Muscle-targeted complexes for treating facioscapulohumeral muscular dystrophy and their use

Abstract

Aspects of the present disclosure relate to oligonucleotides (e.g., RNAi oligonucleotides such as siRNA) designed to target DUX4 RNA, and targeting complexes for delivering such oligonucleotides to cells (e.g., muscle cells), and their use, 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) of U.S. Provisional Application No. 63 / 367,783, filed on July 6, 2022, with the title "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY", and U.S. Provisional Application No. 63 / 477,160, filed on December 23, 2022, with the title "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY", the contents of each of which are hereby incorporated by reference in their entirety.

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

[0003] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (D082470078WO00-SEQ-CBD.xml; size: 339,190 bytes; and creation date June 30, 2023) are hereby incorporated by reference in their entirety.

Background Art

[0004] Background Muscular dystrophy (MD) is a group of diseases characterized by progressive weakness and loss of muscle mass. These diseases are caused by mutations in genes that encode proteins required for healthy muscle tissue. Facioscapulohumeral muscular dystrophy (FSHD) is a dominant genetic form of MD that primarily affects the muscles of the face, scapula, and upper arm. Other symptoms of FSHD include weakness of the abdominal muscles, retinal abnormalities, hearing loss, and joint pain and inflammation. FSHD is the most commonly seen of nine types of MD that affect both adults and children, with an incidence worldwide of approximately 1 in 8,300 people. FSHD is caused by the abnormal production of double homeobox 4 (DUX4), a protein that regulates gene expression. The DUX4 gene, which encodes the DUX4 protein, is located in the D4Z4 repeat region on chromosome 4 and is typically expressed in adult testes and thymus, as well as in two-cell stage embryos, after which it is suppressed by hypermethylation of the D4Z4 repeats that surround and compress the DUX4 gene. Two types of FSHD, type 1 and type 2, are described. Type 1, which accounts for approximately 95% of cases, is associated with a deletion of the D4Z4 repeats in the subtelomeric region of chromosome 4. Unaffected individuals generally have more than 10 repeats that align in the subtelomeric region of chromosome 4, while the most common form of FSHD (FSHD1) is caused by a contraction of the alignment of fewer than 10 repeats and is associated with reduced epigenetic suppression and variegated expression of DUX4 in skeletal muscle. Two alleles of chromosome 4q (4qA and 4qB) are present in the most distal unit of the D4Z4 repeats. 4qA is in cis with a functional polyadenylation consensus site. Contraction of the 4qA allele is pathogenic because the DUX4 transcript is polyadenylated and stable. Type 2 FSHD, which accounts for approximately 5% of cases, is associated with mutations in the SMCHD1 gene on chromosome 18. Type 2 FSHD may also be associated with the DNMT3B gene or the LRIF1 gene. In addition to supportive care and treatments to address the symptoms of the disease, there is no effective treatment for FSHD.

SUMMARY OF THE INVENTION

[0005] SUMMARY 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 that are useful for reducing the level of DUX4 RNA and / or protein. In some embodiments, the present disclosure provides oligonucleotides complementary to exon 3 of DUX4 RNA that are useful for reducing the level of DUX4 RNA. In some embodiments, the oligonucleotides provided herein are designed to cause RNAi-mediated degradation of DUX4 RNA. In some embodiments, the oligonucleotides are designed to efficiently engage the RNA-induced silencing complex (RISC) for degradation of DUX4 RNA, but also to have a reduced off-target effect. In some embodiments, the oligonucleotides are designed to have desirable bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desirable binding affinity properties. In some embodiments, the oligonucleotides are designed to have desirable toxicity and / or immunogenicity profiles. Aberrant (e.g., increased) expression of DUX4 RNA and / or protein in muscle is associated with features of the pathology of facioscapulohumeral muscular dystrophy (FSHD), which includes muscle atrophy, inflammation, and reduced differentiation ability and oxidative stress. In some embodiments, the oligonucleotides described herein that reduce the level of DUX4 RNA and / or protein are effective in treating FSHD.

[0006] According to some aspects, the present disclosure provides a complex that targets these cells for the purpose of delivering a molecular payload (e.g., an oligonucleotide described herein) to muscle cells (e.g., primary myoblasts). In some embodiments, the complexes provided herein are particularly useful for delivering a molecular payload that inhibits the expression or activity of DUX4, for example, in a subject 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 the purpose of delivering the molecular payload to the muscle cell. In some embodiments, the complex may be taken up into the cell via receptor-mediated endocytosis, and thereafter, the molecular payload may be released to exert its function inside the cell. For example, a complex modified to deliver an oligonucleotide may release the oligonucleotide such that the oligonucleotide can inhibit DUX4 gene expression in muscle cells. In some embodiments, the oligonucleotide is released by endosomal cleavage of a covalent linker that connects the oligonucleotide of the complex to the muscle-targeting agent.

[0007] Some aspects of the present disclosure provide a complex comprising a muscle-targeting agent covalently linked to an oligonucleotide that targets the messenger ribonucleic acid (mRNA) of double homeobox 4 (DUX4) (e.g., mRNA), wherein the oligonucleotide comprises an antisense strand that is 18 to 25 nucleotides in length and comprises a region of complementarity to a target sequence as set forth in SEQ ID NOS: 174 to 235, and wherein the region of complementarity is at least 16 consecutive nucleosides in length.

[0008] Some aspects of the present disclosure provide a complex comprising a muscle targeting agent covalently linked to an oligonucleotide that targets mRNA of double homeobox 4 (DUX4) (e.g., mRNA), wherein the oligonucleotide comprises an antisense strand that is 18 to 25 nucleotides in length and comprises a region of complementarity to a target sequence as set forth in SEQ ID NOs: 200, 191, 189, 186, 190, 174 - 185, 187, 188, 192 - 199, and 201 - 235, and wherein the region of complementarity is at least 16 consecutive nucleosides in length.

[0009] In some embodiments, the muscle targeting agent is an anti-transferrin receptor (TfR) antibody.

[0010] In some embodiments, the oligonucleotide is an RNAi oligonucleotide.

[0011] In some embodiments, the antisense strand comprises any one nucleotide sequence of SEQ ID NOs: 236 - 266.

[0012] In some embodiments, the antisense strand comprises any one nucleotide sequence of SEQ ID NOs: 262, 253, 251, 248, 252, 236 - 247, 249, 250, 254 - 261, 263 - 266.

[0013] In some embodiments, the oligonucleotide further comprises a sense strand comprising at least 18 consecutive nucleosides complementary to the antisense strand.

[0014] In some embodiments, the sense strand comprises 21 consecutive nucleosides complementary to the antisense strand.

[0015] In some embodiments, the sense strand comprises any one nucleotide sequence of SEQ ID NOs: 205 - 235.

[0016] In some embodiments, the sense strand comprises any one nucleotide sequence of SEQ ID NOs: 231, 222, 220, 217, 221, 205 - 216, 218, 219, 223, 230, 232 - 235.

[0017] In some embodiments, the muscle targeting agent is covalently linked to the 5' or 3' end of the sense strand.

[0018] In some embodiments, the antisense strand of the RNAi oligonucleotide further comprises 5'-(E)-vinylphosphonate.

[0019] In some embodiments, the oligonucleotide comprises one or more modified nucleosides.

[0020] In some embodiments, the one or more modified nucleosides are 2'-modified nucleotides, and optionally here, the one or more 2'-modified nucleotides 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).

[0021] In some embodiments, each 2'-modified nucleotide is 2'-O-methyl (2'-O-Me) or 2'-fluoro (2'-F). In some embodiments, the 2'-modified nucleotide is 2'-O-methyl (2'-O-Me). In some embodiments, the 2'-modified nucleotide is 2'-fluoro (2'-F).

[0022] In some embodiments, the oligonucleotide comprises one or more phosphorothioate internucleotide linkages.

[0023] In some embodiments, one or more phosphorothioate internucleoside linkages are present on the antisense strand of the oligonucleotide.

[0024] In some embodiments, the internucleoside linkage at the 3' end of the antisense strand is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage at the 5' end of the antisense strand is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage at the 3' end of the antisense strand and the internucleoside linkage at the 5' end of the antisense strand are phosphorothioate linkages.

[0025] In some embodiments, one or more phosphorothioate internucleoside linkages are present on the sense strand of the oligonucleotide.

[0026] In some embodiments, the internucleoside linkage at the 3' end of the sense strand is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage at the 5' end of the sense strand is a phosphorothioate internucleoside linkage. In some embodiments, the internucleoside linkage at the 3' end of the sense strand and the internucleoside linkage at the 5' end of the sense strand are phosphorothioate linkages.

[0027] In some embodiments, one or more cytidines of the oligonucleotide are 2'-modified 5-methyl-cytidines, optionally where the 2'-modified 5-methyl-cytidine is a 2'-O-Me modified 5-methyl-cytidine or a 2'-F modified 5-methyl-cytidine.

[0028] In some embodiments, the antisense strand of the oligonucleotide described herein is selected from the modified versions of SEQ ID NOs: 236-266 listed in Table 8 (e.g., MAS1-MAS4). For example, in some embodiments, the antisense strand is selected from MAS1-236, MAS1-237, MAS1-238, MAS2-236, MAS2-237, MAS2-238, MAS2-239, MAS2-240, MAS2-241, MAS2-242, MAS2-243, MAS2-244, MAS2-245, MAS2-246, MAS2-247, MAS2-248, MAS2-249, MAS2-250, MAS2-251, MAS2-252, MAS2-253, MAS2-254, MAS2-255, MAS2-256, MAS2-257, MAS3-236, MAS3-237, MAS3-238, MAS3-239, MAS3-240, MAS3-241, MAS3-242, MAS3-243, MAS3-244, MAS3-245, MAS3-246, MAS3-247, MAS3-248, MAS3-249, MAS3-250, MAS3-251, MAS3-252, MAS3-253, MAS3-254, MAS3-255, MAS3-256, MAS3-257, MAS3-258, MAS3-259, MAS3-260, MAS3-261, MAS3-262, MAS3-263, MAS3-264, MAS3-265, MAS3-266, MAS4-236, MAS4-237, MAS4-238, MAS4-239, MAS4-240, MAS4-241, MAS4-242, MAS4-243, MAS4-244, MAS4-245, MAS4-246, MAS4-247, MAS4-248, MAS4-249, MAS4-250, MAS4-251, MAS4-252, MAS4-253, MAS4-254, MAS4-255, MAS4-256, and MAS4-257.

[0029] In some embodiments, the sense strand of the oligonucleotides described herein is selected from the modified versions (e.g., MS1 - MS6) of SEQ ID NOs: 205 - 235 listed in Table 8.For example, in some embodiments, the sense strand is selected from MS1-205, MS1-206, MS1-207, MS2-208, MS2-209, MS2-210, MS2-211, MS2-212, MS2-213, MS2-214, MS2-215, MS2-216, MS2-217, MS2-218, MS2-219, MS2-220, MS2-221, MS2-222, MS2-223, MS2-224, MS2-225, MS2-226, MS3-205, MS3-206, MS3-207, MS3-208, MS3-209, MS3-210, MS3-211, MS3-212, MS3-213, MS3-214, MS3-215, MS3-216, MS3-217, MS3-218, MS3-219, MS3-220, MS3-221, MS3-222, MS3-223, MS3-224, MS3-225, MS3-226, MS3-227, MS3-228, MS3-229, MS3-230, MS3-231, MS3-232, MS3-233, MS3-234, MS3-235, MS4-205, MS4-206, MS4-207, MS4-208, MS4-209, MS4-210, MS4-211, MS4-212, MS4-213, MS4-214, MS4-215, MS4-216, MS4-217, MS4-218, MS4-219, MS4-220, MS4-221, MS4-222, MS4-223, MS4-224, MS4-225, MS4-226, MS5-205, MS5-206, MS5-207, MS5-208, MS5-209, MS5-210, MS5-211, MS5-212, MS5-213, MS5-214, MS5-215, MS5-216, MS5-217, MS5-218, MS5-219, MS5-220, MS5-221, MS5-222, MS5-223, MS5-224, MS5-225, MS5-226, MS6-205, MS6-206, MS6-207, MS6-208, MS6-209, MS6-210, MS6-211, MS6-212, MS6-213, MS6-214, MS6-215, MS6-216, MS6-217, MS6-218, MS6-219, MS6-220, MS6-221, MS6-222, MS6-223, MS6-224, MS6-225, and MS6-226.

[0030] In some embodiments, the antisense strand is selected from the modified versions of SEQ ID NOs: 262, 253, 251, 248, and 252 (e.g., VP-MAS5, VP-MAS6, and VP-MAS7) listed in Table 9, and / or, herein, the sense strand is selected from the modified versions of SEQ ID NOs: 231, 222, 220, 217, and 221 (e.g., MS7-MS9) listed in Table 9.

[0031] In some embodiments, the oligonucleotide is an siRNA molecule selected from the siRNAs listed in Table 8.

[0032] In some embodiments, the oligonucleotide is an siRNA molecule selected from the siRNAs listed in Table 9.

[0033] In some embodiments, the anti-TfR1 antibody comprises any of the 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 the anti-TfR1 antibodies listed in Table 2.

[0034] In some embodiments, the anti-TfR1 antibody comprises any of the heavy chain variable region (VH) and light chain variable region (VL) of the anti-TfR1 antibodies listed in Table 3.

[0035] In some embodiments, the anti-TfR1 antibody is a Fab, and optionally, herein, the Fab comprises any of the heavy and light chains of the anti-TfR1 Fabs listed in Table 5.

[0036] In some embodiments, the anti-TfR1 antibody comprises: (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.

[0037] In some embodiments, the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 75.

[0038] In some embodiments, the anti-TfR1 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.

[0039] In some embodiments, the muscle targeting agent and the antisense oligonucleotide are covalently linked via a linker, optionally where the linker comprises a valine-citrulline sequence.

[0040] Further provided herein is a method of reducing DUX4 expression in muscle cells, the method comprising contacting the muscle cells with an effective amount of the complex described herein to promote internalization of the oligonucleotide into the muscle cells. In some embodiments, reducing DUX4 expression comprises reducing the level of DUX4 protein and / or mRNA.

[0041] Further provided herein is a method of treating facioscapulohumeral muscular dystrophy (FSHD), the method comprising administering to a subject in need thereof an effective amount of the complex provided herein. In some embodiments, the subject has aberrant production of DUX4 protein.

[0042] Another aspect of the disclosure provides an oligonucleotide comprising a siRNA oligonucleotide selected from the siRNA oligonucleotides listed in Table 8.

[0043] Another aspect of the disclosure provides an oligonucleotide comprising a siRNA oligonucleotide selected from the siRNA oligonucleotides listed in Table 9.

[0044] Further provided herein is a method for producing a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide, the method comprising: (i) obtaining a compound comprising the structure of formula (B), wherein the oligonucleotide comprises the sense strand of an siRNA oligonucleotide, optionally wherein the siRNA oligonucleotide targets DUX4 RNA; (ii) annealing the antisense strand of the siRNA oligonucleotide to the sense strand; (iii) reacting the compound comprising the structure of formula (B) with a compound comprising the structure of formula (C) to obtain a compound comprising the structure of formula (D); and (iv) covalently linking an anti-TfR1 antibody to the compound comprising the structure of formula (D), together with a muscle targeting agent, to obtain a compound comprising the structure of formula (E), optionally wherein the annealing of step (ii) is performed at 30° C., and further optionally wherein the method further comprises isolating the compound comprising the structure of formula (D) after step (iii) and before step (iv); and further optionally wherein the anti-TfR1 antibody is covalently linked to the 5′ end of the sense strand of an siRNA oligonucleotide that targets DUX4 RNA.

[0045] Further provided herein is a method for producing a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide, the method comprising: (i) obtaining a compound comprising the structure of formula (B), wherein the oligonucleotide comprises the sense strand of a siRNA oligonucleotide, optionally wherein the siRNA oligonucleotide targets DUX4 RNA; (ii) annealing the antisense strand of the siRNA oligonucleotide to the sense strand; (iii) covalently linking an anti-TfR1 antibody to a compound comprising the structure of formula (C) to obtain a compound comprising the structure of formula (F); and (iv) reacting the compound comprising the structure of formula (F) with the compound comprising the structure of formula (B) to obtain a compound comprising the structure of formula (E), optionally wherein the annealing of step (ii) is performed at 30° C., and further optionally wherein the anti-TfR1 antibody is covalently linked to the 5′ end of the sense strand of a siRNA oligonucleotide that targets DUX4 RNA.

Brief Description of the Drawings

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[0056] 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 that are useful for reducing the level of DUX4 RNA and / or protein. In some embodiments, the oligonucleotides provided herein are designed to cause RNAi-mediated degradation of DUX4 RNA. In some embodiments, the oligonucleotides are designed to efficiently engage the RNA-induced silencing complex (RISC) for degradation of DUX4 RNA, but also to have a reduced off-target effect. In some embodiments, the oligonucleotides are designed to have desirable bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desirable binding affinity properties. In some embodiments, the oligonucleotides are designed to have a desirable toxicity and / or immunogenicity profile. Aberrant (e.g., increased) expression of DUX4 RNA and / or protein in muscle is associated with features of the pathology of facioscapulohumeral muscular dystrophy (FSHD), including muscle atrophy, inflammation, which includes inflammation, and reduced differentiation ability and oxidative stress. In some embodiments, the oligonucleotides described herein that reduce the level of DUX4 RNA and / or protein are effective in treating FSHD.

[0057] In some aspects, the present disclosure provides a complex comprising a muscle targeting agent covalently linked to an oligonucleotide for efficient delivery of the oligonucleotide to muscle cells. In some embodiments, the complex is particularly useful for delivering a molecular payload that inhibits the expression or activity of a target gene in muscle cells, for example, in a subject having or suspected of having a rare muscle disease. For example, in some embodiments, the complex is provided for treating a subject having FSHD. In some embodiments, the complex is provided for treating a subject having FSHD1. In some embodiments, the complex is provided for treating a subject having FSHD2. In some embodiments, the complex is provided for targeting DUX4 for treating a subject having FSHD. In some embodiments, the complex provided herein comprises an oligonucleotide that inhibits the expression of DUX4 in a subject having a deletion of one or more D4Z4 repeats on chromosome 4. In some embodiments, the complex provided herein comprises an oligonucleotide that inhibits the expression of DUX4 in a subject having a mutation in SMCHD1 or another DUX4 regulatory gene.

