with a length of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the complementary region is in the range of 8–20, 10–20, or 15–20 nucleotides in length. In some embodiments, the complementary region is perfectly complementary to all or part of its target sequence. In some embodiments, the complementary region contains one, two, three, or more mismatches.

[0221] In some embodiments, an oligonucleotide is complementary (for example, at least 85%, at least 90%, at least 95%, or 100%) to any one target sequence of the oligonucleotides provided herein (for example, the oligonucleotides listed in Table 8). In some embodiments, such a target sequence is 100% complementary to the oligonucleotides listed in Table 8. In some embodiments, an oligonucleotide is complementary (for example, at least 85%, at least 90%, at least 95%, or 100%) to any one target sequence of the oligonucleotides provided herein (for example, the oligonucleotides listed in Table 9). In some embodiments, such a target sequence is 100% complementary to the oligonucleotides listed in Table 9. In some embodiments, an oligonucleotide is complementary (for example, at least 85%, at least 90%, at least 95%, or 100%) to any one target sequence of the oligonucleotides provided herein (for example, oligonucleotides including any one of sequence numbers 1575-2986 and 3027-3066). In some embodiments, such target sequences are 100% complementary to the oligonucleotides described herein (for example, oligonucleotides comprising any one of sequence numbers 1575-2986 and 3027-3066).

[0222] In some embodiments, methylation of the nucleic acid base uracil at the C5 position should be understood to form thymine. Therefore, in some embodiments, nucleotides or nucleosides having C5-methylated uracil (or 5-methyl-uracil) may be identified equivalently as thymine nucleotides or nucleosides.

[0223] In some embodiments, one or more thymine bases (T) in any one of the oligonucleotides provided herein may be independently and optionally uracil bases (U), and / or one or more U may be independently and optionally T. In some embodiments, one or more thymine bases (T) in any one of the oligonucleotides listed in Table 8 or Table 9 may be independently and optionally uracil bases (U), and / or one or more U may be independently and optionally T.

[0224] b. Oligonucleotide modification: The oligonucleotides described herein may be modified, including, for example, modified sugar moieties, modified nucleoside linkages, modified nucleotides, and / or combinations thereof. In addition, in some embodiments, oligonucleotides may exhibit one or more of the following properties: non-mediating of alternative splicing; non-immunostimulant; nuclease-resistant; having improved cellular uptake compared to unmodified oligonucleotides; non-toxic to cells or mammals; having improved exit into endosomes within cells; minimizing TLR stimulation; or evading pattern recognition receptors. Any modified chemical properties or formats of the oligonucleotides described herein may be combined with each other. For example, 1, 2, 3, 4, 5, or more different types of modifications may be contained within the same oligonucleotide.

[0225] In some embodiments, specific nucleotide modifications may be used to make the oligonucleotide into which the modification is incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide or oligoribonucleotide molecule; these modified oligonucleotides remain intact for longer periods than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those with modified backchains, such as phosphorothioates, phosphotriesters, methylphosphonates, short-chain alkyl or cycloalkyl sugar linkages, or modified nucleoside linkages such as short-chain heteroatom or heterocyclic sugar linkages. Consequently, the oligonucleotides of this disclosure can be stabilized against nucleolysis by modification, such as the incorporation of nucleotide modifications.

[0226] In some embodiments, the oligonucleotide may be an oligonucleotide with a length of up to 50 nucleotides or up to 100 nucleotides, where 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30, 2-40, 2-45 nucleotides, or more, are modified nucleotides. The oligonucleotide may be an oligonucleotide with a length of 8-30 nucleotides, where 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30 nucleotides are modified nucleotides. The oligonucleotide may be an oligonucleotide with a length of 8-15 nucleotides, where 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14 nucleotides are modified nucleotides. Optionally, any oligonucleotide may be modified except for nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Oligonucleotide modifications are further described herein.

[0227] c. Modified nucleoside In some embodiments, the oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of a sugar. In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleoside. In some embodiments, all nucleosides on the oligonucleotide are 2'-modified nucleosides.

[0228] In some embodiments, the oligonucleotides described herein include one or more non-bicyclic 2'-modified nucleotides, for example, 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA) modified nucleosides.

[0229] In some embodiments, the oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleosides, wherein the ribose ring in the nucleoside comprises a bridging moiety connecting two atoms in the ring (e.g., a methylene (LNA) bridge, an ethylene (ENA) bridge, or a (S)-restricted ethyl (cEt) bridge connecting a 2'-O atom to a 4'-C atom). An example of LNA is described in the international patent application publication WO / 2008 / 043753, published on April 17, 2008, entitled "RNA Antagonist Compounds For The Modulation Of PCSK9" (the contents of which are incorporated herein by reference in their entirety). Examples of ENA are provided in the international patent publication WO 2005 / 042777, published on 12 May 2005, titled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006; and Horie et al., Nucleic Acids Symp. Ser(Oxf), 49:171-172, 2005, the entirety of which disclosures are incorporated herein by reference. Examples of cEt are provided in U.S. Patents 7,101,993; 7,399,845 and 7,569,686, each of which is incorporated herein by reference in whole.

[0230] In some embodiments, the oligonucleotides include modified nucleosides disclosed in one of the following U.S. patents or patent application publications: U.S. Patent 7,399,845, issued July 15, 2008, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,741,457, issued June 22, 2010, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 8,022,193, issued September 20, 2011, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,569,686, issued August 4, 2009, titled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,335,765, issued February 26, 2008, titled "Novel Nucleoside And Oligonucleotide" "Analogues"; U.S. Patent 7,314,923, issued January 1, 2008, titled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent 7,816,333, issued October 19, 2010, titled "Oligonucleotide Analogues And Methods Utilizing The Same"; and U.S. Publication No. 2011 / 0009471, currently U.S. Patent 8,957,201, issued February 17, 2015, titled "Oligonucleotide Analogues And Methods Utilizing The Same". For all purposes, the entire contents of each of these are incorporated herein by reference.

[0231] In some embodiments, the oligonucleotide contains at least one modified nucleoside, resulting in an increased Tm of the oligonucleotide in the range of 1°C, 2°C, 3°C, 4°C, or 5°C compared to an oligonucleotide that does not contain at least one modified nucleoside. The oligonucleotide may contain multiple modified nucleosides in total, resulting in an increased Tm of the oligonucleotide in the range of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or above, compared to an oligonucleotide that does not contain modified nucleosides.

[0232] Oligonucleotides may contain a mix of different types of nucleosides. For example, an oligonucleotide may contain a mix of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoromodified nucleosides. An oligonucleotide may contain a mix of deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may contain a mix of 2'-fluoromodified nucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may contain a mix of cross-linked nucleosides and 2'-fluoro or 2'-O-methyl modified nucleosides. An oligonucleotide may contain a mix of non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). Oligonucleotides may comprise a mix of 2'-fluoro-modified nucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may comprise a mix of 2'-4' bicyclic nucleosides and 2'MOE, 2'-fluoro, or 2'-O-Me-modified nucleosides. Oligonucleotides may comprise a mix of non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0233] Oligonucleotides may contain alternating nucleosides of different types. For example, an oligonucleotide may contain alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro-modified nucleosides. An oligonucleotide may contain alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me-modified nucleosides. An oligonucleotide may contain alternating 2'-fluoro-modified nucleosides and 2'-O-Me-modified nucleosides. An oligonucleotide may contain alternating cross-linked nucleosides and 2'-fluoro or 2'-O-methyl-modified nucleosides. An oligonucleotide may contain alternating non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). The oligonucleotide may contain alternating 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. The oligonucleotide may also contain alternating non-bicyclic 2' modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0234] In some embodiments, the oligonucleotides described herein include a 5'-vinylphosphonate modification, one or more debased residues, and / or one or more inverted debased residues.

[0235] d. Internucleoside linkage / main chain In some embodiments, the oligonucleotide may contain phosphorothioate or other modified nucleoside linkages. In some embodiments, the oligonucleotide contains phosphorothioate nucleoside linkages. In some embodiments, the oligonucleotide contains phosphorothioate nucleoside linkages between at least two nucleosides. In some embodiments, the oligonucleotide contains phosphorothioate nucleoside linkages between all nucleosides. For example, in some embodiments, the oligonucleotide contains modified nucleoside linkages at the 5' or 3' end of the nucleotide sequence, at the first, second, and / or (for example, and) third nucleoside linkages.

[0236] Phosphorus-containing linkages that may be used include, but are not limited to, ordinary 3'-5' linkages, their 2'-5' linkage analogs, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, and boranophosphorates, and those having opposite polarity (where adjacent pairs of nucleoside units are 3'-5' to 5'-3' or (connected from 2'-5' to 5'-2') and encompassing; US Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,196; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,39 See issues 9,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0237] In some embodiments, the oligonucleotide may have a heteroatom backbone such as a methylene (methylimino) or MMI backbone; an amide backbone (see De Mesmaeker et al. Ace.Chem.Res. 1995, 28:366-374); a morpholino backbone (see Summerton and Weller, U.S. Patent No. 5,034,506); or a peptide nucleic acid (PNA) backbone (where the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotide is directly or indirectly bonded to the aza nitrogen atom of the polyamide backbone, see Nielsen et al., Science 1991, 254,1497).

[0238] e. Stereospecific oligonucleotides In some embodiments, the phosphorus atoms between nucleotides of the oligonucleotide are chiral, and the properties of the oligonucleotide are tuned based on the stereochemistry of the chiral phosphorus atoms. In some embodiments, suitable methods may be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms. Chem Soc Rev. 2011 Dec;40(12):5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided, comprising nucleoside units linked together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages. In some embodiments, such phosphorothioate oligonucleotides having substantially chiral pure intersugar linkages are prepared by enzymatic or chemical synthesis, for example, as described in U.S. Patent 5,587,261 issued December 12, 1996 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, chiral-controlled oligonucleotides provide selective cleavage patterns for target nucleic acids. For example, in some embodiments, chiral-controlled oligonucleotides provide a single cleavage site within a complementary sequence of nucleic acids, as described, for example, in U.S. Patent Application Publication 20170037399 A1, titled "CHIRAL DESIGN," published on February 2, 2017 (the contents of which are incorporated herein by reference in their entirety).

[0239] f. morpholino In some embodiments, oligonucleotides may be morpholino-based compounds. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510; Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Naseviius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Patent No. 5,034,506 issued July 23, 1991. In some embodiments, morpholino-based oligomeric compounds are phosphorodiamidate morpholino oligomers (PMOs) (for example, as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; these disclosures are incorporated herein by reference in their entirety).

