Muscle-targeting complexes and their uses for treating facioscapulohumeral muscular dystrophy - Patent Application 20070123333

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 under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 133,156, entitled "MUSCLE-TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY," filed on December 31, 2020, and U.S. Provisional Application No. 63 / 181,439, entitled "MUSCLE-TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY," filed on April 29, 2021, the contents of each of which are incorporated by reference in their entirety.

[0002] FIELD OF THE PRESENT APPLICATION This application relates to oligonucleotides designed to target DUX4 RNA and targeting complexes for delivering molecular payloads (e.g., oligonucleotides) to cells, and their uses, particularly for the treatment of disease.

[0003] Reference to a sequence listing submitted as a text file via EFS-WEB This application contains a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on December 30, 2021, is entitled D082470047WO00-SEQ-ZJG and is 708,513 bytes in size. [Background technology]

[0004] background Muscular dystrophies (MD) are a group of diseases characterized by progressive weakness and loss of muscle mass. These diseases are caused by mutations in genes that code for proteins necessary to form healthy muscle tissue. Facioscapulohumeral muscular dystrophy (FSHD) is a dominantly inherited form of MD that primarily affects the muscles of the face, scapula, and upper arm. Other symptoms of FSHD include abdominal muscle weakness, retinal abnormalities, hearing loss, and joint pain and inflammation. FSHD is the most common of nine types of MD that affect both adults and children, with a worldwide incidence of approximately 1 in 8,300. FSHD is caused by abnormal production of double homeobox 4 (DUX4), a protein with unknown function. The DUX4 gene, which codes for the DUX4 protein, is located in the D4Z4 repeat region on chromosome 4 and is typically expressed only during fetal development, after which it is silenced by hypermethylation of the D4Z4 repeats that surround and compact the DUX4 gene. Two types of FSHD have been described: type 1 and type 2. Type 1, which accounts for approximately 95% of cases, is associated with a deletion of the D4Z4 repeat on chromosome 4. Whereas unaffected individuals generally have more than 10 repeats aligned in the subtelomeric region of chromosome 4, the most common form of FSHD (FSHD1) is caused by a contraction of the array to fewer than 10 repeats and is associated with reduced epigenetic repression and variable expression of DUX4 in skeletal muscle. Two allelic variants of chromosome 4q (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 DUX4 transcripts are polyadenylated and translated into stable proteins. Type 2 FSHD, which accounts for approximately 5% of cases, is associated with mutations in the SMCHD1 gene on chromosome 18. There is no effective treatment for FSHD other than supportive care and treatment to address the symptoms of the disease. Summary of the Invention

[0005] overview In some aspects, the present disclosure provides oligonucleotides designed to target DUX4 RNA. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA, which are useful for reducing the level of DUX4 mRNA and / or protein associated with features of facioscapulohumeral muscular dystrophy (FSHD), including muscle 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 engage the RNA-induced silencing complex (RISC) for DUX4 RNA degradation, but also reduce off-target effects. In some embodiments, the oligonucleotides are designed to reduce the level of DUX4 RNA and / or protein. In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desired binding affinity properties. In some embodiments, the oligonucleotides are designed to have desired toxicity and / or immunogenicity profiles.

[0006] According to some aspects, the present disclosure provides a complex for targeting muscle cells (e.g., primary myoblast cells) for delivery of a molecular payload (e.g., a DUX4-targeting oligonucleotide described herein) to those cells. In some embodiments, the complexes provided herein are particularly useful for delivery of a molecular payload that inhibits expression or activity of DUX4 in subjects having or suspected of having facioscapulohumeral muscular dystrophy (FSHD). Thus, 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 a muscle cell for delivery of the molecular payload to the muscle cell. In some embodiments, the complex is taken up into the cell via receptor-mediated internalization, whereupon the molecular payload may be released inside 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 the muscle cell. 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 the disclosure provide a complex that includes a muscle targeting agent covalently linked to an oligonucleotide that targets double homeobox 4 (DUX4) mRNA, where the oligonucleotide includes an antisense strand that is 18 to 25 nucleotides in length and is selected from the group consisting of SEQ ID NOs: 356, 501, 1398, 494, 509, 224, 1320, 561, 225, 226, 261, 265, 320, 341, 343, 388, 466, 470, 472, 474, 476, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552 and wherein the region of complementarity is at least 16 contiguous nucleosides 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 embodiments, the antisense strand comprises the nucleotide sequence of any one 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.

[0011] In some embodiments, the oligonucleotide further comprises a sense strand comprising at least 18 contiguous nucleosides that are complementary to the antisense strand.

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

[0013] In some embodiments, the one or more modified nucleosides are 2'-modified nucleotides, optionally wherein the one or more 2'-modified nucleosides are 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), 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 internucleoside linkages.

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

[0017] In some embodiments, the linkage between the two nucleosides at the 3'-end of the antisense strand is a phosphorothioate internucleoside linkage.

[0018] In some embodiments, one or more cytidines of the oligonucleotide are 2'-modified 5-methyl-cytidines, optionally wherein the 2'-modified 5-methyl-cytidine is a 2'-O-Me modified 5-methyl-cytidine or a 2'-F modified 5-methyl-cytidine.

[0019] In some embodiments, the antisense strand 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 strand is selected from modified versions of SEQ ID NOs: 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 a siRNA molecule selected from the siRNAs listed in Table 8.

[0022] In some embodiments, the antisense strand is selected from modified versions of SEQ ID NOs: 3040, 3061, 3027, 3037, 3039, 3041, 3044, and 3052 listed in Table 9.

[0023] In some embodiments, the sense strand is selected from modified versions of SEQ ID NOs: 3000, 3021, 2987, 2997, 2999, 3001, 3004, and 3012 listed in Table 9.

[0024] In some embodiments, the RNAi oligonucleotide is a 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 a Fab, optionally wherein the Fab comprises the heavy and light chains of any of the anti-TfR Fabs listed in Table 5.

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

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

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

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

[0032] Further provided herein is a method of reducing DUX4 expression in a muscle cell, the method comprising contacting the muscle cell with a complex described herein in an amount effective to promote internalization of an oligonucleotide into the muscle cell.

[0033] Further provided herein is a method for treating facioscapulohumeral muscular dystrophy (FSHD), the method comprising administering an effective amount of a conjugate of any one of claims 1-18 to a subject in need thereof, wherein the subject has abnormal production of DUX4 protein.

[0034] Other aspects of the present disclosure include: Antisense strand: 5'-fCfUmCfUmCfAmUfUmCfUmGfAmAfAmCfCmAfAmAfUmC*fU*mG-3' (SEQ ID NO: 3035) Sense strand: 5'-mGmAfUmUfUmGfGmUfUmUfCmAfGmAfAmUfGmAfGmAfG-3' (SEQ ID NO: 2995); Antisense strand: 5'-fUfGmGfAmAfAmGfCmGfAmUfCmCfUmUfCmUfCmAfAmA*fG*mG-3' (SEQ ID NO: 3040) Sense strand: 5'-mUmUfUmGfAmGfAmAfGmGfAmUfCmGfCmUfUmUfCmCfA-3' (SEQ ID NO: 3000); Antisense strand: 5'-fCfCmGfGmUfAmUfUmCfUmUfCmCfUmCfGmCfUmGfAmG*fG*mG-3' (SEQ ID NO: 3061) Sense strand: 5'-mCmUfCmAfGmCfGmAfGmGfAmAfGmAfAmUfAmCfCmGfG-3' (SEQ ID NO: 3021); Antisense strand: 5'-fCfGmAfUmCfCmUfUmCfUmCfAmAfAmGfGmCfUmCfGmG*fA*mG-3' (SEQ ID NO: 3039) Sense strand: 5'-mCmCfGmAfGmCfCmUfUmUfGmAfGmAfAmGfGmAfUmCfG-3' (SEQ ID NO: 2999); Antisense strand: 5'-fGfCmGfAmUfGmCfCmUfGmGfAmAfAmGfCmGfAmUfCmC*fU*mU-3' (SEQ ID NO: 3041) Sense strand: 5'-mGmGfAmUfCmGfCmUfUmUfCmCfAmGfGmCfAmUfCmGfC-3' (SEQ ID NO: 3001); Antisense strand: 5'-fGfUmCfCmAfAmAfCmGfAmGfUmCfUmCfCmGfUmCfGmC*fC*mG-3' (SEQ ID NO: 3027) Sense strand: 5'-mGmCfGmAfCmGfGmAfGmAfCmUfCmGfUmUfUmGfGmAfC-3' (SEQ ID NO: 2987); Antisense strand: 5'-fUfUmCfUmAfGmGfAmGfAmGfGmUfUmGfCmGfCmCfUmG*fC*mU-3' (SEQ ID NO: 3052) Sense strand: 5'-mCmAfGmGfCmGfCmAfAmCfCmUfCmUfCmCfUmAfGmAfA-3' (SEQ ID NO: 3012); Antisense strand: 5'-fCfUmGfAmAfUmCfCmUfGmGfAmCfUmCfCmGfGmGfAmG*fG*mC-3' (SEQ ID NO: 3044) Sense strand: 5'-mCmUfCmCfCmGfGmAfGmUfCmCfAmGfGmAfUmUfCmAfG-3' (SEQ ID NO: 3004); Antisense strand: 5'-fUfCmCfGmCfUmCfAmAfAmGfCmAfGmGfCmUfCmGfCmA*fG*mG-3' (SEQ ID NO: 3031) Sense strand: 5'-mUmGfCmGfAmGfCmCfUmGfCmUfUmUfGmAfGmCfGmGfA-3' (SEQ ID NO: 2991); Antisense strand: 5'-fAfCmCfAmAfAmUfCmUfGmGfAmCfCmCfUmGfGmGfCmU*fC*mC-3' (SEQ ID NO: 3034) Sense strand: 5'-mAmGfCmCfCmAfGmGfGmUfCmCfAmGfAmUfUmUfGmGfU-3' (SEQ ID NO: 2994); Antisense strand: 5'-fGfGmAfAmUfGmCfCmGfAmUfGmGfCmCfUmGfGmGfCmC*fA*mG-3' (SEQ ID NO: 3032) Sense strand: 5'-mGmGfCmCfCmAfGmGfCmCfAmUfCmGfGmCfAmUfUmCfC-3' (SEQ ID NO: 2992); Antisense strand: 5'-fCfAmAfAmUfCmUfGmGfAmCfCmCfUmGfGmGfCmUfCmC*fG*mG-3' (SEQ ID NO: 3033) Sense strand: 5'-mGmGfAmGfCmCfCmAfGmGfGmUfCmCfAmGfAmUfUmUfG-3' (SEQ ID NO: 2993); Antisense strand: 5'-fGfGmAfCmUfCmCfGmGfGmAfGmGfCmCfCmGfUmCfUmC*fU*mC-3' (SEQ ID NO: 3042) Sense strand: 5'-mGmAfGmAfCmGfGmGfCmCfUmCfCmCfGmGfAmGfUmCfC-3' (SEQ ID NO: 3002); Antisense strand: 5'-fCfUmCfAmAfAmGfCmAfGmGfCmUfCmGfCmAfGmGfGmC*fC*mU-3' (SEQ ID NO: 3030) Sense strand: 5'-mGmCfCmCfUmGfCmGfAmGfCmCfUmGfCmUfUmUfGmAfG-3' (SEQ ID NO: 2990); Antisense strand: 5'-fAfUmUfCmCfCmGfCmCfGmGfUmGfCmUfGmCfCmUfCmA*fG*mC-3' (SEQ ID NO: 3036) Sense strand: 5'-mUmGfAmGfGmCfAmGfCmAfCmCfGmGfCmGfGmGfAmAfU-3' (SEQ ID NO: 2996); Antisense strand: 5'-fAfUmGfCmCfCmAfGmGfAmAfAmGfAmAfUmGfGmCfAmG*fU*mU-3' (SEQ ID NO: 3065) Sense strand: 5'-mCmUfGmCfCmAfUmUfCmUfUmUfCmCfUmGfGmGfCmAfU-3' (SEQ ID NO: 3025); Antisense strand: 5'-fGfUmUfUmCfUmAfGmGfAmGfAmGfGmUfUmGfCmGfCmC*fU*mG-3' (SEQ ID NO: 3054) Sense strand: 5'-mGmGfCmGfCmAfAmCfCmUfCmUfCmCfUmAfGmAfAmAfC-3' (SEQ ID NO: 3014); Antisense strand: 5'-fUfCmCfGmUfUmUfCmUfAmGfGmAfGmAfGmGfUmUfGmC*fG*mC-3' (SEQ ID NO: 3057) Sense strand: 5'-mGmCfAmAfCmCfUmCfUmCfCmUfAmGfAmAfAmCfGmGfA-3' (SEQ ID NO: 3017); Antisense strand: 5'-fGfAmAfAmCfUmCfCmGfGmGfCmUfCmGfCmCfAmGfGmA*fG*mC-3' (SEQ ID NO: 3049) Sense strand: 5'-mUmCfCmUfGmGfCmGfAmGfCmCfCmGfGmAfGmUfUmUfC-3' (SEQ ID NO: 3009); Antisense strand: 5'-fAfAmGfAmAfUmGfGmCfAmGfUmUfCmUfCmCfGmCfGmG*fU*mG-3' (SEQ ID NO: 3064) Sense strand: 5'-mCmCfGmCfGmGfAmGfAmAfCmUfGmCfCmAfUmUfCmUfU-3' (SEQ ID NO: 3024); Antisense strand: 5'-fCfGmUfUmUfCmUfAmGfGmAfGmAfGmGfUmUfGmCfGmC*fC*mU-3' (SEQ ID NO: 3055) Sense strand: 5'-mGmCfGmCfAmAfCmCfUmCfUmCfCmUfAmGfAmAfAmCfG-3' (SEQ ID NO: 3015); Antisense strand: 5'-fGfGmUfCmCfAmAfAmCfGmAfGmUfCmUfCmCfGmUfCmG*fC*mC-3' (SEQ ID NO: 3028) Sense strand: 5'-mCmGfAmCfGmGfAmGfAmCfUmCfGmUfUmUfGmGfAmCfC-3' (SEQ ID NO: 2988); Antisense strand: 5'-fGfCmGfGmUfGmUfGmGfAmGfUmCfUmCfUmCfAmCfCmG*fG*mG-3' (SEQ ID NO: 3063) Sense strand: 5'-mCmGfGmUfGmAfGmAfGmAfCmUfCmCfAmCfAmCfCmGfC-3' (SEQ ID NO: 3023); Antisense strand: 5'-fUfAmUfUmCfUmUfCmCfUmCfGmCfUmGfAmGfGmGfGmU*fG*mC-3' (SEQ ID NO: 3059) Sense strand: 5'-mAmCfCmCfCmUfCmAfGmCfGmAfGmGfAmAfGmAfAmUfA-3' (SEQ ID NO: 3019); Antisense strand: 5'-fGfGmGfUmCfCmAfAmAfCmGfAmGfUmCfUmCfCmGfUmC*fG*mC-3' (SEQ ID NO: 3029) Sense strand: 5'-mGmAfCmGfGmAfGmAfCmUfCmGfUmUfUmGfGmAfCmCfC-3' (SEQ ID NO: 2989); Antisense strand: 5'-fUfUmUfCmUfAmGfGmAfGmAfGmGfUmUfGmCfGmCfCmU*fG*mC-3' (SEQ ID NO: 3053) Sense strand: 5'-mAmGfGmCfGmCfAmAfCmCfUmCfUmCfCmUfAmGfAmAfA-3' (SEQ ID NO: 3013); Antisense strand: 5'-fCfAmGfAmAfAmCfUmCfCmGfGmGfCmUfCmGfCmCfAmG*fG*mA-3' (SEQ ID NO: 3050) Sense strand: 5'-mCmUfGmGfCmGfAmGfCmCfCmGfGmAfGmUfUmUfCmUfG-3' (SEQ ID NO: 3010); Antisense strand: 5'-fAfAmAfGmGfCmUfCmGfGmAfGmGfAmGfCmAfGmGfGmC*fG*mG-3' (SEQ ID NO: 3038) Sense strand: 5'-mGmCfCmCfUmGfCmUfCmCfUmCfCmGfAmGfCmCfUmUfU-3' (SEQ ID NO: 2998); Antisense strand: 5'-fGfCmUfUmUfUmGfCmCfCmGfGmGfUmGfCmGfGmAfGmG*fC*mC-3' (SEQ ID NO: 3047) Sense strand: 5'-mCmCfUmCfCmGfCmAfCmCfCmGfGmGfCmAfAmAfAmGfC-3' (SEQ ID NO: 3007); Antisense strand: 5'-fCfCmUfGmUfCmCfCmGfGmGfUmGfCmCfUmGfGmCfCmC*fU*mU-3' (SEQ ID NO: 3045) Sense strand: 5'-mGmGfGmCfCmAfGmGfCmAfCmCfCmGfGmGfAmCfAmGfG-3' (SEQ ID NO: 3005); Antisense strand: 5'-fUfGmAfAmUfCmCfUmGfGmAfCmUfCmCfGmGfGmAfGmG*fC*mC-3' (SEQ ID NO: 3043) Sense strand: 5'-mCmCfUmCfCmCfGmGfAmGfUmCfCmAfGmGfAmUfUmCfA-3' (SEQ ID NO: 3003); Antisense strand: 5'-fGfGmGfAmUfGmCfCmCfAmGfGmAfAmAfGmAfAmUfGmG*fC*mA-3' (SEQ ID NO: 3066) Sense strand: 5'-mCmCfAmUfUmCfUmUfUmCfCmUfGmGfGmCfAmUfCmCfC-3' (SEQ ID NO: 3026); Antisense strand: 5'-fGfAmGfUmCfUmCfUmCfAmCfCmGfGmGfCmCfUmAfGmA*fC*mC-3' (SEQ ID NO: 3062) Sense strand: 5'-mUmCfUmAfGmGfCmCfCmGfGmUfGmAfGmAfGmAfCmUfC-3' (SEQ ID NO: 3022); Antisense strand: 5'-fCfCmGfUmUfUmCfUmAfGmGfAmGfAmGfGmUfUmGfCmG*fC*mC-3' (SEQ ID NO: 3056) Sense strand: 5'-mCmGfCmAfAmCfCmUfCmUfCmCfUmAfGmAfAmAfCmGfG-3' (SEQ ID NO: 3016); Antisense strand: 5'-fCfGmGfUmCfCmUfCmCfCmGfGmCfUmUfUmUfGmCfCmC*fG*mG-3' (SEQ ID NO: 3048) Sense strand: 5'-mGmGfGmCfAmAfAmAfGmCfCmGfGmGfAmGfGmAfCmCfG-3' (SEQ ID NO: 3008); Antisense strand: 5'-fCfCmAfGmCfGmAfGmGfAmGfCmCfUmGfAmGfGmGfUmG*fG*mG-3' (SEQ ID NO: 3046) Sense strand: 5'-mCmAfCmCfCmUfCmAfGmGfCmUfCmCfUmCfGmCfUmGfG-3' (SEQ ID NO: 3006); Antisense strand: 5'-fGfCmUfUmCfCmAfGmCfGmAfGmGfCmGfGmCfCmUfCmU*fU*mC-3' (SEQ ID NO: 3058) Sense strand: 5'-mAmGfAmGfGmCfCmGfCmCfUmCfGmCfUmGfGmAfAmGfC-3' (SEQ ID NO: 3018); Antisense strand: 5'-fGfGmGfCmGfGmUfCmUfGmGfGmAfUmCfCmGfGmUfGmA*fC*mG-3' (SEQ ID NO: 3037) Sense strand: 5'-mUmCfAmCfCmGfGmAfUmCfCmCfAmGfAmCfCmGfCmCfC-3' (SEQ ID NO: 2997); Antisense strand: 5'-fGfUmAfUmUfCmUfUmCfCmUfCmGfCmUfGmAfGmGfGmG*fU*mG-3' (SEQ ID NO: 3060) Sense strand: 5'-mCmCfCmCfUmCfAmGfCmGfAmGfGmAfAmGfAmAfUmAfC-3' (SEQ ID NO: 3020); and Antisense strand: 5'-fUfGmCfUmGfCmAfGmAfAmAfCmUfCmCfGmGfGmCfUmC*fG*mC-3' (SEQ ID NO: 3051) Sense strand: 5'-mGmAfGmCfCmCfGmGfAmGfUmUfUmCfUmGfCmAfGmCfA-3' (SEQ ID NO: 3011) providing an oligonucleotide comprising an siRNA oligonucleotide selected from: Here, "m" indicates a 2'-O-methyl (2'-O-Me) modified nucleoside; "f" indicates a 2'-fluoro (2'-F) modified nucleoside; "*" indicates a phosphorothioate internucleoside linkage; and the absence of an "*" between two nucleosides indicates a phosphodiester internucleoside linkage.