[0058] Further aspects of the disclosure, including a description of defined terms, are provided below.

[0059] I. Definitions Administering: As used herein, the term “administering” or “administration” means providing a complex to a subject in a physiologically and / or (by way of example, and) pharmacologically useful manner (e.g., treating a disease of the subject).

[0060] Approximately: As used herein, the term "approximately" or "about" when applied to one or more values of interest refers to a value similar to the defined reference value. In one aspect, the term "approximately" or "about" refers to a value in the range of 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than 1%, above or below (higher than the described reference value, or lower than the described reference value) of the described reference value, unless otherwise stated or apparent from the context (except where such number would exceed 100% of an executable value).

[0061] 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, such as a paratope, that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, an Fv fragment, or a 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 diabody. In some embodiments, the antibody includes a framework having human germline sequences. In another embodiment, the antibody includes a heavy chain constant domain selected from the group consisting of the constant domains of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. In some embodiments, the antibody includes a heavy (H) chain variable region (abbreviated herein as VH) and / or (e.g., and) a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody includes a constant domain, such as an Fc region. The immunoglobulin constant domain refers to the constant domain of the heavy or light chain. The amino acid sequences of the constant domains of the heavy and light chains of human IgG, as well as their functional variations, are known. With respect to the heavy chain, in some embodiments, the heavy chain of the antibody described herein 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 include a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In a specific embodiment, the antibody described herein includes the CH1 domain, CH2 domain, and / or (e.g., and) CH3 domain of human gamma 1.In some embodiments, the amino acid sequence of the VH domain includes the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any known in the art. Non-limiting examples of human constant region sequences are described in the art, for example, in U.S. Patent No. 5,693,780 and the above Kabat E A et al., (1991). 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, for example, modified via glycosylation, phosphorylation, SUMOylation, and / or (by way of example, 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 (by way of example, and) phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or lipid phosphate units. In some embodiments, the antibody is a construct comprising a polypeptide that includes a linker polypeptide or one or more antigen-binding fragments of the disclosure linked to an immunoglobulin constant domain. The linker polypeptide includes two or more amino acid residues joined by peptide bonds and is used to link one or more antigen-binding portions.Examples of linker polypeptides have been reported (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., 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, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101), as well as the use of cysteine residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent biotinylated scFv molecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).

[0062] CDR: As used herein, the term "CDR" refers to the complementarity-determining regions within an antibody variable sequence. A typical antibody molecule includes a heavy-chain variable region (VH) and a light-chain variable region (VL), which are usually involved in antigen binding. The VH region and the VL region can be further subdivided into hypervariable regions, also known as "complementarity-determining regions" ("CDRs"), which are interspersed with more conserved regions known as "framework regions" ("FRs"). Each VH and VL typically consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxy terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The ranges of the framework regions and CDRs can be accurately identified using methodologies known in the art, such as the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (by way of example, and) the contact definition, all of which are well-known in the art.As an example, see Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242; IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark) www.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 (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol.Recognit. 17:132-143 (2004). Also see hgmp.mrc.ac.uk and bioinf.org.uk / abs.As used herein, CDR may refer to a CDR defined by any method known in the art. Two antibodies having the same CDR, when determined by the same method, e.g., the IMGT definition, means that the two antibodies have the same amino acid sequence for that CDR.

[0063] In each of the variable regions of the heavy and light chains, there are three CDRs, which are designated CDR1, CDR2, and CDR3 for each of the variable regions. The term "CDR set", as used herein, refers to a group of three CDRs that occur in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently by various systems. Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides the exact residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Sub-portions of the CDRs may sometimes be designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light and heavy chain regions, respectively. These regions may also be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries that define 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 follow one of the above systems, but may still overlap with the Kabat CDRs. However, based on predictions or experimental findings that certain residues, groups of residues, or even entire CDRs do not significantly affect antigen binding, they may be shortened or extended. The methods used herein may utilize CDRs defined according to any of these systems. Examples of systems for defining CDRs are provided in Table 1.

Table 1

[0064] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody that contains the sequences of the heavy and light chain variable regions from one species, but in which the sequence of one or more of the CDR regions of its VH and / or VL has been replaced with the CDR sequence of another species. For example, an antibody that has mouse heavy and light chain variable regions, but in which one or more of the mouse CDRs (e.g., CDR3) has been replaced with a human CDR sequence.

[0065] Chimeric antibody: The term "chimeric antibody" refers to an antibody that contains the sequences of the heavy and light chain variable regions from one species and the sequences of the constant regions from another species. For example, an antibody that has mouse heavy and light chain variable regions linked to human constant regions.

[0066] Complementary: As used herein, the term "complementary" refers to the ability for exact base pairing between two nucleotides or two sets of nucleotides. In particular, complementary is a term that characterizes the degree of hydrogen bond base pairing that results in a bond between two nucleosides or two sets of nucleosides. For example, if the base at one position of an oligonucleotide is capable of hydrogen bonding with the base of a target nucleic acid (e.g., mRNA) at the corresponding position, then the bases are considered to be complementary to each other at that position. Base pairing may include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairs, the adenosine-type base (A) is complementary to the thymidine-type base (T) or the uracil-type base (U), the cytosine-type base (C) is complementary to the guanosine-type base (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize to any A, C, U, or T and are considered to be complementary to them. 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.

[0067] Conservative amino acid substitutions: As used herein, "conservative amino acid substitutions" refer to amino acid substitutions that do 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 known to those of skill in the art for changing polypeptide sequences, e.g., as described in references that compile 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, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made between amino acids within 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.

[0068] Covalently linked: As used herein, the term "covalently linked" refers to the characteristic of two or more molecules that are linked together through at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond (e.g., a disulfide bond or disulfide bridge) that serves as an intermolecular linker. However, in some embodiments, two or more molecules can be covalently linked together through a molecule that serves as a linker that joins the two or more molecules 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.

[0069] Cross-reacting: As used herein, and in the context of a targeting agent (e.g., an antibody), the term "cross-reacting" refers to the property of an agent that is capable of specifically binding to more than one antigen of the same type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or binding activity. For example, in some embodiments, an antibody that cross-reacts with antigens of the same type or class of human and non-human primate animals (e.g., the human transferrin receptor and the non-human primate transferrin receptor) is capable of binding to the human antigen and the non-human primate antigen with similar affinity or binding activity. In some embodiments, the antibody is cross-reactive with antigens of the same type or class of human and rodent antigens. In some embodiments, the antibody cross-reacts with antigens of the same type or class of rodent and non-human primate antigens. In some embodiments, the antibody cross-reacts with antigens of the same type or class of human, non-human primate, and rodent antigens.

[0070] 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 the testes of adult male (male) individuals. In some embodiments, DUX4 may be a human (Gene ID: 100288687), non-human primate animal (e.g., Gene ID: 750891, Gene ID: 100405864), or rodent gene (e.g., Gene ID: 306226). In humans, expression of the DUX4 gene outside of fetal development and the testes is associated with facioscapulohumeral muscular dystrophy. In addition, multiple human transcript variants encoding different protein isoforms (e.g., as annotated under GenBank RefSeq accession numbers: NM_001293798.2, NM_001306068.2, NM_001363820.1) have been characterized.

[0071] Facioscapulohumeral muscular dystrophy (FSHD): As used herein, the term "facioscapulohumeral muscular dystrophy (FSHD)" refers to a genetic disorder caused by a mutation in the DUX4 gene, SMCHD1 gene, DNMT3B gene, or LRIF1 gene, characterized primarily by a reduction in muscle mass and muscle atrophy in the muscles of the face, scapula, and upper arm. Two types of the disease, type 1 and type 2, are described. Type 1 is associated with a deletion in the D4Z4 repeat region on chromosome 4 containing the DUX4 gene. In some embodiments, type 1 is associated with a deletion in the D4Z4 repeat region on the 4qA variant of the chromosome 4 allele containing the DUX4 gene. Type 2 is associated with a mutation in the SMCHD1 gene, DNMT3B gene, or LRIF1 gene (see, for example, Jia et al., "Facioscapulohumeral muscular dystrophy type 2: an update on the clinical, genetic, and molecular findings" Neuromuscul Disord. (2021), 31(11): 1101-1112). Both type 1 and type 2 FSHD are characterized by abnormal production of the DUX4 protein outside the testis after fetal development. Facioscapulohumeral dystrophy, the genetic basis of the disease, and related symptoms are described in the art (see, for example, Campbell, A.E., 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). Type 1 FSHD is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 158900.FSHD2 is associated with OMIM Entry #158901.

[0072] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence after subtracting the CDRs from the variable region. Since the exact definition of the CDR sequences 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 regions on the light and heavy chains into four smaller regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is positioned between FR1 and FR2, CDR2 is between FR2 and FR3, and CDR3 is between FR3 and FR4. Without specifying a particular smaller region as FR1, FR2, FR3, or FR4, the framework region, when otherwise referred to, represents the combined FR(s) within the variable region of a naturally occurring single immunoglobulin chain. As used herein, FR represents one of the four smaller regions, and FR(s) represents two or more of the four smaller regions that make up the framework region. The acceptor sequences of human heavy and light chains are known in the art. In one aspect, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0073] Human antibody: As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random mutagenesis or site-directed mutagenesis in vitro or by somatic mutation in vivo), for example, in the CDRs, particularly CDR3. However, as used herein, the term "human antibody" 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.

[0074] Humanized antibody: The term "humanized antibody" refers to an antibody that includes 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 have been introduced into the non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one aspect, humanized anti-TfR1 antibodies and antigen-binding portions are provided. Such antibodies may be generated by obtaining a mouse anti-TfR1 monoclonal antibody using classical hybridoma technology and then humanizing it using in vitro genetic engineering (such as that disclosed in PCT Publication No. WO 2005 / 123126 A2 by Kasaian et al.).

[0075] 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, for example, in response to an external stimulus (such as the binding of a ligand 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, caveola- 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 may optionally further comprise a ligand-binding domain. In some embodiments, the cell surface receptor becomes internalized by the cell after ligand binding. In some embodiments, the ligand may be a muscle targeting agent or a muscle targeting antibody. In some embodiments, the internalizing cell surface receptor is the transferrin receptor.

[0076] Isolated antibody: As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., 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 a 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 materials and / or (e.g., and) chemicals.

[0077] Kabat numbering: The terms "Kabat numbering", "Kabat definition", and "Kabat labeling" are used interchangeably herein. These terms, as recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the variable regions of the heavy and light chains of an antibody or their antigen-binding portions (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E. A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable regions are in the range of amino acid positions 31 to 35 for CDR1, 50 to 65 for CDR2, and 95 to 102 for CDR3. For the light chain variable region, the hypervariable regions are in the range of amino acid positions 24 to 34 for CDR1, 50 to 56 for CDR2, and 89 to 97 for CDR3.

[0078] In some embodiments, the molecular payload functions to modulate protein expression, modulate transcription of a DNA sequence, or 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.

[0079] Muscle targeting agent: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on muscle cells. The antigen in or on the muscle cells may be a membrane protein, such as an integral membrane protein or a surface membrane protein. Typically, the muscle targeting agent specifically binds to an antigen on the muscle cells that facilitates the internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cells. In some embodiments, the muscle targeting agent can specifically bind to an internalizing cell surface receptor on the muscle and be internalized into the muscle cells through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, protein, peptide, nucleic acid (e.g., an aptamer), or antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0080] Muscle targeting antibody: As used herein, the term "muscle targeting antibody" refers to a muscle targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, the muscle targeting antibody specifically binds to an antigen on the muscle cells that facilitates the internalization of the muscle targeting antibody (and any associated molecular payload) into the muscle cells. In some embodiments, the muscle targeting antibody specifically binds to an internalizing cell surface receptor present on the muscle cells. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0081] Oligonucleotide: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound having a length of up to 200 nucleotides. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNAs, gapmers, mixmers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), and the like. The oligonucleotide may be single-stranded or double-stranded. In some embodiments, the oligonucleotide may contain one or more modified nucleosides (e.g., 2'-O-methyl sugar modification, purine modification, or pyrimidine modification). In some embodiments, the oligonucleotide may contain one or more modified internucleoside linkages. In some embodiments, the oligonucleotide may contain one or more phosphorothioate linkages, which may be in the Rp or Sp stereochemical configuration.

[0082] Recombinant antibody: As used herein, the term "recombinant human antibody" refers to all human antibodies prepared, expressed, created or isolated by recombinant means, for example, antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail in the present disclosure), antibodies isolated from recombination, combinatorial human antibody libraries (Hoogenboom H. R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W. E., (2002) Clin. Biochem. 35:425-445; Gavilondo J. V., and Larrick J. W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see, for example, Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L. L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or antibodies prepared, expressed, created or isolated by other means involving splicing human immunoglobulin gene sequences to other DNA sequences.

[0083] Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to mutagenesis in vitro (or, if transgenic animals are used for human Ig sequences, somatic mutagenesis in vivo), and thus the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to the VH and VL sequences of the human germline, while on the other hand, sequences that may not naturally exist within the germline repertoire of human antibodies in vivo. However, in some embodiments, the complementary regions are partially complementary to the cognate nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95%, or 99% complementary). In some embodiments, the complementary regions contain 1, 2, 3, or 4 mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0084] Specifically bind: As used herein, the term "specifically binds" refers to the ability of a molecule to bind to its binding partner with an affinity or binding activity such that the molecule can be used to distinguish the binding partner from appropriate controls in a binding assay or other binding context. With respect to an antibody, the term "specifically binds" refers to the ability of the antibody to bind to a specific antigen with an affinity or binding activity such that the antibody can be used to distinguish the specific antigen from other antigens (e.g., to the extent of allowing preferential targeting to a particular cell (e.g., a muscle cell) through binding to the antigen, as described herein). In some embodiments, the antibody has, for binding to the target, at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13When it has a KD equal to or lower than M, the antibody specifically binds to the target. In some embodiments, the antibody specifically binds to a transferrin receptor, for example, an epitope of the tip domain of the transferrin receptor.

[0085] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient 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.

[0086] Transferrin receptor: As used herein, the term "transferrin receptor" (also known as TFRC, CD71, p90, or TFR1) refers to an internalizing cell surface receptor that binds transferrin and promotes iron uptake by endocytosis. In some embodiments, the transferrin receptor may be of human origin (NCBI Gene ID 7037), of non-human primate origin (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or of rodent origin (e.g., NCBI Gene ID 22042). In addition, multiple human transcript variants have been characterized as encoding different isoforms of the receptor (e.g., as noted under GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0087] 2'-Modified Nucleoside: 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, where the 2'- and 4'-positions of the sugar are bridged (e.g., via a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is an acyclic 2'-modified nucleoside, where, for example, 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'-O-N-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 containing 2'-modified nucleosides have an increased affinity for a target sequence compared to unmodified oligonucleotides. Examples of the structure of 2'-modified nucleosides are provided below: [Chemical Formula] These examples are shown with phosphate groups, but any internucleoside linkage is contemplated between 2'-modified nucleosides.

[0088] II. Complex Provided herein is a complex comprising a targeting agent (such as an antibody) covalently linked to a molecular payload. In some embodiments, the complex comprises a muscle targeting antibody covalently linked to an oligonucleotide. The molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and / or (such as, and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets DUX4 in muscle cells.

[0089] In some embodiments, the complex comprises a muscle targeting agent (such as an anti-transferrin receptor antibody) covalently linked to a molecular payload (such as an antisense oligonucleotide that targets DUX4). In some embodiments, the complex comprises a muscle targeting agent (such as an anti-transferrin receptor antibody) covalently linked to a molecular payload (such as siRNA that targets DUX4).

[0090] In some embodiments, the complex comprises a muscle targeting agent (such as an anti-transferrin receptor antibody) covalently linked to a molecular payload (such as an antisense oligonucleotide that targets DUX4). In some embodiments, the complex comprises a muscle targeting agent (such as an anti-transferrin receptor antibody) covalently linked to a molecular payload (such as siRNA that targets DUX4).

[0091] A. Muscle targeting agent Some aspects of the present disclosure provide myotargeting agents, such as myotargeting agents for delivering a molecular payload to muscle cells. In some embodiments, such myotargeting agents are capable of binding to muscle cells, and delivering an associated molecular payload to muscle cells, for example, via specific binding to an antigen on the muscle cell. In some embodiments, the molecular payload is bound to (e.g., covalently bound to) the myotargeting agent and internalized into muscle cells by binding of the myotargeting agent to an antigen on the muscle cell (e.g., via endocytosis). It should be understood that a variety of types of myotargeting agents may be used in accordance with the present disclosure. For example, the myotargeting 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). Exemplary myotargeting agents are described in more detail herein, however, it should be understood that the exemplary myotargeting agents provided herein are not intended to be limiting.

[0092] Some aspects of the present disclosure provide myotargeting agents that specifically bind to an antigen on muscle, such as skeletal muscle, smooth muscle, or cardiac muscle. In some embodiments, any of the myotargeting agents provided herein bind to (e.g., specifically bind to) an antigen on skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells.

[0093] By interaction with a muscle-specific cell surface recognition element (e.g., a cell membrane protein), both tissue localization to and selective uptake into muscle cells can be achieved. In some embodiments, a molecule that is a substrate for a muscle uptake transporter is useful for delivering a molecular payload into muscle tissue. Binding to a muscle surface recognition element and subsequent endocytosis can allow even macromolecules such as antibodies to penetrate muscle cells. As another example, a molecular payload conjugated to transferrin or an anti-TfR1 antibody can be taken up by muscle cells via binding to the transferrin receptor, which may then be endocytosed, for example, via clathrin-mediated endocytosis.