[0240] h.Gapmer In some embodiments, the oligonucleotides described herein are gapmers. Gapmer oligonucleotides generally have the formula 5'-XYZ-3', with X and Z as flanking regions surrounding a gap region Y. In some embodiments, the flanking region X of formula 5'-XYZ-3' is also referred to as the X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, the flanking region Z of formula 5'-XYZ-3' is also referred to as the Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, the gap region Y of formula 5'-XYZ-3' is also referred to as the Y region, Y segment, or gap segment Y. In some embodiments, each nucleoside in the gap region Y is a 2'-deoxyribonucleoside, and neither the 5' wing region X nor the 3' wing region Z contains any 2'-deoxyribonucleoside.

[0241] In some embodiments, the Y region is a sequence of nucleotides, e.g., a region of six or more DNA nucleotides, capable of recruiting an RNAse such as RNAse H. In some embodiments, a gapmer binds to a target nucleic acid, in which case an RNAse is recruited and then cleaved from the target nucleic acid. In some embodiments, the Y region is flanked at both the 5' and 3' ends by regions X and Z containing high-affinity modified nucleosides, e.g., one to six high-affinity modified nucleosides. Examples of high-affinity modified nucleosides include, but are not limited to, 2'-modified nucleosides (e.g., 2'-MOE, 2'O-Me, 2'-F) or 2'-4' bicyclic nucleosides (e.g., LNA, cEt, ENA). In some embodiments, the flanking sequences X and Z may be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides in length. The flanking sequences X and Z may be of similar or dissimilar lengths. In some embodiments, the gap segment Y may be a nucleotide sequence having a length of 5–20 nucleotides, 5–15, 12 nucleotides, or 6–10 nucleotides.

[0242] In some embodiments, the gap region of the gapmer oligonucleotide may contain, in addition to DNA nucleotides, modified nucleotides known to be acceptable for efficient RNase H action, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-type nucleotides. In some embodiments, the gap region contains one or more unmodified internucleoside links. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate internucleoside links (e.g., phosphorothioate internucleoside links or other links) between at least two, at least three, at least four, at least five, or more nucleotides. In some embodiments, the gap region and the two flanking regions each independently contain modified internucleoside links (e.g., phosphorothioate internucleoside links or other links) between at least two, at least three, at least four, at least five, or more nucleotides.

[0243] Gapmers can be produced using appropriate methods. Representative U.S. patents, U.S. patent publications, and PCT publications teaching the preparation of gapmers are U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,6 No. 23,065; No. 5,652,355; No. 5,652,356; No. 5,700,922; No. 5,898,031; No. 7,015,315; No. 7,1 No. 01,993; No. 7,399,845; No. 7.432,250; No. 7,569,686; No. 7,683,036; No. 7,750,131; No. 8,5 No. 80,756; No. 9,045,754; No. 9,428,534; No. 9,695,418; No. 10,017,764; No. 10,260,069; No. 9,428,534; No. 8,580,756; U.S. Patent Publications US20050074801, US20090221685, US200902869 This includes, but is not limited to, Patent Nos. 69, US20100197762, and US20110112170; PCT Publications W02004069991; W02005023825; W02008049085 and W02009090182; and European Patent No. EP2,149,605. Each of these is incorporated herein by reference in its entirety.

[0244] In some aspects, the gapmer has a length of 10–40 nucleosides. For example, the gapmer may have a length of 10–40, 10–35, 10–30, 10–25, 10–20, 10–15, 15–40, 15–35, 15–30, 15–25, 15–20, 20–40, 20–35, 20–30, 20–25, 25–40, 25–35, 25–30, 30–40, 30–35, or 35–40 nucleosides. In some embodiments, the gapmer has a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleosides.

[0245] In some embodiments, the gap region Y on the gapmer has a length of 5–20 nucleosides. For example, the gap region Y may have a length of 5–20, 5–15, 5–10, 10–20, 10–15, or 15–20 nucleosides. In some embodiments, the gap region Y has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In some embodiments, each nucleoside in the gap region Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides in the gap region Y are 2'-deoxyribonucleosides. In some embodiments, one or more nucleosides in the gap region Y are modified nucleosides (for example, 2'-modified nucleosides, e.g., those described herein). In some embodiments, one or more cytosines in gap region Y are optionally 5-methylcytosine. In some embodiments, each cytosine in gap region Y is 5-methylcytosine.

[0246] In some embodiments, the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) are independently 1 to 20 nucleoside lengths. For example, the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) can independently be 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, 2 to 5, 2 to 7, 3 to 5, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleoside lengths. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are independently the lengths of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are the same length. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are of different lengths. In some embodiments, the 5' wing region of gapmer (X in the 5'-XYZ-3' equation) is longer than the 3' wing region of gapmer (Z in the 5'-XYZ-3' equation). In some embodiments, the 5' wing region of gapmer (X in the 5'-XYZ-3' equation) is shorter than the 3' wing region of gapmer (Z in the 5'-XYZ-3' equation).

[0247] In some aspects, gapmer is 5-10-5, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3, 2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1, 2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12- 5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14 -1, 2-14-3, 3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-1 1-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1- 13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4 -11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4,1-19-1、1-18-2、2-18-1、1-17-3、3-17-1、2-17-2、1-16-4、4-16-1、2-16-3、3-16-2、1-15-5、2-15-4、4-15-2、3-15-3、1-14-6、6-14-1、2-14-5、5-14-2、3-14-4、4-14-3、1-13-7、7-13-1、2-13-6、6-13-2、3-13-5、5-13-3、4-13-4、1-12-8、8-12-1、2-12-7、7-12-2、3-12-6、6-12-3、4-12-5、5-12-4、2-11-8、8-11-2、3-11-7、7-11-3、4-11-6、6-11-4、5-11-5、1-20-1、1-19-2、2-19-1、1-18-3、3-18-1、2-18-2、1-17-4、4-17-1、2-17-3、3-17-2、1-16-5、2-16-4、4-16-2、3-16-3、1-15-6、6-15-1、2-15-5、5-15-2、3-15-4、4-15-3、1-14-7、7-14-1、2-14-6、6-14-2、3-14-5、5-14-3、4-14-4、1-13-8、8-13-1、2-13-7、7-13-2、3-13-6、6-13-3、4-13-5、5-13-4、2-12-8、8-12-2、3-12-7、7-12-3、4-12-6、6-12-4、5-12-5、3-11-8、8-11-3、4-11-7、7-11-4、5-11-6、6-11-5、1-21-1、1-20-2、2-20-1、1-20-3、3-19-1、2-19-2、1-18-4、4-18-1、2-18-3、3-18-2、1-17-5、2-17-4、4-17-2、3-17-3、1-16-6、6-16-1、2-16-5、5-16-2、3-16-4、4-16-3、1-15-7、7-15-1、2-15-6、6-15-2、3-15-5、5-15-3、4-15-4、1-14-8、8-14-1、2-14-7、7-14-2、3-14-6、6-14-3、4-14-5、5-14-4、2-13-8、8-13-2、3-13-7、7-13-3、4-13-6、6-13-4、5-13-5、1-12-10、10-12-1、2-12-9、9-12-2、3-12-8、8-12-3、4-12-7、7-12-4、5-12-6、6-12-5、4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1 Includes 5'-XYZ-3' of -15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6. The numbers indicate the nucleoside numbers in the X, Y, and Z regions of the 5'-XYZ-3' gapmer.

[0248] In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) or the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are modified nucleosides (e.g., high-affinity modified nucleosides). In some embodiments, the modified nucleosides (e.g., high-affinity modified nucleosides) are 2' modified nucleosides. In some embodiments, the 2' modified nucleosides are 2'-4' bicyclic nucleosides or non-bicyclic 2' modified nucleosides. In some embodiments, high affinity modified nucleosides are 2'-4' bicyclic nucleosides (e.g., LNA, cEt, or ENA) or non-bicyclic 2'-modified nucleosides (e.g., 2'-fluoro(2'-F), 2'-O-methyl(2'-O-Me), 2'-O-methoxyethyl(2'-MOE), 2'-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'-O-DMAOE), 2'-O-dimethylaminopropyl(2'-ODMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O-DMAEOE), or 2'-ON-methylacetamide(2'-O-NMA)).

[0249] In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) are high-affinity modified nucleosides, and one or more nucleosides in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are high-affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is a high-affinity modified nucleoside, and each nucleoside in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a high-affinity modified nucleoside.

[0250] In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) contains the same high-affinity nucleoside as the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5' wing region (X in the 5'-XYZ-3' formula) and the 3' wing region (Z in the 5'-XYZ-3' formula) of the gapmer may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me). In another example, the 5' wing region (X in the 5'-XYZ-3' formula) and the 3' wing region (Z in the 5'-XYZ-3' formula) of the gapmer may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XY-Z-3' formula) is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt).

[0251] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 1–7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6–10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X and Z is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), and each nucleoside of Y is a 2' deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 1–7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6–10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X and Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2' deoxyribonucleoside. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) contains high-affinity nucleosides that are different from those in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0252] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 1–7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6–10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), each nucleoside of Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 1–7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6–10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), each nucleoside of Z is a non-bicyclic 2' modified nucleoside (e.g., 2'MOE or 2'-O-Me), and each nucleoside of Y is a 2'-deoxyribonucleoside.

[0253] In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) includes one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-OMe) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) includes one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-OMe) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0254] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 2-7 (e.g., 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6-10 (e.g., 6, 7, 8, 9, or 10), where at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) of positions 1, 2, 3, 4, 5, 6, or 7 of X (the highest 5' position being position 1) is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 2-7 (e.g., 2, 3, 4, 5, 6, or 7), Y is a nucleoside of length 6-10 (e.g., 6, 7, 8, 9, or 10), at least one (e.g., 1, 2, 3, 4, 5, 6, or 7) of Z (the most 5' position being position 1) but not all of them are non-bicyclic 2' modified nucleosides (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside in Y is a 2' deoxyribonucleoside.In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently nucleosides of length 2-7 (e.g., 2, 3, 4, 5, 6, or 7), and Y is a nucleoside of length 6-10 (e.g., 6, 7, 8, 9, or 10), where X is at least one of positions 1, 2, 3, 4, 5, 6, or 7 (e.g., 1, 2, 3, 4, 5, or 6) and Z (most of the 5') Not all of the positions (where position 1 is) but at least one of positions 1, 2, 3, 4, 5, 6, or 7 (for example, 1, 2, 3, 4, 5, or 6) are non-bicyclic 2'-modified nucleosides (for example, 2'-MOE or 2'-O-Me), the rest of the nucleosides in both X and Z are 2'-4' bicyclic nucleosides (for example, LNA or cEt), and each nucleoside in Y is a 2'-deoxyribonucleoside.