[0035] Other aspects of the present invention include: Antisense strand: 5'-fUfGmGfAmAfAmGfxCmGfAmUfCmCfUmUfCmUfCmAfAmA*fG*mG-3' (SEQ ID NO: 3040) Sense strand: 5'-mUmUfUmGfAmGfAmAfGmGfAmUfxCmGfCmUfUmUfCmCfA-3' (SEQ ID NO: 3000); Antisense strand: 5'-fCfxCmGfGmUfAmUfUmCfUmUfCmCfUmxCfGmCfUmGfAmG*fG*mG-3' (SEQ ID NO: 3061) Sense strand: 5'-mCmUfCmAfGmxCfGmAfGmGfAmAfGmAfAmUfAmCfxCmGfG-3' (SEQ ID NO: 3021); Antisense strand: 5'-fGfUmCfCmAfAmAfxCmGfAmGfUmCfUmCfxCmGfUmxCfGmC*fxC*mG-3' (SEQ ID NO: 3027) Sense strand: 5'-mGmxCfGmAfxCmGfGmAfGmAfCmUfxCmGfUmUfUmGfGmAfC-3' (SEQ ID NO: 2987); Antisense strand: 5'-fGfGmGfxCmGfGmUfCmUfGmGfGmAfUmCfxCmGfGmUfGmA*fxC*mG-3' (SEQ ID NO: 3037) Sense strand: 5'-mUmCfAmCfxCmGfGmAfUmCfCmCfAmGfAmCfxCmGfCmCfC-3' (SEQ ID NO: 2997); Antisense strand: 5'-fxCfGmAfUmCfCmUfUmCfUmCfAmAfAmGfGmCfUmxCfGmG*fA*mG-3' (SEQ ID NO: 3039) Sense strand: 5'-mCmxCfGmAfGmCfCmUfUmUfGmAfGmAfAmGfGmAfUmxCfG-3' (SEQ ID NO: 2999); Antisense strand: 5'-fGfxCmGfAmUfGmCfCmUfGmGfAmAfAmGfxCmGfAmUfCmC*fU*mU-3' (SEQ ID NO: 3041) Sense strand: 5'-mGmGfAmUfxCmGfCmUfUmUfCmCfAmGfGmCfAmUfxCmGfC-3' (SEQ ID NO: 3001); Antisense strand: 5'-fCfUmGfAmAfUmCfCmUfGmGfAmCfUmCfxCmGfGmGfAmG*fG*mC-3' (SEQ ID NO: 3044) Sense strand: 5'-mCmUfCmCfxCmGfGmAfGmUfCmCfAmGfGmAfUmUfCmAfG-3' (SEQ ID NO: 3004); and Antisense strand: 5'-fUfUmCfUmAfGmGfAmGfAmGfGmUfUmGfxCmGfCmCfUmG*fC*mU-3' (SEQ ID NO: 3052) Sense strand: 5'-mCmAfGmGfxCmGfCmAfAmCfCmUfCmUfCmCfUmAfGmAfA-3' (SEQ ID NO: 3012) providing an oligonucleotide comprising an siRNA oligonucleotide selected from: where "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-methyl-cytidine; "fxC" indicates a 2'-F modified 5-methyl-cytidine; "*" indicates a phosphorothioate internucleoside linkage; and the absence of an "*" between two nucleosides indicates a phosphodiester internucleoside linkage.

[0036] Another aspect of the 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 strand 18-25 nucleotides in length and comprises a region of complementarity to a target sequence as represented by SEQ ID NOs: 163-1574, and wherein the region of complementarity is at least 16 contiguous nucleosides in length. 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 strand comprises the nucleotide sequence of any one of SEQ ID NOs: 1575-2986. [Brief description of the drawings]

[0037] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 depicts a non-limiting schematic diagram showing the effects of transfecting cells with siRNA.

[0038] [Diagram 2] FIG. 2 depicts a non-limiting schematic showing the activity of a muscle-targeting complex containing siRNA.

[0039] [Diagram 3]3A-3B depict non-limiting schematic diagrams showing the activity of muscle-targeting complexes containing siRNA in mouse muscle tissues (gastrocnemius and heart) in vivo compared to vehicle-treated controls. (N=4 C57BL / 6 WT mice).

[0040] [Figure 4A-B] 4A-4E depict non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing siRNA. [Figure 4C-D] 4A-4E depict non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing siRNA. [Figure 4E] 4A-4E depict 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-targeting siRNAs listed in Table 8 in knocking down DUX4 mRNA expression in Hepa1-6 cells. Figure 5A shows the activity of the siRNAs in knocking down DUX4 mRNA when Hepa1-6 cells were treated with 2 nM or 10 nM of each of the indicated siRNAs. [Figure 5B] FIG. 5B shows a dose-response curve for siRNA9, which yielded 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 at various concentrations with specific DUX4-targeting siRNAs listed in Table 8. 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 at various concentrations with specific DUX4-targeting siRNAs listed in Table 8. 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 AB1080-immortalized FSHD patient-derived myotubes after incubation with siRNA conjugates containing siRNA9, siRNA14, or siRNA35 (corresponding to siRNA9, siRNA14, siRNA35 in Table 8) covalently linked to 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. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] Detailed Description Some aspects of the present disclosure provide oligonucleotides designed to target DUX4 RNA. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA, which are useful for reducing the level of DUX4 mRNA and / or protein associated with features of facioscapulohumeral muscular dystrophy (FSHD), including muscle atrophy, in 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 engage the RNA-induced silencing complex (RISC) for DUX4 RNA degradation, but also reduce off-target effects. In some embodiments, the oligonucleotides are designed to reduce the level of DUX4 RNA and / or protein. In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desired binding affinity properties. In some embodiments, the oligonucleotides are designed to have desired toxicity and / or immunogenicity profiles.

[0044] In some aspects, the present disclosure provides a complex comprising a muscle targeting agent covalently linked to a DUX4 targeting oligonucleotide for effective delivery of 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 with or suspected of having a rare muscle disease.For example, in some embodiments, the complex is provided for targeting DUX4 to treat subjects with FSHD.In some embodiments, the complex provided herein comprises an oligonucleotide that inhibits the expression of DUX4 in subjects with one or more deletions of D4Z4 repeats on chromosome 4.

[0045] Further aspects of the disclosure, including descriptions of defined terms, are provided below.

[0046] I. Definition Administering: As used herein, the terms "administering" or "administration" mean providing a conjugate to a subject in a physiologically and / or pharmacologically useful manner (e.g., treating a disease in a subject).

[0047] about: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to a specified reference value. In some embodiments, the term "approximately" or "about" refers to a broad range of values ​​that fall within plus or minus (greater or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the specified reference value, unless otherwise stated or clear from the context (except when such number exceeds 100% of a feasible value).

[0048] antibody: As used herein, the term "antibody" refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., 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, a F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody, 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 human germline sequences. In another embodiment, the antibody comprises a heavy chain constant region selected from the group consisting of the 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. Immunoglobulin constant region refers to a heavy or light chain constant region. Human IgG heavy and light chain constant region amino acid sequences and their functional variations are known. With respect to the heavy chain, the heavy chain of the antibody described herein in some embodiments can 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 can 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, CH2 domain, and / or (for example and) a CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as 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) supra. 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 (e.g., and) methylation). 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, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include 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 linker polypeptide or a polypeptide comprising one or more antigen-binding fragments of the present disclosure linked to an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues linked together by peptide bonds and is used to link one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Furthermore, an antibody may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate 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 generate bivalent 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 in an antibody variable sequence. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: 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 the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (for example and) the contact definition, 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. al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDRs may refer to CDRs defined by any method known in the art.Two antibodies having the same CDR, as determined by the same method, for example the IMGT definition, means that the two antibodies have the same amino acid sequence of that CDR.

[0050] There are three CDRs in each of the heavy and light chain variable regions, which are designated 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 exact boundaries of these CDRs are defined differently according to various systems. The system described by Kabat (Kabat et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Sub-portions of the CDRs are sometimes designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Still other CDR boundary definitions may not strictly adhere to one of the above systems, but may still overlap with the Kabat CDRs, and may be shortened or extended in light of predictions or experimental findings that specific residues or groups of residues, or even entire CDRs, 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-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species but in which the sequence of one or more of the CDR regions of its VH and / or VL have been replaced with CDR sequences from another species, such as an antibody having murine heavy and light chain variable regions but in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0052] Chimeric antibodies: The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy 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 binding between two nucleotides or two sets of nucleotides. The term "complementary" can 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 binding between two nucleosides or two sets of nucleosides. For example, if a base of an oligonucleotide at a position is capable of hydrogen bonding with a base of a target nucleic acid (e.g., mRNA) at a corresponding position, then the bases are considered to be complementary to each other at that position. Base pairing may include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing).For example, in some embodiments, as complementary base pairing, adenosine-based base (A) is complementary to thymidine-based base (T) or uracil-based base (U), cytosine-based base (C) is complementary to guanosine-based base (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U, or T and are considered to be complementary thereto.Inosine (I) is also considered to be a universal base in the art and is considered to be complementary to any A, C, U, or T.