[0094] The use of a muscle targeting agent can also be useful for concentrating a molecular payload (e.g., an oligonucleotide) in muscle while reducing the toxicity associated with the effect in other tissues. In some embodiments, the muscle targeting agent concentrates the conjugated molecular payload in muscle cells as compared to another cell type in the subject. In some embodiments, the muscle targeting agent concentrates the conjugated molecular payload 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 more than the amount in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or fat cells). In some embodiments, the toxicity of the molecular payload in the subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when it is conjugated to a muscle targeting agent and delivered to the subject.

[0095] In some embodiments, a muscle recognition element (e.g., a muscle cell antigen) may be required to achieve muscle selectivity. As an 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 transporter-based approaches provide a direct route to cell entry, while receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0096] i. Muscle targeting antibody In some embodiments, the muscle targeting agent is an antibody. Generally, the high specificity of antibodies for 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 the present disclosure. For example, antibodies targeting 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 K.S., 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 R.H. et al., “Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb” Mol Immunol. 2003 Mar, 39(13):78309, the entire contents of each of which are incorporated herein by reference.

[0097] 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, such as anti-transferrin receptor antibodies, can target muscle cells. In some embodiments, the binding protein that binds to the transferrin receptor is internalized into muscle cells along with any molecular payload to which it is bound. As used herein, an antibody that binds to the transferrin receptor may be referred to interchangeably as a transferrin receptor antibody, an anti-transferrin receptor antibody, or an anti-TfR1 antibody. An antibody that binds to, e.g., specifically binds to, the transferrin receptor may be internalized into cells upon binding to the transferrin receptor, e.g., through receptor-mediated endocytosis.

[0098] It should be understood that anti-TfR1 antibodies may be produced, synthesized, and / or (by way of example, and) derivatized using several known methodologies, such as library design using phage display. Exemplary methodologies are characterized in the art and incorporated by reference (Diez, P. et al. “High-throughput phage-display screening in array format”, Enzyme and microbial technology, 2015, 79, 34-41.; Christoph M. H. and Stanley, J.R. “Antibody Phage Display: Technique and Applications” J Invest Dermatol. 2014, 134:2.; Engleman, Edgar (Ed.) “Human Hybridomas and Monoclonal Antibodies.” 1985, Springer.). In other embodiments, anti-TfR1 antibodies have been previously characterized or disclosed.Antibodies that specifically bind to the transferrin receptor are known in the art (see, for example, U.S. Patent No. 4,364,934, filed December 4, 1979, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Patent No. 8,409,573, filed June 14, 2006, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Patent No. 9,708,406, filed May 20, 2014, "Anti-Transferrin receptor antibodies and methods of use"; US 9,611,323, filed December 19, 2014, "Low affinity blood brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed December 24, 2014, "Novel anti-Transferrin receptor antibody that passes through blood-brain barrier"; Schneider C. et al. "Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9." J Biol Chem. 1982, 257:14, 8516-8522.; Lee et al. "Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse" 2000, J Pharmacol. Exp. Ther., 292: 1048-1052).

[0099] In some embodiments, the anti-TfR1 antibodies described herein bind to the transferrin receptor with high specificity and affinity. In some embodiments, the anti-TfR1 antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor or an epitope that becomes exposed to the antibody. In some embodiments, the anti-TfR1 antibodies provided herein specifically bind to transferrin receptors from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR1 antibodies provided herein bind to the human transferrin receptor. In some embodiments, the anti-TfR1 antibodies described herein bind to the amino acid segments of the human or non-human primate transferrin receptor as provided in SEQ ID NOs: 105-108. In some embodiments, the anti-TfR1 antibodies described herein bind to the amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor as represented by SEQ ID NO: 105, which is not in the tip domain of the transferrin receptor. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to TfR1 but not to TfR2.

[0100] 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 comprises 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 comprising 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 comprising one or more of the residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1 as set forth in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope comprising the residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1 as set forth in SEQ ID NO: 105.

[0101] In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and its variants in Table 2 below) bind to an epitope in TfR1, where the epitope 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 its variants in Table 2 below) bind to an epitope that includes 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 its variants in Table 2 below) bind to an epitope that includes one or more of the residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as set forth 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 that includes the residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as set forth in SEQ ID NO: 105.

[0102] An example of the human transferrin receptor amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 105).

[0103] An example of the transferrin receptor amino acid sequence of a non-human primate corresponding to NCBI sequence NP_001244232.1 (transferrin receptor protein 1, Macaca mulatta) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 106).

[0104] An example of the amino acid sequence of the transferrin receptor of a non - human primate corresponding to NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKANGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF(SEQ ID NO: 107).

[0105] An example of the amino acid sequence of the mouse transferrin receptor corresponding to NCBI sequence NP_001344227.1 (transferrin receptor protein 1, Mus musculus) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYVKIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKNVLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTRLDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF (SEQ ID NO: 108).

[0106] In some embodiments, the anti-TfR1 antibody is directed against the following receptor amino acid segments: FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) binds to and does not inhibit the binding interaction between the transferrin receptor and transferrin and / or (by way of example and) human hemochromatosis protein (alias HFE). In some embodiments, the anti-TfR1 antibodies described herein do not bind to the epitope at SEQ ID NO: 109.

[0107] Suitable methodologies may be used to obtain and / or (by way of 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 creation 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 in any form or entity, for example, in recombinant or naturally occurring form or entity. Hybridomas are screened using standard methods, such as ELISA screening, to find at least one hybridoma that produces an antibody that targets a specific antigen. Antibodies may also be produced through the screening of a protein expression library that expresses the antibody (for example, a phage display library). Phage display library design may also be used in some embodiments (see, for example, U.S. Patent 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”; WO 1992 / 15679, filed 2 / 28 / 1992, and “Improved epitope displaying phage”). In some embodiments, the antigen of interest may be used to immunize a non-human animal, such as a rodent or a goat.In some embodiments, the antibodies are then obtained from non-human animals and may optionally be modified using a number of methodologies, using recombinant DNA techniques as an example. Further examples and methodologies of antibody production are known in the art (see, for example, Harlow et al. “Antibodies: A Laboratory Manual”, Cold Spring Harbor Laboratory, 1988).

[0108] In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation, and / or (by way of example 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 (by way of example and) phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or lipid phosphate units. In some embodiments, there are about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 3, or about 2 sugar molecules present. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by a chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell, which may optionally lack enzymes (such as glycosyltransferases) 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, titled "Modified antibody, antibody-conjugate and process for the preparation thereof".

[0109] In some embodiments, the anti-TfR1 antibodies of the present disclosure comprise any one of the VL domains and / or (by way of example, and) VH domains of any one of the anti-TfR1 antibodies selected from any one of Tables 2-7, and further comprise a constant region comprising the amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule. Non-limiting examples of human constant regions are described in the art. See, e.g., Kabat E A et al., (1991) supra.

[0110] In some embodiments, an agent that binds to the transferrin receptor, e.g., an anti-TfR1 antibody, can target muscle cells and / or (by way of example, and) mediate the transport of an agent that crosses the blood-brain barrier. The transferrin receptor is an internalizing cell surface receptor that transports transferrin across the cell membrane and is involved 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, e.g., through receptor-mediated endocytosis, when bound to the transferrin receptor.

[0111] In some embodiments, the anti-TFR1 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 -13It binds specifically to TfR1 (e.g., human or non-human primate TfR1) with an affinity equal to or lower than M (as indicated by Kd for example). In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 with a KD in the sub-nanomolar range. In some embodiments, the anti-TfR1 antibodies described herein selectively bind to transferrin receptor 1 (TfR1), but do not bind to transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR1 antibodies described herein bind to human TfR1 and cynomolgus (cyno) TfR1 (e.g., at 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or lower Kd), but do not bind to mouse TfR1. The affinity and kinetics of binding of the anti-TfR1 antibodies can be tested using any suitable method including, but not limited to, biosensor technologies (e.g., OCTET or BIACORE). In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit the binding of transferrin to TfR1. In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit the binding of HFE-beta-2-microglobulin to TfR1.

[0112] Non-limiting examples of anti-TfR1 antibodies are provided in Table 2.

Table 2-1

Table 2-2

Table 2-3

Table 2-4

[0113] In some embodiments, the anti-TfR1 antibody of the present disclosure is a variant of any one of the anti-TfR1 antibodies provided in Table 2. In some embodiments, the anti-TfR1 antibody of the present disclosure comprises CDR-H1, CDR-H2, and CDR-H3 that are the same as those in any one of the anti-TfR1 antibodies provided in Table 2, and further comprises CDR-L1, CDR-L2, and CDR-L3, and a humanized heavy chain variable region and / or (by way of example, and) a humanized light chain variable region.

[0114] Examples of the amino acid sequences of the anti-TfR1 antibodies described herein are provided in Table 3.

Table 3-1

Table 3-2

[0115] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VH that includes CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 3, and includes one or more (by way of example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework region as compared to each of the VHs provided in Table 3. Alternatively or in addition (by way of example, in addition), the anti-TfR1 antibody of the present disclosure comprises a VL that includes CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 3, and includes one or more (by way of example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework region as compared to each of the VLs provided in Table 3.

[0116] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VH comprising any one of the CDR-H1, CDR-H2, and CDR-H3 of the anti-TfR1 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 region as compared to each of the VHs provided in Table 3. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibody of the present disclosure comprises a VL comprising any one of the CDR-L1, CDR-L2, and CDR-L3 of the anti-TfR1 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 region as compared to each of the VLs provided in Table 3.

[0117] In some embodiments, the anti-TfR1 antibody of the present 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.

[0118] In some embodiments, the anti-TfR1 antibody of the present 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.

[0119] In some embodiments, the anti-TfR1 antibody of the present 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.

[0120] In some embodiments, the anti-TfR1 antibody of the present 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.

[0121] In some embodiments, the anti-TfR1 antibody of the present 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.

[0122] In some embodiments, the anti-TfR1 antibody of the present 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.

[0123] In some embodiments, the anti-TfR1 antibody of the present 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.

[0124] In some embodiments, the anti-TfR1 antibody of the present 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.

[0125] In some embodiments, the anti-TfR1 antibody of the present 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.

[0126] In some embodiments, the anti-TfR1 antibody of the present 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.

[0127] In some embodiments, the anti-TfR1 antibody of the present 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.

[0128] In some embodiments, the anti-TfR1 antibodies described herein are full-length IgG, which may include the heavy-chain constant region and the light-chain constant region from human antibodies. In some embodiments, the heavy chain of any of the anti-TfR1 antibodies as described herein may include the heavy-chain constant region (CH) or a portion thereof (by way of example, CH1, CH2, CH3, or combinations thereof). The heavy-chain constant region may be of any suitable origin, by way of example, human, mouse, rat, or rabbit. In one specific example, the heavy-chain constant region is from human IgG (gamma heavy chain), by way of example, IgG1, IgG2, or IgG4. An example of the human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81).

[0129] In some embodiments, any heavy chain of the anti-TfR1 antibodies described herein comprises a mutant human IgG1 constant region. For example, the introduction of the LALA mutation in the CH2 domain of human IgG1 (a mutant derived from mAb b12 mutated to replace the residues Leu234 and Leu235 of the lower hinge 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 are in bold and underlined): [Number]

[0130] In some embodiments, any light chain of the anti-TfR1 antibodies described herein may further comprise a light chain constant region (CL), which can 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 and its sequence is provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83).

[0131] The constant regions of the heavy and light chains of other antibodies are well known in the art and include, by way of example, those provided in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php, all of which are hereby incorporated by reference into this specification.

[0132] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variant thereof as listed in Table 3 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-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variant thereof as listed in Table 3 and a heavy chain constant region that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variant thereof as listed in Table 3 and a heavy chain constant region as represented by SEQ ID NO: 81. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variant thereof as listed in Table 3 and a heavy chain constant region as represented by SEQ ID NO: 82.

[0133] In some embodiments, the anti-TfR1 antibodies described herein include a light chain that includes any one of the VLs or any variant 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-TfR1 antibodies described herein include a light chain that includes any one of the VLs or any variant thereof as listed in Table 3, and a light chain constant region that contains 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein include a light chain that includes any one of the VLs or any variant thereof as listed in Table 3, and a light chain constant region represented by SEQ ID NO: 83.

[0134] Examples of the amino acid sequences of the IgG heavy and light chains of the anti-TfR1 antibodies described are provided in Table 4 below.

Table 4-1

Table 4-2

Table 4-3

[0135] In some embodiments, the anti-TfR1 antibodies of the present disclosure comprise a heavy chain having 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) as compared to the heavy chain represented in any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibodies of the present disclosure comprise a light chain having 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) as compared to the light chain represented in any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0136] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or in addition (e.g., in addition), the anti-TfR1 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.

[0137] In some embodiments, the anti-TfR1 antibody of the present 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.

[0138] In some embodiments, the anti-TfR1 antibody of the present 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.

[0139] In some embodiments, the anti-TfR1 antibody of the present 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.

[0140] In some embodiments, the anti-TfR1 antibody of the present 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.

[0141] In some embodiments, the anti-TfR1 antibody of the present 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.

[0142] In some embodiments, the anti-TfR1 antibody of the present 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.

[0143] In some embodiments, the anti-TfR1 antibody of the present 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.

[0144] In some embodiments, the anti-TfR1 antibody of the present 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.

[0145] In some embodiments, the anti-TfR1 antibody of the present 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.

[0146] In some embodiments, the anti-TfR1 antibody of the present 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.

[0147] In some embodiments, the anti-TfR1 antibody of the present 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.

[0148] In some embodiments, the anti-TfR1 antibody is a Fab fragment, Fab’ fragment, or F(ab’)2 fragment of an intact antibody (full-length antibody). The antigen-binding fragment of an intact antibody (full-length antibody) can be prepared by conventional methods (e.g., recombinantly or by digesting the heavy chain constant region of full-length IgG using an enzyme such as papain). For example, an F(ab’)2 fragment can be produced by pepsin or papain digestion of an antibody molecule, and a Fab fragment can be generated by reducing the disulfide bridges of an F(ab’)2 fragment. In some embodiments, the heavy chain constant region in the Fab fragment of the anti-TfR1 antibody described herein comprises the following amino acid sequence: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96).

[0149] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any of their variants as listed in Table 3, 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-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any of their variants as listed in Table 3, and a heavy chain constant region containing 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any of their variants as listed in Table 3, and a heavy chain constant region as represented by SEQ ID NO: 96.

[0150] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any of their variants 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-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any of their variants as listed in Table 3, and a light chain constant region containing 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any of their variants as listed in Table 3, and a light chain constant region as represented by SEQ ID NO: 83.

[0151] Examples of the amino acid sequences of the Fab heavy and light chains of the anti-TfR1 antibodies described are provided in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3]

[0152] In some embodiments, the anti-TfR1 antibodies of the present disclosure have 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the heavy chain as represented by any one of SEQ ID NOs: 97-103, 158, and 159, and include a heavy chain. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibodies of the present disclosure have 25 or fewer amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the light chain as represented by any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157, and include a light chain.

[0153] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence that is at least 75% (by way of example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively or in addition (by way of example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence that is at least 75% (by way of example, 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-TfR1 antibodies described herein comprise a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively or in addition (by way of example, in addition), the anti-TfR1 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.

[0154] In some embodiments, the anti-TfR1 antibody of the present 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.

[0155] In some embodiments, the anti-TfR1 antibody of the present 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.

[0156] In some embodiments, the anti-TfR1 antibody of the present 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.

[0157] In some embodiments, the anti-TfR1 antibody of the present 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.

[0158] In some embodiments, the anti-TfR1 antibody of the present 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.

[0159] In some embodiments, the anti-TfR1 antibody of the present 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.

[0160] In some embodiments, the anti-TfR1 antibody of the present 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.

[0161] In some embodiments, the anti-TfR1 antibody of the present 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.

[0162] In some embodiments, the anti-TfR1 antibody of the present 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.

[0163] In some embodiments, the anti-TfR1 antibody of the present 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.

[0164] In some embodiments, the anti-TfR1 antibody of the present 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.

[0165] In some embodiments, the anti-TfR1 antibody of the present 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.

[0166] Other known anti-TfR1 antibodies Any other suitable anti-TfR1 antibody known in the art may be used as a muscle targeting agent in the conjugate disclosed herein. Examples of known anti-TfR1 antibodies (including related references and binding epitopes) are listed in Table 6. In some embodiments, the anti-TfR1 antibody comprises any one of the complementarity determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the anti-TfR1 antibodies provided herein (such as the anti-TfR1 antibodies listed in Table 6 as examples).

[0167] Table 6 - List of anti-TfR1 antibody clones including related references and binding epitope information

Table 6-1

Table 6-2

[0168] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises one or more of the amino acid sequences of CDR-H (such as CDR-H1, CDR-H2, and CDR-H3) from any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the anti-TfR1 antibody comprises CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the anti-TfR1 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-TfR1 antibodies selected from Table 6.

[0169] In some embodiments, the anti-TfR1 antibodies of the present disclosure include any antibody that includes the heavy chain variable domain and / or (by way of example, and) the light chain variable domain of any anti-TfR1 antibody, such as any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the anti-TfR1 antibodies of the present disclosure include any antibody that includes the heavy chain variable and light chain variable pair of any anti-TfR1 antibody, such as any one of the anti-TfR1 antibodies selected from Table 6.

[0170] Aspects of the present disclosure provide anti-TfR1 antibodies having amino acid sequences of heavy chain variable (VH) and / or (by way of example, and) light chain variable (VL) domains that are homologous to any of those described herein. In some embodiments, the anti-TfR1 antibody includes a heavy chain variable sequence and / or a light chain variable sequence that is at least 75% (by way of 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-TfR1 antibody, such as any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the homologous heavy chain variable and / or (by way of example, and) light chain variable amino acid sequences do not vary within any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (by way of example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) can occur within the heavy chain variable and / or (by way of example, and) light chain variable sequences that exclude any of the CDR sequences provided herein. In some embodiments, any of the anti-TfR1 antibodies provided herein includes a heavy chain variable sequence and a light chain variable sequence that includes a framework sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequence of any anti-TfR1 antibody, such as any one of the anti-TfR1 antibodies selected from Table 6.