[0255] Non-cyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and 2'-4'-bicyclic nucleosides (e.g., LNA or cEt) in the 5'-wing region (X in the 5'-X-Y-Z-3' form) and / or 3'-wing region (Z in the 5'-X-Y-Z-3' form) of the gapmer. Non-limiting examples of the gapmer configuration are as follows: BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BABA-(D)n-ABAB; KAKA-(D)n-AKAK; LALA-(D)n-ALAL; BEBE-(D)n-EBEB; KEKE-(D)n-EKEK; LELE-(D)n-ELEL; BABA-(D)n-ABAB; KAKA-(D)n-AKAK; LALA-(D)n-ALAL; BEBE-(D)n-EBEB; KEKE-(D)n-EKEK; LELE-(D)n-ELEL; ABAB-(D)n-ABAB; AKAK-(D)n-AKAK; ALAL-(D)n-ALAL; EBEB-(D)n-EBEB; EKEK-(D)n-EKEK; ELEL-(D)n-ELEL; ABAB-(D)n-ABAB; AKAK-(D)n-AKAK; ALAL-(D)n-ALAL; EBEB-(D)n-EBEB; EKEK-(D)n-EKEK; ELEL-(D)n-ELEL; AABB-(D)n-BBAA; BBAA-(D)n-AABB; AAKK-(D)n-KKAA; AALL-(D)n-LLAA; EEBB-(D)n-BBEE; EEKK-(D)n-KKEE;EELL-(D)n-LLEE;AABB-(D)n-BBAA;AAKK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;BBB-(D)n-BBA;KKK-(BBA; D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE; E;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;ABBB-(D)n-BBBA;ACC-KK-KKA; ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK-(D)n-KKKE;ELLL-(D)n-LLLE;ABBB-(D)n-BBBA;AKKK-(D)n-KKKA;ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK -(D)n-CCKE;ELLL-(D)n-LLLE;ABBB-(D)n-BBBAA;ACC-(D)n-CCKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;ECC-(D)n-CCKEE;ELLL-(D)n-LLLEE;ACC-(D)n-LLLAA; BBB-(D)n-BBBAA;ACC-(D)n-CCKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;CCK-(D)n-CCKEE;ELLL-(D)n-LLLEE;AABBB-(D)n-BBB;AACCC-(D)n-CCKKK" ;AALLL-(D)n-LLL;EEBBB-(D)n-BBB;EEKKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBB;AACKK-(D)n-KKK;AALLL-(D)n-LLL; KKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBBA;AACKK-(D)n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKK-(D)n-KKKE;EELLL-(D)n-LLLE;AABBB-(D)n-BBBA;AAKKK-(D)n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE;ABBAABB-(D)n-BB;AKKAA ALLAALL-( D)n-LL;EBBEEBB-(D)n-BB;EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALLL-(D)n-LLL;EBBEBB-(D)n-B BB;EKKEKK-(D)n-KKK;ELLELL-(D)n-LLL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALL-(D)n-LLL;EBBEBB-(D)n-BBB;EKKEKK-(D)n-KKK;ELL ELL-(D)n-LLL;EEEK-(D)n-EEEEEEEE;EEK-(D)n-EEEEEEEEEE;EK-(D)n-EEEEEEEEEE;EK-(D)n-EEEKK;K-(D)n-EEEKEKE;K-(D)n-EEEKEKEE;K- (D)n-EEKEK;EK-(D)n-EEEEKEKE;EK-(D)n-EEEKEK;EEK-(D)n-KEEKE;EK-(D)n-EEKEK;EK-(D)n-KEEK;EEK-(D)n-EEEEKEK;EK-(D)n-KEEEKEE;E This includes K-(D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK; "A" nucleosides include 2'-modified nucleosides; "B" represents 2'-4' bicyclic nucleosides; "K" represents --bound ethyl nucleosides (cEt); "L" represents LNA nucleosides; "E" represents 2'-MOE modified ribonucleosides; "D" represents 2'-deoxyribonucleosides; and "n" represents the length of the gap segment (Y in the 5'-XYZ-3' configuration), which is an integer between 1 and 20.

[0256] In some embodiments, any one of the gapmers described herein contains one or more modified nucleoside linkages (e.g., phosphorothioate linkages) in each of the X, Y, and Z regions. In some embodiments, each internucleoside linkage in any one of the gapmers described herein is a phosphorothioate linkage. In some embodiments, each of the X, Y, and Z regions independently contains a mix of phosphorothioate linkages and phosphodiester linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate linkage, the 5' wing region X contains a mix of phosphorothioate linkages and phosphodiester linkages, and the 3' wing region Z contains a mix of phosphorothioate linkages and phosphodiester linkages.

[0257] i. RNA interference (RNAi) In some embodiments, the DUX4-targeting oligonucleotides provided herein may be in the form of small interfering RNA (siRNA), also known as small interfering RNA or silencing RNA. siRNA is a type of double-stranded RNA molecule, typically about 20–25 base pairs long, that targets nucleic acids (e.g., mRNA) for degradation via the RNA interference (RNAi) pathway in cells. The specificity of an siRNA molecule may be determined by the binding of the antisense strand molecule to its target RNA. Effective siRNA molecules are generally less than 30–35 base pairs long, although longer siRNAs may also be effective, to prevent the triggering of nonspecific RNA interference pathways in cells via interferon responses. In some embodiments, siRNA molecules are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 base pairs long, or more. In some embodiments, siRNA molecules are 8–30 base pairs long, 10–15 base pairs long, 10–20 base pairs long, 15–25 base pairs long, 19–21 base pairs long, or 21–23 base pairs long. In some embodiments, the siRNA molecule has a length of 8–32 base pairs, 8–29 base pairs, 8–27 base pairs, 15–32 base pairs, 15–29 base pairs, 15–27 base pairs, 21–31 base pairs, 21–29 base pairs, 21–27 base pairs, 21–23 base pairs, 23–32 base pairs, 23–29 base pairs, or 23–27 base pairs.

[0258] Following the selection of an appropriate target RNA sequence, an siRNA molecule containing nucleotide sequences complementary to all or some of the target sequences, i.e., antisense sequences, can be designed and prepared using appropriate methods (see, for example, PCT publication WO2004 / 016735; and U.S. Patent Publications 2004 / 0077574 and 2008 / 0081791).

[0259] siRNA molecules can be double-stranded (i.e., dsRNA molecules containing an antisense strand and a complementary sense strand) or single-stranded (i.e., ssRNA molecules containing only an antisense strand). siRNA molecules may include double-stranded, asymmetric double-stranded, hairpin, or asymmetric hairpin secondary structures having self-complementary sense and antisense strands. In some embodiments, the DUX4-targeted oligonucleotides described herein are siRNAs containing an antisense strand and a sense strand.

[0260] In some embodiments, the antisense chain of the siRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 nucleotides, or more. In some embodiments, the antisense chain is 8–50 nucleotides, 8–40 nucleotides, 8–30 nucleotides, 10–15 nucleotides, 10–20 nucleotides, 15–25 nucleotides, 19–21 nucleotides, or 21–23 nucleotides. In some embodiments, the antisense chain has a length of 8-32 nucleotides, 8-29 nucleotides, 8-27 nucleotides, 15-32 nucleotides, 15-29 nucleotides, 15-27 nucleotides, 21-31 nucleotides, 21-29 nucleotides, 21-27 nucleotides, 21-23 nucleotides, 23-32 nucleotides, 23-29 nucleotides, or 23-27 nucleotides.

[0261] In some embodiments, the sense strand of the siRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 nucleotides, or more. In some embodiments, the sense strand is 8–50 nucleotides, 8–40 nucleotides, 8–30 nucleotides, 10–15 nucleotides, 10–20 nucleotides, 15–25 nucleotides, 19–21 nucleotides, or 21–23 nucleotides. In some embodiments, the sense strand is 8-32 nucleotides long, 8-29 nucleotides long, 8-27 nucleotides long, 15-32 nucleotides long, 15-29 nucleotides long, 15-27 nucleotides long, 21-31 nucleotides long, 21-29 nucleotides long, 21-27 nucleotides long, 21-23 nucleotides long, 23-32 nucleotides long, 23-29 nucleotides long, or 23-27 nucleotides long.

[0262] In some embodiments, the siRNA molecule comprises an antisense strand containing a region complementary to a target region on DUX4 mRNA. In some embodiments, the complementary region is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target region on DUX4 mRNA. In some embodiments, the target region is a sequence of nucleotides on DUX4 mRNA. In some embodiments, the complementary nucleotide sequence does not need to be 100% complementary to that of its target in order to be specifically hybridizable or specific to the target RNA sequence.

[0263] In some embodiments, the siRNA molecule comprises an antisense strand containing a region complementary to the DUX4 RNA sequence, the complementary region having a length in the range of 8–15, 8–30, 8–40, or 10–50, or 5–50, or 5–40 nucleotides. In some embodiments, the complementary region has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides. In some embodiments, the complementary region is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the DUX4 RNA sequence. In some embodiments, the complementary region comprises a nucleotide sequence containing 1, 2, 3, 4, or 5 or fewer mismatches compared to the complementary portion of the DUX4 RNA sequence. In some embodiments, the complementary region comprises a nucleotide sequence having up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

[0264] In some embodiments, the siRNA molecule comprises an antisense chain containing a nucleotide sequence that is complementary to the target sequence as represented by any one of SEQ ID NOs: 163-1574 (e.g., at least 85%, at least 90%, at least 95%, or 100%). In some embodiments, the siRNA molecule comprises an antisense chain of 18-25 nucleotides in length and contains a complementary region of at least 15 nucleotides (e.g., at least 15, at least 16, at least 17, at least 18, or at least 19 nucleotides) to the target sequence as represented by any one of SEQ ID NOs: 163-1574.

[0265] In some embodiments, the siRNA molecule comprises an antisense strand containing a nucleotide sequence that is complementary to the target sequence as represented by any one of SEQ ID NOs. 2987-3026 (e.g., at least 85%, at least 90%, at least 95%, or 100%). In some embodiments, the siRNA molecule comprises an antisense strand of 18-25 nucleotides in length and contains a complementary region of at least 15 nucleotides (e.g., at least 15, at least 16, at least 17, at least 18, or at least 19 nucleotides) to the target sequence as represented by any one of SEQ ID NOs. 2987-3026.

[0266] In some embodiments, the siRNA molecule comprises an antisense chain containing a nucleotide sequence identical to, at least 85%, at least 90%, at least 95%, or 100% of, the oligonucleotide represented by any one of SEQ ID NOs. 1575-2986 and 3027-3066. In some embodiments, the siRNA molecule comprises an antisense chain of 18-25 nucleotides in length and contains at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of the oligonucleotide represented by any one of SEQ ID NOs. 1575-2986 and 3027-3066.

[0267] In some embodiments, the siRNA molecule comprises an antisense chain containing a nucleotide sequence identical to, at least 85%, at least 90%, at least 95%, or 100%, the oligonucleotide represented by any one of SEQ ID NOs: 3027-3066. In some embodiments, the siRNA molecule comprises an antisense chain containing at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or at least 23 consecutive nucleotides of the oligonucleotide represented by any one of SEQ ID NOs: 3027-3066.