[0054] Conservative Amino Acid Substitutions: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, references that summarize 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 those 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 being linked together through 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 a disulfide bridge) that acts as an intermolecular linker. However, in some embodiments, two or more molecules may be covalently linked together through a molecule that acts as a linker that connects 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 a non-cleavable linker.

[0056] Cross-reacting: As used herein, and in the context of targeting agents (e.g., antibodies), the term "cross-reacting" refers to the property of an agent that can specifically bind to more than one antigen of the same type or class (e.g., multiple homologues, paralogues, or orthologs of antigens) with similar affinity or avidity. For example, in some embodiments, an antibody that cross-reacts to a similar type or class of human and non-human primate antigens (e.g., human transferrin receptor and non-human primate transferrin receptor) can bind to a human antigen and a non-human primate antigen with similar affinity or avidity. In some embodiments, the antibody cross-reacts to a similar type or class of human antigen and rodent antigen. In some embodiments, the antibody cross-reacts to a similar type or class of rodent antigen and non-human primate antigen. In some embodiments, the antibody cross-reacts to a similar type or class of human antigen, non-human primate antigen, and rodent antigen.

[0057] DUX4: As used herein, the term "DUX4" refers to the gene encoding double homeobox 4, a protein that is generally expressed during fetal development and in adult male testes. In some embodiments, DUX4 can be a human (Gene ID: 100288687), a non-human primate (e.g., Gene ID: 750891, Gene ID: 100405864), or a rodent (e.g., Gene ID: 306226) gene. In humans, expression of the DUX4 gene outside fetal development and testis is associated with facioscapulohumeral muscular dystrophy. In addition, multiple human transcript variants that code for different protein isoforms have been characterized (e.g., as annotated with GenBank RefSeq accession numbers: 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 disease caused by a mutation in the DUX4 gene or the SMCHD1 gene, characterized by reduced muscle mass and muscle atrophy, mainly in the muscles of the face, scapula, and upper arm. Two types of the disease, type 1 and type 2, have been described. Type 1 is associated with a deletion in the D4Z4 repeat region on chromosome 4, which contains the DUX4 gene. In some embodiments, type 1 is associated with a deletion in the D4Z4 repeat region on the variant 4qA of the chromosome 4 allele, which contains 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 DUX4 protein after fetal development and outside the testis. Facioscapulohumeral muscular dystrophy, the genetic basis of the disease, and associated symptoms have been described in the art. (See, e.g., 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.) FSHD1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 158900. FSHD2 is associated with OMIM Entry #158901.

[0059] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of the CDR sequence can be determined by various systems, the meaning of the framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain, and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework region on the light chain and the heavy chain into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. The framework region that does not specify a specific subregion as FR1, FR2, FR3, or FR4, when referred to by others, represents the combined FR(s) in the variable region of a naturally occurring single immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that contain framework regions. 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: The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may include, for example, in the CDRs, particularly CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[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 and / or (e.g., and) VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced onto non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen-binding portions are provided. Such antibodies may be produced by obtaining a mouse anti-transferrin receptor monoclonal antibody using existing hybridoma technology, followed by humanization using in vitro genetic engineering (such as that disclosed in PCT Publication No. WO 2005 / 123126 A2 to Kasaian et al.).

[0062] Internalizing cell surface receptors: As used herein, the term "internalizing cell surface receptor" refers to a cell surface receptor that is internalized by a cell upon an external stimulus (e.g., a ligand binding 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 a clathrin-independent pathway, 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 a 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: An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (for example, an isolated antibody that specifically binds to the transferrin receptor is substantially free of 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. Moreover, 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, which are recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., more variable) than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof (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, USDepartment of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable region spans amino acid positions 31-35 for CDR1, amino acid positions 50-65 for CDR2, and amino acid positions 95-102 for CDR3. In the light chain variable region, the hypervariable region spans 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 linked or otherwise associated with a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, a protein, a peptide, a nucleic acid, or an 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 that includes a strand having a region of complementarity 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 a muscle cell may be a membrane protein, such as an integral or peripheral membrane protein. Typically, a muscle targeting agent specifically binds to an antigen on a muscle cell that facilitates internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle and is capable of being internalized into the muscle cell through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an 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 a muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an antigen on a muscle cell that facilitates internalization of the muscle-targeting antibody (and any attached molecular payload) into the muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an internalizing cell surface receptor present on a muscle cell. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0068] Oligonucleotides: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, gapmer, mixmer, phosphorodiamidite morpholino, peptide nucleic acid, aptamer, guide nucleic acid (e.g., Cas9 guide RNA), and the like. Oligonucleotides may be single-stranded or double-stranded. In some embodiments, oligonucleotides may contain one or more modified nucleotides (e.g., 2'-O-methyl sugar modification, purine or pyrimidine modification). In some embodiments, oligonucleotides may contain one or more modified nucleosides (e.g., 2'-O-methyl sugar modification, purine or pyrimidine modification). In some embodiments, oligonucleotides may contain one or more modified internucleoside linkages. In some embodiments, oligonucleotides may contain one or more modified internucleoside linkages. In some embodiments, oligonucleotides may contain one or more phosphorothioate linkages that may be in Rp or Sp stereochemical configuration.

[0069] Recombinant antibodies: The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (as described in more detail in this disclosure), antibodies isolated from a recombinant combinatorial human antibody library (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 a human immunoglobulin gene transgenic animal (e.g., a mouse) (see, e.g., Taylor, LD, et al., J. Immunology 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), or antibodies isolated from a human immunoglobulin gene transgenic animal (e.g., a mouse) (see, e.g., Taylor, LD, et al., J. Immunology 29:128-145; Hoogenboom ... 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), or any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, although derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur within the germline repertoire of human antibodies in vivo.One aspect of the disclosure provides fully human antibodies capable of binding to the human transferrin receptor that can be generated using techniques well known in the art, such as, but not limited to, techniques using human Ig phage libraries (e.g., those disclosed in PCT Publication No. WO 2005 / 007699 A2 to Jermutus et al.).

[0070] Regions of complementarity: As used herein, the term "region of complementarity" 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 are capable of annealing to each other under physiological conditions (e.g., in a cell). In some embodiments, the region of complementarity is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the region of complementarity is partially complementary (e.g., at least 80%, 90%, 95%, or 99% complementary) to the cognate nucleotide sequence of the target nucleic acid. In some embodiments, the region of complementarity contains 1, 2, 3, or 4 mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0071] Specific binding to: As used herein, the term "specifically binds" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that can be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context. With respect to an antibody, the term "specifically binds" refers to the ability of an antibody to bind to a particular antigen, as compared to an appropriate reference antigen, or an antigen that can be used to distinguish the particular antigen from other antigens, with a degree of affinity or avidity (e.g., a degree that allows preferential targeting to certain cells (e.g., muscle cells) through binding to the antigen, as described herein). In some embodiments, the antibody binds to a target with at least about 10% affinity. -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 less than this K D In some embodiments, the antibody specifically binds to a transferrin receptor, e.g., an epitope in the apical domain of the transferrin receptor.

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

[0073] Transferrin Receptor: As used herein, the term "transferrin receptor" (also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalizing 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, multiple human transcript variants encoding different isoforms of the receptor have been characterized (e.g., as annotated with GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0074] 2' Modified Nucleosides: As used herein, the terms "2'-modified nucleoside" and "2'-modified ribonucleoside" are used interchangeably and refer to a nucleoside having a sugar moiety modified at the 2'-position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, in which the 2' and 4' positions of the sugar are bridged (e.g., by a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, e.g., in which the 2' position of the sugar moiety is substituted. Non-limiting examples of 2' modified nucleosides include the following: 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 acid (LNA, methylene bridged nucleic acid), ethylene bridged nucleic acid (ENA), and (S)-constrained ethyl bridged nucleic acid (cEt). In some embodiments, the 2' modified nucleosides described herein are high affinity modified nucleosides, and oligonucleotides comprising the 2' modified nucleosides have increased affinity for target sequences compared to unmodified oligonucleotides. Examples of 2' modified nucleosides structures are provided below: [ka] The examples are shown with a phosphate group, however, any internucleoside linkage is contemplated between 2'-modified nucleosides.

[0075] II. Complex Provided herein is a conjugate comprising a targeting agent, for example an antibody, covalently linked to a molecular payload. In some embodiments, the conjugate comprises a muscle-targeting antibody covalently linked to an oligonucleotide. The conjugate may comprise 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 gene, protein, and / or (for example, and) nucleic acid. The molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity that can modulate the activity or function of gene, protein, and / or (for example, and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets DUX4 in muscle cells.

[0077] In some embodiments, the conjugate 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 Some aspects of the present disclosure provide muscle targeting agents, e.g., muscle targeting agents for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents are capable of binding to muscle cells and delivering the associated molecular payload to muscle cells, e.g., via specific binding to an antigen on the muscle cell. In some embodiments, the molecular payload is attached (e.g., covalently attached) to the muscle targeting agent and is internalized into the muscle cell, e.g., via endocytosis, when the muscle targeting agent binds to the antigen on the muscle cell. It should be understood that various types of muscle targeting agents may be used in accordance with the present disclosure. It should also be understood that any muscle target (e.g., muscle surface protein) may be targeted by any type of muscle targeting agent described herein. For example, the muscle targeting agent may include or consist of a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide). In some embodiments, the muscle targeting agent may comprise or consist of a small molecule. Exemplary muscle targeting agents are described in further detail herein; however, it should be understood that the exemplary muscle targeting agents provided herein are not intended to be limiting.

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

[0080] Through interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, molecules that are substrates for 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, molecular payloads conjugated to transferrin or anti-transferrin receptor antibodies can be taken up by muscle cells via binding to the transferrin receptor and then endocytosed, e.g., via clathrin-mediated endocytosis.

[0081] The use of muscle targeting agents can be useful to concentrate molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells when compared to other cell types in a subject. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells (e.g., skeletal muscle cells, smooth muscle cells, or cardiomyocytes) at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than the amount in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or adipocytes). In some embodiments, the toxicity in a subject of a molecular payload 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 delivered to a subject.

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

[0083] i. Muscle targeting antibody In some embodiments, the muscle targeting agent is an antibody. In general, the high specificity of antibodies to their target antigens provides the potential to selectively target muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and / or (e.g., and) 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 which are incorporated herein by reference.

[0084] a. Anti-transferrin receptor (TfR) antibody Some aspects of the present disclosure are based on the recognition that agents that bind to the transferrin receptor, e.g., anti-transferrin receptor antibodies, can target muscle cells. The transferrin receptor is an internalizing cell surface receptor that transports transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to the transferrin receptor. Accordingly, aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to the transferrin receptor. In some embodiments, the binding proteins that bind to the transferrin receptor are internalized into muscle cells along with any molecular payloads attached. As used herein, antibodies that bind to the transferrin receptor may be interchangeably referred to as transferrin receptor antibodies, anti-transferrin receptor antibodies, or anti-TfR antibodies. Antibodies that bind, e.g., specifically bind, to the transferrin receptor may be internalized into cells upon binding to the transferrin receptor, e.g., 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, for example library design using phage display. Exemplary methodologies have been characterized in the art and are 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, the anti-TfR antibodies have been previously characterized or disclosed.Antibodies that specifically bind to the transferrin receptor are known in the art (see, e.g., U.S. Pat. No. 4,364,934, filed Dec. 4, 1979, entitled "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Pat. No. 8,409,573, filed Jun. 14, 2006, entitled "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Pat. No. 9,708,406, filed May 20, 2014, entitled "Anti-transferrin receptor antibodies and methods of use"; U.S. Pat. No. 9,611,323, filed Dec. 19, 2014, entitled "Low affinity blood brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed Dec. 24, 2014, entitled "Novel anti-Transferrin receptor antibody that passes through blood-brain barrier"; Schneider C. et al., "Structural features of 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 epitope that becomes exposed to the antibody. In some embodiments, the anti-TfR antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, 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 an amino acid segment of the human or non-human primate transferrin receptor provided in SEQ ID NOs: 105-108. In some embodiments, the anti-TfR antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor as represented in SEQ ID NO: 105 that is not in the apical domain of the transferrin receptor.

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

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

[0090] An example of a primate non-human transferrin receptor amino acid sequence, which corresponds to the NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Macaca mulatta), is as follows: (Sequence number 106).

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

[0092] An example of a mouse transferrin receptor amino acid sequence, which corresponds 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, the 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 (by way of 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] Suitable methodologies may be used to obtain and / or (for example and) produce antibodies, antibody fragments, or antigen-binding agents, for example through the use of recombinant DNA protocols. 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 that targets a specific antigen. Antibodies may also be produced through screening of protein expression libraries (for example, phage display libraries) that express antibodies. Phage display library design may also be used in some embodiments (see, e.g., U.S. Pat. No. 5,223,409, filed 3 / 1 / 1991, "Directed evolution of novel binding proteins"; WO 1992 / 18619, filed 4 / 10 / 1992, "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, filed 5 / 1 / 1991, "Recombinant library screening methods"; WO 1992 / 20791, filed 5 / 15 / 1992, "Methods for producing members of specific binding pairs"; and WO 1992 / 15679, filed 2 / 28 / 1992, "Improved epitope displaying phage"). In some embodiments, the antigen of interest may be used to immunize a non-human animal, e.g., a rodent animal or a goat.In some embodiments, once the antibody is obtained from the non-human animal, it may then be optionally modified using a number of methodologies, for example using recombinant DNA techniques. Additional examples of antibody production and methodologies 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 (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, optionally deficient in an enzyme (e.g., glycosyltransferase) in the N- or O-glycosylation pathway. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0096] In some embodiments, the anti-TfR antibody of the present disclosure comprises a VL domain and / or (for example and) a VH domain of any one of the anti-TfR antibodies selected from any one of Tables 2-7, and comprises 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 are described in the art, see, for example, Kabat EA et al. (1991), supra.