[0171] Examples of transferrin receptor antibodies that may be used in accordance with the present disclosure are described in International Application Publication WO 2016 / 081643, which is incorporated herein by reference. The amino acid sequences of this antibody are provided in Table 7. [Table 7-1] [Table 7-2]

[0172] In some embodiments, the anti-TfR1 antibodies of the present disclosure include CDR-H1, CDR-H2, and CDR-H3 that are the same as CDR-H1, CDR-H2, and CDR-H3 as shown in Table 7. Alternatively or additionally (by way of example, additionally), the anti-TfR1 antibodies of the present disclosure include CDR-L1, CDR-L2, and CDR-L3 that are the same as CDR-L1, CDR-L2, and CDR-L3 as shown in Table 7.

[0173] In some embodiments, the anti-TfR1 antibodies of the present disclosure include a CDR-L3 that contains up to 3 amino acid variations (e.g., 3, 2, or 1 or fewer amino acid variations) compared to the CDR-L3 as shown in Table 7. In some embodiments, the anti-TfR1 antibodies of the present disclosure include a CDR-L3 that contains 1 amino acid variation compared to the CDR-L3 as shown in Table 7. In some embodiments, the anti-TfR1 antibodies of the present disclosure include a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition systems) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system). In some embodiments, the anti-TfR1 antibodies of the present disclosure include 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 include a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition systems) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system).

[0174] In some embodiments, the anti-TfR1 antibodies of the present disclosure include heavy chain CDRs that are at least 80% identical (e.g., 80%, 85%, 90%, 95%, or 98%) in combination with the heavy chain CDRs as shown in Table 7. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibodies of the present disclosure include light chain CDRs that are at least 80% identical (e.g., 80%, 85%, 90%, 95%, or 98%) in combination with the light chain CDRs as shown in Table 7.

[0175] In some embodiments, the anti-TfR1 antibodies of the present disclosure include a VH that contains the amino acid sequence of SEQ ID NO: 124. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibodies of the present disclosure include a VL that contains the amino acid sequence of SEQ ID NO: 125.

[0176] In some embodiments, the anti-TfR1 antibodies of the present disclosure comprise a VH comprising the amino acid sequence of SEQ ID NO: 128. Alternatively or in addition (by way of example, in addition), the anti-TfR1 antibodies of the present disclosure comprise a VL comprising the amino acid sequence of SEQ ID NO: 129.

[0177] In some embodiments, the anti-TfR1 antibodies of the present disclosure comprise a VH that contains 25 or fewer amino acid variations (by way of example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the VH as represented by SEQ ID NO: 128. Alternatively or in addition (by way of example, in addition), the anti-TfR1 antibodies of the present disclosure comprise a VL that contains 15 or fewer amino acid variations (by way of example, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) compared to the VL as represented by SEQ ID NO: 129.

[0178] In some embodiments, the anti-TfR1 antibodies of the present disclosure are full-length IgG1 antibodies that may comprise heavy chain constant regions and light chain constant regions from human antibodies. In some embodiments, the heavy chain of any of the anti-TfR1 antibodies as described herein may comprise a heavy chain constant region (CH) or a portion thereof (by way of example, CH1, CH2, CH3, or combinations thereof). The heavy chain constant region can be of any suitable origin, by way of example, human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is from human IgG (gamma heavy chain), by way of example, IgG1, IgG2, or IgG4. Examples of the human IgG1 constant region are given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81)

[0179] In some embodiments, any light chain of the anti-TfR1 antibodies described herein may further comprise a light chain constant region (CL), which can be any CL known in the art. In some examples, the CL is a kappa light chain. In some embodiments, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain and its sequence is provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)

[0180] In some embodiments, the anti-TfR1 antibodies described herein are chimeric antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 132. Alternatively or additionally (by way of example, additionally), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0181] In some embodiments, the anti-TfR1 antibodies described herein are fully human antibodies that include a heavy chain comprising the amino acid sequence of SEQ ID NO: 134. Alternatively or additionally (by way of example, additionally), the anti-TfR1 antibodies described herein include a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0182] In some embodiments, the anti-TfR1 antibody is an antigen-binding fragment (Fab) of an intact antibody (full-length antibody). In some embodiments, the anti-TfR1 Fab described herein includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 136. Alternatively or additionally (by way of example, additionally), the anti-TfR1 Fab described herein includes a light chain comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-TfR1 Fab described herein includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 137. Alternatively or additionally (by way of example, additionally), the anti-TfR1 Fab described herein includes a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0183] The anti-TfR1 antibodies described herein can be in any antibody form, including but not limited to intact (i.e., full-length) antibodies, their antigen-binding fragments (Fab, Fab’, F(ab’)2, Fv, etc.), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the anti-TfR1 antibodies described herein are scFv. In some embodiments, the anti-TfR1 antibodies described herein are scFv-Fab (e.g., scFv fused to a portion of a constant region). In some embodiments, the anti-TfR1 antibodies described herein are scFv fused to a constant region (e.g., a human IgG1 constant region as represented by SEQ ID NO: 81).

[0184] In some embodiments, conservative mutations can be introduced into the antibody sequence (e.g., CDR or framework regions) at positions where the residue is unlikely to be involved in the interaction with the target antigen (e.g., transferrin receptor as an example), as determined (e.g., based on crystal structure). In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the anti-TfR1 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 cytotoxicity to cells (e.g., according to the Kabat numbering system (e.g., Kabat's EU index), in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region).

[0185] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region (CH1 domain) of the Fc region such that the number of cysteine residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains, to alter the stability of the antibody (e.g., increase or decrease), or to facilitate conjugation of a linker.

[0186] In some embodiments, one, two, or more amino acid mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeting antibodies described herein (e.g., by numbering according to the Kabat numbering system (e.g., EU index in Kabat), in the CH2 domain (residues 231-340 of human IgG1) and / or (e.g., and) CH3 domain (residues 341-447 of human IgG1) and / or (e.g., and) hinge region) to increase or reduce the affinity of the antibody for Fc receptors (e.g., activating Fc receptors) on the surface of effector cells. Techniques for introducing mutations in the Fc region of an antibody that reduce or increase the affinity of the antibody for an Fc receptor, and such mutations into an Fc receptor or a 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 for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0187] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or an FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the in vivo half-life of the antibody. Examples of mutations that would alter (e.g., increase or decrease) the in vivo half-life of an antibody are described, for example, in International Publication Nos. 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.

[0188] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably, the Fc or hinge-Fc domain fragment) to decrease the in vivo half-life of the anti-TfR1 antibody. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably, the Fc or hinge-Fc domain fragment) to increase the in vivo half-life of the antibody. 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 (by way of example, and) the third constant (CH3) domain (residues 341-447 of human IgG1) according to the numbering following the Kabat's EU index (Kabat E A et al., (1991) supra). In some embodiments, the IgG1 constant region of the antibodies described herein includes a substitution of methionine (M) to tyrosine (Y) at position 252, a substitution of serine (S) to threonine (T) at position 254, and a substitution of threonine (T) to glutamic acid (E) 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, designated the "YTE mutant," has been shown to exhibit a 4-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua W F et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody includes an IgG constant domain 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 Kabat's EU index as in Kabat.

[0189] In some embodiments, one, two or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to alter the effector function(s) of the anti-TfR1 antibody. Effector ligands whose affinity for it is altered can be, for example, Fc receptors 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 (through point mutations or other means) of the constant region domain can reduce binding of the circulating antibody to Fc receptors, thereby increasing tumor localization. For a description of mutations that delete or inactivate the constant domain and thereby increase tumor localization, see, for example, U.S. Pat. Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region (which may also reduce binding to Fc receptors) (see, for example, Shields R L et al.,(2001)J Biol Chem 276:6591-604).

[0190] In some embodiments, one or more amino acid residues in the constant region of the anti-TfR1 antibodies described herein can be replaced with different amino acid residues such that the antibody can have altered C1q binding and / or (by way of example, and) reduced or abrogated complement-dependent cytotoxicity (CDC). This approach is described in more 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 antibodies described herein are altered, thereby altering the complement binding ability of the antibody. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibodies described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or (by way of example, and) to increase the affinity of the antibody for the Fcγ receptor. This approach is further described in International Publication No. WO 00 / 42072.

[0191] In some embodiments, the sequence(s) of the heavy chain and / or (by way of example, and) light chain variable domain(s) of the antibodies provided herein are, for example, CDR grafted, chimeric, humanized, or bispecific human antibodies or antigen-binding fragments as described herein. As would be understood by one of skill in the art, any variant, CDR-grafted antibody, chimeric antibody, humanized antibody, or bispecific antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, provided that the variant, CDR-grafted antibody, chimeric antibody, humanized antibody, or bispecific antibody maintains specific binding ability to the transferrin receptor such that it can 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 is derived.

[0192] In some embodiments, the antibodies provided herein include mutations that confer desired properties to the antibody. For example, to avoid potential complications resulting from Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include the stabilizing "Adair" mutation.

[0193] In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation, and / or (by way of example 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 (by way of example and) phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or lipid phosphate units. In some embodiments, there are about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 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 a chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell (which may optionally lack enzymes (such as glycosyltransferases) 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".

[0194] In some embodiments, any one of the anti-TfR1 antibodies described herein may include a signal peptide (e.g., an N-terminal signal peptide) on the heavy chain and / or (by way of example, and) light chain sequence. 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 F(ab’) 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).

[0195] 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 pyroglutamate formation (pyroGlu), may occur in the antibody at the N-terminal glutamate (Glu) and / or glutamine (Gln) residues during production. Thus, it should be understood that antibodies identified as having a sequence that includes an N-terminal glutamate residue or glutamine residue encompass antibodies that have undergone pyroglutamate formation as a result of post-translational modification. In some embodiments, pyroglutamate formation occurs in the heavy chain sequence. In some embodiments, pyroglutamate formation occurs in the light chain sequence.

[0196] b. Other muscle-targeting antibodies In some embodiments, the muscle-targeted antibody is an antibody that specifically binds to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin IIb, or CD63. In some embodiments, the muscle-targeted antibody is an antibody that specifically binds to a myogenic precursor protein. Exemplary myogenic precursor proteins include, without limitation, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha7, integrin alpha7beta1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle-targeted 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 alpha7, integrin alpha7beta1, integrin beta1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, the muscle-targeted 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 list of exemplary targets provided herein is not intended to be limiting.

[0197] c. Features / Modifications of the Antibody In some embodiments, conservative mutations can be introduced into the antibody sequence (e.g., CDR or framework regions) at positions where the residue is unlikely to be involved in the interaction with the target antigen (e.g., transferrin receptor as an example), as determined (e.g., based on crystal structure). In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies described herein (e.g., numbered according to the Kabat numbering system (e.g., Kabat's EU index), in the CH2 domain (residues 231 - 340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341 - 447 of human IgG1), and / or (e.g., and) in the hinge region) to 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.

[0198] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region (CH1 domain) of the Fc region such that the number of cysteine residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of the CH1 domain can be altered, for example, to modify the stability of the antibody (e.g., increase or decrease) to facilitate the assembly of the light and heavy chains, or to facilitate the conjugation of linkers.

[0199] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeting antibodies described herein to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the effector cell surface (e.g., by numbering according to the Kabat numbering system (e.g., Kabat's EU index) in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region). Techniques for introducing mutations in the Fc region of an antibody that reduce or increase the affinity of the antibody for an Fc receptor, and such mutations into an Fc receptor or a 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 for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0200] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or an FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the in vivo half-life of the antibody. For examples of mutations that would alter (e.g., increase or decrease) the in vivo half-life of an antibody, see, for example, International Publication Nos. 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.

[0201] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably, the Fc or hinge-Fc domain fragment) to decrease the in vivo half-life of an anti-transferrin receptor antibody. In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably, the Fc or hinge-Fc domain fragment) to increase the in vivo half-life of an antibody. 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 (by way of example, and) in the third constant (CH3) domain (residues 341-447 of human IgG1) according to the numbering following the Kabat's EU index (Kabat E A et al., (1991) supra). In some embodiments, the IgG1 constant region of the antibodies described herein comprises substitutions of methionine (M) to tyrosine (Y) at position 252, serine (S) to threonine (T) at position 254, and threonine (T) to glutamic acid (E) 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, designated the "YTE mutant," has been shown to exhibit a 4-fold increased half-life compared to the wild-type version of the same antibody (see Dall’Acqua W F et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant domain 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.

[0202] In some embodiments, one, two, or more amino acid substitutions are introduced into the Fc region of the IgG constant region to alter the effector function(s) of the anti-transferrin receptor antibody. Effector ligands whose affinity for it is altered can be, for example, Fc receptors 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 (through point mutations or other means) of the constant region domain can reduce the binding of the circulating antibody to Fc receptors, thereby increasing tumor localization. For a description of mutations that delete or inactivate the constant domain and thereby increase tumor localization, see, for example, U.S. Pat. Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region (which may also reduce binding to Fc receptors) (see, for example, Shields R L et al., (2001) J Biol Chem 276:6591-604).

[0203] In some embodiments, one or more amino acid residues in the constant region of the muscle-targeting antibodies described herein can be replaced with different amino acid residues such that the antibody can have altered Clq binding and / or (by way of example, and) reduced or abolished complement-dependent cytotoxicity (CDC). This approach is described in more 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 antibodies described herein are altered, thereby altering the complement-binding ability of the antibody. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibodies described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or (by way of example, and) to increase the affinity of the antibody for Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0204] In some embodiments, the heavy and / or (by way of example, and) light chain variable domains of the antibodies provided herein can be used, for example, to generate CDR grafted, chimeric, humanized, or bispecific human antibodies or antigen-binding fragments as described elsewhere herein. As would be understood by one of skill in the art, any variant, CDR grafted, chimeric, humanized, or bispecific antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and the variant, CDR grafted, chimeric, humanized, or bispecific antibody will maintain the ability to specifically bind to the transferrin receptor such that it has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or higher binding to the transferrin receptor compared to the original antibody from which it was derived.

[0205] In some embodiments, the antibodies provided herein include mutations that confer desired properties to the antibody. For example, to avoid potential complications resulting from Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may include the stabilizing “Adair” mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include the stabilizing “Adair” mutation.

[0206] As provided herein, the antibodies of the disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be attached at its C-terminal end to a light chain constant region such as Cκ or Cλ. Similarly, the VH domain or a portion thereof may be attached to all or a portion of any of the heavy chains, including IgA, IgD, IgE, IgG, and IgM, and to any isotype subclass. The antibody may include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of the disclosure may include the VH and VL domains, or antigen-binding portion thereof, in combination with any suitable constant region.

[0207] ii. Muscle-targeting peptide Some aspects of the present disclosure provide myotargeting peptides as myotargeting agents. Short peptide sequences that bind to specific cell types (e.g., peptide sequences 5 to 20 amino acids in length) are described. For example, peptides that target cells are described in Vines e., et al., A. “Cell-penetrating and cell-targeting peptides in drug delivery” Biochim Biophys Acta 2008, 1786: 126-38; Jarver P., et al., “In vivo biodistribution and efficacy of peptide mediated delivery” Trends Pharmacol Sci 2010;31: 528-35; Samoylova T.I., et al., “Elucidation of muscle-binding peptides by phage display screening” Muscle Nerve 1999;22: 460-6; U.S. Patent No. 6,329,501, issued December 11, 2001, titled “METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE”; and Samoylov A.M., 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. Selectivity for a desired tissue (e.g., muscle) can be achieved by designing the peptide to interact with a specific cell surface antigen (e.g., a receptor). Targeting of skeletal muscle has been studied and can deliver a wide range of molecular payloads. These approaches can have a high degree of selectivity for muscle tissue and are not accompanied by many of the substantial disadvantages of large antibodies or viral particles.Thus, in some embodiments, the muscle targeting agent is a muscle targeting peptide having a length of 4 to 50 amino acids. 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. The muscle targeting peptide can be generated using several methods such as phage display.

[0208] In some embodiments, the muscle-targeting peptide may bind to an internalizing cell surface receptor (e.g., the transferrin receptor) that is overexpressed or relatively highly expressed in muscle cells compared to other cells. In some embodiments, the muscle-targeting peptide may target the transferrin receptor (e.g., may bind to the transferrin receptor). In some embodiments, the peptide that targets the transferrin receptor may include a segment of a naturally occurring ligand, such as transferrin. In some embodiments, the peptide that targets the transferrin receptor is as described in U.S. Patent No. 6,743,893, filed November 30, 2000, "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR". In some embodiments, the peptide that targets the transferrin receptor 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 peptide that targets the transferrin receptor is as described in U.S. Patent No. 8,399,653, filed May 20, 2011, "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY".

[0209] As discussed above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides were identified using a phage display library presenting surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 167) bound to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. Thus, in some embodiments, the muscle targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 167). This peptide showed improved specificity for binding to myocardial and skeletal muscle tissues after intravenous injection into mice, with reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have also been identified using phage display. For example, in the context of treatment of DMD, a 12-amino acid peptide was identified by a phage display library for muscle targeting. See Yoshida D., et al., “Targeting of salicylate to skin and muscle following topical injections in rats.” Int J Pharm 2002;231: 177-84, the entire contents of which are incorporated herein by reference. Here, a 12-amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO: 168) was identified, and this muscle-targeting peptide showed improved binding to C2C12 cells compared to the ASSLNIA (SEQ ID NO: 167) peptide.

[0210] Additional methods for identifying peptides that are selective for muscle (e.g., skeletal muscle) over other cell types also involve 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 content of which is incorporated herein by reference. Nonspecific cell binders were selected and excluded by pre-incubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After several rounds of selection, the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 169) emerged most frequently. Accordingly, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 169).