[0268] Double-stranded siRNA may contain sense RNA strands and antisense RNA strands of the same or different lengths. Double-stranded siRNA molecules can also be associated with single oligonucleotides in a stem-loop structure (where the self-complementary sense and antisense regions of the siRNA molecule are linked using one or more nucleic acid-based or non-nucleic acid-based linkers), as well as circular single-stranded RNA having two or more loop structures and a stem containing self-complementary sense and antisense strands (where the circular RNA can be processed either in vivo or in vitro to produce an active siRNA molecule capable of mediating RNAi). Thus, small hairpin RNA (shRNA) molecules are also contemplated herein. These molecules contain a specific antisense sequence in addition to a reverse-complementary (sense) sequence, typically separated by a spacer or loop sequence. Breaking a spacer or loop provides a single-stranded RNA molecule and its reverse complement (optionally, through an additional processing step which may result in the addition or removal of one, two, or three or more nucleotides from the 3' and / or (for example, and) 5' ends of one or both strands) so that they can anneal to form a dsRNA molecule. The spacer may be long enough to anneal the antisense and sense sequences to form a double-stranded structure (or stem) prior to the break of the spacer (and optionally, a subsequent processing step which may result in the addition or removal of one, two, three, or four or more nucleotides from the 3' and / or (for example, and) 5' ends of one or both strands). The spacer sequence may be an unrelated nucleotide sequence placed between two complementary nucleotide sequence regions, which will contain shRNA once annealed to form a double-stranded nucleic acid.

[0269] The overall length of an siRNA molecule can vary from approximately 14 to 100 nucleotides, depending on the type of siRNA molecule designed. Generally, these nucleotides between approximately 14 and 50 are complementary to the RNA target sequence, i.e., constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is double-stranded or single-stranded, the length can vary from approximately 14 to 50 nucleotides, while when the siRNA is shRNA or a circular molecule, the length can vary from approximately 40 to 100 nucleotides.

[0270] An siRNA molecule may contain a 3' overhang at one end of the molecule. The other end may be blunt-ended or may also have an overhang (5' or 3'). When an siRNA molecule contains overhangs at both ends of the molecule, the lengths of the overhangs may be the same or different. In one embodiment, the siRNA molecule of this disclosure contains 3' overhangs of about 1 to about 3 (e.g., 1, 2, 3) nucleotides on both ends of the molecule. In some embodiments, an siRNA molecule contains 3' overhangs of about 1 to about 3 (e.g., 1, 2, 3) nucleotides on the sense strand. In some embodiments, an siRNA molecule contains 3' overhangs of about 1 to about 3 (e.g., 1, 2, 3) nucleotides on the antisense strand. In some embodiments, an siRNA molecule contains 3' overhangs of about 1 to about 3 nucleotides on both the sense and antisense strands.

[0271] In some embodiments, the siRNA molecule comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the siRNA molecule comprises one or more modified nucleotides and / or (e.g., and) one or more modified nucleoside linkages. In some embodiments, the modified nucleotide is a modified sugar moiety (e.g., a 2' modified nucleotide). In some embodiments, the siRNA molecule comprises one or more 2'-modified nucleotides, for example, 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA). In some embodiments, each nucleotide of the siRNA molecule is a modified nucleotide (for example, a 2'-modified nucleotide). In some embodiments, the siRNA molecule comprises one or more 2'-O-methyl modified nucleotides. In some embodiments, the siRNA molecule comprises one or more 2'-F modified nucleotides. In some embodiments, the siRNA molecule comprises one or more 2'-O-methyl and 2'-F modified nucleotides.

[0272] In some embodiments, the siRNA molecule contains phosphorothioate or other modified internucleotide linkages. In some embodiments, the siRNA molecule contains phosphorothioate nucleoside linkages. In some embodiments, the siRNA molecule contains phosphorothioate nucleoside linkages between at least two nucleotides. In some embodiments, the siRNA molecule contains phosphorothioate nucleoside linkages between all nucleotides. For example, in some embodiments, the siRNA molecule contains modified internucleotide linkages at the 5' end of the siRNA molecule. end or end of 3' endIt includes the first, second, and / or (for example, and) third internucleoside linkages.

[0273] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, the phosphorus-containing linkage that can be used is a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotryester, aminoalkylphosphotryester, methyl and other alkylphosphonates including 3'alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotryesters, and boranophosphorates, their 2'-5' linked analogues, and a reverse polarity in which adjacent pairs of nucleoside units are linked in a 3'-5' to 5'-3' or 2'-5' to 5'-2' configuration. This includes, but is not limited to, those having: U.S. Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,196; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,399, See issues 676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0274] Any of the modified chemistry or formats of the siRNA molecules described herein can be combined with one another. For example, 1, 2, 3, 4, 5, or more different types of modifications may be incorporated on the same siRNA molecule.

[0275] In some embodiments, the antisense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the antisense strand comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleotide links. In some embodiments, the modified nucleotide comprises a modified sugar moiety (e.g., a 2' modified nucleotide). In some embodiments, the antisense chain comprises one or more 2'-modified nucleotides, for example, 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAE0E), or 2'-ON--methylacetamide (2'-O--NMA). In some embodiments, each nucleotide in the antisense chain is a modified nucleotide (for example, a 2'-modified nucleotide). In some embodiments, the antisense chain comprises one or more 2'-O-methyl modified nucleotides. In some embodiments, the antisense chain comprises one or more 2'-F modified nucleotides. In some embodiments, the antisense chain contains one or more 2'-O-methyl and 2'-F modified nucleotides.

[0276] In some embodiments, the antisense chain contains phosphorothioate or other modified internucleotide linkages. In some embodiments, the antisense chain contains phosphorothioate nucleoside linkages. In some embodiments, the antisense chain contains phosphorothioate nucleoside linkages between at least two nucleotides. In some embodiments, the antisense chain contains phosphorothioate nucleoside linkages between all nucleotides. For example, in some embodiments, the antisense chain contains modified internucleotide linkages at the 5' end of the siRNA molecule. end or end of 3' endThis includes the first, second, and / or (for example, and) third nucleoside linkages. In some embodiments, a linkage between two nucleosides at the 3' end of an antisense chain is a phosphorothioate nucleoside linkage.

[0277] In some embodiments, the modified nucleotide linkage is a phosphorus-containing linkage. In some embodiments, the phosphorus-containing linkage that can be used is a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotryester, aminoalkylphosphotryester, methyl and other alkylphosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotryesters, and boranophosphorates, their 2'-5' linked analogues, and a reverse polarity in which adjacent pairs of nucleoside units are linked in a 3'-5' to 5'-3' or 2'-5' to 5'-2' configuration. This includes, but is not limited to, those having: U.S. Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,196; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,399, See issues 676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0278] Any of the modified chemistry or formats of antisense chains described herein can be combined with each other. For example, 1, 2, 3, 4, 5, or more different types of modifications may be included on the same antisense chain.

[0279] In some embodiments, the sense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the sense strand comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleotide links. In some embodiments, the modified nucleotide comprises a modified sugar moiety (e.g., a 2' modified nucleotide). In some embodiments, the sense chain comprises one or more 2'-modified nucleotides, for example, 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAE0E), or 2'-ON--methylacetamide (2'-O--NMA). In some embodiments, each nucleotide in the sense chain is a modified nucleotide (for example, a 2'-modified nucleotide). In some embodiments, the sense chain comprises one or more phosphorodiamidate morpholino oligomers (PMOs). In some embodiments, the sense strand contains one or more 2'-O-methyl modified nucleotides. In some embodiments, the sense strand contains one or more 2'-F modified nucleotides. In some embodiments, the sense strand contains one or more 2'-O-methyl and 2'-F modified nucleotides.

[0280] In some embodiments, the sense strand contains phosphorothioate or other modified internucleotide linkages. In some embodiments, the sense strand contains phosphorothioate nucleoside linkages. In some embodiments, the sense strand contains phosphorothioate nucleoside linkages between at least two nucleotides. In some embodiments, the sense strand contains phosphorothioate nucleoside linkages between all nucleotides. For example, in some embodiments, the sense strand contains modified internucleotide linkages at the 5' or 3' end of the siRNA molecule, at the first, second, and / or (for example, and) third nucleoside linkages. In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage.In some embodiments, phosphorus-containing linkages that may be used include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, and boranophosphorates, their 2'-5' linked analogues, and reverse polarity in which adjacent pairs of nucleoside units are linked in a 3'-5' to 5'-3' or 2'-5' to 5'-2' configuration. This includes, but is not limited to, those having: U.S. Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,196; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,399, See issues 676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0281] Any of the modified chemistry or formats of sense chains described herein can be combined with each other. For example, 1, 2, 3, 4, 5, or more different types of modifications may be included on the same sense chain.

[0282] In some embodiments, the antisense or sense strand of an siRNA molecule includes modifications that enhance or reduce RNA-induced silencing complex (RISC) loading. In some embodiments, the antisense strand of an siRNA molecule includes modifications that enhance RISC loading. In some embodiments, the sense strand of an siRNA molecule includes modifications that reduce RISC loading and reduce off-target effects. In some embodiments, the antisense strand of an siRNA molecule includes 2'-O-methoxyethyl (2'-MOE) modifications. As described in whole by reference in Song et al., (2017) Mol Ther Nucleic Acids 9:242-250, the addition of a 2'-O-methoxyethyl (2'-MOE) group at the cleavage site improves both the specificity and silencing activity of the siRNA by facilitating targeted RNA-induced silencing complex (RISC) loading of the modified strand. In some embodiments, the antisense chain of the siRNA molecule includes a 2'-OMe-phosphodithioate modification, which increases RISC loading, as described in Wu et al. (2014) Nat Commun 5:3459, the whole of which is incorporated herein by reference.