[0097] In some embodiments, agents that bind to the transferrin receptor, e.g., anti-TfR antibodies, can target muscle cells and / or mediate transport of agents across the blood-brain barrier (e.g., and). The transferrin receptor is an internalizing cell surface receptor that transports transferrin across cell membranes and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to the transferrin receptor. Antibodies that bind, e.g., specifically bind, to the transferrin receptor may be internalized into cells upon binding to the transferrin receptor, e.g., through receptor-mediated endocytosis.

[0098] In some aspects, provided herein are antibodies that bind with high specificity and affinity to the transferrin receptor. In some embodiments, the anti-TfR antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor or epitopes that become exposed to the antibody. In some embodiments, the anti-TfR antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR antibodies provided herein bind to human transferrin receptor. In some embodiments, the anti-TfR antibodies described herein bind to an amino acid segment of the human or non-human primate transferrin receptor provided in SEQ ID NOs: 105-108. In some embodiments, the anti-TfR antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor as represented in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor. In some embodiments, 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 -13In some embodiments, the anti-TfR antibodies described herein bind to TfR1 (e.g., human or non-human primate TfR1) with a binding affinity (e.g., as indicated by a Kd) of 10 M or less. In some embodiments, the anti-TfR antibodies described herein bind to TfR1 with a Kd in the sub-nanomolar 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 cynomolgus TfR1 (e.g., as indicated by a Kd) of 10 M or less. -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or smaller Kd) to mouse TfR1. The affinity and binding kinetics of the anti-TfR antibodies can be tested using any suitable method, including but not limited to biosensor technology (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-beta2-microglobulin binding to TfR1.

[0100] Non-limiting examples of anti-TfR antibodies are provided in Table 2. Table 2. Examples of anti-TfR antibodies [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] *The positions of the mutations are according to the Kabat numbering of each VH sequence containing the mutation.

[0101] In some embodiments, an anti-TfR antibody of the disclosure is a variant of any one of the anti-TfR antibodies provided in Table 2. In some embodiments, an anti-TfR antibody of the disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that are the same as the 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 (by way of 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 positions of the mutations are according to the Kabat numbering of each VH sequence containing the mutation. ** The CDRs according to the Kabat numbering system are in bold.

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

[0104] In some embodiments, an anti-TfR antibody of the disclosure comprises a VH comprising CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR antibodies provided in Table 3, and comprises an amino acid sequence that is 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%) identical in the framework regions when compared to the respective VHs provided in Table 3. Alternatively or in addition (e.g., in addition), an anti-TfR antibody of the disclosure comprises a VL comprising CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR antibodies provided in Table 3, and comprises an amino acid sequence that is 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%) identical in the framework regions when compared to the respective VLs provided in Table 3.

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

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

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

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

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

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

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

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

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

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

[0115] In some embodiments, an anti-TfR antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:154 and a VL comprising 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 include heavy and light chain constant regions from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR antibodies described herein may include 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 be of any suitable origin, e.g., human, mouse, rat, or rabbit. In one 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 number 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 LALA mutations 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)). The mutant human IgG1 constant region is provided below (mutations in bold and underlined): [ka]

[0118] In some embodiments, the 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 number 83).

[0119] Heavy and light chain constant regions of other antibodies are well known in the art and include, for example, those provided 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 comprise a heavy chain comprising a VH as listed in Table 3, or any one of variants thereof, and 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 comprise a heavy chain comprising a VH as listed in Table 3, or any one of variants thereof, and a heavy chain constant region that contains no more than 25 amino acid variations (e.g., no more than 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 variations) compared to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising a VH as listed in Table 3 or any one of their variants, and 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 a VH as listed in Table 3 or any one of their variants, and a heavy chain constant region as represented by SEQ ID NO: 82.

[0121] In some embodiments, the anti-TfR antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and 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 antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and a light chain constant region that contains no more than 25 amino acid variations (e.g., no more than 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 variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and 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 example anti-TfR Fabs [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] * The positions of the mutations are according to the Kabat numbering of each VH sequence containing the mutation. ** The CDRs according to the Kabat numbering system are in bold; the VH / VL sequences are underlined.

[0123] In some embodiments, an anti-TfR antibody of the disclosure comprises a heavy chain that contains no more than 25 amino acid variations (e.g., no more than 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) when compared to a heavy chain set forth in any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or additionally (for example, in addition), an anti-TfR antibody of the disclosure comprises a light chain that contains no more than 25 amino acid variations (for example, no more than 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) when compared to a light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0124] In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising an amino acid sequence 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 additionally (e.g., in addition), the anti-TfR antibodies described herein comprise a light chain comprising an amino acid sequence 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 antibodies described herein comprise a heavy chain comprising an amino acid sequence 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 additionally (by way of example, in addition), the anti-TfR antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

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

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

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

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

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

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

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

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

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

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

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

[0136] In some embodiments, the anti-TfR antibody is a Fab fragment, a Fab' fragment, or a F(ab') fragment of an intact antibody (full-length antibody). 2 Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods (e.g., recombinantly or by digesting the heavy chain constant region of full-length IgG with an enzyme such as papain). For example, F(ab') 2 Fragments can be produced by pepsin or papain digestion of antibody molecules, with Fab fragments being F(ab') 2 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 antibodies described herein comprise a heavy chain comprising a VH as listed in Table 3 or any one of its variants, and 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 antibodies described herein comprise a heavy chain comprising a VH as listed in Table 3 or any one of its variants, and a heavy chain constant region that contains 25 or less 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 less amino acid variations) compared to SEQ ID NO: 96. In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising a VH as listed in Table 3 or any one of its variants, and a heavy chain constant region as represented by SEQ ID NO: 96.

[0138] In some embodiments, the anti-TfR antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and a light chain constant region 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 antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and a light chain constant region containing 25 or less 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 less amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and 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 example anti-TfR Fabs [Table 5-1] [Table 5-2] [Table 5-3] * The positions of the mutations are according to the Kabat numbering of each VH sequence containing the mutation. ** The CDRs according to the Kabat numbering system are in bold; the VH / VL sequences are underlined.

[0140] In some embodiments, an anti-TfR antibody of the disclosure comprises a heavy chain that contains 25 or fewer amino acid variations (e.g., no more than 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) when compared to a heavy chain as set forth in any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively or additionally (for example, in addition), an anti-TfR antibody of the disclosure comprises a light chain that contains no more than 25 amino acid variations (for example, no more than 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 a light chain as set forth in any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0141] In some embodiments, the anti-TfR antibodies described herein comprise a heavy chain comprising an amino acid sequence 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 additionally (e.g., in addition), the anti-TfR antibodies described herein comprise a light chain comprising an amino acid sequence 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 antibodies described herein comprise a heavy chain comprising an amino acid sequence 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 additionally (by way of example, in addition), the anti-TfR antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

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

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

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

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

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

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

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

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

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

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

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

[0153] In some embodiments, an anti-TfR antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:159 and a light chain comprising 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 can be used as a muscle targeting agent in the conjugates disclosed herein. Examples of known anti-TfR antibodies (including related references and binding epitopes) are listed in Table 6. In some embodiments, the anti-TfR antibody comprises the complementarity 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, the anti-TfR antibodies listed in Table 6.

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

[0156] In some embodiments, an anti-TfR antibody of the disclosure includes one or more of the 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 embodiments, an anti-TfR antibody includes CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-TfR antibodies selected from Table 6. In some embodiments, an anti-transferrin antibody includes 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 antibodies of the disclosure include any antibody that includes a heavy chain variable domain and / or (by way of example and) a 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 antibodies of the disclosure include any antibody that includes a 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 the present disclosure provide anti-TfR antibodies having heavy chain variable (VH) and / or (for example and) light chain variable (VL) domain amino acid sequences homologous to any of those described herein. In some embodiments, the anti-TfR antibody comprises a heavy chain variable sequence or a light chain variable sequence that is at least 75% (for example, 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 (for example and) light chain variable amino acid sequence does not vary in any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur within the heavy chain variable and / or (for example 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 comprise heavy and light chain variable sequences that comprise framework sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequences of any anti-TfR antibody, such as any one of the anti-TfR antibodies selected from Table 6.

[0159] An example of a transferrin receptor antibody that may be used in accordance with the present disclosure is described in International Application Publication WO 2016 / 081643, which is incorporated herein by reference. The amino acid sequence of this antibody is 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 embodiments, an anti-TfR antibody of the disclosure comprises a CDR-H1, CDR-H2, and CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. Alternatively or additionally (for example, in addition), an anti-TfR antibody of the disclosure comprises a CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

[0161] In some embodiments, an anti-TfR antibody of the disclosure comprises a CDR-L3 that contains no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) as compared to a CDR-L3 as shown in Table 7. In some embodiments, an anti-TfR antibody of the disclosure comprises a CDR-L3 that contains 1 amino acid variation as compared to a CDR-L3 as shown in Table 7. In some embodiments, an anti-TfR antibody of the disclosure comprises a 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, an anti-TfR antibody of the disclosure comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7, and comprises a 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, anti-TfR antibodies of the disclosure comprise heavy chain CDRs that, together, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs as shown in Table 7. Alternatively or additionally (e.g., in addition), anti-TfR antibodies of the disclosure comprise light chain CDRs that, together, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the light chain CDRs as shown in Table 7.

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

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

[0165] In some embodiments, an anti-TfR antibody of the disclosure comprises a VH that contains 25 or fewer amino acid variations (e.g., no more than 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 variations) when compared to a VH as set forth in SEQ ID NO: 128. Alternatively or additionally (e.g., in addition), an anti-TfR antibody of the disclosure comprises a VL that contains 15 or fewer amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) when compared to a VL as set forth in SEQ ID NO: 129.

[0166] In some embodiments, the anti-TfR antibodies of the present disclosure are full-length IgG1 antibodies that may include heavy and light chain constant regions from a human antibody. In some embodiments, any heavy chain of an anti-TfR antibody as described herein may include 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 suitable origin, e.g., human, mouse, rat, or rabbit. In one 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 number 81).

[0167] In some embodiments, the 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 number 83).

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

[0169] In some embodiments, the anti-TfR antibodies described herein are fully human antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 134. Alternatively or additionally (for example, in addition), the anti-TfR antibodies described herein comprise a light chain comprising 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 comprising the amino acid sequence of SEQ ID NO: 136. Alternatively or additionally (for example, in addition), the anti-TfR Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-TfR Fab described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 137. Alternatively or additionally (for example, in addition), the anti-TfR Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 135.

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

[0172] In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where the residue is unlikely to be involved in interactions with the target antigen (e.g., the transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (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) of a muscle-targeting antibody described herein 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) antigen-dependent cellular cytotoxicity to cells.

[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, e.g., in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, 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 a muscle-targeting 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, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to 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 Nos. WO 02 / 060919; WO 98 / 23289; and 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 FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. See, for example, International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that will alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0176] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to decrease 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 FcRn-binding fragment thereof (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) in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al. (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, termed "YTE mutant", has been shown to exhibit a 4-fold increased half-life when 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 comprises an IgG constant region comprising one, two, 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 in Kabat.

[0177] In some embodiments, one or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the anti-TfR antibody. The effector ligand with altered affinity to itself can be, for example, an Fc receptor or the 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 of a constant region domain (through point mutation or other means) can reduce binding of circulating antibodies to Fc receptors, thereby increasing tumor localization. See, for example, U.S. Pat. Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., Shields RL 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 a different amino acid residue, such that the antibody may have altered Clq binding and / or (for example and) reduced or eliminated 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 altered, thereby altering the antibody's ability to fix complement. This approach is further described in International Publication No. 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 cellular cytotoxicity (ADCC) to cells and / or (for example and) to increase the affinity of the antibody to Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0179] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein may be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be appreciated by one of skill in the art, any variant, CDR-grafted, chimeric, humanized, or composite antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and will retain specific binding ability to the transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it was derived.

[0180] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibody. For example, to avoid potential complications due to Fab-arm exchange that is known to occur in native IgG4 mAbs, the antibodies provided herein may contain 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), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include a stabilizing "Adair" mutation.

[0181] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, optionally deficient in an enzyme (e.g., glycosyltransferase) in the N- or O-glycosylation pathway. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 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 include a signal peptide (e.g., an N-terminal signal peptide) on the heavy and / or (e.g., and) light chain sequences. In some embodiments, the anti-TfR1 antibodies described herein include any one of the VH and VL sequences, any one of the IgG heavy and light chain sequences, or any one of the Fab heavy and light chain sequences described herein, and further include a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide includes the amino acid sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

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

[0184] b. Other muscle-targeting antibodies In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin IIb, or CD63. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a myogenic precursor protein. Exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha7, integrin alpha7 beta1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a skeletal muscle protein. Exemplary skeletal muscle proteins include, without limitation, alpha-sarcoglycan, beta-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycan, 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, the muscle-targeting antibody is an antibody that specifically binds to a smooth muscle protein. Exemplary smooth muscle proteins include, without limitation, alpha-smooth muscle actin, VE-cadherin, caldesmon / CALD1, calponin 1, desmin, histamine H2 R, motilin R / GPR38, Transgelin / TAGLN, and vimentin, however, it should be understood that antibodies to additional targets are within the scope of the present disclosure and that the exemplary list of targets provided herein is not intended to be limiting.

[0185] c. Antibody Features / Modifications In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where the residue is unlikely to be involved in interactions with the target antigen (e.g., the transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (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) of a muscle-targeting antibody described herein 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) antigen-dependent cellular cytotoxicity to cells.

[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, e.g., in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, 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 a muscle-targeting 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, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to 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 Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which 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 FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. See, for example, International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that will 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 FcRn-binding fragment thereof (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 FcRn-binding fragment thereof (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) in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al. (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, termed "YTE mutant", has been shown to exhibit a 4-fold increased half-life when 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 comprises an IgG constant region comprising one, two, 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 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(s) of the anti-transferrin receptor antibody. The effector ligand with altered affinity to itself can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce binding of circulating antibodies to Fc receptors, thereby increasing tumor localization. See, for example, U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., 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 a different amino acid residue, such that the antibody may have altered Clq binding and / or (for example and) reduced or eliminated 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 altered, thereby altering the antibody's ability to fix complement. This approach is further described in International Publication No. 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 cellular cytotoxicity (ADCC) to cells and / or (for example and) to increase the affinity of the antibody to Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0192] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein may be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be appreciated by one of skill in the art, any variant, CDR-grafted, chimeric, humanized, or composite antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and will retain specific binding ability to the transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it was derived.

[0193] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibody. For example, to avoid potential complications due to Fab-arm exchange that is known to occur in native IgG4 mAbs, the antibodies provided herein may contain 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), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include a stabilizing "Adair" mutation.