[0211] The muscle targeting agent may be a molecule or peptide containing an amino acid. The muscle targeting peptide may correspond to the sequence of a protein that preferentially binds to a protein receptor found in muscle cells. In some embodiments, the muscle targeting peptide contains a high proportion of the properties of hydrophobic amino acids (such as valine) so that the peptide can preferentially target muscle cells. In some embodiments, the muscle targeting peptides have not been previously characterized or disclosed. These peptides may be generated, synthesized, and / or (such as and) derivatized using any of several methodologies, such as 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 incorporated by reference (Gray, B.P. and Brown, K.C. “Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides” Chem Rev. 2014, 114:2, 1020-1081.; Samoylova, T.I. and Smith, B.F. “Elucidation of muscle-binding peptides by phage display screening.” Muscle Nerve, 1999, 22:4. 460-6.).In some embodiments, the muscle-targeting peptide has been previously disclosed (see, for example, Writer M.J. et al. “Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptide 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, M.J. 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 amino acid sequences of the following groups: CQAQGQLVC (SEQ ID NO: 170), CSERSMNFC (SEQ ID NO: 171), CPKTRRVPC (SEQ ID NO: 130), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 172), ASSLNIA (SEQ ID NO: 167), CMQHSMRVC (SEQ ID NO: 173), and DDTRHWG (SEQ ID NO: 131). In some embodiments, the muscle-targeting peptide may comprise 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. The muscle-targeting peptide 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 muscle - targeting peptide may be linear; in other embodiments, the muscle - targeting peptide may be cyclic (e.g., bicyclic) (see, e.g., Silvana, M. G. et al. Mol. Therapy, 2018, 26:1, 132 - 147).

[0212] iii. Muscle - targeting receptor ligand 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 that binds to an internalizing cell - surface receptor expressed by muscle cells, e.g., transferrin. Consequently, 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 compared to other cell types. Exemplary lipophilic small molecules that may target muscle cells include cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolenic acid, linoleic acid, myristic acid, sterol, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and compounds containing alkoxy acids.

[0213] iv. Muscle - targeting aptamer The muscle-targeting agent may be an aptamer, such as an RNA aptamer, that preferentially targets muscle cells compared to other cell types. In some embodiments, the muscle-targeting aptamers have not been previously characterized or disclosed. These aptamers may be recalled, generated, synthesized, and / or (e.g., and) derivatized using any of several methodologies, such as Systematic Evolution of Ligands by Exponential Enrichment. Exemplary methodologies are characterized in the art and are incorporated by reference (Yan, A. C. 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, the 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 less than this.

[0214] 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 muscle fiber sheath. In some embodiments, the muscle targeting agent is a substrate of an influx transporter specific for muscle tissue. In some embodiments, the influx transporter is specific for skeletal muscle tissue. Two major classes of transporters expressed on the muscle fiber sheath of skeletal muscle are: (1) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue, and (2) the solute carrier (SLC) superfamily, which can facilitate 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, e.g., a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0215] In some embodiments, the muscle targeting agent is any of the muscle targeting agents described herein that target 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 equilibrium or use a proton or sodium ion gradient created across the membrane to drive substrate transport. Exemplary SLC transporters having high expression in skeletal muscle include, without limitation, the SATT transporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7 transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), LAT3 transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-J transporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2 transporter (FLJ46769; SLC22A5), ENT transporter (ENT1; SLC29A1 and ENT2; SLC29A2), PAT2 transporter (SLC36A2), and SAT2 transporter (KIAA1382; SLC38A2). These transporters can provide an opportunity for muscle targeting by facilitating the influx of substrates into skeletal muscle.

[0216] In some embodiments, the muscle targeting agent is a substrate of the equilibrative nucleoside transporter 2 (ENT2) transporter. Compared to other transporters, ENT2 has one of the highest expressed mRNAs in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs such as the 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 gradients. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. The hENT2 transporter has a low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except inosine. As a result, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, without limitation, inosine, 2’,3’-dideoxyinosine, and clofarabine. In some embodiments, any of the muscle targeting agents provided herein are related to a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular payload.

[0217] In some embodiments, the muscle targeting agent is a substrate of 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 derivatives thereof are covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0218] The muscle targeting agent may be a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be the protein hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein) that is 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 the functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack N-terminal signaling, and / or (by way of example and) may lack the C-terminal anchoring domain. In some embodiments, hemojuvelin may be annotated with GenBank RefSeq accession numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It should be understood that hemojuvelin may be of human, non-human primate, or rodent origin.

[0219] B. Molecular Payload Some aspects of the present disclosure provide oligonucleotides designed to target DUX4 RNA to modulate the expression or activity of a molecular payload, such as DUX4. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA that are useful for reducing the levels of DUX4 mRNA and / or protein associated with the hallmarks of the pathological state of facioscapulohumeral muscular dystrophy (FSHD), including muscle atrophy, inflammation, as well as reduced differentiation capacity 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 to reduce off-target effects. In some embodiments, the oligonucleotides are designed to have properties of desired bioavailability and / or serum stability. In some embodiments, the oligonucleotides are designed to have properties of desired binding affinity. In some embodiments, the oligonucleotides are designed to have a desired toxicity and / or immunogenicity profile.

[0220] In some embodiments, the 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.

[0221] i. Oligonucleotide In some embodiments, the oligonucleotides provided herein are designed to cause RNAi-mediated degradation of DUX4 mRNA. In some embodiments, the DUX4-targeting oligonucleotides provided herein comprise an antisense strand that is complementary to DUX4 mRNA. In some embodiments, the oligonucleotides provided herein further comprise a sense strand that forms a double-stranded oligonucleotide (e.g., siRNA). It should be understood that in some embodiments, an oligonucleotide of one format (e.g., an antisense oligonucleotide) may be suitably adapted to another format (e.g., an siRNA oligonucleotide) by incorporating a functional sequence (e.g., an antisense strand sequence) from one format into the other format.

[0222] Any suitable oligonucleotide may be used as a molecular payload as described herein. Examples of oligonucleotides useful for targeting DUX4 are provided in U.S. Patent No. 9,988,628, issued February 2, 2017, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. Patent No. 9,469,851, issued October 30, 2014, entitled "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4"; U.S. Patent Application Publication No. 20120225034, published September 6, 2012, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; PCT Patent Application Publication No. WO 2013 / 120038, published August 15, 2013, entitled "MORPHOLINO TARGETING DUX4 FOR TREATING FSHD"; Chen et al., "Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics," Molecular Therapy, 2016, 24:8, 1405-1411.; and Ansseau et al., "Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy (FSHD)," Genes, 2017, 8, 93., the entire contents of each of which are incorporated herein by reference. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA, or another oligonucleotide that hybridizes to the target DUX4 gene or mRNA. In some embodiments, the oligonucleotide is an siRNA oligonucleotide.

[0223] In some embodiments, the oligonucleotides described herein have regions of complementarity to sequences represented as follows: human DUX4 corresponding to the following NCBI sequences: NM_001293798.2 (SEQ ID NO: 160) or NCBI sequence: NM_001306068.3 (SEQ ID NO: 161), and / or (by way of example, and) mouse DUX4 corresponding to the following NCBI sequence: NM_001081954.1 (SEQ ID NO: 162). Other non-limiting exemplary human DUX4 mRNAs include NCBI sequences: 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 and HM190160, HM190161, HM190162, HM190163, HM190164, HM190165, HM190166, HM190167, HM190168, HM190169, HM190170, HM190171, HM190172, HM190173, HM190174, HM190175, HM190176, HM190177, HM190178, HM190179, HM190180, HM190181, HM190182, HM190183, HM190184, HM190185, HM190186, HM190187, HM190188, HM190189, HM190190, HM190191, HM190192, HM190193, HM190194, HM190195, HM190196, each of which is incorporated herein by reference.In some embodiments, the oligonucleotide may have a region of complementarity to hypomethylated and reduced D4Z4 repeats, as described in Daxinger, et al., "Genetic and Epigenetic Contributors to FSHD," Lim J-W, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1;24(17):4817-4828 (the entire contents of each of which are incorporated herein by reference), published in Curr Opin Genet Dev in 2015.

[0224] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence represented as follows, which is an example of the human DUX4 gene sequence (NM_001293798.2) (SEQ ID NO: 160):

[0225] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence represented as follows, which is an example of the human DUX4 gene sequence (NM_001306068.3) (SEQ ID NO: 161):

[0226] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence represented as follows, which is an example of the mouse DUX4 gene sequence (SEQ ID NO: 162) (NM_001081954.1):

[0227] In some embodiments, the oligonucleotide may have regions of complementarity to DUX4 gene sequences of multiple species selected, by way of example, from human, mouse, and non-human species. In some embodiments, the non-human species is a cynomolgus monkey.

[0228] i. Size / sequence of the oligonucleotide The oligonucleotide may be of various different lengths, depending, by way of example, on the format. In some embodiments, the oligonucleotide 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, 75 nucleotides in length or longer. In some embodiments, the oligonucleotide is 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, and so on. In some embodiments, the oligonucleotide is 8-32 nucleotides in length, 15-29 nucleotides in length, 15-27 nucleotides in length, 15-20 nucleotides in length, 20-25 nucleotides in length, 21-27 nucleotides in length, 23-27 nucleotides in length, 25-30 nucleotides in length, or 25-32 nucleotides in length.

[0229] In some embodiments, for the purposes of the present disclosure, a complementary nucleic acid sequence of an oligonucleotide is such that when the binding of the sequence to a target molecule (e.g., mRNA) of the sequence interferes with the normal function of the target (e.g., mRNA) causing a loss of activity (e.g., inhibition of translation) or a loss of expression (e.g., degradation of the target mRNA), and under conditions where avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatments and in the case of in vitro assays, and under conditions where the assay is performed under suitable conditions of stringency, the oligonucleotide is capable of specifically hybridizing to the target nucleic acid or is specific for the target nucleic acid when there is a degree of complementarity sufficient to avoid non-specific binding to non-target sequences of the said sequence. 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 consecutive nucleotides of the target nucleic acid. In some embodiments, the complementary nucleotide sequence need not be 100% complementary to the sequence of its target that is capable of specifically hybridizing to the target nucleic acid or is specific for the target nucleic acid. In some embodiments, the oligonucleotide contains one or more mismatched nucleobases compared to the target nucleic acid. In some embodiments, the activity with respect to the target is reduced by such a mismatch, but the activity with respect to non-targets is reduced by a greater amount (i.e., the selectivity for the target nucleic acid is increased and the off-target effect is decreased). In some embodiments, the target nucleic acid is a pre-mRNA molecule or an mRNA molecule.

[0230] In some embodiments, the oligonucleotide comprises a region of complementarity to a target nucleic acid having a length in the range of 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides. In some embodiments, the oligonucleotide comprises a region of complementarity to a target nucleic acid having a length in the range of 8 to 32, 15 to 29, 15 to 27, 21 to 27, 23 to 27 nucleotides. In some embodiments, the oligonucleotide comprises a region of complementarity to a target nucleic acid having a length in the range of 15 to 29, 15 to 27, 15 to 20, 20 to 25, 21 to 27, 23 to 27, 25 to 27, or 25 to 32 nucleotides. 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 region of complementarity is complementary to at least 12 consecutive nucleotides of the target nucleic acid. In some embodiments, the region of complementarity is complementary to at least 16 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain 1, 2, or 3 base mismatches compared to a portion of the 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.

[0231] 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: 236 - 266. In some embodiments, the oligonucleotide comprises a sequence comprising any one of SEQ ID NOs: 236 - 266. In some embodiments, the oligonucleotide comprises at least 12 or at least 15 consecutive nucleotides of any one of SEQ ID NOs: 236 - 266 and a sequence sharing at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity.

[0232] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence as set forth by any one of SEQ ID NOs: 174 - 235. In some embodiments, the oligonucleotide comprises at least 12 or at least 15 consecutive nucleotides of a target sequence as set forth by any one of SEQ ID NOs: 174 - 235 and a region of complementarity that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementary. 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 is in the range of 8 - 20, 10 - 20, or 15 - 20 nucleotides in length. In some embodiments, the region of complementarity is completely complementary to all or part of its target sequence. In some embodiments, the region of complementarity encompasses 1, 2, 3, or more mismatches.

[0233] In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to the target sequence of any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 8). In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to the target sequence of any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 9). In some embodiments, such target sequence is 100% complementary to the oligonucleotides listed in Table 8. In some embodiments, such target sequence is 100% complementary to the oligonucleotides listed in Table 9. In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to the target sequence of any one of the oligonucleotides provided herein (e.g., the oligonucleotide comprising any one of SEQ ID NOs: 236 to 266). In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to the target sequence of any one of the oligonucleotides provided herein (e.g., the oligonucleotide comprising any one of SEQ ID NOs: 248, 251 to 253, and 262). In some embodiments, such target sequence is 100% complementary to the oligonucleotides described herein (e.g., the oligonucleotide comprising any one of SEQ ID NOs: 236 to 266). In some embodiments, such target sequence is 100% complementary to the oligonucleotides described herein (e.g., the oligonucleotide comprising any one of SEQ ID NOs: 248, 251 to 253, and 262).

[0234] In some embodiments, it should be understood that methylation at the C5 position of the nucleobase uracil forms thymine. Thus, in some embodiments, a nucleotide or nucleoside having C5-methylated uracil (or 5-methyl-uracil) may be equivalently identified as a thymine nucleotide or nucleoside.

[0235] In some embodiments, one or more of the thymine bases (T) in any one of the oligonucleotides provided herein may independently and optionally be uracil bases (U), and / or any one or more of the Us 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 may independently and optionally be uracil bases (U), and / or any one or more of the Us 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 9 may independently and optionally be uracil bases (U), and / or any one or more of the Us may independently and optionally be T.

[0236] b. Oligonucleotide modification: The oligonucleotides described herein may be modified and include, by way of example, modified sugar moieties, modified internucleoside linkages, modified nucleotides or nucleosides, and / or combinations thereof. Additionally, in some embodiments, the oligonucleotides may exhibit one or more of the following properties: not mediate alternative splicing; not be immunostimulatory; be nuclease resistant; have improved cellular uptake compared to unmodified oligonucleotides; be non-toxic to cells or mammals; have improved exit from endosomes into the cell; minimize TLR stimulation; or avoid pattern recognition receptors. Any of the modified chemical properties or formats of the oligonucleotides described herein may be combined with each other. For example, 1, 2, 3, 4, 5, or more different types of modifications may be incorporated within the same oligonucleotide.

[0237] In some embodiments, certain nucleotide or nucleoside modifications may be used that render the oligonucleotide into which the modification is incorporated more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides persist intact for longer periods of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those with modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl sugar linkages, or short chain heteroatomic or heterocyclic sugar linkages, including those with modified internucleoside linkages. As a result, the oligonucleotides of the present disclosure may be stabilized against nucleic acid degradation by modifications, such as incorporation of nucleotide or nucleoside modifications.

[0238] In some embodiments, the oligonucleotide may be an oligonucleotide up to 50 nucleotides in length or up to 100 nucleotides in length, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The oligonucleotide may be an oligonucleotide 8 to 30 nucleotides in length, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The oligonucleotide may be an oligonucleotide 8 to 15 nucleotides in length, wherein 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. Optionally, the oligonucleotide may be modified at any nucleotide or nucleoside except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides / nucleosides. Oligonucleotide modifications are further described herein.

[0239] c. Modified nucleoside In some embodiments, the oligonucleotides described herein include at least one nucleoside modified at the 2'-position of the sugar. In some embodiments, the oligonucleotide includes at least one 2'-modified nucleoside. In some embodiments, all of the nucleosides on the oligonucleotide are 2'-modified nucleosides.

[0240] 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-methylacetamide (2'-O-NMA) modified nucleosides.

[0241] In some embodiments, the oligonucleotides described herein include one or more 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the oligonucleotides described herein include at least two 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the oligonucleotides described herein include at least four 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the oligonucleotides described herein include at least six 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the oligonucleotides described herein include one or more 2'-O-methyl (2'-O-Me) modified nucleosides.

[0242] In some embodiments, the oligonucleotides described herein include one or more 2'-4' bicyclic nucleosides, wherein the ribose ring in the nucleoside includes a bridging moiety that connects 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 LNA are described in the international patent application publication WO / 2008 / 043753 entitled "RNA Antagonist Compounds For The Modulation Of PCSK9", published on April 17, 2008, the contents of which are incorporated herein by reference in their entirety. Examples of ENA are provided in the international patent publication No. WO 2005 / 042777 entitled "APP / ENA Antisense", published on May 12, 2005; 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 entirety. Examples of cEt are provided in U.S. Patents 7,101,993; 7,399,845 and 7,569,686, each of which is incorporated herein by reference in its entirety.

[0243] 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, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,741,457, issued June 22, 2010, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 8,022,193, issued September 20, 2011, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,569,686, issued August 4, 2009, titled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,335,765, issued February 26, 2008, titled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent 7,314,923, issued January 1, 2008, titled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent 7,816,333, issued October 19, 2010, titled "Oligonucleotide Analogues And Methods Utilizing The Same" and U.S. Publication No. 2011 / 0009471, now U.S. Patent 8,957,201, issued February 17, 2015, titled "Oligonucleotide Analogues And Methods Utilizing The Same", in their entireties for all purposes, the entire contents of each of which are hereby incorporated by reference.

[0244] In some embodiments, the 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 as compared to an oligonucleotide that has no modified nucleosides. The oligonucleotide may have a plurality of modified nucleosides that result in an increase in the Tm of the oligonucleotide in the range of a total 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 as compared to an oligonucleotide that has no modified nucleosides.

[0245] The oligonucleotide may contain a mix of different types of nucleosides. For example, the oligonucleotide may contain a mix of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. The oligonucleotide may contain a mix of deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. The oligonucleotide may contain a mix of 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. The oligonucleotide may contain a mix of crosslinked nucleosides and 2'-fluoro or 2'-O-methyl modified nucleosides. The 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 modified nucleosides 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).

[0246] An oligonucleotide may contain alternating different types of nucleosides. For example, an oligonucleotide may contain alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro-modified nucleosides. An oligonucleotide may contain alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me-modified nucleosides. An oligonucleotide may contain alternating 2'-fluoro-modified nucleosides and 2'-O-Me-modified nucleosides. An oligonucleotide may contain alternating cross-linked nucleosides and 2'-fluoro or 2'-O-methyl-modified nucleosides. An oligonucleotide may contain alternating non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4'-bicyclic nucleosides (e.g., LNA, ENA, cEt). An oligonucleotide may contain alternating 2'-4'-bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me-modified nucleosides. An oligonucleotide 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).