[0283] In some embodiments, the sense strand of the siRNA molecule contains a 5' morpholino, which reduces RISC loading of the sense strand and improves antisense strand selection and RNAi activity, as described in Kumar et al., (2019) Chem Commun (Camb) 55(35):5139-5142, which is incorporated herein by reference in whole. In some embodiments, the sense strand of the siRNA molecule is modified with locked nucleic acid (LNA), a synthetic RNA-like high-affinity nucleotide analog. This reduces RISC loading of the sense strand and further enhances antisense strand integration into RISC, as described in Elman et al., (2005) Nucleic Acids Res. 33(1):439-447, which is incorporated herein by reference in whole. In some embodiments, the sense strand of the siRNA molecule includes a 5' unlocked nucleic acid (UNA) modification, which reduces RISC loading of the sense strand and improves the silencing potency of the antisense strand, as described in Snead et al., (2013) Mol Ther Nucleic Acids 2(7):e103, which is incorporated herein by reference in its entirety. In some embodiments, the sense strand of the siRNA molecule includes a 5-nitroindole modification, which reduces the RNAi potency of the sense strand and mitigates off-target effects, as described in Zhang et al., (2012) Chembiochem 13(13):1940-1945, which is incorporated herein by reference in its entirety. In some embodiments, the sense chain includes a 2'-O'methyl (2'-O-Me) modification, which reduces RISC loading and off-target effects of the sense chain, as described in Zheng et al., FASEB (2013) 27(10):4017-4026, which is incorporated herein by reference in whole.In some embodiments, the sense strand of the siRNA molecule is completely substituted with morpholino, 2'-MOE, or 2'-O-Me residues, and the whole is not recognized by RISC, as described in Kole et al., (2012) Nature reviews. Drug Discovery 11(2):125-140, which is incorporated herein by reference. In some embodiments, the antisense strand of the siRNA molecule contains a 2'-MOE modification and the sense strand contains a 2'-O-Me modification (see, for example, Song et al., (2017) Mol Ther Nucleic Acids 9:242-250). In some embodiments, at least one (e.g., at least two, at least three, at least four, at least five, at least ten) siRNA molecules are ligated (e.g., covalently) to a muscle targeting agent. In some embodiments, the muscle targeting agent may or may consist of nucleic acids (e.g., DNA or RNA), peptides (e.g., antibodies), lipids (e.g., microvesicles), or sugar moieties (e.g., polysaccharides). In some embodiments, the muscle targeting agent is an antibody. In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody (for example, one of the anti-TfR antibodies provided in Tables 2-7). In some embodiments, the muscle targeting agent may be ligated to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be ligated to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be ligated internally to the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be ligated to the 5' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be ligated to the 3' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be ligated internally to the antisense strand of the siRNA molecule.

[0284] Non-limiting examples of DUX4-targeted siRNAs are provided in Table 8. Table 8. DUX4-targeted oligonucleotides † [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] "m" indicates a 2'-O-methyl (2'-O-Me) modified nucleoside; "f" indicates a 2'-fluoro (2'-F) modified nucleoside; "*" indicates a phosphorothioate nucleoside linkage; and the absence of "*" between two nucleosides indicates a phosphodiester nucleoside linkage. †Each uracil base (U) in any one of the oligonucleotides and / or target sequences provided in Table 8 may be independently and optionally replaced with a thymine base (T), and / or each T may be independently and optionally replaced with a U. The target sequences listed in Table 8 contain T, but the binding of DUX4-targeted oligonucleotides to RNA and / or DNA is intended. ^The target sequence start position is in NM_001293798.2 (sequence number 160).

[0285] Additional non-limiting examples of further modified DUX4-targeted siRNAs are provided in Table 9. Table 9. Additional DUX4-targeted oligonucleotides † [Table 9-1] [Table 9-2] "m" indicates a 2'-O-methyl(2'-O-Me) modified nucleoside; "f" indicates a 2'-fluoro(2'-F) modified nucleoside; "mxC" indicates a 2'-O-Me modified 5-methylcytidine; "fxC" indicates a 2'-F modified 5-methylcytidine; "*" indicates a phosphorothioate nucleoside linkage; and the absence of "*" between two nucleosides indicates a phosphodiester nucleoside linkage. †Each uracil base (U) in any one of the oligonucleotides and / or target sequences provided in Table 8 may be independently and optionally replaced with a thymine base (T), and / or each T may be independently and optionally replaced with a U. The target sequences listed in Table 9 contain T, but the binding of DUX4-targeted oligonucleotides to RNA and / or DNA is intended. ^The target sequence start position is in NM_001293798.2 (sequence number 160).

[0286] In some embodiments, the DUX4-targeted oligonucleotide comprises an antisense chain having a length of 18–25 nucleosides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides), and includes SEQ ID NOs. 224–226, 261, 265, 320, 341, 343, 356, 388, 466, 483, 494, 501, 509, 552, 560, 561, 601, The sequence includes a complementary region to the target sequence as represented by one of the following: 921, 942, 953, 1294, 1296, 1301, 1320-1325, 1373, 1394, 1395, 1398, 1523, 1531, 1548, 1558, and 1561, where the complementary region is at least 16 nucleotides long (e.g., 16, 17, 18, or 19 nucleotides). In some embodiments, the antisense strand is 21 nucleotides long and includes a complementary region to the target sequence as represented by one of the sequence numbers 224-226, 261, 265, 320, 341, 343, 356, 388, 466, 483, 494, 501, 509, 552, 560, 561, 601, 921, 942, 953, 1294, 1296, 1301, 1320-1325, 1373, 1394, 1395, 1398, 1523, 1531, 1548, 1558, and 1561, where the complementary region is 19 nucleotides long. In some embodiments, the complementary region is perfectly complementary to all or part of its target sequence. In some embodiments, the complementary region contains one, two, three, or more mismatches.

[0287] In some embodiments, the DUX4-targeted oligonucleotide comprises an antisense chain containing at least 15 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) consecutive nucleosides of any one nucleotide sequence from SEQ ID NOs. 3027-3066. In some embodiments, the DUX4-targeted oligonucleotide further comprises a sense chain containing at least 15 (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) consecutive nucleosides of any one nucleotide sequence from SEQ ID NOs. 2987-3026.

[0288] In some embodiments, the DUX4-targeted oligonucleotide comprises an antisense strand containing one nucleotide sequence from SEQ ID NOs. 3027 to 3066. In some embodiments, the DUX4-targeted oligonucleotide further comprises a sense strand containing one nucleotide sequence from SEQ ID NOs. 2987 to 3026.

[0289] In some embodiments, the DUX4-targeted oligonucleotide is a double-stranded oligonucleotide (e.g., siRNA) comprising an antisense strand containing one nucleotide sequence from SEQ ID NOs. 3027 to 3066 and a sense strand that hybridizes to the antisense strand containing one nucleotide sequence from SEQ ID NOs. 2987 to 3026, where the antisense strand and / or (e.g., and) comprises one or more modified nucleosides (e.g., 2'-modified nucleosides). In some embodiments, the one or more modified nucleosides are selected from 2'-O-Me and 2'-F modified nucleosides.

[0290] In some embodiments, the DUX4-targeted oligonucleotide is a double-stranded oligonucleotide (e.g., siRNA) comprising an antisense strand containing one nucleotide sequence of any one of SEQ ID NOs: 3027-3066 and a sense strand that hybridizes to the antisense strand containing one nucleotide sequence of any one of SEQ ID NOs: 2987-3026, where each nucleoside in the antisense strand and / or (e.g., and) each nucleoside in the sense strand is a 2'-modified nucleoside selected from 2'-O-Me and 2'-F modified nucleosides.

[0291] In some embodiments, the DUX4-targeted oligonucleotide is a double-stranded oligonucleotide (e.g., siRNA) comprising an antisense strand containing one nucleotide sequence from SEQ ID NOs. 3027 to 3066 and a sense strand hybridizing to the antisense strand containing one nucleotide sequence from SEQ ID NOs. 2987 to 3026, wherein each nucleoside in the antisense strand and each nucleoside in the sense strand are 2'-modified nucleosides selected from 2'-O-Me and 2'-F modified nucleosides, and wherein the antisense strand and / or (e.g., and) the sense strand each contain one or more phosphorothioate nucleoside linkages. In some embodiments, the sense strand contains no phosphorothioate nucleoside linkages (all nucleoside linkages in the sense strand are phosphodiester nucleoside linkages), and the antisense strand contains one, two, or three phosphorothioate nucleoside linkages. In some embodiments, the antisense chain comprises two phosphorothioate nucleoside linkages, optionally where the two nucleoside linkages at the 3' end of the antisense chain are phosphorothioate nucleoside linkages, and the remaining nucleoside linkages in the antisense chain are phosphodiester nucleoside linkages.

[0292] In some embodiments, the antisense strand of a DUX4-targeted oligonucleotide is (5'→3'): fNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmN*fN*mN The structure includes, where "mN" refers to a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" refers to a 2'-fluoro (2'-F) modified nucleoside; "*" refers to a phosphorothioate nucleoside linkage; and the absence of "*" between two nucleosides refers to a phosphodiester nucleoside linkage.

[0293] In some embodiments, the sense strand of a DUX4-targeted oligonucleotide is (5'→3'): mNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfN The structure includes, where "mN" refers to a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" refers to a 2'-fluoro (2'-F) modified nucleoside; and the absence of "*" between the two nucleosides indicates a phosphodiester nucleoside linkage.

[0294] In some embodiments, in any one of the DUX-targeted siRNs described herein, one or more cytidine(C) atoms in the sense and antisense chains (e.g., 1, 2, 3, 4, 5, 6, 7, or more) are 2'-modified 5-methylcytidine (e.g., 2'-O-Me-modified 5-methylcytidine or 2'-F-modified 5-methylcytidine). In some embodiments, in any one of the DUX-targeted siRNs described herein, one or more cytidine(C) atoms in the sense chain (e.g., 1, 2, 3, 4) and / or one or more cytidine(C) atoms in the antisense chain (e.g., 1, 2, 3, 4) are 2'-modified 5'-methylcytidine (e.g., 2'-O-Me-modified 5-methylcytidine or 2'-F-modified 5-methylcytidine).

[0295] In some embodiments, the cytidine CG motif of the sense and / or antisense chain is 2'-modified 5-methylcytidine (e.g., 2'-O-Me modified 5-methylcytidine or 2'-F modified 5-methylcytidine). In some embodiments, the cytidine of one or more (e.g., 1, 2, 3, 4) CG motifs of the sense chain is 2'-modified 5-methylcytidine (e.g., 2'-O-Me modified 5-methylcytidine or 2'-F modified 5-methylcytidine). In some embodiments, the cytidine of one or more (e.g., 1, 2, 3, 4) CG motifs of the antisense chain is 2'-modified 5-methylcytidine (e.g., 2'-O-Me modified 5-methylcytidine or 2'-F modified 5-methylcytidine). In some embodiments, the cytidines of one or more (e.g., 1, 2, 3, 4) CG motifs of the sense chain are 2'-modified 5-methylcytidines (e.g., 2'-O-Me modified 5-methylcytidine or 2'-F modified 5-methylcytidine); and the cytidines of one or more (e.g., 1, 2, 3, 4) CG motifs of the antisense chain are 2'-modified 5-methylcytidines (e.g., 2'-O-Me modified 5-methylcytidine or 2'-F modified 5-methylcytidine).

[0296] In some embodiments, the antisense strand of the DUX4-targeted oligonucleotide is selected from modified versions of SEQ ID NOs. 3027–3066 listed in Table 8. In some embodiments, the sense strand of the DUX4-targeted oligonucleotide is selected from modified versions of SEQ ID NOs. 2987–3026 listed in Table 8. In some embodiments, the DUX4-targeted oligonucleotide is an siRNA selected from the siRNAs listed in Table 8.