[0194] As provided herein, the antibodies of the present disclosure may optionally include a constant region or a portion thereof. For example, a VL domain may be attached at its C-terminus to a light chain constant region like Cκ or Cλ. Similarly, a VH domain or a portion thereof may be attached to all or a portion of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any subclass of isotype. The antibody may include a suitable 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, an antibody within the scope of the present disclosure may include VH and VL domains, or antigen-binding portions thereof, combined with any suitable constant region.

[0195] ii. Muscle-targeting peptides Some aspects of the disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences (e.g., peptide sequences between 5 and 20 amino acids in length) that bind to specific cell types have been described. For example, cell-targeting peptides can be found in the following publications: 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, entitled "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 Eng 2002;18:269-72; the entire contents of each of which are incorporated herein by reference. By designing peptides to interact with specific cell surface antigens (e.g., receptors), selectivity to desired tissues, e.g., muscle, can be obtained. 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. As a result, in some embodiments, the muscle targeting agent is a muscle targeting peptide that is 4 to 50 amino acids in length.In some embodiments, the muscle-targeting peptide is 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 in length. Muscle-targeting peptides can be generated using any of several methods, such as phage display.

[0196] In some embodiments, the muscle targeting peptide may bind to an internalizing cell surface receptor (e.g., transferrin receptor) that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells. In some embodiments, the muscle targeting peptide may target (e.g., bind to) the transferrin receptor. In some embodiments, the transferrin receptor targeting peptide may include a segment of a naturally occurring ligand, e.g., transferrin. In some embodiments, the transferrin receptor targeting peptide is as described in U.S. Patent No. 6,743,893, 11 / 30 / 2000 application, "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, filed 5 / 20 / 2011, "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY."

[0197] As mentioned above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides have been identified using phage display libraries that display surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 3071) binds 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 exhibits improved specificity for binding to cardiac 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 for DMD, a 12-amino acid peptide has been 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 contents of which are hereby incorporated by reference. Therein, 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 for muscle (e.g., skeletal muscle) over other cell types includes in vitro selection, as 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 contents of which are incorporated herein by reference. Non-specific cell binders were selected by pre-incubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After undergoing rounds of selection, the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 3073) emerged most frequently. Consequently, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 3073).

[0199] The muscle targeting agent may be an amino acid-containing molecule or peptide. The muscle targeting peptide may correspond to a sequence of a protein that preferentially binds to a protein receptor found on muscle cells. In some embodiments, the muscle targeting peptide contains a high propensity of hydrophobic amino acids (e.g., valine) such that the peptide may preferentially target muscle cells. In some embodiments, the muscle targeting peptide has not been previously characterized or disclosed. These peptides may be conceived, produced, synthesized, and / or derivatized (e.g., and) using any of several methodologies, e.g., phage-displayed peptide libraries, one-bead one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. Exemplary methodologies are characterized in the art 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 some embodiments, muscle-targeting peptides have been previously disclosed (see, e.g., Writer MJ et 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, MJ et al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004,342:1,171-82). Exemplary muscle-targeting peptides include the following groups of 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, muscle-targeting peptides may include about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. Muscle-targeting peptides may include naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring amino acids, or modified amino acids.Non-naturally occurring amino acids 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 peptides can be linear; in other embodiments, the muscle-targeting peptides can be cyclic (e.g., bicyclic) (see, e.g., Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

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

[0201] iv. Muscle-targeting aptamers The muscle targeting agent may be an aptamer, e.g., an RNA aptamer, that preferentially targets muscle cells over other cell types. In some embodiments, the muscle targeting aptamer has not been previously characterized or disclosed. These aptamers may be conceived, produced, synthesized, and / or (e.g., and) derivatized using any of several methodologies, e.g., Systematic Evolution of Ligands by Exponential Enrichment. Exemplary methodologies have been characterized in the art and are 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-targeting aptamers have been previously disclosed (see, e.g., Phillippou, S. et al. "Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers." Mol Ther Nucleic Acids. 2018, 10:199-214.; Thiel, W H et al. "Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation." Mol Ther. 2016, 24:4, 779-87). Exemplary muscle-targeting 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 about 5-15 kDa, about 5-10 kDa, about 10-15 kDa, about 1-5 Da, about 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 a substrate of a muscle transporter protein, such as a transporter protein expressed on the sarcolemma. In some embodiments, the muscle targeting agent is a substrate of an influx transporter specific to muscle tissue. In some embodiments, the influx transporter is specific to skeletal muscle tissue. The two major classes of transporters expressed on the sarcolemma 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, the muscle targeting agent is a substrate that binds to the ABC or SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a naturally occurring substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-naturally occurring substrate, such as a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0203] In some embodiments, the muscle targeting agent is any muscle targeting agent described herein that targets the SLC superfamily of transporters (e.g., antibodies, nucleic acids, small molecules, peptides, aptamers, lipids, sugar moieties). In some embodiments, the muscle targeting agent is a substrate of the SLC superfamily of transporters. SLC transporters are either equilibria or use the proton or sodium ion gradient created across the membrane to drive transport of the substrate. Exemplary SLC transporters with high expression in skeletal muscle include, without limitation, the SATT transporter (ASCT1; SLC1A4), the GLUT4 transporter (SLC2A4), the GLUT7 transporter (GLUT7; SLC2A7), the ATRC2 transporter (CAT-2; SLC7A2), the LAT3 transporter (KIAA0245; SLC7A6), the PHT1 transporter (PTR4; SLC15A4), the OATP-J transporter (OATP5A1; SLC21A15), the OCT3 transporter (EMT; SLC22A3), the OCTN2 transporter (FLJ46769; SLC22A5), the ENT transporters (ENT1; SLC29A1 and ENT2; SLC29A2), the PAT2 transporter (SLC36A2), and the SAT2 transporter (KIAA1382; SLC38A2). These transporters may facilitate the entry of substrates into skeletal muscle, providing opportunities for muscle targeting.

[0204] In some embodiments, the muscle targeting agent is a substrate for equilibrative nucleoside transporter 2 (ENT2) transporter. Compared to other transporters, ENT2 has one of the highest mRNA expression in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs, such as brain, heart, placenta, thymus, pancreas, prostate, and kidney, but is particularly abundant in skeletal muscle. Human ENT2 facilitates the uptake of its substrates according to their concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. hENT2 transporter has low affinity for all nucleosides except inosine (adenosine, guanosine, uridine, thymidine, and cytidine). Consequently, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and clofarabine. In some embodiments, any of the muscle targeting agents provided herein are associated with 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 non-covalently linked to the molecular payload.

[0205] In some embodiments, the muscle targeting agent is a substrate for the organic cation / carnitine transporter (OCTN2), 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 a derivative thereof is covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0206] A muscle targeting agent may be a protein, which is a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, hemojuvelin may be full-length or a fragment 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 to a functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack the N-terminal signaling and / or (by way of example and) may lack the C-terminal anchoring domain. In some embodiments, the hemojuvelin may be annotated as GenBank RefSeq Accession No. NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It is to be understood that the hemojuvelin may be of human, non-human primate, or rodent origin.

[0207] B. Molecular Payload Some aspects of the present disclosure provide oligonucleotides designed to target DUX4 RNA to modulate molecular payloads, such as expression or activity of DUX4. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA, which are useful for reducing the level of DUX4 mRNA and / or protein associated with features of facioscapulohumeral muscular dystrophy (FSHD), including muscle 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 engage the RNA-induced silencing complex (RISC) for DUX4 RNA degradation, but also reduce off-target effects. In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desired binding affinity properties. In some embodiments, the oligonucleotides are designed to have desired toxicity and / or immunogenicity profiles.

[0208] In some embodiments, the DUX4 targeting oligonucleotide comprises a strand having a region of complementarity to DUX4 RNA. Exemplary oligonucleotides are described in more detail herein; however, it should be understood that the exemplary oligonucleotides provided herein are not intended to be limiting.

[0209] i. Oligonucleotides In some embodiments, the DUX4 targeting oligonucleotide provided herein is designed to cause RNAi-mediated degradation of DUX4 mRNA.In some embodiments, the DUX4 targeting oligonucleotide provided herein comprises an antisense strand complementary to DUX4 mRNA.In some embodiments, the oligonucleotide provided herein further comprises a sense strand to form a double-stranded oligonucleotide (for example, siRNA).It should be understood that in some embodiments, the oligonucleotide of one format (for example, antisense oligonucleotide) can be suitably adapted to another format (for example, siRNA oligonucleotide) by incorporating a functional sequence (for example, antisense strand sequence) from one format into the other format.

[0210] Any suitable oligonucleotide may be used as a molecular payload as described herein. Examples of oligonucleotides useful for targeting DUX4 include those described in U.S. Patent No. 9,988,628, published on February 2, 2017, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. Patent No. 9,469,851, published on October 30, 2014, entitled "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4"; U.S. Patent Application Publication No. 20120225034, published on September 6, 2012, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. Patent Application Publication No. 20120225034, published on September 6, 2012, entitled "MORPHOLINO TARGETING DUX4 FOR TREATING 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 which are incorporated herein. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA, or another oligonucleotide that hybridizes to the target DUX4 gene or mRNA.

[0211] In some embodiments, the oligonucleotides described herein have regions of complementarity to sequences as represented 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 below, and / or (by way of example and) mouse DUX4 corresponding to NCBI sequence NM_001081954.1 (SEQ ID NO: 162) as below. Other non-limiting exemplary human DUX4 The mRNAs are listed in NCBI sequence: NM_033178, GenBank accession numbers FJ439133, AF117653, HM101229, HM101230, HM101232, HM101233, HM101234, HM101235, HM101240, HM101241, HM101242, HM101243, HM101244, HM101245, HM101246, HM101247, HM101248, HM101249, HM101250, HM101251, HM101252, HM101253, HM101254, HM101255, HM101256, HM101257, HM101258, HM101259, HM101260, HM101261, HM101262, HM101263, HM101264, HM101265, HM101266, HM101267, HM101268, HM101269, HM101270, HM101271, HM101272, HM101273, HM101274, HM101275, HM101276, HM101277, HM101278, HM101279, HM101280, HM101281, HM101282, HM101283, HM101284, HM101285 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 HM190194, HM190195, HM190196, each of which is incorporated herein by reference.In some embodiments, the oligonucleotide may have a region of complementarity to hypomethylated, shortened D4Z4 repeats as per 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, published in Curr Opin Genet Dev in 2015 (the entire contents of each of which are incorporated herein).

[0212] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence depicted 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 the sequence depicted 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 the sequence depicted 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, for example selected from human, mouse and non-human species. In some embodiments, the non-human species is cynomolgus monkey.

[0216] i. Oligonucleotide size / sequence Oligonucleotides may be of a variety of different lengths, for example depending on the format. In some embodiments, the 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 in length or longer. In some embodiments, the oligonucleotides are 8-50 nucleotides in length, 8-40 nucleotides in length, 8-32 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 21-23 nucleotides in length, etc. In some embodiments, the oligonucleotide is 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 in length.

[0217] In some embodiments, for purposes of the present disclosure, a complementary nucleic acid sequence of an oligonucleotide is specifically hybridizable to or specific for a target nucleic acid when the binding of said sequence to a target molecule (e.g., an mRNA) interferes with the normal function of the target (e.g., an mRNA) causing loss of activity (e.g., inhibition of translation) or loss of expression (e.g., degradation of the target mRNA) and when there is a sufficient degree of complementarity to avoid non-specific binding of said sequence to non-target sequences under conditions where avoidance of non-specific binding is desired, e.g., physiological conditions in the case of in vivo assays or therapeutic treatments and in the case of in vitro assays, where the assay is performed under suitable conditions of stringency. Thus, 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 100% complementary to the sequence of the target that is specifically hybridizable to or specific to the target nucleic acid. In some embodiments, the oligonucleotide comprises one or more mismatched nucleobases compared to the target nucleic acid. In some embodiments, the activity of the target is reduced by such mismatch, while the activity of the non-target is reduced by a greater amount (i.e., the selectivity for the target nucleic acid is increased and the off-target effect is reduced). In some embodiments, the target nucleic acid is a pre-mRNA molecule or an mRNA molecule.

[0218] In some embodiments, the oligonucleotide comprises a region of complementarity to a target nucleic acid ranging from 8-15, 8-30, 8-40, or 10-50, or 5-50, or 5-40 nucleotides in length. In some embodiments, the oligonucleotide comprises a region of complementarity to a target nucleic acid ranging from 8-32, 15-29, 15-27, 21-27, or 23-27 nucleotides in length. In some embodiments, the oligonucleotide comprises a region of complementarity to a target nucleic acid ranging from 15-29, 15-27, 15-20, 20-25, 21-27, 23-27, 25-27, or 25-32 nucleotides in length. In some embodiments, the region of complementarity of the oligonucleotide to the target nucleic acid is 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 length. In some embodiments, the region of complementarity 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 comprising any one of SEQ ID NOs: 1575-2986 and 3027-3066. In some embodiments, the oligonucleotide comprises a sequence comprising any one of SEQ ID NOs: 1575-2986 and 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 any one of SEQ ID NOs: 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 comprising any one of SEQ ID NOs: 3027-3066. In some embodiments, the oligonucleotide comprises a sequence comprising any one of SEQ ID NOs: 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 contiguous nucleotides of any one of SEQ ID NOs: 3027-3066.

[0220] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence as represented by any one of SEQ ID NOs: 163-1574. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence as represented by any one of SEQ ID NOs: 2987-3026. In some embodiments, the oligonucleotide comprises 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 contiguous nucleotides of the target sequence as represented by any one of SEQ ID NOs: 163-1574. In some embodiments, the oligonucleotide comprises 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 contiguous nucleotides of the target sequence as represented by any one of SEQ ID NOs: 2987-3026. In some embodiments, the region of complementarity is 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 length. In some embodiments, the region of complementarity is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the region of complementarity ranges from 8-20, 10-20, or 15-20 nucleotides in length. In some embodiments, the region of complementarity is fully complementary to all or a portion of its target sequence. In some embodiments, the region of complementarity includes one, two, three, or more mismatches.

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

[0222] It should be understood that in some embodiments, methylation at the C5 position of the nucleobase uracil forms thymine. Thus, in some embodiments, a nucleotide or nucleoside having a C5 methylated uracil (or 5-methyl-uracil) may be equivalently identified as a thymine nucleotide or nucleoside.

[0223] In some embodiments, one or more of the thymine bases (T) in any one of the oligonucleotides provided herein may independently and optionally be a uracil base (U), and / or any one or more of the U's may independently and optionally be T. In some embodiments, one or more of the thymine bases (T) in any one of the oligonucleotides listed in Table 8 or Table 9 may independently and optionally be a uracil base (U), and / or any one or more of the U's may independently and optionally be T.