[0247] In some embodiments, the oligonucleotides described herein contain 5'-vinylphosphonate modifications, one or more abasic residues, and / or one or more inverted abasic residues. In some embodiments, the oligonucleotides described herein are siRNA oligonucleotides comprising a sense strand and an antisense strand, where the antisense strand or the sense strand contains a 5'-vinylphosphonate (e.g., 5'-(E)-vinylphosphonate modification). In some embodiments, the oligonucleotides described herein are siRNA oligonucleotides comprising a sense strand and an antisense strand, where the antisense strand contains a 5'-vinylphosphonate (e.g., 5'-(E)-vinylphosphonate modification).

[0248] d. Inter-nucleoside linkage / backbone In some embodiments, the oligonucleotide may contain phosphorothioate or other modified internucleoside linkages. In some embodiments, the oligonucleotide contains phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide contains phosphorothioate internucleoside linkages between at least two nucleosides. In some embodiments, the oligonucleotide contains phosphorothioate internucleoside linkages between all nucleosides. For example, in some embodiments, the oligonucleotide contains modified internucleoside linkages at the first, second, and / or (by way of example and) third internucleoside linkages at the 5' or 3' end of the nucleotide sequence.

[0249] Linkages containing phosphorus that may be used include, but are not limited to, normal 3'-5' linkages, phosphorothioates having analogs of these 2'-5' linkages, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3' alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates, and those having the opposite polarity (where the adjacent pair of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'); see 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; 5,399,676; 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.

[0250] In some embodiments, the oligonucleotide may have a heteroatom backbone such as a methylene(methylimino) or MMI backbone; an amide backbone (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28: 366-374); a morpholino backbone (see Summerton and Weller, U.S. Patent No. 5,034,506); or a peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone and the nucleotides are directly or indirectly attached to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

[0251] e. Stereospecific oligonucleotides In some embodiments, the phosphorus atom between nucleotides of the oligonucleotide is chiral, and the properties of the oligonucleotide are modulated 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 (such as, for example, 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 internucleoside linkages or substantially all Rp phosphorothioate internucleoside linkages. In some embodiments, such phosphorothioate oligonucleotides having substantially enantiopure internucleoside linkages are prepared by enzymatic synthesis or chemical synthesis, as described, for example, 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, chirally controlled oligonucleotides provide a selective cleavage pattern of a target nucleic acid. For example, in some embodiments, chirally controlled oligonucleotides provide a single cleavage site within a complementary sequence of a nucleic acid, as described, for example, in U.S. Patent Application Publication 20170037399 A1, entitled "CHIRAL DESIGN," published February 2, 2017, the contents of which are incorporated herein by reference in their entirety.

[0252] 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 July 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (as described, for example, 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 herein by reference in their entirety).

[0253] h. Gapmer In some embodiments, the oligonucleotides described herein are gapmers. Gapmer oligonucleotides generally have the formula 5'-X-Y-Z-3' with flanking regions X and Z surrounding a gap region Y. In some embodiments, the flanking region X of the formula 5'-X-Y-Z-3' is also referred to as the X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, the flanking region Z of the formula 5'-X-Y-Z-3' is also referred to as the Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, the gap region Y of the formula 5'-X-Y-Z-3' is also referred to as the Y region, Y segment, or gap segment Y. In some embodiments, each nucleoside of the gap region Y is a 2'-deoxyribonucleoside, and neither the 5' wing region X nor the 3' wing region Z contains any 2'-deoxyribonucleosides.

[0254] In some embodiments, the Y region is a continuous stretch of nucleotides, such as a region of 6 or more DNA nucleotides, that can recruit an RNase, such as RNase H. In some embodiments, the gapmer binds to a target nucleic acid, at which point the RNase is recruited and can then cleave the target nucleic acid. In some embodiments, the Y region is flanked on both the 5' and 3' sides by the flanking regions X and Z that contain 1 to 6 high affinity modified nucleosides, such as 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 to 20 nucleotides in length, 1 to 8 nucleotides in length, or 1 to 5 nucleotides in length. The flanking sequences X and Z can be of similar length or dissimilar length. In some embodiments, the gap segment Y can be a nucleotide sequence that is 5 to 20 nucleotides in length, 5 to 15, 12 nucleotides in length, or 6 to 10 nucleotides in length.

[0255] In some embodiments, the gap region of the gapmer oligonucleotide can contain modified nucleotides, such as C4'-substituted nucleotides, acyclic nucleotides, and arabinonucleotides, in addition to DNA nucleotides, which are known to be tolerated for efficient RNase H activity. In some embodiments, the gap region includes one or more unmodified internucleoside linkages. In some embodiments, one or both of the flanking regions each independently include at least two, at least three, at least four, at least five, or more internucleotide phosphorothioate linkages (by way of example, internucleotide phosphorothioate linkages or other linkages). In some embodiments, the gap region and the two flanking regions each independently include at least two, at least three, at least four, at least five, or more internucleotide modified linkages (by way of example, internucleotide phosphorothioate linkages or other linkages).

[0256] Gapmers can be produced using appropriate methods. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, 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; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,015,315; 7,101,993; 7,399,845; 7,432,250; 7,569,686; 7,683,036; 7,750,131; 8,580,756; 9,045,754; 9,428,534; 9,695,418; 10,017,764; 10,260,069; 9,428,534; 8,580,756; U.S. Patent Publication Nos. US20050074801, US20090221685, US20090286969, US20100197762, and US20110112170; PCT Publication Nos. WO2004069991, WO2005023825, WO2008049085, and WO2009090182; and European Patent No. EP2,149,605. Each of these is hereby incorporated by reference in its entirety.

[0257] In some embodiments, the gapmer is 10 to 40 nucleosides in length. For example, the gapmer can be 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 40, 25 to 35, 25 to 30, 30 to 40, 30 to 35, or 35 to 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.

[0258] In some embodiments, the gap region Y on the gapmer has a length of 5 to 20 nucleosides. For example, the gap region Y can have a length of 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleosides. In some embodiments, the gap region Y has a length of 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides. In some embodiments, each nucleoside of the gap region Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides of the gap region Y are 2'-deoxyribonucleosides. In some embodiments, one or more of the nucleosides of the gap region Y are modified nucleosides (by way of example, 2'-modified nucleosides, such as those described herein). In some embodiments, one or more cytosines of the gap region Y are optionally 5-methylcytosine. In some embodiments, each cytosine of the gap region Y is 5-methylcytosine.

[0259] In some embodiments, the 5' wing region of the gapmer (X in the 5'-X-Y-Z-3' format) and the 3' wing region of the gapmer (Z in the 5'-X-Y-Z-3' format) are independently 1 to 20 nucleosides in length. For example, the 5' wing region of the gapmer (X in the 5'-X-Y-Z-3' format) and the 3' wing region of the gapmer (Z in the 5'-X-Y-Z-3' format) 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 in the 5'-X-Y-Z-3' format) and the 3' wing region of the gapmer (Z in the 5'-X-Y-Z-3' format) 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 in the 5'-X-Y-Z-3' format) and the 3' wing region of the gapmer (Z in the 5'-X-Y-Z-3' format) are of the same length. In some embodiments, the 5' wing region of the gapmer (X in the 5'-X-Y-Z-3' format) and the 3' wing region of the gapmer (Z in the 5'-X-Y-Z-3' format) are of different lengths. In some embodiments, the 5' wing region of the gapmer (X in the 5'-X-Y-Z-3' format) is longer than the 3' wing region of the gapmer (Z in the 5'-X-Y-Z-3' format). In some embodiments, the 5' wing region of the gapmer (X in the 5'-X-Y-Z-3' format) is shorter than the 3' wing region of the gapmer (Z in the 5'-X-Y-Z-3' format).

[0260] In some embodiments, the gapmer is 5-10-5, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3, 2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1, 2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12-5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14-1, 2-14-3, 3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-11-5, 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、It contains 5'-X-Y-Z-3' of 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. The numbers indicate the number of nucleotides in the X, Y, and Z regions of the 5'-X-Y-Z-3' gapmer.,

[0261] In some embodiments, one or more nucleosides of the 5’ wing region (X in the 5’-X-Y-Z-3’ format) of the gapmer or the 3’ wing region (Z in the 5’-X-Y-Z-3’ format) of the gapmer 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’-O-N-methylacetamide (2’-O-NMA)).

[0262] In some embodiments, one or more nucleosides of the 5' wing region (X in the 5'-X-Y-Z-3' format) of the gapmer are high-affinity modified nucleosides. In some embodiments, each nucleoside of the 5' wing region (X in the 5'-X-Y-Z-3' format) of the gapmer is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides of the 3' wing region (Z in the 5'-X-Y-Z-3' format) of the gapmer are high-affinity modified nucleosides. In some embodiments, each nucleoside of the 3' wing region (Z in the 5'-X-Y-Z-3' format) of the gapmer is a high-affinity modified nucleoside. In some embodiments, one or more nucleosides of the 5' wing region (X in the 5'-X-Y-Z-3' format) of the gapmer are high-affinity modified nucleosides, and one or more nucleosides of the 3' wing region (Z in the 5'-X-Y-Z-3' format) of the gapmer are high-affinity modified nucleosides. In some embodiments, each nucleoside of the 5' wing region (X in the 5'-X-Y-Z-3' format) of the gapmer is a high-affinity modified nucleoside, and each nucleoside of the 3' wing region (Z in the 5'-X-Y-Z-3' format) of the gapmer is a high-affinity modified nucleoside.

[0263] In some embodiments, the 5' wing region (X in the 5'-X-Y-Z-3' format) of the gapmer comprises the same high-affinity nucleosides as the 3' wing region (Z in the 5'-X-Y-Z-3' format) of the gapmer. For example, the 5' wing region (X in the 5'-X-Y-Z-3' format) of the gapmer and the 3' wing region (Z in the 5'-X-Y-Z-3' format) of the gapmer may comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me). In another example, the 5' wing region (X in the 5'-X-Y-Z-3' format) of the gapmer and the 3' wing region (Z in the 5'-X-Y-Z-3' format) of the gapmer may comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, each nucleoside of the 5' wing region (X in the 5'-X-Y-Z-3' format) of the gapmer and the 3' wing region (Z in the 5'-X-Y-Z-3' format) of the gapmer is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me). In some embodiments, each nucleoside of the 5' wing region (X in the 5'-X-Y-Z-3' format) of the gapmer and the 3' wing region (Z in the 5'-X-Y-Z-3' format) of the gapmer is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt).

[0264] In some embodiments, the gapmer comprises a 5’-X-Y-Z-3’ configuration, where X and Z are independently nucleosides having a length of 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside having a length of 6 to 10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X and Z is a non-bicyclic 2’-modified nucleoside (e.g., 2’-MOE or 2’-O-Me), and each nucleoside of Y is a 2’-deoxyribonucleoside. In some embodiments, the gapmer comprises a 5’-X-Y-Z-3’ configuration, where X and Z are independently nucleosides having a length of 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside having a length of 6 to 10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X and Z is a 2’-4’-bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2’-deoxyribonucleoside. In some embodiments, the 5’ wing region (X of the 5’-X-Y-Z-3’ formula) of the gapmer comprises a different high-affinity nucleoside from the 3’ wing region (Z of the 5’-X-Y-Z-3’ formula) of the gapmer. For example, the 5’ wing region (X of the 5’-X-Y-Z-3’ formula) of the gapmer may comprise one or more non-bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-O-Me), and the 3’ wing region (Z of the 5’-X-Y-Z-3’ formula) of the gapmer may comprise one or more 2’-4’-bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3’ wing region (Z of the 5’-X-Y-Z-3’ formula) of the gapmer may comprise one or more non-bicyclic 2’-modified nucleosides (e.g., 2’-MOE or 2’-O-Me), and the 5’ wing region (X of the 5’-X-Y-Z-3’ formula) of the gapmer may comprise one or more 2’-4’-bicyclic nucleosides (e.g., LNA or cEt).

[0265] In some embodiments, the gapmer comprises a 5'-X-Y-Z-3' configuration, where X and Z are independently nucleosides having a length of 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside having a length of 6 to 10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), each nucleoside of Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-X-Y-Z-3' configuration, where X and Z are independently nucleosides having a length of 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7), Y is a nucleoside having a length of 6 to 10 (e.g., 6, 7, 8, 9, or 10), each nucleoside of X is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), each nucleoside of Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), and each nucleoside of Y is a 2'-deoxyribonucleoside.

[0266] In some embodiments, the 5' wing region (X in the 5'-X-Y-Z-3' formula) of the gapmer 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 (Z in the 5'-X-Y-Z-3' formula) of the gapmer 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 (X in the 5'-X-Y-Z-3' formula) and the 3' wing region (Z in the 5'-X-Y-Z-3' formula) of the gapmer 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).

[0267] In some embodiments, the gapmer comprises a 5'-X-Y-Z-3' configuration, where X and Z are independently nucleosides having a length of 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleotides, Y is a nucleoside having a length of 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleotides, and at least one (e.g., 1, 2, 3, 4, 5, or 6), but not all, of positions 1, 2, 3, 4, 5, 6, or 7 of X (where the most 5'-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'-X-Y-Z-3' configuration, where X and Z are independently nucleosides having a length of 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleotides, Y is a nucleoside having a length of 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleotides, and at least one (e.g., 1, 2, 3, 4, 5, or 6), but not all, of positions 1, 2, 3, 4, 5, 6, or 7 of Z (where the most 5'-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’-X-Y-Z-3’ configuration, where X and Z are, independently, from 2 to 7 (by way of example, 2, 3, 4, 5, 6, or 7) nucleotides in length, Y is from 6 to 10 (by way of example, 6, 7, 8, 9, or 10) nucleotides in length, where at least one (by way of example, 1, 2, 3, 4, 5, or 6) but not all of positions 1, 2, 3, 4, 5, 6, or 7 of X and at least one of positions 1, 2, 3, 4, 5, 6, or 7 (by way of example, 1, 2, 3, 4, 5, or 6) of Z (where the most 5’ position is position 1) are acyclic 2’-modified nucleotides (by way of example, 2’-MOE or 2’-O-Me), the remainder of the nucleotides of both X and Z are 2’-4’ bicyclic nucleotides (by way of example, LNA or cEt), and each nucleotide of Y is a 2’-deoxyribonucleotide.

[0268] Non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and 2'-4'-bicyclic nucleosides (e.g., LNA or cEt) in the 5'-wing region of a gapmer (X in the 5'-X-Y-Z-3' form) and / or the 3'-wing region of a gapmer (Z in the 5'-X-Y-Z-3' form) non-limiting examples of gapmer constructs having 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-(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; KKK-(D)n-KKE; 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; AKKK-(D)n-KKKA; 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-KKKE; ELLL-(D)n-LLLE; ABBB-(D)n-BBBAA; AKKK-(D)n-KKKAA; ALLL-(D)n-LLLAA; EBBB-(D)n-BBBEE; EKKK-(D)n-KKKEE; ELLL-(D)n-LLLEE; ABBB-(D)n-BBBAA; AKKK-(D)n-KKKAA; ALLL-(D)n-LLLAA; EBBB-(D)n-BBBEE; EKKK-(D)n-KKKEE; ELLL-(D)n-LLLEE; AABBB-(D)n-BBB; AAKKK-(D)n-KKK; AALL-(D)n-LLL; EEBBB-(D)n-BBB; EEKKK-(D)n-KKK; EELLL-(D)n-LLL; AABBB-(D)n-BBB; AAKKK-(D)n-KKK; AALL-(D)n-LLL; EEBBB-(D)n-BBB; EEKKK-(D)n-KKK; EELLL-(D)n-LLL; AABBB-(D)n-BBBA; AAKKK-(D)n-KKKA; AALL-(D)n-LLLA; EEBBB-(D)n-BBBE; EEKKK-(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; AKKAAKK-(D)n-KK; ALLAALLL-(D)n-LL; EBBEEBB-(D)n-BB; EKKEEKK-(D)n-KK; ELLEELL-(D)n-LL; ABBAABB-(D)n-BB; AKKAAKK-(D)n-KK; 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-BBB; 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; ELLELL-(D)n-LLL; EEEK-(D)n-EEEEEEEE; EEK-(D)n-EEEEEEEEE; 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-EEEKEK; EK-(D)n-KEEEKEE; EK-(D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK; wherein the "A" nucleoside includes 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; and "n" represents the length of the gap segment (Y in the 5'-X-Y-Z-3' configuration), which is an integer between 1 and 20.;

[0269] In some embodiments, any one of the gapmers described herein includes 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 includes a mix of phosphorothioate linkages and phosphodiester linkages. In some embodiments, each internucleoside linkage in the gap region Y is a phosphorothioate linkage, the 5' wing region X includes a mix of phosphorothioate linkages and phosphodiester linkages, and the 3' wing region Z includes a mix of phosphorothioate linkages and phosphodiester linkages.

[0270] i.RNA interference (RNAi) In some embodiments, the oligonucleotides provided herein may be in the form of small interfering RNAs (siRNAs), also known as short interfering RNAs or silencing RNAs. siRNAs are a class of double-stranded RNA molecules that target nucleic acids (e.g., mRNA) for degradation via the RNA interference (RNAi) pathway in cells and are typically about 20-25 base pairs in length. The specificity of an siRNA molecule may be determined by the binding of the antisense strand molecule to its target RNA. Effective siRNA molecules generally have less than 30-35 base pairs in length to prevent triggering of non-specific RNA interference pathways in cells via the interferon response, although longer siRNAs may also be effective. In some embodiments, 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 base pairs in length, or more. In some embodiments, the siRNA molecule is 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.

[0271] Upon selection of a suitable target RNA sequence, siRNA molecules comprising nucleotide sequences complementary to all or a portion of the target sequence, i.e., antisense sequences, can be designed and prepared using suitable methods (see, e.g., PCT Publication No. WO2004 / 016735; and U.S. Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791).