[0297] In some embodiments, the antisense strand of the DUX4-targeted oligonucleotide is selected from one modified version of SEQ ID NOs. 3027, 3037, 3039, 3040, 3041, 3044, 3052, and 3061 listed in Table 9. In some embodiments, the sense strand of the DUX4-targeted oligonucleotide is selected from one modified version of SEQ ID NOs. 2987, 2997, 2999, 3000, 3001, 3004, 3012, and 3021 listed in Table 9. In some embodiments, the DUX4-targeted oligonucleotide is an siRNA selected from the siRNAs listed in Table 9.

[0298] In some embodiments, any one of the DUX4-targeted oligonucleotides (for example, DUX4-targeted siRNAs selected from the siRNAs in Table 8) may be in the form of a salt, for example, as a salt of sodium, potassium, or magnesium. In some embodiments, any one of the DUX4-targeted oligonucleotides (for example, DUX4-targeted siRNAs selected from the siRNAs in Table 9) may be in the form of a salt, for example, as a salt of sodium, potassium, or magnesium.

[0299] In some embodiments, one 5' or 3' nucleoside (e.g., a terminal nucleoside) of any oligonucleotide described herein (e.g., oligonucleotides listed in Table 8) is optionally conjugated to an amine group via a spacer. In some embodiments, one 5' or 3' nucleoside (e.g., a terminal nucleoside) of any oligonucleotide described herein (e.g., oligonucleotides listed in Table 9) is optionally conjugated to an amine group via a spacer. In some embodiments, the spacer comprises an aliphatic moiety. In some embodiments, the spacer comprises a polyethylene glycol moiety. In some embodiments, a phosphodiester linkage exists between the oligonucleotide spacer and the 5' or 3' nucleoside. In some embodiments, a 5' or 3' nucleoside (e.g., a terminal nucleoside) of any of the oligonucleotides described herein (e.g., oligonucleotides listed in Table 8) is conjugated to a spacer, wherein the spacer 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)2NR A -, -NR A S(O)2-, or a combination thereof; each R AThese are independently hydrogen, or a substituted or unsubstituted alkyl group. In some embodiments, a 5' or 3' nucleoside (e.g., a terminal nucleoside) of any of the oligonucleotides described herein (e.g., oligonucleotides listed in Table 9) is conjugated to a spacer, wherein the spacer is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, a 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)2NR A -, -NR A S(O)2-, or a combination thereof; each R A In one embodiment, the spacer is a substituted or unsubstituted alkyl, a substituted or unsubstituted heterocyclene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )-, or -C(=O)N(R A )2, or a combination thereof.

[0300] In some embodiments, one 5' or 3' nucleoside of any of the oligonucleotides described herein (for example, the oligonucleotides, sense or antisense chains listed in Table 8) is of the formula -NH2-(CH2) nIt is conjugated to a compound represented by the formula -NH2-(CH2) (wherein n is an integer from 1 to 12). In some embodiments, a 5' or 3' nucleoside of any one of the oligonucleotides described herein (e.g., oligonucleotides, sense or antisense chains listed in Table 9) is conjugated to a compound represented by the formula -NH2-(CH2) n It is conjugated to a compound represented by the formula NH2-(CH2) (wherein n is an integer from 1 to 12). In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the phosphodiester linkage is an oligonucleotide (for example, oligonucleotides, sense or antisense chains listed in Table 8) of the formula NH2-(CH2) n It exists between the compound represented by - and the 5' or 3' nucleoside. In some embodiments, the phosphodiester linkage is of the oligonucleotide (e.g., oligonucleotides, sense or antisense chains listed in Table 9) of the formula NH2-(CH2) n The compound represented by the formula NH2-(CH2)6- exists between the compound and the 5' or 3' nucleoside. In some embodiments, the compound represented by the formula NH2-(CH2)6- is conjugated to the oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide (e.g., the 5' phosphate of the sense or antisense chain). In some embodiments, the compound represented by the formula NH2-(CH2)6- is conjugated to the oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3' phosphate of the oligonucleotide (e.g., the 3' phosphate of the sense or antisense chain). In some embodiments, the oligonucleotide is conjugated to a targeting agent, such as a muscle targeting agent, such as an anti-TfR antibody, via an amine group, for example.

[0301] C. Linker The complexes described herein generally include a linker that connects one of the anti-TfR antibodies described herein to a molecular payload. The linker includes at least one covalent bond. In some embodiments, the linker may be a single bond, e.g., a disulfide bond or disulfide crosslink, connecting the anti-TfR antibody to the molecular payload. However, in some embodiments, the linker may connect one of the anti-TfR antibodies described herein to the molecular payload through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be an incleavable linker. The linker is generally stable in vitro and in vivo and may be stable in a given cellular environment. In addition, the linker generally does not negatively affect the functional properties of either the anti-TfR antibody or the molecular payload. Examples and methods of linker synthesis are known in the art (see, for example, Kline, T. et al. "Methods to Make Homogenous Antibody Drug Conjugates." Pharmaceutical Research, 2015, 32:11, 3480-3493.; Jain, N. et al. "Current ADC Linker Chemistry." Pharm Res. 2015, 32:11, 3526-3540.; McCombs, JR and Owen, SC "Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry." AAPS J. 2015, 17:2, 339-351.).

[0302] The linker precursor will typically contain two different highly reactive species capable of attaching to both the anti-TfR antibody and the molecular payload. In some embodiments, the two different highly reactive species may be a nucleophile and / or (for example, and) an electrophile. In some embodiments, the linker is attached to the anti-TfR antibody via conjugation to a lysine or cysteine ​​residue of the anti-TfR antibody. In some embodiments, the linker is attached to a cysteine ​​residue of the anti-TfR antibody via a maleimide-containing linker, where optionally the maleimide-containing linker contains a maleimide-caproyl or maleimide-methylcyclohexane-1-carboxylate group. In some embodiments, the linker is attached to a cysteine ​​residue or a thiol-functionalized molecular payload of the anti-TfR antibody via a 3-arylpropionitrile functional group. In some embodiments, the linker is attached to a lysine residue of the anti-TfR antibody. In some embodiments, the linker is connected to the anti-TfR antibody and / or (for example, and) the molecular payload via an amide bond, carbamate bond, hydrazide, triazole, thioether, or disulfide bond.

[0303] i. Cuttable linker The cleavable linker may be a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. These linkers are generally cleavable only intracellularly and are preferably stable in the extracellular environment, for example, outside the cells of muscle cells.

[0304] Protease-sensitive linkers are cleavable by protease enzyme activity. These linkers typically contain peptide sequences, which may be 2–10 amino acids, about 2–5 amino acids, about 5–10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length. In some embodiments, the peptide sequences may contain naturally occurring amino acids, e.g., cysteine, alanine, or amino acids that are not naturally occurring or are modified. Amino acids that are not naturally occurring include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the protease-sensitive linkers contain valine-citrulline sequences or alanine-citrulline sequences. In some embodiments, the protease-sensitive linkers can be cleaved by lysosomal proteases, e.g., cathepsin B, and / or endosomal proteases.

[0305] A pH-sensitive linker is a covalent linkage that is readily degraded in a high or low pH environment. In some embodiments, the pH-sensitive linker may be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved within an endosome or lysosome.

[0306] In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with a glutathione species inside the cell. In some embodiments, the disulfide moiety further comprises at least one amino acid, for example, a cysteine ​​residue.

[0307] In some embodiments, the linker is a Val-cit linker (as described, for example, in U.S. Patent 6,214,345, incorporated herein by reference). In some embodiments, the pre-conjugation Val-cit linker has the following structure: [ka] It has.

[0308] In some embodiments, the conjugated val-cit linker has the following structure: [ka] It has.

[0309] In some embodiments, the Val-cit linker is attached to a reactive chemical moiety (e.g., a SPAAC for click chemistry conjugation). In some embodiments, the Val-cit linker attached to the highly reactive chemical moiety (e.g., a SPAAC for click chemistry conjugation) before click chemistry conjugation has the following structure: [ka] The formula has such that n is a number between 0 and 10. In some embodiments, n is 3.

[0310] In some embodiments, a val-cit linker attached to a highly reactive chemical moiety (e.g., a SPAAC for click chemistry conjugation) is conjugated to a molecular payload (e.g., an oligonucleotide) (e.g., via a different chemical moiety). In some embodiments, a val-cit linker attached to a highly reactive chemical moiety (e.g., a SPAAC for click chemistry conjugation) and conjugated to a molecular payload (e.g., an oligonucleotide) has a structure represented by formula (A) (before click chemistry conjugation): [ka] The formula has such a property that n is a number between 0 and 10. In some embodiments, n is 3.

[0311] In some embodiments, after conjugation to a molecular payload (e.g., an oligonucleotide), the val-cit linker has a structure represented by formula (B): [ka] The formula includes n, where n is a number between 0 and 10, and m is a number between 0 and 10. In some embodiments, n is 3 and m is 4.

[0312] ii. Linker that cannot be severed In some embodiments, an uncleavable linker may be used. Generally, an uncleavable linker cannot be readily degraded in the cellular or physiological environment. In some embodiments, the uncleavable linker comprises an optionally substituted alkyl group, where substitution may include halogens, hydroxyl groups, oxygen species, and other common substitutions. In some embodiments, the linker may comprise an optionally substituted alkyl group, an optionally substituted alkylene, an optionally substituted arylene, a optionally substituted heteroarylene, a peptide sequence comprising at least one non-natural amino acid, a truncated glycan, an enzymatically undegradable sugar (one or more), an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, biotin, biphenyl, polyethylene glycol or equivalent compounds of repeating units, an acid ester, an acid amide, a sulfamide, and / or (for example, and), an alkoxy-amine linker. In some embodiments, sortase-mediated ligation may be used to ligate an anti-TfR antibody containing an LPXT sequence to a molecular payload containing a (G)n sequence (see, for example, Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1):1-10).

[0313] In some embodiments, the linker may include a substituted alkylene, optionally substituted alkenylene, optionally substituted alkylene, optionally substituted cycloalkylene, optionally substituted cycloalkenylene, optionally substituted arylene, optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; imino, optionally substituted nitrogen species, optionally substituted oxygen species O, optionally substituted sulfur species, or poly(alkylene oxide), for example, polyethylene oxide or polypropylene oxide.

[0314] In some embodiments, the linker has the following structure: [ka] It may also contain a bis-PFP monodidispersed PEG having n, where n is 1 to 10.