[0224] b. Oligonucleotide Modification: The oligonucleotides described herein may be modified, including, for example, modified sugar moieties, modified internucleoside linkages, modified nucleotides, and / or combinations thereof.In addition, in some embodiments, the oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immunostimulatory; are nuclease resistant; have improved cellular uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; have improved endosomal exit into cells; minimize TLR stimulation; or avoid pattern recognition receptors.Any of the modified chemistries 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 included in the same oligonucleotide.

[0225] In some embodiments, certain nucleotide modifications may be used that make the oligonucleotides that incorporate the modifications more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides remain intact longer than unmodified oligonucleotides.Specific examples of modified oligonucleotides include those that include modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or modified internucleoside linkages, such as short heteroatom or heterocyclic intersugar linkages.As a result, the oligonucleotides of the present disclosure can be stabilized against nucleic acid degradation by modifications, such as the incorporation of nucleotide modifications.

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

[0227] C modified nucleosides In some embodiments, the oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of the sugar. In some embodiments, the oligonucleotide comprises at least one 2' modified nucleoside. In some embodiments, all of the 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, such as 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-methylacetamido (2'-O-NMA) modified nucleosides.

[0229] In some embodiments, the oligonucleotides described herein include one or more 2'-4' bicyclic nucleosides in which the ribose ring contains a bridging moiety connecting two atoms in the ring (e.g., connecting the 2'-O atom to the 4'-C atom via a methylene (LNA) bridge, an ethylene (ENA) bridge, or an (S)-constrained ethyl (cEt) bridge). Examples of LNAs are described in International Patent Application Publication WO / 2008 / 043753, published 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 ENAs are provided in International Patent Publication No. WO 2005 / 042777, published May 12, 2005, entitled "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 disclosures of which are incorporated herein by reference in their entireties. Examples of cEt are provided in US Patents 7,101,993; 7,399,845 and 7,569,686, each of which is incorporated herein by reference in its entirety.

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

[0231] In some embodiments, an oligonucleotide comprises at least one modified nucleoside that results in an increase in the 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 have at least one modified nucleoside. An oligonucleotide may have multiple modified nucleosides that, in total, result in an increase in the 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 more, compared to an oligonucleotide that does not have the modified nucleoside.

[0232] An oligonucleotide may contain a mix of different types of nucleosides. For example, an oligonucleotide may contain a mix of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified 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'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may contain a mix of bridged 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). The oligonucleotide may contain a mix of 2'-fluoro and 2'-O-Me modified nucleosides. The oligonucleotide may contain a mix of 2'-4' bicyclic nucleosides and 2'MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. The oligonucleotide may contain 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] The oligonucleotide may contain different types of alternating nucleosides. For example, the oligonucleotide may contain alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. The oligonucleotide may contain alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. The oligonucleotide may contain alternating 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. The oligonucleotide may contain alternating bridged nucleosides and 2'-fluoro or 2'-O-methyl modified nucleosides. The 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 oligonucleotides may contain alternating 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. The oligonucleotides may 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 contain a 5'-vinylphosphonate modification, one or more abasic residues, and / or one or more inverted abasic residues.

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

[0236] Phosphorus-containing linkages which may be used include, but are not limited to, regular 3'-5' linkages, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3' alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, as well as those of reverse polarity (where adjacent pairs of nucleoside units are 3'-5' to 5'-3' or 6'-7' to 8'-9'). 2'-5' to 5'-2') and the like; U.S. Patents Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,39 See Nos. 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, oligonucleotides may have heteroatom backbones such as methylene (methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleic acid (PNA) backbones (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone and the nucleotides are attached directly or indirectly to aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

[0238] e. Stereospecific oligonucleotides In some embodiments, the phosphorus atom between oligonucleotides of oligonucleotides is chiral, and the properties of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atom. 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 that include 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 with substantially chiral pure intersugar linkages are prepared by enzymatic synthesis 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, the chiral controlled oligonucleotide provides a selective cleavage pattern of the target nucleic acid.For example, in some embodiments, the chiral controlled oligonucleotide provides a single cleavage site within the complementary sequence of the nucleic acid, as described, for example, in U.S. Patent Application Publication No. 20170037399 A1, entitled "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, the oligonucleotide may be a morpholino-based compound.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;Nasevicius 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 on July 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated by reference in their entireties herein).

[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 gap region Y. In some embodiments, flanking region X of formula 5'-XYZ-3' is also referred to as X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, flanking region Z of formula 5'-XYZ-3' is also referred to as Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, gap region Y of formula 5'-XYZ-3' is also referred to as Y region, Y segment, or gap segment Y. In some embodiments, each nucleoside of gap region Y is a 2'-deoxyribonucleoside, and neither 5' wing region X nor 3' wing region Z contains any 2'-deoxyribonucleosides.

[0241] In some embodiments, the Y region is a stretch of nucleotides, e.g., a region of six or more DNA nucleotides, that can recruit an RNAse, such as RNAse H. In some embodiments, the gapmer binds to the target nucleic acid, at which point an RNAse can be recruited and then cleave the target nucleic acid. In some embodiments, the Y region is flanked on both 5' and 3' by regions X and Z that contain high affinity modified nucleosides, e.g., 1 to 6 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 can be 1-20 nucleotides, 1-8 nucleotides, or 1-5 nucleotides in length. The flanking sequences X and Z can be of similar or dissimilar lengths. In some embodiments, the gap segment Y can be a nucleotide sequence between 5 and 20 nucleotides, between 5 and 15, 12 nucleotides, or between 6 and 10 nucleotides in length.

[0242] In some embodiments, the gap region of the gapmer oligonucleotide may contain, in addition to DNA nucleotides, modified nucleotides known to be permissive 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 linkages. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least 2, at least 3, at least 4, at least 5, or more nucleotides. In some embodiments, the gap region and the two flanking regions each independently contain modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least 2, at least 3, at least 4, at least 5, or more nucleotides.

[0243] Gapmers may be produced using any suitable method. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include 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 Publication Nos. US20050074801, US20090221685; US200902869 69, US20100197762, and US20110112170; PCT Publication Nos. W02004069991; W02005023825; W02008049085 and W02009090182; and European Patent No. EP 2,149,605, each of which is incorporated herein by reference in its entirety.

[0244] In some embodiments, gapmers are 10-40 nucleosides in length. For example, gapmers can be 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 length. In some embodiments, the gapmer is 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 in length.

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

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

[0247] In some embodiments, the gapmers are 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 -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 5'-XYZ-3'. The numbers indicate the number of nucleosides 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 nucleoside (e.g., high affinity modified nucleoside) is a 2' modified nucleoside. In some embodiments, the 2' modified nucleoside is a 2'-4' bicyclic nucleoside or a non-bicyclic 2' modified nucleoside. In some embodiments, the high affinity modified nucleoside is a 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2'-modified nucleoside (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 of the 5'-XYZ-3' formula) comprises the same high affinity nucleosides as the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula). For example, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) can comprise one or more non-bicyclic 2' modified nucleosides (e.g., 2'-MOE or 2'-O-Me). In another example, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) can comprise 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 formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z in the formula 5'-XY-Z-3') 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 formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z in the formula 5'-XYZ-3') 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 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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 of the 5'-XYZ-3' formula) comprises a different high affinity nucleoside than the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula). For example, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) can include one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) can include one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) can include one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) can include 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 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, 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 of the 5'-XYZ-3' formula) comprises 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 of the 5'-XYZ-3' formula) comprises 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 of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') comprise 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 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 of X (the 5'-most position is position 1) is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (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 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 of Z (the 5'-most position is position 1) is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside of Y is a 2' deoxyribonucleoside.In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where at least one, but not all (e.g., 1, 2, 3, 4, 5, 6, or 7) of positions 1, 2, 3, 4, 5, 6, or 7 of X and Z (the 5'-most At least one, but not all, of positions 1, 2, 3, 4, 5, 6, or 7 (e.g., positions 1, 2, 3, 4, 5, or 6) of both X and Z are non-bicyclic 2' modified nucleosides (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside of Y is a 2' deoxyribonucleoside.

[0255] Non-cyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and a mix of 2'-4' cyclic nucleosides (e.g., LNA or cEt) in the 5'-wing region (X in the 5'-X-Y-Z-3' format) and / or the 3'-wing region (Z in the 5'-X-Y-Z-3' format) of a gapmer. Non-limiting examples of gapmer constructs 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 K-(D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK; "A" nucleosides include 2' modified nucleosides; "B" represents a 2'-4' bicyclic nucleoside; "K" represents a -constrained ethyl nucleoside (cEt); "L" represents an LNA nucleoside; "E" represents a 2'-MOE modified ribonucleoside; "D" represents a 2' deoxyribonucleoside; "n" represents the length of the gap segment (Y in the 5'-XYZ-3' configuration) and is an integer between 1 and 20;

[0256] In some embodiments, any one of the gapmers described herein comprises 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 comprises a mix of phosphorothioate and phosphodiester linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate linkage, 5' wing region X comprises a mix of phosphorothioate and phosphodiester linkages, and 3' wing region Z comprises a mix of phosphorothioate 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 class of double-stranded RNA molecules, typically about 20-25 base pairs in length, that target 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 in length to prevent triggering non-specific RNA interference pathways in cells via the interferon response, although longer siRNAs may also be effective. In some embodiments, the 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 or more base pairs in length, in some embodiments, the siRNA molecules are 8-30 base pairs in length, 10-15 base pairs in length, 10-20 base pairs in length, 15-25 base pairs in length, 19-21 base pairs in length, 21-23 base pairs in length. In some embodiments, the siRNA molecule is 8-32 base pairs in length, 8-29 base pairs in length, 8-27 base pairs in length, 15-32 base pairs in length, 15-29 base pairs in length, 15-27 base pairs in length, 21-31 base pairs in length, 21-29 base pairs in length, 21-27 base pairs in length, 21-23 base pairs in length, 23-32 base pairs in length, 23-29 base pairs in length, or 23-27 base pairs in length.

[0258] Upon selection of an appropriate target RNA sequence, an siRNA molecule containing a nucleotide sequence complementary to all or a portion of the target sequence, i.e., an antisense sequence, can be designed and prepared using suitable methods (see, for example, PCT Publication No. WO2004 / 016735; and U.S. Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791).

[0259] siRNA molecule can be double-stranded (i.e., dsRNA molecule that comprises antisense strand and complementary sense strand) or single-stranded (i.e., ssRNA molecule that comprises only antisense strand). siRNA molecule can comprise duplex, asymmetric duplex, hairpin, or asymmetric hairpin secondary structure with self-complementary sense and antisense strand. In some embodiments, the DUX4 targeting oligonucleotide described herein is siRNA that comprises antisense strand and sense strand.

[0260] In some embodiments, the antisense 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, or more nucleotides in length. In some embodiments, the antisense strand is 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 19-21 nucleotides in length, 21-23 nucleotides in length. In some embodiments, the antisense strand is 8-32 nucleotides in length, 8-29 nucleotides in length, 8-27 nucleotides in length, 15-32 nucleotides in length, 15-29 nucleotides in length, 15-27 nucleotides in length, 21-31 nucleotides in length, 21-29 nucleotides in length, 21-27 nucleotides in length, 21-23 nucleotides in length, 23-32 nucleotides in length, 23-29 nucleotides in length, or 23-27 nucleotides in length.

[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, or more nucleotides in length, in some embodiments, the sense strand is 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 19-21 nucleotides in length, 21-23 nucleotides in length. In some embodiments, the sense strand is 8-32 nucleotides in length, 8-29 nucleotides in length, 8-27 nucleotides in length, 15-32 nucleotides in length, 15-29 nucleotides in length, 15-27 nucleotides in length, 21-31 nucleotides in length, 21-29 nucleotides in length, 21-27 nucleotides in length, 21-23 nucleotides in length, 23-32 nucleotides in length, 23-29 nucleotides in length, or 23-27 nucleotides in length.

[0262] In some embodiments, the siRNA molecule comprises an antisense strand that comprises a region of complementarity to the target region on DUX4 mRNA.In some embodiments, the region of complementarity 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 region of a stretch of nucleotides on DUX4 mRNA.In some embodiments, the complementary nucleotide sequence does not need to be 100% complementary to the 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 that includes a region of complementarity to a DUX4 RNA sequence, and the region of complementarity ranges from 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, the region of complementarity is 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 length. In some embodiments, the region of complementarity 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 region of complementarity comprises a nucleotide sequence that contains no more than 1, 2, 3, 4, or 5 bases mismatched compared to the complementary portion of the DUX4 RNA sequence. In some embodiments, the region of complementarity comprises a nucleotide sequence that has 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 strand comprising a nucleotide sequence that is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to a target sequence as set forth in any one of SEQ ID NOs: 163-1574. In some embodiments, the siRNA molecule comprises an antisense strand that is 18-25 nucleotides in length and comprises a region of complementarity of at least 15 nucleotides (e.g., at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, or at least 19 nucleotides) to a target sequence as set forth in any one of SEQ ID NOs: 163-1574.

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

[0266] In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence at least 85%, at least 90%, at least 95%, or 100% identical to an oligonucleotide as set forth in any one of SEQ ID NOs: 1575-2986 and 3027-3066. In some embodiments, the siRNA molecule comprises an antisense strand having a length of 18-25 nucleotides and comprising 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 an oligonucleotide as set forth in any one of SEQ ID NOs: 1575-2986 and 3027-3066.

[0267] In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence at least 85%, at least 90%, at least 95%, or 100% identical to an oligonucleotide as set forth in any one of SEQ ID NOs: 3027-3066. In some embodiments, the siRNA molecule comprises an antisense strand comprising 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 an oligonucleotide as set forth in any one of SEQ ID NOs: 3027-3066.

[0268] Double-stranded siRNA may comprise sense and antisense RNA strands of the same or different length.Double-stranded siRNA molecules can also be assembled from a single oligonucleotide of stem-loop structure (wherein the self-complementary sense and antisense regions of siRNA molecules are linked using nucleic acid-based or non-nucleic acid-based linker(s)), as well as from circular single-stranded RNA with two or more loop structures and a stem that comprises self-complementary sense and antisense strands (wherein the circular RNA can be processed either in vivo or in vitro to generate active siRNA molecules that can mediate RNAi).Therefore, small hairpin RNA (shRNA) molecules are also contemplated herein.These molecules contain specific antisense sequences in addition to reverse-complementary (sense) sequences, typically separated by spacer or loop sequences. Cleavage of the spacer or loop provides the single-stranded RNA molecule and its reverse complement (optionally with additional processing steps that may result in the addition or removal of one, two, three or more nucleotides from the 3'-end and / or (for example and) the 5'-end of one or both strands) such that they can anneal to form a dsRNA molecule. The spacer can be of sufficient length to allow the antisense and sense sequences to anneal to form a double-stranded structure (or stem) prior to cleavage of the spacer (and optionally with subsequent processing steps that may result in the addition or removal of one, two, three, four or more nucleotides from the 3'-end and / or (for example and) the 5'-end of one or both strands). The spacer sequence can be an unrelated nucleotide sequence located between two complementary nucleotide sequence regions that, once annealed to the double-stranded nucleic acid, will comprise the shRNA.