[0272] The siRNA molecule can be double-stranded (i.e., a dsRNA molecule containing an antisense strand and a complementary sense strand) or single-stranded (i.e., an ssRNA molecule containing only the antisense strand). The siRNA molecule can contain a duplex, asymmetric duplex, hairpin, or asymmetric hairpin secondary structure having self-complementary sense and antisense strands. In some embodiments, the oligonucleotides described herein are siRNAs containing an antisense strand and a sense strand.

[0273] 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 nucleotides in length, or more. 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.

[0274] In some embodiments, the sense strand of the siRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 nucleotides in length, or more. 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.

[0275] In some embodiments, the siRNA molecule comprises an antisense strand that includes a region complementary to a target region on DUX4 mRNA. In some embodiments, the complementary region is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target region on DUX4 mRNA. In some embodiments, the target region is a region of a contiguous stretch of nucleotides on DUX4 mRNA. In some embodiments, for a complementary nucleotide sequence to be specifically hybridizable or specific for a target RNA sequence, it need not be 100% complementary to that of its target.

[0276] In some embodiments, the siRNA molecule comprises an antisense strand comprising a region of complementarity to the DUX4 RNA sequence, the region of complementarity ranging in length from 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides. 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 containing 1, 2, 3, 4, or 5 bases or fewer mismatches compared to the complementary portion of the DUX4 RNA sequence. In some embodiments, the region of complementarity comprises a nucleotide sequence having up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

[0277] In some embodiments, the siRNA molecule comprises an antisense strand that comprises a nucleotide sequence that is (e.g., at least 85%, at least 90%, at least 95%, or 100%) complementary to a target RNA sequence as set forth in any one of SEQ ID NOs: 174 - 235. In some embodiments, the siRNA molecule comprises an antisense strand that comprises a nucleotide sequence that is (e.g., at least 85%, at least 90%, at least 95%, or 100%) complementary to a target RNA sequence as set forth in any one of SEQ ID NOs: 186, 189 - 191, and 200. In some embodiments, the siRNA molecule is 18 - 25 nucleotides in length and comprises an antisense strand that 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 RNA sequence as set forth in any one of SEQ ID NOs: 174 - 235. In some embodiments, the siRNA molecule is 18 - 25 nucleotides in length and comprises an antisense strand that 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 RNA sequence as set forth in any one of SEQ ID NOs: 186, 189 - 191, and 200.

[0278] In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to the oligonucleotide set forth in any one of SEQ ID NOs: 236 - 266. In some embodiments, the siRNA molecule comprises an antisense strand having a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to the oligonucleotide set forth in any one of SEQ ID NOs: 248, 251 - 253, and 262. In some embodiments, the siRNA molecule is 18 - 25 nucleotides in length and comprises an antisense strand that includes at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of the oligonucleotide set forth in any one of SEQ ID NOs: 236 - 266. In some embodiments, the siRNA molecule is 18 - 25 nucleotides in length and comprises an antisense strand that includes at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, or at least 19 consecutive nucleotides of the oligonucleotide set forth in any one of SEQ ID NOs: 248, 251 - 253, and 262.

[0279] Double-stranded siRNA may comprise sense and antisense RNA strands of the same or different lengths. Double-stranded siRNA molecules can also be assembled from single oligonucleotides having a stem-loop structure, where the self-complementary sense and antisense regions of the siRNA molecule are linked using nucleic acid-based or non-nucleic acid-based linker(s), as well as circular single-stranded RNAs having two or more loop structures and a stem comprising self-complementary sense and antisense strands, where the circular RNA can be processed either in vivo or in vitro to generate active siRNA molecules capable of mediating RNAi. Thus, small hairpin RNA (shRNA) molecules are also contemplated herein. These molecules contain a specific antisense sequence in addition to the reverse complementary (sense) sequence and are typically separated by a spacer or loop sequence. Cleavage of the spacer or loop provides single-stranded RNA molecules and their reverse complements (by additional processing steps that may also result in the addition or removal of one, two, three or more nucleotides from the 3'-end and / or (by way of example, and) 5'-end, optionally single-stranded or double-stranded) such that they can anneal to form a dsRNA molecule. The spacer can be of sufficient length to allow annealing of the antisense and sense sequences to form a duplex structure (or stem) prior to cleavage of the spacer (and optionally, subsequent processing steps that may also result in the addition or removal of one, two, three, four or more nucleotides from the 3'-end and / or (by way of example, and) 5'-end, optionally single-stranded or double-stranded). The spacer sequence can be an unrelated nucleotide sequence placed between two regions of complementary nucleotide sequences, which regions will anneal to form a double-stranded nucleic acid that will contain the shRNA when formed.

[0280] The overall length of the siRNA molecule can vary from about 14 nucleotides to about 100 nucleotides, depending on the type of siRNA molecule designed. Generally, these nucleotides between about 14 and about 50 are complementary to the RNA target sequence, that is, they constitute a specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double-stranded siRNA or a single-stranded siRNA, the length can vary from about 14 nucleotides to about 50 nucleotides, while when the siRNA is a shRNA or a circular molecule, the length can vary from about 40 nucleotides to about 100 nucleotides.

[0281] The siRNA molecule may contain a 3' overhang at one end of the molecule. The other end may be blunt-ended or may also have an overhang (5' or 3'). When the siRNA molecule contains overhangs at both ends of the molecule, the lengths of the overhangs may be the same or different. In one aspect, the siRNA molecules of the present disclosure contain 3' overhangs of about 1 to about 3 (by way of example, 1, 2, 3) nucleotides on both ends of the molecule. In some aspects, the siRNA molecule contains a 3' overhang of about 1 to about 3 (by way of example, 1, 2, 3) nucleotides in the sense strand. In some aspects, the siRNA molecule contains a 3' overhang of about 1 to about 3 (by way of example, 1, 2, 3) nucleotides in the antisense strand. In some aspects, the siRNA molecule contains a 3' overhang of about 1 to about 3 nucleotides in both the sense strand and the antisense strand.

[0282] 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, e.g., 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'-O-N-methylacetamide (2'-O-NMA). In some embodiments, each nucleotide of the siRNA molecule is a modified nucleotide (e.g., 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. In some embodiments, the siRNA molecule comprises one or more modified nucleosides (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 nucleosides and / or (e.g., and) one or more modified internucleoside linkages. In some embodiments, the modified nucleoside is a modified sugar moiety (e.g., 2'-modified nucleoside).In some embodiments, the siRNA molecule comprises one or more 2'-modified nucleosides, such as, by way of example, 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA). In some embodiments, each nucleoside of the siRNA molecule is a modified nucleotide (such as, by way of example, a 2'-modified nucleoside). In some embodiments, the siRNA molecule comprises one or more 2'-O-methyl modified nucleosides. In some embodiments, the siRNA molecule comprises one or more 2'-F modified nucleosides. In some embodiments, the siRNA molecule comprises one or more 2'-O-methyl and 2'-F modified nucleosides.

[0283] In some embodiments, the siRNA molecule contains phosphorothioate or other modified internucleotide linkages. In some embodiments, the siRNA molecule contains phosphorothioate or other modified inter-nucleoside linkages. In some embodiments, the siRNA molecule includes phosphorothioate internucleoside linkages. In some embodiments, the siRNA molecule includes phosphorothioate inter-nucleotide linkages. In some embodiments, the siRNA molecule includes phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the siRNA molecule includes phosphorothioate internucleoside linkages between at least two nucleosides. In some embodiments, the siRNA molecule includes phosphorothioate inter-nucleotide linkages between at least two nucleotides. In some embodiments, the siRNA molecule includes phosphorothioate internucleoside linkages between all nucleotides. In some embodiments, the siRNA molecule includes phosphorothioate internucleoside linkages between all nucleosides. For example, in some embodiments, the siRNA molecule includes modified internucleoside linkages at the 5' or 3' end of the siRNA molecule, in the first, second, and / or (by way of example, and) third internucleoside linkages. In some embodiments, the siRNA molecule includes phosphorothioate inter-nucleotide linkages between all nucleotides. For example, in some embodiments, the siRNA molecule includes modified inter-nucleotide linkages at the 5' or 3' end of the siRNA molecule, in the first, second, and / or (by way of example, and) third inter-nucleotide linkages. For example, in some embodiments, the siRNA molecule includes modified inter-nucleotide linkages at the 5' or 3' end of the siRNA molecule, in the first, second, and / or (by way of example, and) third internucleoside linkages.

[0284] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, the modified inter-nucleoside linkage is a phosphorus-containing linkage. In some embodiments, phosphorus-containing linkages that may be used include, but are not limited to, the following: phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'-amino phosphoramidate and aminoalkyl phosphoramidate, thiono phosphoramidate, thionoalkyl phosphonate, thionoalkyl phosphotriester, and borano phosphate, 2'-5'-linked analogs thereof, and those having reversed polarity, where the pair of adjacent nucleoside units are linked 3'-5' to 5'-3', or 2'-5' to 5'-2'; see 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; 5,399,676; 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.

[0285] Any of the modified chemistries or formats of the siRNA molecules described herein can be combined with each other. For example, 1, 2, 3, 4, 5, or more different types of modifications can be included within the same siRNA molecule.

[0286] 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 nucleotide). In some embodiments, the antisense strand comprises one or more 2'-modified nucleotides, e.g., 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'-O-N-methylacetamide (2'-O-NMA). In some embodiments, each nucleotide of the antisense strand is a modified nucleotide (e.g., 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 comprises one or more 2'-O-methyl and 2'-F modified nucleotides. In some embodiments, the antisense strand comprises one or more modified nucleosides (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 nucleosides and / or (e.g., and) one or more modified internucleoside linkages. In some embodiments, the modified nucleoside comprises a modified sugar moiety (e.g., 2'-modified nucleoside).In some embodiments, the antisense strand comprises one or more 2'-modified nucleosides, such as, for example, 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA). In some embodiments, each nucleoside of the antisense strand is a modified nucleoside (such as, for example, a 2'-modified nucleoside). In some embodiments, the antisense strands described herein comprise one or more 2'-F modified nucleosides. In some embodiments, the antisense strands described herein comprise at least two 2'-F modified nucleosides. In some embodiments, the antisense strands described herein comprise at least four 2'-F modified nucleosides. In some embodiments, the antisense strands described herein comprise at least six 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the antisense strands described herein comprise one or more 2'-O-methyl modified nucleosides. In some embodiments, the antisense strand comprises one or more 2'-O-methyl and 2'-F modified nucleosides.

[0287] In some embodiments, the antisense strand contains phosphorothioate or other modified internucleotide linkages. In some embodiments, the antisense strand contains phosphorothioate or other modified nucleoside linkages. In some embodiments, the antisense strand contains phosphorothioate internucleoside linkages. In some embodiments, the antisense strand contains phosphorothioate inter-nucleotide linkages. In some embodiments, the antisense strand contains phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the antisense strand contains phosphorothioate internucleoside linkages between at least two nucleosides. In some embodiments, the antisense strand contains phosphorothioate inter-nucleotide linkages between at least two nucleotides. In some embodiments, the antisense strand contains phosphorothioate internucleoside linkages between all nucleotides. In some embodiments, the antisense strand contains phosphorothioate internucleoside linkages between all nucleosides. For example, in some embodiments, the antisense strand contains modified internucleoside linkages at the 5' or 3' end of the siRNA molecule, at the first, second, and / or (by way of example, and) third internucleoside linkages. In some embodiments, the internucleoside linkage between two nucleosides at the 3' end of the antisense strand is a phosphorothioate internucleoside linkage. In some embodiments, the antisense strand contains phosphorothioate inter-nucleotide linkages between all nucleotides. For example, in some embodiments, the antisense strand contains modified inter-nucleotide linkages at the 5' or 3' end of the siRNA molecule, at the first, second, and / or (by way of example, and) third inter-nucleotide linkages. In some embodiments, the inter-nucleotide linkage between two nucleotides at the 3' end of the antisense strand is a phosphorothioate inter-nucleotide linkage. For example, in some embodiments, the antisense strand contains modified inter-nucleotide linkages at the 5' or 3' end of the siRNA molecule, at the first, second, and / or (by way of example, and) third internucleoside linkages.In some embodiments, the internucleoside linkage at the 3' end of the antisense strand is a phosphorothioate internucleoside linkage.

[0288] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, the modified internucleoside linkage is a phosphorus-containing linkage. In some embodiments, phosphorus-containing linkages that may be used include, but are not limited to, the following: phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkyl phosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkyl phosphoramidate, thionophosphoramidate, thionoalkyl phosphonate, thionoalkyl phosphotriester, and boranophosphate, analogs thereof linked 2'-5', and those having reversed polarity, where the pair of adjacent nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see 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; 5,399,676; 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.

[0289] Any of the modified chemistries or formats of the antisense strand described herein can be combined with each other. For example, 1, 2, 3, 4, 5, or more different types of modifications can be incorporated within the same antisense strand.

[0290] 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 nucleotide comprises a modified sugar moiety (e.g., a 2'-modified nucleotide). In some embodiments, the sense strand comprises one or more 2'-modified nucleotides, e.g., 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'-O-N-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 modified nucleosides (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 internucleoside linkages. In some embodiments, the modified nucleoside comprises a modified sugar moiety (e.g., a 2'-modified nucleoside). In some embodiments, the sense strand comprises one or more 2'-modified nucleosides, e.g., 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'-O-N-methylacetamide (2'-O-NMA). In some embodiments, each nucleoside of the sense strand is a modified nucleoside (e.g., a 2'-modified nucleoside).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.

[0291] In some embodiments, the sense strands described herein comprise one or more 2'-F modified nucleosides. In some embodiments, the sense strands described herein comprise at least two 2'-F modified nucleosides. In some embodiments, the sense strands described herein comprise at least four 2'-F modified nucleosides. In some embodiments, the sense strands described herein comprise at least six 2'-F modified nucleosides. In some embodiments, the sense strands described herein comprise one or more 2'-O-methyl modified nucleosides. In some embodiments, the sense strand comprises one or more 2'-O-methyl and 2'-F modified nucleosides.

[0292] In some embodiments, the sense strand comprises phosphorothioate or other modified internucleotide linkages. In some embodiments, the sense strand comprises phosphorothioate or other modified inter-nucleoside linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises phosphorothioate inter-nucleotide 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 at least two nucleosides. In some embodiments, the sense strand comprises phosphorothioate inter-nucleotide linkages between at least two nucleotides. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between all nucleotides. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between all nucleosides. For example, in some embodiments, the sense strand comprises modified internucleoside linkages at the 5' or 3' end of the sense strand, in the first, second, and / or (by way of example, and) third internucleoside linkages. In some embodiments, the sense strand comprises phosphorothioate inter-nucleotide linkages between all nucleotides. For example, in some embodiments, the sense strand comprises modified inter-nucleotide linkages at the 5' or 3' end of the sense strand, in the first, second, and / or (by way of example, and) third inter-nucleotide linkages. For example, in some embodiments, the sense strand comprises modified inter-nucleotide linkages at the 5' or 3' end of the sense strand, in the first, second, and / or (by way of example, and) third internucleoside linkages. In some embodiments, the sense strand comprises phosphodiester internucleoside linkages. In some embodiments, the sense strand does not comprise phosphorothioate internucleoside linkages. In some embodiments, the modified internucleotide linkages are phosphorus-containing linkages. In some embodiments, the modified inter-nucleoside linkages are phosphorus-containing linkages.In some embodiments, phosphor-containing linkages that may be used include, but are not limited to, the following: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates, 2'-5'-linked analogs thereof, and those having inverted polarity, wherein the pair of adjacent nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see 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; 5,399,676; 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.

[0293] Any of the modified chemistries or formats of the sense strand described herein can be combined with each other. For example, one, two, three, four, five, or more different types of modifications can be included within the same sense strand.

[0294] In some embodiments, the antisense or sense strand of the siRNA molecule comprises a modification that enhances or reduces the loading of the RNA-induced silencing complex (RISC). In some embodiments, the antisense strand of the siRNA molecule comprises a modification that enhances the loading of RISC. In some embodiments, the sense strand of the siRNA molecule comprises a modification that reduces the loading of RISC 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 (incorporated by reference in its entirety), the addition of a 2'-O-methoxyethyl (2'-MOE) group at the cleavage site improves both the specificity and silencing activity of the siRNA by facilitating the loading of the modified strand into the oriented RNA-induced silencing complex (RISC). In some embodiments, as described in Wu et al., (2014) Nat Commun 5:3459 (incorporated by reference in its entirety), the antisense strand of the siRNA molecule comprises a 2'-OMe-phosphorodithioate modification, which increases the loading of RISC.

[0295] In some embodiments, the sense strand of the siRNA molecule comprises a 5'-morpholino, which reduces the loading of the sense strand into RISC and improves the selection of the antisense strand and the activity of RNAi, as described in Kumar et al., (2019) Chem Commun (Camb) 55(35):5139-5142 (incorporated by reference in its entirety). In some embodiments, the sense strand of the siRNA molecule is modified with locked nucleic acid (LNA), a synthetic RNA-like high-affinity nucleotide analog, which reduces the loading of the sense strand into RISC and further enhances the incorporation of the antisense strand into RISC, as described in Elman et al., (2005) Nucleic Acids Res. 33(1): 439-447 (incorporated by reference in its entirety). In some embodiments, the sense strand of the siRNA molecule comprises a 5'-unlocked nucleic acid (UNA) modification, which reduces the loading of the sense strand into RISC and improves the silencing efficacy of the antisense strand, as described in Snead et al., (2013) Mol Ther Nucleic Acids 2(7):e103 (incorporated by reference in its entirety). In some embodiments, the sense strand of the siRNA molecule comprises a 5-nitroindole modification, which reduces the RNAi efficacy of the sense strand and reduces the off-target effect, as described in Zhang et al., (2012) Chembiochem 13(13):1940-1945 (incorporated by reference in its entirety). In some embodiments, the sense strand comprises a 2'-O-methyl (2'-O-Me) modification, which reduces the loading of the sense strand into RISC and the off-target effect, as described in Zheng et al., FASEB (2013) 27(10): 4017-4026 (incorporated by reference in its entirety).In some embodiments, the sense strand of the siRNA molecule is completely 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 (incorporated by reference in its entirety). In some embodiments, the antisense strand of the siRNA molecule contains 2'-MOE modifications and the sense strand contains 2'-O-Me modifications (see, for example, Song et al., (2017) Mol Ther Nucleic Acids 9:242-250). In some embodiments, at least 1 (for example, at least 2, at least 3, at least 4, at least 5, at least 10) siRNA molecules are linked (for example, covalently) to a muscle targeting agent. In some embodiments, the muscle targeting agent may comprise or consist of a nucleic acid (for example, DNA or RNA), a peptide (for example, an antibody), a lipid (for example, a microvesicle), or a sugar moiety (for example, 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 (for example, any one of the anti-TfR1 antibodies provided in Tables 2-7). In some embodiments, the muscle targeting agent may be linked to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be covalently linked to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be linked to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be covalently linked to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be linked internally to the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be covalently linked internally to the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be linked to the 5' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be covalently linked to the 5' end of the antisense strand of the siRNA molecule.In some embodiments, the muscle targeting agent may be linked to the 3' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be covalently linked to the 3' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be linked internally to the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be covalently linked internally to the antisense strand of the siRNA molecule.