[0315] iii. Linker conjugation In some embodiments, the linker is linked to the anti-TfR antibody and / or (for example, and) the molecular payload via a phosphate, thioether, ether, carbon-carbon, carbamate, or amide bond. In some embodiments, the linker is linked to the oligonucleotide via a phosphate or phosphorothioate group, for example, a phosphate at the terminal of the oligonucleotide backbone. In some embodiments, the linker is linked to the anti-TfR antibody via a lysine residue or cysteine ​​residue present on the anti-TfR antibody.

[0316] In some embodiments, the linker is linked to an anti-TfR antibody and / or (for example, and) a molecular payload by a cycloaddition reaction between an azide and an alkyne forming a triazole, where the azide and alkyne may be positioned on the anti-TfR antibody, molecular payload, or linker. In some embodiments, the alkyne may be a cyclic alkyne, for example, cyclooctane. In some embodiments, the alkyne may be bicyclononine (also known as bicyclo[6.1.0]nonine or BCN) or a substituted bicyclononine. In some embodiments, cyclooctane is as described in the international patent application publication WO2011136645, published on November 3, 2011, titled "Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions". In some embodiments, the azide may be a sugar or carbohydrate molecule containing the azide. In some embodiments, the azide may be 6-azide-6-deoxygalactose or 6-azide-N-acetylgalactosamine. In some embodiments, sugar or carbohydrate molecules containing azides are as described in the international patent application publication WO2016170186, published on 27 October 2016, titled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase".In some embodiments, the cycloaddition reaction between an azide and an alkyne that forms a triazole (where the azide and alkyne may be positioned on an anti-TfR antibody, molecular payload, or linker) is described in the international patent application publication WO2014065661, published on May 1, 2014, titled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or the international patent application publication WO2016170186, published on October 27, 2016, titled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase".

[0317] In some embodiments, the linker further includes spacers, e.g., polyethylene glycol spacers or acyl / carbamoyl sulfamide spacers, e.g., HydraSpace® spacers. In some embodiments, the spacers are as described in Verkade, JMM et al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturing Capability, Stability, and Therapeutic Index of Antibody-Drug Conjugates", Antibodies, 2018, 7, 12.

[0318] In some embodiments, the linker is ligated to the anti-TfR antibody and / or (for example, and) molecular payload by a Diels-Alder reaction between the dienephile and the diene / heterodiene, where the dienephile and the diene / heterodiene may be located on the anti-TfR antibody, molecular payload, or linker. In some embodiments, the linker is ligated to the anti-TfR antibody and / or (for example, and) molecular payload by other pericyclic reactions, for example, an ene reaction. In some embodiments, the linker is ligated to the anti-TfR antibody and / or (for example, and) molecular payload by an amide, thioamide, or sulfonamide bonding reaction. In some embodiments, the linker is ligated to the anti-TfR antibody and / or (for example, and) molecular payload by a condensation reaction that forms an oxime group, hydrazone group, or semicarbazide group between the linker and the anti-TfR antibody and / or (for example, and) molecular payload.

[0319] In some embodiments, the linker is linked to an anti-TfR antibody and / or (for example, and) a molecular payload by a conjugate addition reaction between a nucleophile (e.g., an amine group or a hydroxyl group) and an electrophile (e.g., a carboxylic acid, a carbonate, or an aldehyde). In some embodiments, prior to the reaction between the linker and the anti-TfR antibody or molecular payload, the nucleophile may be present on the linker and the electrophile may be present on the anti-TfR antibody or molecular payload. In some embodiments, prior to the reaction between the linker and the anti-TfR antibody or molecular payload, the electrophile may be present on the linker and the nucleophile may be present on the anti-TfR antibody or molecular payload. In some embodiments, the electrophile may be an azide, pentafluorophenyl, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimidyl ester, sulfosuccinimidyl ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center, and / or (for example, and), activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, optionally substituted alkyne, optionally substituted aryl, optionally substituted heterocyclyl, hydroxyl group, amino group, alkylamino group, anilide group, or thiol group.

[0320] In some embodiments, the val-cit linker attached to a highly reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) has the following structure: [ka] The anti-TfR antibody is conjugated by a compound, where m is a number between 0 and 10. In some embodiments, m is 4.

[0321] In some embodiments, the val-cit linker attached to a highly reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) has a structure represented by formula (G): [ka] (G) It is conjugated to an anti-TfR antibody having m, where m is a number from 0 to 10. In some embodiments, m is 4. It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (G) is produced as a result of the reaction of the anti-TfR1 antibody with an amine such as lysine epsilonamine.

[0322] In some embodiments, the val-cit linker, which is attached to a highly reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) and conjugated to an anti-TfR antibody, has a structure represented by formula (F): [ka] (F) The formula has the following characteristics, where n is a number from 0 to 10, and m is a number from 0 to 10. In some embodiments, n is 3 and / or (for example, and) m is 4. In some embodiments, the oligonucleotide is covalently linked to a compound containing the structure represented by formula (F), thereby forming a complex containing the structure represented by formula (D). It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (F) is a result of the reaction of the anti-TfR1 antibody with an amine such as lysine epsilonamine.

[0323] In some embodiments, the val-cit linker that connects the antibody and the molecular payload has a structure represented by formula (C): [ka] The formula has a value where n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3 and / or (for example, and) m is 4. In some embodiments, n is 3 and / or (for example, and) m is 4. In some embodiments, X is NH of the antibody (for example, NH from the amine group of lysine), S (for example, S from the thiol group of cysteine), or O (for example, O from the hydroxyl group of serine, threonine, or tyrosine).

[0324] In some embodiments, the composite described herein has a structure represented by formula (D): [ka] The formula has such that n is a number between 0 and 10, where m is a number between 0 and 10. In some embodiments, n is 3 and / or (for example, and) m is 4.

[0325] In structural formulas (A), (B), (C), and (D), L1 is a spacer which in some embodiments is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, -O-, -N(RA)-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NRA-, -NRAC(=O)-, -NRAC(=O)RA-, -C(=O)RA-, -NRAC(=O)O-, -NRAC(=O)N(RA)-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(RA)-, -S(O)2NRA-, -NRAS(O)2-, or a combination thereof, where each R A L1 is independently hydrogen, or a substituted or unsubstituted alkyl group. In some embodiments, L1 is [ka] And here L2 is, [ka] Here, a labels the site directly linked to the carbamate moiety represented by formulas (A), (B), (C), and (D); and b labels the site covalently linked (directly or via an additional chemical moiety) to the oligonucleotide.

[0326] In some forms, L1 is as follows: [ka] Here, a labels the site directly linked to the carbamate moiety represented by formulas (A), (B), (C), and (D); and b labels the site covalently linked (directly or via an additional chemical moiety) to the oligonucleotide.

[0327] In some forms, L1 is [ka] Here, x is between 0 and 10. For example, in some embodiments, x is 3, 4, 5, or 6. In some embodiments, L1 is [ka] In some forms, L1 is [ka] That is the case.

[0328] In some forms, L1 is [ka] [ka] That is the case.

[0329] In some forms, L1 is [ka] Here, y is between 0 and 10. For example, in some embodiments, y is 3, 4, 5, or 6.

[0330] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide. In some embodiments, the linkage of L1 to the 5' phosphate of the oligonucleotide forms a phosphodiester bond between L1 and the oligonucleotide.

[0331] In some embodiments, L1 is linked to the 3' phosphate of the oligonucleotide. In some embodiments, the linkage of L1 to the 3' phosphate of the oligonucleotide forms a phosphodiester bond between L1 and the oligonucleotide.

[0332] In some embodiments, L1 is optional (for example, it does not need to exist).

[0333] In some embodiments, any one of the complexes described herein has a structure represented by formula (E): [ka] The formula has such that n is between 0 and 15 (for example, 3) and m is between 0 and 15 (for example, 4).

[0334] C. Examples of antibody-molecule payload complexes Furthermore, non-limiting examples of complexes comprising any one anti-TfR antibody as described herein, covalently linked to any of the molecular payloads (e.g., oligonucleotides) described herein. In some embodiments, the anti-TfR antibody (e.g., any one of the anti-TfR antibodies provided in Tables 2-7) is covalently linked via a linker to a molecular payload (e.g., an oligonucleotide comprising at least 12 consecutive nucleotides (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19) of any one nucleotide sequence of SEQ ID NOs. 163-3066). In some embodiments, the anti-TfR antibody (e.g., any one of the anti-TfR antibodies provided in Tables 2-7) is covalently linked via a linker to a molecular payload (e.g., an oligonucleotide such as the oligonucleotide provided in Table 8). In some embodiments, the anti-TfR antibody (e.g., any one of the anti-TfR antibodies provided in Tables 2-7) is covalently linked via a linker to a molecular payload (e.g., an oligonucleotide such as the oligonucleotide provided in Table 9). Any of the linkers described herein may be used. In some embodiments, when the molecular payload is an oligonucleotide, the linker is ligated to the 5' end, 3' end, or internally to the sense or antisense strand. In some embodiments, the molecular payload is an siRNA, and the linker is ligated to the 5' end of the sense strand. In some embodiments, the linker is ligated to an anti-TfR antibody via thiol-reactive ligation (e.g., via cysteine ​​in the anti-TfR antibody). In some embodiments, the linker (e.g., a Val-cit linker) is ligated to an antibody (e.g., an anti-TfR antibody described herein) via an amine group (e.g., via lysine in the antibody). In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., DUX4-targeted oligonucleotides listed in Table 8).In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (for example, the DUX4-targeted oligonucleotides listed in Table 9). In some embodiments, the molecular payload is the sense strand of a DUX4-targeted siRNA. In some embodiments, the molecular payload is the antisense strand of a DUX4-targeted siRNA. In some embodiments, the molecular payload is a DUX4-targeted siRNA comprising both a sense strand and an antisense strand.

[0335] An example of the structure of a complex containing an anti-TfR antibody covalently linked to a molecular payload via a Val-cit linker is provided below: [ka] Here, the linker is linked to the antibody via thiol-reactive linkage (e.g., via cysteine ​​in the antibody). In some embodiments, the molecular payload is an oligonucleotide comprising at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19) consecutive nucleotides of any one nucleotide sequence of SEQ ID NOs. In some embodiments, the molecular payload is an oligonucleotide comprising at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (e.g., target sequences listed in Table 8), wherein optionally the antisense strand comprises at least 16 consecutive nucleotides of any one antisense sequence listed in Table 8, and optionally the DUX4-targeted oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), wherein optionally the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally the DUX4-targeted oligonucleotide further comprises a sense strand that hybridizes to the antisense strand.