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

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

[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 internucleoside linkages. In some embodiments, the modified nucleotide is a modified sugar moiety (e.g., 2' modified nucleotide). In some embodiments, the siRNA molecule comprises one or more 2' modified nucleotides, such as 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 (such as 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 comprises phosphorothioate internucleoside linkages. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the siRNA molecule comprises modified internucleoside linkages at the 5' end of the siRNA molecule. end or 3' end endin the first, second, and / or (by way of example and) third internucleoside linkages.

[0273] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, phosphorus-containing linkages that can be used include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates, 2'-5' linked analogs of these, as well as reverse polarity nucleoside linkages in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. and the like; including, but not limited to, those having U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399, See Nos. 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 chemistries or formats of the siRNA molecules described herein can be combined with each other, for example, one, two, three, four, five, or more different types of modifications can be included 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 linkages. In some embodiments, the modified nucleotide comprises a modified sugar moiety (e.g., 2' modified nucleotides). In some embodiments, the antisense strand comprises one or more 2' modified nucleotides, such as 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 of the antisense strand is a modified nucleotide (such as a 2' modified nucleotide). In some embodiments, the antisense strand comprises one or more 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand comprises one or more 2'-F modified nucleotides. In some embodiments, the antisense strand contains one or more 2'-O-methyl and 2'-F modified nucleotides.

[0276] In some embodiments, the antisense strand contains phosphorothioate or other modified internucleotide linkages. In some embodiments, the antisense strand includes phosphorothioate internucleoside linkages. In some embodiments, the antisense strand includes phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the antisense strand includes phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the antisense strand includes modified internucleotide linkages at the 5' end of the siRNA molecule. end or 3' end endIn some embodiments, the first, second, and / or (by way of example and) third internucleoside linkages at the 3'-end of the antisense strand are phosphorothioate internucleoside linkages.

[0277] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, phosphorus-containing linkages that can be used include phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates, including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates, including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates, their 2'-5' linked analogs, and reverse polarity nucleoside linkages in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. and the like; including, but not limited to, those having U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399, See Nos. 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 antisense strand modified chemistries or formats described herein can be combined with each other, for example, one, two, three, four, five, or more different types of modifications can be included on the same antisense strand.

[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 linkages. In some embodiments, the modified nucleotides comprise modified sugar moieties (e.g., 2' modified nucleotides). In some embodiments, the sense strand comprises one or more 2' modified nucleotides, such as 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 of the sense strand is a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the sense strand comprises one or more phosphorodiamidate morpholinos. In some embodiments, the sense strand is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the sense strand comprises one or more 2'-O-methyl modified nucleotides. In some embodiments, the sense strand comprises one or more 2'-F modified nucleotides. In some embodiments, the sense strand comprises one or more 2'-O-methyl and 2'-F modified nucleotides.

[0280] In some embodiments, the sense strand contains phosphorothioate or other modified internucleotidic linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the sense strand comprises modified internucleotidic linkages at the first, second, and / or (for example and) third internucleoside linkages at the 5'-end or 3'-end of the siRNA molecule. In some embodiments, the modified internucleotidic linkages are phosphorus-containing linkages.In some embodiments, phosphorus-containing linkages that may be used include phosphorothioates having normal 3'-5' linkages, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, 2'-5' linked analogs thereof, as well as reverse polarity phosphates in which adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. and the like; including, but not limited to, those having U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399, See Nos. 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 sense strand modification chemistries or formats described herein can be combined with each other, for example, one, two, three, four, five, or more different types of modifications can be included on the same sense strand.

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

[0283] In some embodiments, the sense strand of the siRNA molecule comprises 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, the entirety of which is incorporated herein by reference. In some embodiments, the sense strand of the siRNA molecule is modified with a synthetic RNA-like high affinity nucleotide analogue, locked nucleic acid (LNA). This reduces RISC loading of the sense strand and further enhances antisense strand incorporation into RISC, as described in Elman et al., (2005) Nucleic Acids Res. 33 (1): 439-447, the entirety of which is incorporated herein by reference. In some embodiments, the sense strand of the siRNA molecule comprises a 5'unlocked nucleic acid (UNA) modification, which reduces the 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, the entirety of which is incorporated herein by reference. In some embodiments, the sense strand of the siRNA molecule comprises a 5-nitroindole modification, which reduces the RNAi potency of the sense strand and reduces off-target effects, as described in Zhang et al., (2012) Chembiochem 13(13):1940-1945, the entirety of which is incorporated herein by reference. In some embodiments, the sense strand contains a 2'-O' methyl (2'-O-Me) modification, which reduces RISC loading and off-target effects of the sense strand, as described in Zheng et al., FASEB (2013) 27(10):4017-4026, which is incorporated by reference in its entirety.In some embodiments, the sense strand of the siRNA molecule is fully substituted with morpholino, 2'-MOE, or 2'-O-Me residues and is not recognized by RISC, as described in Kole et al., (2012) Nature reviews. Drug Discovery 11(2):125-140, which is incorporated by reference in its entirety. In some embodiments, the antisense strand of the siRNA molecule comprises a 2'-MOE modification and the sense strand comprises a 2'-O-Me modification (see, e.g., Song et al., (2017) Mol Ther Nucleic Acids 9:242-250). In some embodiments, at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 10) siRNA molecule is linked (e.g., covalently) to a muscle targeting agent. In some embodiments, the muscle targeting agent may comprise or consist of a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide). In some embodiments, the muscle targeting agent is an antibody. In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody (e.g., any one of the anti-TfR antibodies provided in Tables 2-7). In some embodiments, the muscle targeting agent can be linked to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked internally to the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked to the 5' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked to the 3' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked internally to the antisense strand of the siRNA molecule.

[0284] Non-limiting examples of siRNAs targeting DUX4 are provided in Table 8. Table 8. DUX4 targeting 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 internucleoside linkage; and the absence of an "*" between two nucleosides indicates a phosphodiester internucleoside 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. Although the target sequences listed in Table 8 contain T, binding of DUX4-targeting oligonucleotides to RNA and / or DNA is contemplated. ^ Target sequence start position is in NM_001293798.2 (SEQ ID NO: 160)

[0285] Additional non-limiting examples of further modified DUX4-targeting siRNAs are provided in Table 9. Table 9. Additional DUX4-targeting 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-methyl-cytidine; "fxC" indicates a 2'-F modified 5-methyl-cytidine; "*" indicates a phosphorothioate internucleoside linkage; and the absence of a "*" between two nucleosides indicates a phosphodiester internucleoside 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. Although the target sequences listed in Table 9 contain T, binding of DUX4-targeted oligonucleotides to RNA and / or DNA is contemplated. ^ Target sequence start position is in NM_001293798.2 (SEQ ID NO: 160)

[0286] In some embodiments, the DUX4-targeting oligonucleotide comprises an antisense strand that is 18-25 nucleosides in length (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) and is selected from the group consisting of SEQ ID NOs: 224-226, 261, 265, 320, 341, 343, 356, 388, 466, 483, 494, 501, 509, 552, 560, 561, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 921, 942, 953, 1294, 1296, 1301, 1320-1325, 1373, 1394, 1395, 1398, 1523, 1531, 1548, 1558, and 1561, wherein the region of complementarity is at least 16 nucleotides in length (e.g., 16, 17, 18, or 19 nucleotides). In some embodiments, the antisense strand is 21 nucleotides in length and comprises a region of complementarity to a target sequence as represented by any one of SEQ ID NOs: 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, wherein the region of complementarity is 19 nucleotides in length. In some embodiments, the region of complementarity is fully complementary to all or a portion of the target sequence. In some embodiments, the region of complementarity encompasses one, two, three, or more mismatches.

[0287] In some embodiments, the DUX4 targeting oligonucleotide comprises an antisense strand comprising 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 of the nucleotide sequences of SEQ ID NOs: 3027-3066. In some embodiments, the DUX4 targeting oligonucleotide further comprises a sense strand comprising 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 of the nucleotide sequences of SEQ ID NOs: 2987-3026.

[0288] In some embodiments, the DUX4 targeting oligonucleotide comprises an antisense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 3027-3066. In some embodiments, the DUX4 targeting oligonucleotide further comprises a sense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 2987-3026.

[0289] In some embodiments, the DUX4 targeting oligonucleotide is a double-stranded oligonucleotide (e.g., siRNA) comprising an antisense strand comprising any one of the nucleotide sequences of SEQ ID NOs: 3027-3066 and a sense strand hybridizing to the antisense strand comprising any one of the nucleotide sequences of SEQ ID NOs: 2987-3026, wherein the antisense strand and / or (e.g., and) comprise 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-targeting oligonucleotide is a double-stranded oligonucleotide (e.g., siRNA) comprising an antisense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 3027-3066 and a sense strand that hybridizes to the antisense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 2987-3026, wherein 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 targeting oligonucleotide is a double-stranded oligonucleotide (for example, siRNA) comprising an antisense strand comprising any one of the nucleotide sequences of SEQ ID NOs: 3027-3066 and a sense strand hybridizing to the antisense strand comprising any one of the nucleotide sequences of SEQ ID NOs: 2987-3026, wherein each nucleoside in the antisense strand and each nucleoside in the sense strand is a 2'-modified nucleoside selected from 2'-O-Me and 2'-F modified nucleosides, and wherein the antisense strand and / or (for example and) the sense strand each comprise one or more phosphorothioate internucleoside linkages. In some embodiments, the sense strand does not comprise any phosphorothioate internucleoside linkages (all internucleoside linkages in the sense strand are phosphodiester internucleoside linkages), and the antisense strand comprises one, two, or three phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises two phosphorothioate internucleoside linkages, optionally wherein the two internucleoside linkages at the 3'-end of the antisense strand are phosphorothioate internucleoside linkages and the remaining internucleoside linkages in the antisense strand are phosphodiester internucleoside linkages.

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

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

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

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

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

[0297] In some embodiments, the antisense strand of the DUX4 targeting oligonucleotide is selected from any one of the modified versions 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 targeting oligonucleotide is selected from any one of the modified versions of SEQ ID NOs: 2987, 2997, 2999, 3000, 3001, 3004, 3012, and 3021 listed in Table 9. In some embodiments, the DUX4 targeting oligonucleotide is an siRNA selected from the siRNAs listed in Table 9.

[0298] In some embodiments, any one of the DUX4 targeting oligonucleotides (e.g., DUX4 targeting siRNAs selected from the siRNAs in Table 8) can be in the form of a salt, e.g., sodium, potassium, magnesium salt. In some embodiments, any one of the DUX4 targeting oligonucleotides (e.g., DUX4 targeting siRNAs selected from the siRNAs in Table 9) can be in the form of a salt, e.g., sodium, potassium, magnesium salt.

[0299] In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8) is conjugated to an amine group, optionally via a spacer. In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 9) is conjugated to an amine group, optionally 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 is present between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8) is conjugated to a spacer, which is selected from the group consisting of substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O) 2 NR A -, -NR A S(O) 2 -, or a combination thereof; each R Aare independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 9) is conjugated to a spacer, which is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O) 2 NR A -, -NR A S(O) 2 -, or a combination thereof; each R A are independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, the spacer is a substituted or unsubstituted alkylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )-, or -C(=O)N(R A ) 2 , or a combination thereof.

[0300] In some embodiments, the 5' or 3' nucleoside of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8, sense or antisense strands) is of the formula -NH 2 -(CH 2 ) n-, where n is an integer from 1 to 12. In some embodiments, the 5' or 3' nucleoside of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 9, sense or antisense strands) is conjugated to a compound of the formula -NH 2 -(CH 2 ) n -, where 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 conjugated to a compound of the formula NH 2 -(CH 2 ) n - and the 5' or 3' nucleoside. In some embodiments, the phosphodiester linkage is between the compound of formula NH 2 -(CH 2 ) n - and the 5' or 3' nucleoside. 2 -(CH 2 ) 6 The compound represented by - is 6-amino-1-hexanol (NH 2 -(CH 2 ) 6 In some embodiments, the 5' phosphate of the oligonucleotide is conjugated to the oligonucleotide via a reaction between the 5' phosphate of the oligonucleotide (e.g., the 5' phosphate of the sense or antisense strand) and the 5' phosphate of the oligonucleotide. 2 -(CH 2 ) 6 The compound represented by - is 6-amino-1-hexanol (NH 2 -(CH 2 ) 6-OH) and the 3' phosphate of the oligonucleotide (e.g., the 3' phosphate of the sense or antisense strand). In some embodiments, the oligonucleotide is conjugated to a targeting agent, e.g., a muscle targeting agent such as an anti-TfR antibody, e.g., via an amine group.

[0301] C. Linker The conjugates described herein generally include a linker that connects any 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, such as a disulfide bond or a disulfide bridge, that connects the anti-TfR antibody to the molecular payload. However, in some embodiments, the linker may connect any 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 a non-cleavable linker. The linker is generally stable in vitro and in vivo, and may be stable in a certain 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, e.g., 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 precursor of the linker will typically contain two different reactive species that can be attached to both the anti-TfR antibody and the molecular payload. In some embodiments, the two different reactive species may be nucleophiles and / or (by way of example and) electrophiles. In some embodiments, the linker is connected to the anti-TfR antibody via conjugation to a lysine or cysteine ​​residue of the anti-TfR antibody. In some embodiments, the linker is connected to the cysteine ​​residue of the anti-TfR antibody via a maleimide-containing linker, where optionally the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some embodiments, the linker is connected to the cysteine ​​residue of the anti-TfR antibody or a thiol-functionalized molecular payload via a 3-arylpropionitrile functional group. In some embodiments, the linker is connected to the lysine residue of the anti-TfR antibody. In some embodiments, the linker is connected to the anti-TfR antibody and / or the molecular payload (by way of example and not limitation) via an amide bond, a carbamate bond, a hydrazide, a triazole, a thioether, or a disulfide bond.

[0303] i. Cleavable Linker The cleavable linker may be a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. These linkers are generally only cleavable intracellularly and are preferably stable in the extracellular environment, e.g., outside of a muscle cell.

[0304] Protease-sensitive linkers are cleavable by protease enzyme activity. These linkers typically comprise peptide sequences and 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 sequence may comprise naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids 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 linker comprises a valine-citrulline sequence or an alanine-citrulline sequence. In some embodiments, the protease-sensitive linker may be cleaved by lysosomal proteases, such as cathepsin B, and / or endosomal proteases.