[0336] Another example of the structure of a complex containing an anti-TfR antibody covalently linked to a molecular payload via a Val-cit linker is provided below: [ka] Here, n is a number between 0 and 10, where m is a number between 0 and 10, where the linker is linked to the antibody via an amine group (e.g., on a lysine residue) and / or (e.g., and), where the linker is linked to the sense or antisense strand (e.g., at the 5' end, 3' end, or internally). In some embodiments, the linker is linked to the antibody via lysine, the linker is linked to the oligonucleotide at the 5' end, where n is 3 and m is 4. In some embodiments, the molecular payload is an oligonucleotide containing at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19) nucleotides of any one nucleotide sequence of SEQ ID NOs. 163-3066. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, L1 is one of the spacers described herein.

[0337] It should be understood that antibodies can be conjugated to molecular payloads in various stoichiometric ways, and this property is sometimes referred to as the drug-antibody ratio (DAR), where "drug" is the molecular payload. In some embodiments, one molecular payload is conjugated to the antibody (DAR=1). In some embodiments, two molecular payloads are conjugated to the antibody (DAR=2). In some embodiments, three molecular payloads are conjugated to the antibody (DAR=3). In some embodiments, four molecular payloads are conjugated to the antibody (DAR=4). In some embodiments, a mixture of different complexes is provided, each having a different DAR. In some embodiments, the average DAR of the complexes in such a mixture may be in the range of 1-3, 1-4, or 1-5 or more. The DAR may be increased by conjugating molecular payloads to various sites on the antibody and / or (for example, and) by conjugating multimers to one or more sites on the antibody. For example, DAR in 2 may be achieved by conjugating a single-molecule payload to two different sites on the antibody, or by conjugating a dimeric molecular payload to a single site on the antibody.

[0338] In some embodiments, the complex described herein comprises an anti-TfR antibody described herein (for example, an antibody provided in Tables 2-7) covalently linked to a molecular payload. In some embodiments, the complex described herein comprises an anti-TfR antibody described herein (for example, an antibody provided in Tables 2-7) covalently linked to a molecular payload via a linker (for example, a Val-cit linker). In some embodiments, the linker (for example, a Val-cit linker) is linked to the antibody (for example, an anti-TfR antibody described herein) via thiol-reactive linkage (for example, via cysteine ​​in the antibody). In some embodiments, the linker (for example, a Val-cit linker) is linked to the antibody (for example, an anti-TfR antibody described herein) via an amine group (for example, via lysine in the antibody). In some embodiments, the molecular payload is an oligonucleotide comprising at least 12 (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19) nucleotides of any one nucleotide sequence of SEQ ID NOs. In some embodiments, the molecular payload is an oligonucleotide comprising at least 16 nucleotides of complementary region to a target sequence in DUX4 mRNA (e.g., a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1586, and optionally wherein the DUX4 targeted oligonucleotide further comprises a sense strand that hybridizes to the antisense strand.In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0339] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises any one of the antibodies listed in Table 2: CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0340] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH containing the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 71, or SEQ ID NO: 72, and a VL containing the amino acid sequence of SEQ ID NO: 70. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0341] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH containing the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL containing the amino acid sequence of SEQ ID NO: 74. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0342] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH containing the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL containing the amino acid sequence of SEQ ID NO: 75. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0343] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises VH containing the amino acid sequence of SEQ ID NO: 77 and VL containing the amino acid sequence of SEQ ID NO: 78. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0344] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH containing the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 79, and a VL containing the amino acid sequence of SEQ ID NO: 80. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0345] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises VH containing the amino acid sequence of SEQ ID NO: 154 and VL containing the amino acid sequence of SEQ ID NO: 155. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0346] In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (for example, the DUX4-targeted oligonucleotides listed in Table 8). In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (for example, the DUX4-targeted oligonucleotides listed in Table 9).

[0347] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 86, or SEQ ID NO: 87, and a light chain containing the amino acid sequence of SEQ ID NO: 85. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0348] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91 and a light chain containing the amino acid sequence of SEQ ID NO: 89. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0349] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain comprising a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs: 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0350] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 94 and a light chain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain comprising a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs: 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0351] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 92 and a light chain containing the amino acid sequence of SEQ ID NO: 93. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0352] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 156 and a light chain containing the amino acid sequence of SEQ ID NO: 157. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0353] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99, and a VL containing the amino acid sequence of SEQ ID NO: 85. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs. 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence as represented by any one of SEQ ID NOs. 1575-1986, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense chain that hybridizes to the antisense chain. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 8, and optionally further comprising a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, the target sequences listed in Table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in Table 9, and optionally further comprising a sense strand that hybridizes to the antisense strand.

[0354] In some embodiments, the complex described herein comprises an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a heavy chain containing the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101 and a light chain containing the amino acid sequence of SEQ ID NO: 89. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense chain containing a region of at least 16 nucleotides complementary to a target sequence in DUX4 mRNA (for example, a target sequence as represented by any one of SEQ ID NOs: 163-1574), optionally wherein the antisense chain comprises at least 16 consecutive nucleotides of any one antisense sequence a...

Claims

1. A complex comprising an anti-transferrin receptor (TfR) antibody covalently linked to a siRNA oligonucleotide, wherein the siRNA oligonucleotide comprises an antisense chain of 15–25 nucleotides in length and a complementary region to DUX4 RNA which is at least 16 consecutive nucleosides in length, and here: (i) CDR-H1 containing the amino acid sequence of SEQ ID NO: 27, CDR-H2 containing the amino acid sequence of SEQ ID NO: 28, CDR-H3 containing the amino acid sequence of SEQ ID NO: 29, CDR-L1 containing the amino acid sequence of SEQ ID NO: 30, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32; (ii) CDR-H1 containing the amino acid sequence of SEQ ID NO: 33, CDR-H2 containing the amino acid sequence of SEQ ID NO: 34, CDR-H3 containing the amino acid sequence of SEQ ID NO: 35, CDR-L1 containing the amino acid sequence of SEQ ID NO: 36, CDR-L2 containing the amino acid sequence of SEQ ID NO: 37, and CDR-L3 containing the amino acid sequence of SEQ ID NO: 32; or (iii) The complex comprising CDR-H1 containing the amino acid sequence of SEQ ID NO: 38, CDR-H2 containing the amino acid sequence of SEQ ID NO: 39, CDR-H3 containing the amino acid sequence of SEQ ID NO: 40, CDR-L1 containing the amino acid sequence of SEQ ID NO: 41, CDR-L2 containing the amino acid sequence of SEQ ID NO: 31, and CDR-L3 containing the amino acid sequence of SEQ ID NO:

42.

2. The complex according to claim 1, wherein the anti-transferrin receptor (TfR) antibody comprises VH containing the amino acid sequence of SEQ ID NO: 76 and VL containing the amino acid sequence of SEQ ID NO:

75.

3. The complex according to claim 1 or 2, wherein the anti-TfR antibody is Fab, and comprises a heavy chain having the amino acid sequence of SEQ ID NO: 101 and a light chain having the amino acid sequence of SEQ ID NO:

90.

4. The complex according to any one of claims 1 to 3, wherein the heavy chain of the antibody contains an N-terminal pyroglutamic acid.

5. The complex according to any one of claims 1 to 4, wherein the siRNA oligonucleotide further comprises a sense strand containing at least 18 consecutive nucleosides complementary to the antisense strand.

6. The complex according to claim 5, wherein the antisense strand has a length of 21 to 23 nucleotides, and / or the sense strand has a length of 19 to 21 nucleotides.

7. The complex according to any one of claims 1 to 6, wherein the siRNA oligonucleotide includes a 3' overhang of 1 to 3 nucleosides on the antisense strand.

8. The complex according to any one of claims 1 to 7, wherein the siRNA oligonucleotide comprises one or more modified nucleosides.

9. The complex according to any one of claims 5 to 8, wherein the sense chain and / or antisense chain comprises one or more modified nucleosides.

10. The complex according to claim 8 or 9, wherein one or more modified nucleosides are 2'-modified nucleosides.

11. The complex according to claim 10, wherein one or more 2'-modified nucleosides are selected from: 2'-fluoro(2'-F), 2'-O-methyl(2'-O-Me), 2'-O-methoxyethyl(2'-MOE), 2'-O-aminopropyl(2'-O-AP), 2'-O-dimethylaminoethyl(2'-O-DMAOE), 2'-O-dimethylaminopropyl(2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl(2'-O-DMAEOE), and 2'-O-N-methylacetamide(2'-O-NMA).

12. The complex according to any one of claims 5 to 11, wherein each nucleoside of the sense chain and the antisense chain is a 2'-modified nucleoside.

13. The complex according to claim 12, wherein each 2'-modified nucleoside is a 2'-O-methyl (2'-O-Me) modified nucleoside or a 2'-fluoro (2'-F) modified nucleoside.

14. The complex according to any one of claims 1 to 13, wherein the oligonucleotide comprises one or more modified nucleoside links.

15. The complex according to claim 14, wherein one or more modified nucleoside linkages include phosphorothioate nucleoside linkages.

16. The complex according to any one of claims 5 to 15, wherein the sense chain comprises a phosphorothioate nucleoside linkage between at least two nucleosides, and / or the antisense chain comprises a phosphorothioate nucleoside linkage between at least two nucleosides.

17. The complex according to claim 15 or 16, wherein the sense chain and / or antisense chain include phosphorothioate nucleoside linkages in the first, second, and / or third nucleoside linkages at the 5' or 3' terminus.

18. The complex according to any one of claims 1 to 17, wherein the siRNA oligonucleotide comprises a 5'-vinylphosphonate modification.

19. The complex according to any one of claims 1 to 18, wherein the oligonucleotide is in the form of a salt.

20. The composite according to claim 19, wherein the salt is a salt of sodium, potassium, or magnesium.

21. The complex according to any one of claims 1 to 20, wherein an anti-TfR antibody and a siRNA oligonucleotide are covalently linked via a linker.

22. The complex according to claim 21, wherein the linker comprises a valine-citrulline sequence.

23. The anti-TfR antibody is ligated to the 5' end of the sense strand of the siRNA oligonucleotide. The composite according to claim 21 or 22.

24. A complex according to any one of claims 21-23, wherein an anti-TfR antibody is covalently linked to a siRNA oligonucleotide via a lysine residue of the antibody.

25. A complex according to any one of claims 21-23, wherein an anti-TfR antibody is covalently linked to a siRNA oligonucleotide via a cysteine ​​residue of the antibody.

26. A complex for use in a method of treating a disease or condition that can be improved or defended against by reducing DUX4 expression in cells, wherein the method comprises bringing the cells into contact with the complex, according to any one of claims 1 to 25.

27. The complex for use according to claim 26, wherein the cells are muscle cells.

28. A complex for use in claim 26 or 27, comprising reducing DUX4 expression, which reduces DUX4 protein and / or mRNA levels.

29. A complex according to any one of claims 1 to 25 for use in a method for treating facioscapulohumeral dystrophy (FSHD), wherein the method comprises administering the complex to a subject having abnormal production of the DUX4 protein.

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