[0305] A pH-sensitive linker is a covalent linkage that is easily 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 in 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 disulfide exchange reaction with 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 (e.g., as described in US Pat. No. 6,214,345, incorporated herein by reference). In some embodiments, the val-cit linker prior to conjugation has the following structure: [ka] has.

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

[0309] In some embodiments, the Val-cit linker is attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation). In some embodiments, the val-cit linker attached to a highly reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) prior to click chemistry conjugation has the following structure: [ka] where n is any number from 0 to 10. In some embodiments, n is 3.

[0310] In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) is conjugated (e.g., via a different chemical moiety) to a molecular payload (e.g., an oligonucleotide). In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) and conjugated to a molecular payload (e.g., an oligonucleotide) (prior to click chemistry conjugation) has a structure represented by formula (A): [ka] where n is any number from 0 to 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] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and m is 4.

[0312] ii. Non-cleavable linker In some embodiments, a non-cleavable linker may be used. In general, a non-cleavable linker cannot be easily degraded in a cellular or physiological environment. In some embodiments, a non-cleavable linker comprises an optionally substituted alkyl group, where the substitution may include halogen, hydroxyl group, oxygen species, and other common substitutions. In some embodiments, a linker may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one unnatural amino acid, a truncated glycan, a sugar or sugars that cannot be enzymatically degraded, an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, a biotin, a biphenyl, a repeating unit of polyethylene glycol or an equivalent compound, an acid ester, an acid amide, a sulfamide, and / or (for example, and) an alkoxy-amine linker. In some embodiments, sortase-mediated ligation will be utilized to link 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 comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), such as polyethylene oxide or polypropylene oxide.

[0314] In some embodiments, the linker has the following structure: [ka] where n is 1-10.

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

[0316] In some embodiments, the linker is connected to the anti-TfR antibody and / or (for example and) molecular payload by a cycloaddition reaction between an azide and an alkyne forming a triazole, where the azide and alkyne may be located on the anti-TfR antibody, the molecular payload, or the linker. In some embodiments, the alkyne may be a cyclic alkyne, for example, a cyclooctyne. In some embodiments, the alkyne may be a bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctane is as described in International Patent Application Publication WO2011136645, entitled "Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions," published on November 3, 2011. In some embodiments, the azide may be a sugar or carbohydrate molecule containing an azide. In some embodiments, the azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the azide-containing sugar or carbohydrate molecule is as described in International Patent Application Publication WO2016170186, published on October 27, 2016, entitled "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 to form a triazole (wherein the azide and alkyne may be located on the anti-TfR antibody, the molecular payload, or the linker) is as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or International Patent Application Publication WO2016170186, published on October 27, 2016, entitled "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 comprises a spacer, e.g., a polyethylene glycol spacer or an acyl / carbamoyl sulfamide spacer, e.g., a HydraSpace™ spacer. In some embodiments, the spacer is as described in Verkade, JMM et al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturing Ability, Stability, and Therapeutic Index of Antibody-Drug Conjugates", Antibodies, 2018, 7, 12.

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

[0319] In some embodiments, the linker is connected to the anti-TfR antibody and / or (by way of example) the 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, carbonate, or aldehyde). In some embodiments, the nucleophile may be present on the linker and the electrophile may be present on the anti-TfR antibody or the molecular payload prior to the reaction between the linker and the anti-TfR antibody or the molecular payload. In some embodiments, the electrophile may be present on the linker and the nucleophile may be present on the anti-TfR antibody or the molecular payload prior to the reaction between the linker and the anti-TfR antibody or the 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 (by way of example and) activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilide group, or a thiol group.

[0320] In some embodiments, the val-cit linker attached to the highly reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) has the following structure: [ka] where m is any number from 0 to 10. In some embodiments, m is 4.

[0321] In some embodiments, the val-cit linker attached to the reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) has the structure represented by formula (G): [ka] (G) where m is any 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) results from reaction of the anti-TfR1 antibody with an amine, such as the lysine epsilon amine.

[0322] In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) and conjugated to an anti-TfR antibody has the structure represented by formula (F): [ka] (F) 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, an oligonucleotide is covalently linked to a compound comprising a structure represented by formula (F), thereby forming a conjugate comprising a structure represented by formula (D). It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (F) results from reaction of the anti-TfR1 antibody with an amine, such as a lysine epsilon amine.

[0323] In some embodiments, the val-cit linker linking the antibody and the molecular payload has the structure represented by formula (C): [ka] wherein 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 (for example, NH from an amine group of lysine), S (for example, S from a thiol group of cysteine), or O (for example, O from a hydroxyl group of serine, threonine, or tyrosine) of the antibody.

[0324] In some embodiments, the conjugates described herein have a structure represented by formula (D): [ka] where n is any number from 0 to 10, and where m is any number from 0 to 10. In some embodiments, n is 3 and / or (by way of example and) m is 4.

[0325] L in Structural Formulas (A), (B), (C), and (D), in some embodiments, is a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(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, wherein each R A is independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L is [ka] where L2 is [ka] where a labels a site that is directly linked to a carbamate moiety represented by formulas (A), (B), (C), and (D); and b labels a site that is covalently linked (directly or via an additional chemical moiety) to an oligonucleotide.

[0326] In some embodiments, L1 is one of the following: [ka] where a labels a site that is directly linked to a carbamate moiety represented by formulas (A), (B), (C), and (D); and b labels a site that is covalently linked (directly or via an additional chemical moiety) to an oligonucleotide.

[0327] In some embodiments, L1 is [ka] where x is 0 to 10. For example, in some embodiments, x is 3, 4, 5, or 6. In some embodiments, L1 is [ka] In some embodiments, L1 is [ka] It is.

[0328] In some embodiments, L1 is [ka] [ka] It is.

[0329] In some embodiments, L1 is [ka] where y is 0 to 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 (eg, does not have to be present).

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

[0334] C. Examples of Antibody-Molecular Payload Conjugates Further provided herein are non-limiting examples of conjugates comprising any one of the anti-TfR antibodies described herein covalently linked to any one 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 to a molecular payload (e.g., 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 of the nucleotide sequences of SEQ ID NOs: 163-3066) via a linker. In some embodiments, the anti-TfR antibody (e.g., any one of the anti-TfR antibodies provided in Tables 2-7) is covalently linked to a molecular payload (e.g., an oligonucleotide such as an oligonucleotide provided in Table 8) via a linker. In some embodiments, the anti-TfR antibody (e.g., any one of the anti-TfR antibodies provided in Tables 2-7) is covalently linked to a molecular payload (e.g., an oligonucleotide such as an oligonucleotide provided in Table 9) via a linker. Any of the linkers described herein may be used. In some embodiments, when the molecular payload is an oligonucleotide, the linker is linked to the 5' end, 3' end, or internal end of the sense strand or antisense strand. In some embodiments, the molecular payload is an siRNA, and the linker is linked to the 5' end of the sense strand. In some embodiments, the linker is linked to the anti-TfR antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the anti-TfR antibody). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an anti-TfR antibody described herein) via an amine group (e.g., via a lysine in the antibody). In some embodiments, the molecular payload is a DUX4-targeting oligonucleotide (e.g., a DUX4-targeting oligonucleotide listed in Table 8).In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide (for example, the DUX4 targeting oligonucleotide listed in Table 9). In some embodiments, the molecular payload is the sense strand of a DUX4 targeting siRNA. In some embodiments, the molecular payload is the antisense strand of a DUX4 targeting siRNA. In some embodiments, the molecular payload is a DUX4 targeting siRNA that comprises a sense strand and an antisense strand.

[0335] An example structure of a conjugate comprising an anti-TfR antibody covalently linked to a molecular payload via a Val-cit linker is provided below: [ka] wherein the linker is linked to the antibody via a thiol-reactive linkage (e.g., via a 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 of the nucleotide sequences of SEQ ID NOs: 163-3066. In some embodiments, the molecular payload is an oligonucleotide comprising any one of the nucleotide sequences of SEQ ID NOs: 163-3066. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in DUX4 mRNA (e.g., a target sequence 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 wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide comprising an antisense strand that comprises a region of complementarity of at least 16 nucleotides to a target sequence in a DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4-targeted oligonucleotide further comprises a sense strand that hybridizes to the antisense strand.

[0336] Another example of the structure of a conjugate comprising an anti-TfR antibody covalently linked to a molecular payload via a Val-cit linker is provided below: [ka] wherein n is a number between 0 and 10, wherein m is a number between 0 and 10, wherein the linker is linked to the antibody via an amine group (e.g., on a lysine residue) and / or (e.g., and) wherein the linker is linked to the sense strand or antisense strand (e.g., at the 5' end, 3' end, or internally). In some embodiments, the linker is linked to the antibody via a lysine, the linker is linked to the oligonucleotide at the 5' end, n is 3, and m is 4. 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 of SEQ ID NOs: 163-3066. In some embodiments, the molecular payload is an oligonucleotide comprising any one of SEQ ID NOs: 163-3066. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., 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 wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., 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 wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, L1 is any one of the spacers described herein.

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

[0338] In some embodiments, the conjugates described herein comprise an anti-TfR antibody described herein (e.g., an antibody provided in Tables 2-7) covalently linked to a molecular payload. In some embodiments, the conjugates described herein comprise an anti-TfR antibody described herein (e.g., an antibody provided in Tables 2-7) covalently linked to a molecular payload via a linker (e.g., a Val-cit linker). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an anti-TfR antibody described herein) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an anti-TfR antibody described herein) via an amine group (e.g., via a 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 of the nucleotide sequences of SEQ ID NOs: 163-3066. In some embodiments, the molecular payload is an oligonucleotide comprising any one of the nucleotide sequences of SEQ ID NOs: 163-3066. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides 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 contiguous nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs: 1575-986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

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

[0340] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 71, or SEQ ID NO: 72, and a VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in DUX4 mRNA (e.g., a target sequence as set forth in any one of SEQ ID NOs: 163-1574), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences as set forth in any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

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

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

[0343] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77, and a VL comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides 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 contiguous nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

[0344] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 79, and a VL comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in DUX4 mRNA (e.g., a target sequence as set forth in any one of SEQ ID NOs: 163-1574), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences as set forth in any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

[0345] In some embodiments, the conjugates described herein comprise an anti-TfR antibody covalently linked to a molecular payload, wherein the anti-TfR antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 154, and a VL comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides 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 contiguous nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

[0346] In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in Table 8). In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in Table 9).

[0347] In some embodiments, the conjugates described herein comprise 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: 84, SEQ ID NO: 86, or SEQ ID NO: 87, and a light chain comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides 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 contiguous nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

[0348] In some embodiments, the conjugates described herein comprise 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: 89. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides 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 contiguous nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

[0349] In some embodiments, the conjugates described herein comprise 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 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides 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 contiguous nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

[0350] In some embodiments, the conjugates described herein comprise 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 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides 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 contiguous nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

[0351] In some embodiments, the conjugates described herein comprise 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, and a light chain comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides 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 contiguous nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 9), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 9, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense strand.

[0352] In some embodiments, the conjugates described herein comprise 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: 156, and a light chain comprising the amino acid sequence of SEQ ID NO: 157. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides 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 contiguous nucleotides of any one of the antisense sequences as represented by any one of SEQ ID NOs: 1575-1986, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand that hybridizes to the antisense strand. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide comprising an antisense strand comprising a region of at least 16 nucleotides of complementarity to a target sequence in DUX4 mRNA (e.g., a target sequence listed in Table 8), optionally wherein the antisense strand comprises at least 16 contiguous nucleotides of any one of the antisense sequences listed in Table 8, and optionally wherein the DUX4 targeting oligonucleotide further comprises a sense strand hybridizing to the antisense ...

Claims

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

42.

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

75.

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

90.

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

5. The conjugate of any one of claims 1 to 4, wherein the siRNA oligonucleotide further comprises a sense strand comprising at least 18 consecutive nucleosides that are complementary to the antisense strand.

6. The complex of claim 5, wherein the antisense strand is 21-23 nucleotides in length and / or the sense strand is 19-21 nucleotides in length.

7. The conjugate of any one of claims 1 to 6, wherein the siRNA oligonucleotide comprises a 3' overhang of about 1 to about 3 nucleosides on the antisense strand.

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

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

10. 10. The conjugate of claim 8 or 9, wherein the one or more modified nucleosides is a 2' modified nucleoside.

11. 11. The conjugate of claim 10, wherein the one or more 2' modified nucleosides are 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), 2'-O-N-methylacetamide (2'-O-NMA).

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

13. 13. The conjugate of 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 conjugate of any one of claims 1 to 13, wherein the oligonucleotide comprises one or more modified internucleoside linkages.

15. 15. The conjugate of claim 14, wherein the one or more modified internucleoside linkages comprises a phosphorothioate internucleoside linkage.

16. 16. The conjugate of claim 5, wherein the sense strand comprises phosphorothioate internucleoside linkages between at least two nucleosides and / or the antisense strand comprises phosphorothioate internucleoside linkages between at least two nucleosides.

17. 17. The conjugate of claim 15 or 16, wherein the sense strand and / or the antisense strand comprises a phosphorothioate internucleoside linkage at the first, second, and / or third internucleoside linkage at the 5'-terminus or 3'-terminus.

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

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

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

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

22. The conjugate of claim 21 , wherein the linker comprises a valine-citrulline sequence.

23. The anti-TfR antibody is linked to the 5' end of the sense strand of the siRNA oligonucleotide.

23. The complex of claim 21 or 22.

24. The conjugate of any one of claims 21 to 23, wherein the anti-TfR antibody is covalently linked to the siRNA oligonucleotide via conjugation to a lysine residue of the antibody.

25. The conjugate of any one of claims 21 to 23, wherein the anti-TfR antibody is covalently linked to the siRNA oligonucleotide via conjugation to a cysteine ​​residue of the antibody.

26. 26. The conjugate of any one of claims 1 to 25 for use in a method of treating a disease or condition that can be ameliorated or prevented by reducing DUX4 expression in a cell, said method comprising contacting said cell with said conjugate in an amount effective to promote internalization of an siRNA oligonucleotide into said cell.

27. 27. The complex for use according to claim 26, wherein the cell is a muscle cell.

28. 28. The conjugate for use of claim 26 or 27, wherein reducing DUX4 expression comprises reducing DUX4 protein and / or mRNA levels.

29. 26. The conjugate of any one of claims 1 to 25 for use in treating facioscapulohumeral muscular dystrophy (FSHD), said method comprising administering an effective amount of the conjugate to a subject in need thereof, wherein the subject has abnormal production of DUX4 protein.

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