[0296] Non-limiting examples of siRNAs described herein are provided in Table 8. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6] [Table 8-7] [Table 8-8] [Table 8-9] [Table 8-10] [Table 8-11] [Table 8-12]

Table 8-13

Table 8-14

Table 8-15

Table 8-16

Table 8-17

Table 8-18

Table 8-19

Table 8-20

Table 8-21

Table 9-1

Table 9-2

[0297] In some embodiments, the oligonucleotides described herein include an antisense strand that is 18 to 25 nucleosides in length (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) and includes a region of complementarity to a target sequence as set forth in any one of SEQ ID NOs: 174-235, 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 23 nucleotides in length and includes a region of complementarity to a target sequence as set forth in any one of SEQ ID NOs: 174-235, wherein the region of complementarity is 20 nucleotides in length. In some embodiments, the region of complementarity is completely complementary to all or part of its target sequence. In some embodiments, the region of complementarity encompasses 1, 2, 3, or more mismatches.

[0298] In some embodiments, the oligonucleotides described herein include an antisense strand that includes 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: 236-266. In some embodiments, the oligonucleotides described herein further include a sense strand that includes 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: 205-235.

[0299] In some embodiments, the oligonucleotides described herein include an antisense strand that includes any one of the nucleotide sequences of SEQ ID NOs: 236-266. In some embodiments, the oligonucleotides described herein further include a sense strand that includes any one of the nucleotide sequences of SEQ ID NOs: 205-235.

[0300] In some embodiments, the oligonucleotides described herein are double-stranded oligonucleotides (e.g., siRNA) comprising an antisense strand comprising any one of the nucleotide sequences of SEQ ID NOs: 236-266 and a sense strand that hybridizes to the antisense strand and comprises any one of the nucleotide sequences of SEQ ID NOs: 205-235, wherein the antisense strand and / or (e.g., and) comprises one or more modified nucleosides (e.g., 2'-modified nucleosides). In some embodiments, the one or more modified nucleosides are selected from 2'-O-Me and 2'-F modified nucleosides.

[0301] In some embodiments, the oligonucleotides described herein are double-stranded oligonucleotides (e.g., siRNA) comprising an antisense strand comprising any one of the nucleotide sequences of SEQ ID NOs: 236-266 and a sense strand that hybridizes to the antisense strand and comprises any one of the nucleotide sequences of SEQ ID NOs: 205-235, 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.

[0302] In some embodiments, the oligonucleotides described herein are double-stranded oligonucleotides (e.g., siRNA) comprising an antisense strand comprising any one of the nucleotide sequences of SEQ ID NOs: 236-266 and a sense strand that hybridizes to the antisense strand and comprises any one of the nucleotide sequences of SEQ ID NOs: 205-235, wherein each nucleoside in the antisense strand and each nucleoside in the sense strand are 2'-modified nucleosides selected from 2'-O-Me and 2'-F modified nucleosides, and wherein the antisense strand and / or (e.g., and) the sense strand each comprise one or more phosphorothioate internucleoside linkages. In some embodiments, the sense strand does not contain any phosphorothioate internucleoside linkages (all internucleoside linkages in the sense strand are phosphodiester internucleoside linkages), and the antisense strand contains 1, 2, or 3 phosphorothioate internucleoside linkages. In some embodiments, the sense strand contains 2 phosphorothioate internucleoside linkages, and the antisense strand contains 4 phosphorothioate internucleoside linkages. In some embodiments, the sense strand contains 4 phosphorothioate internucleoside linkages, and the antisense strand contains 4 phosphorothioate internucleoside linkages.

[0303] In some embodiments, the antisense strand comprises two phosphorothioate internucleoside linkages, and optionally here, the internucleoside linkage between two nucleosides at the 3' end of the antisense strand is a phosphorothioate internucleoside linkage, and the remainder of the internucleoside linkages in the antisense strand are phosphodiester internucleoside linkages. In some embodiments, the antisense strand comprises four phosphorothioate internucleoside linkages, and optionally here, the internucleoside linkages between two nucleosides at the 3' end and the 5' end of the antisense strand are phosphorothioate internucleoside linkages, and the remainder of the internucleoside linkages in the antisense strand are phosphodiester internucleoside linkages. In some embodiments, the sense strand comprises two phosphorothioate internucleoside linkages, and optionally here, the internucleoside linkage between two nucleosides at the 5' end of the sense strand is a phosphorothioate internucleoside linkage, and the remainder of the internucleoside linkages in the sense strand are phosphodiester internucleoside linkages. In some embodiments, the sense strand comprises four phosphorothioate internucleoside linkages. In some embodiments, the internucleoside linkages between two nucleosides at the 5' end and the 3' end of the sense strand are phosphorothioate internucleoside linkages, and the remainder of the internucleoside linkages in the sense strand are phosphodiester internucleoside linkages.

[0304] In some embodiments, the antisense strand of the oligonucleotide described herein has the following structure (from 5' to 3'; referred to herein as "MAS1"): fN*fN*mNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmN*fN*mN comprising, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the antisense strands MAS1-236, MAS1-237, and MAS1-238 contain this structure.

[0305] In some embodiments, the antisense strand of the oligonucleotide described herein has the following structure (from 5' to 3'; referred to herein as "MAS2"): fN*fN*mNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmN*mN*mN comprising, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the antisense strands MAS2-236, MAS2-237, MAS2-238, MAS2-239, MAS2-240, MAS2-241, MAS2-242, MAS2-243, MAS2-244, MAS2-245, MAS2-246, MAS2-247, MAS2-248, MAS2-249, MAS2-250, MAS2-251, MAS2-252, MAS2-253, MAS2-254, MAS2-255, MAS2-256, and MAS2-257 contain this structure.

[0306] In some embodiments, the antisense strand of the oligonucleotide described herein has the following structure (from 5' to 3'; referred to herein as "MAS3"): fN*fN*mNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmN*mN*mN comprising, wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the antisense strands MAS3-236, MAS3-237, MAS3-238, MAS3-239, MAS3-240, MAS3-241, MAS3-242, MAS3-243, MAS3-244, MAS3-245, MAS3-246, MAS3-247, MAS3-248, MAS3-249, MAS3-250, MAS3-251, MAS3-252, MAS3-253, MAS3-254, MAS3-255, MAS3-256, MAS3-257, MAS3-258, MAS3-259, MAS3-260, MAS3-261, MAS3-262, MAS3-263, MAS3-264, MAS3-265, and MAS3-266 include this structure.

[0307] In some embodiments, the antisense strand of the oligonucleotide described herein has the following structure (5' to 3'; referred to herein as "MAS4"): fN*fN*mNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN comprising, wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the antisense strands MAS4-236, MAS4-237, MAS4-238, MAS4-239, MAS4-240, MAS4-241, MAS4-242, MAS4-243, MAS4-244, MAS4-245, MAS4-246, MAS4-247, MAS4-248, MAS4-249, MAS4-250, MAS4-251, MAS4-252, MAS4-253, MAS4-254, MAS4-255, MAS4-256, and MAS4-257 contain this structure.

[0308] In some embodiments, the antisense strand of the oligonucleotide described herein has the following structure (5' to 3'; referred to herein as "MAS5"): mN*fN*mNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN comprising, wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0309] In some embodiments, the MAS5 structure further comprises a 5'-vinylphosphonate (e.g., 5'-(E)-vinylphosphonate) modification (5' to 3'; referred to herein as "VP-MAS5"): VP- mN*fN*mNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN Here, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage; and, VP represents 5'-(E)-vinylphosphonate. For example, in Table 9, the antisense strand VP-MAS5-248 contains this structure.

[0310] In some embodiments, the antisense strand of the oligonucleotide described herein has the following structure (from 5' to 3'; referred to herein as "MAS6"): mN*fN*mNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0311] In some embodiments, the MAS6 structure further comprises a 5' vinylphosphonate (e.g., 5'-(E)-vinylphosphonate) modification (from 5' to 3'; referred to herein as "VP-MAS6"): VP-mN*fN*mNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage; and, VP represents 5'-(E)-vinylphosphonate. For example, in Table 9, the antisense strands VP-MAS6-251, VP-MAS6-253, and VP-MAS6-248 contain this structure.

[0312] In some embodiments, the antisense strand of the oligonucleotides described herein has the following structure (from 5' to 3'; referred to herein as "MAS7"): mN*fN*mNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmN*mN*mN wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0313] In some embodiments, the MAS7 structure further comprises a 5'-vinylphosphonate (e.g., 5'-(E)-vinylphosphonate) modification (from 5' to 3'; referred to herein as "VP-MAS7"): VP-mN*fN*mNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmN*mN*mN wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage; and VP represents 5'-(E)-vinylphosphonate. For example, in Table 9, the antisense strands VP-MAS7-252 and VP-MAS7-262 have this structure.

[0314] In some embodiments, the sense strand of the oligonucleotides described herein has the following structure (from 5' to 3'; referred to herein as "MS1"): mN*mN*fNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfN comprising, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, sense strands MS1-205, MS1-206, and MS1-207 contain this structure.

[0315] In some embodiments, the sense strand of the oligonucleotide described herein has the following structure (5' to 3'; referred to herein as "MS2"): mN*mN*mNmNfNmNfNmNfNfNfNmNmNmNmNfNmNmNmNmNmN comprising, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, sense strands MS2-208, MS2-209, MS2-210, MS2-211, MS2-212, MS2-213, MS2-214, MS2-215, MS2-216, MS2-217, MS2-218, MS2-219, MS2-220, MS2-221, MS2-222, MS2-223, MS2-224, MS2-225, and MS2-226 contain this structure.

[0316] In some embodiments, the sense strand of the oligonucleotide described herein has the following structure (5' to 3'; referred to herein as "MS3"): mN*mN*mNmNfNmNmNmNfNfNfNmNmNfNmNmNmNmNfNmNmN including, where "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, sense strands MS3-205, MS3-206, MS3-207, MS3-208, MS3-209, MS3-210, MS3-211, MS3-212, MS3-213, MS3-214, MS3-215, MS3-216, MS3-217, MS3-218, MS3-219, MS3-220, MS3-221, MS3-222, MS3-223, MS3-224, MS3-225, MS3-226, MS3-227, MS3-228, MS3-229, MS3-230, MS3-231, MS3-232, MS3-233, MS3-234, and MS3-235 include this structure.

[0317] In some embodiments, the sense strand of the oligonucleotide described herein has the following structure (5' to 3'; referred to herein as "MS4"): mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmN including, where "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, sense strands MS4-205, MS4-206, MS4-207, MS4-208, MS4-209, MS4-210, MS4-211, MS4-212, MS4-213, MS4-214, MS4-215, MS4-216, MS4-217, MS4-218, MS4-219, MS4-220, MS4-221, MS4-222, MS4-223, MS4-224, MS4-225, and MS4-226 include this structure.

[0318] In some embodiments, the sense strand of the oligonucleotides described herein has the following structure (5' to 3'; referred to herein as "MS5"): mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNfNfNmNmNmNmN wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the sense strands MS5-205, MS5-206, MS5-207, MS5-208, MS5-209, MS5-210, MS5-211, MS5-212, MS5-213, MS5-214, MS5-215, MS5-216, MS5-217, MS5-218, MS5-219, MS5-220, MS5-221, MS5-222, MS5-223, MS5-224, MS5-225, and MS5-226 include this structure.

[0319] In some embodiments, the sense strand of the oligonucleotides described herein includes the following structure (5' to 3'; referred to herein as "MS6"):

[0320] mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNfNmNmNfNmNmN Here, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, sense strands MS6-205, MS6-206, MS6-207, MS6-208, MS6-209, MS6-210, MS6-211, MS6-212, MS6-213, MS6-214, MS6-215, MS6-216, MS6-217, MS6-218, MS6-219, MS6-220, MS6-221, MS6-222, MS6-223, MS6-224, MS6-225, and MS6-226 contain this structure.

[0321] In some embodiments, the sense strand of the oligonucleotide described herein has the following structure (5' to 3'; referred to herein as "MS7"): mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmN*mN*mN wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 9, sense strands MS7-220, MS7-222, and MS7-217 contain this structure.

[0322] In some embodiments, the sense strand of the oligonucleotide described herein has the following structure (5' to 3'; referred to herein as "MS8"): mN*mN*mNmNfNmNmNmNfNfNfNmNmNfNmNmNmNmNfN*mN*mN comprising, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 9, sense strands MS8-221 and MS8-231 comprise this structure.

[0323] In some embodiments, the sense strand of the oligonucleotide described herein has the following structure (5' to 3'; referred to herein as "MS9"): mN*mN*mNmNfNmNfNmNfNfNfNmNmNmNmNfNmNmNmN*mN*mN comprising, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 9, sense strand MS9-217 comprises this structure.

[0324] In some embodiments, the antisense strand of the oligonucleotides described herein is selected from the modified versions of SEQ ID NOs: 236-266 listed in Table 8 (e.g., MAS1-MAS4). For example, in some embodiments, the antisense strand is selected from: MAS1-236, MAS1-237, MAS1-238, MAS2-236, MAS2-237, MAS2-238, MAS2-239, MAS2-240, MAS2-241, MAS2-242, MAS2-243, MAS2-244, MAS2-245, MAS2-246, MAS2-247, MAS2-248, MAS2-249, MAS2-250, MAS2-251, MAS2-252, MAS2-253, MAS2-254, MAS2-255, MAS2-256, MAS2-257, MAS3-236, MAS3-237, MAS3-238, MAS3-239, MAS3-240, MAS3-241, MAS3-242, MAS3-243, MAS3-244, MAS3-245, MAS3-246, MAS3-247, MAS3-248, MAS3-249, MAS3-250, MAS3-251, MAS3-252, MAS3-253, MAS3-254, MAS3-255, MAS3-256, MAS3-257, MAS3-258, MAS3-259, MAS3-260, MAS3-261, MAS3-262, MAS3-263, MAS3-264, MAS3-265, MAS3-266, MAS4-236, MAS4-237, MAS4-238, MAS4-239, MAS4-240, MAS4-241, MAS4-242, MAS4-243, MAS4-244, MAS4-245, MAS4-246, MAS4-247, MAS4-248, MAS4-249, MAS4-250, MAS4-251, MAS4-252, MAS4-253, MAS4-254, MAS4-255, MAS4-256, and MAS4-257.

[0325] In some embodiments, the antisense strand of the oligonucleotides described herein is selected from the modified versions (e.g., MAS5 - MAS7) of SEQ ID NOs: 236 - 266 listed in Table 8. In some embodiments, the antisense strand of the oligonucleotides described herein is selected from the modified versions (e.g., MAS5, MAS6, MAS7, VP - MAS5, VP - MAS6, and VP - MAS7) of SEQ ID NOs: 251, 253, 262, 248, and 252 listed in Table 9. For example, in some embodiments, the antisense strand is selected from VP - MAS6 - 251, VP - MAS7 - 252, VP - MAS6 - 253, VP - MAS7 - 262, VP - MAS6 - 248, and VP - MAS5 - 248.

[0326] In some embodiments, the sense strand of the oligonucleotides described herein is selected from modified versions of SEQ ID NOs: 205-235 (e.g., MS1-MS6) listed in Table 8.For example, in some embodiments, the sense strand is selected from: MS1-205, MS1-206, MS1-207, MS2-208, MS2-209, MS2-210, MS2-211, MS2-212, MS2-213, MS2-214, MS2-215, MS2-216, MS2-217, MS2-218, MS2-219, MS2-220, MS2-221, MS2-222, MS2-223, MS2-224, MS2-225, MS2-226, MS3-205, MS3-206, MS3-207, MS3-208, MS3-209, MS3-210, MS3-211, MS3-212, MS3-213, MS3-214, MS3-215, MS3-216, MS3-217, MS3-218, MS3-219, MS3-220, MS3-221, MS3-222, MS3-223, MS3-224, MS3-225, MS3-226, MS3-227, MS3-228, MS3-229, MS3-230, MS3-231, MS3-232, MS3-233, MS3-234, MS3-235, MS4-205, MS4-206, MS4-207, MS4-208, MS4-209, MS4-210, MS4-211, MS4-212, MS4-213, MS4-214, MS4-215, MS4-216, MS4-217, MS4-218, MS4-219, MS4-220, MS4-221, MS4-222, MS4-223, MS4-224, MS4-225, MS4-226, MS5-205, MS5-206, MS5-207, MS5-208, MS5-209, MS5-210, MS5-211, MS5-212, MS5-213, MS5-214, MS5-215, MS5-216, MS5-217, MS5-218, MS5-219, MS5-220, MS5-221, MS5-222, MS5-223, MS5-224, MS5-225, MS5-226, MS6-205, MS6-206, MS6-207, MS6-208, MS6-209, MS6-210, MS6-211, MS6-212, MS6-213, MS6-214, MS6-215, MS6-216, MS6-217, MS6-218, MS6-219, MS6-220, MS6-221, MS6-222, MS6-223, MS6-224, MS6-225, and MS6-226.

[0327] In some embodiments, the sense strand of the oligonucleotides described herein is selected from the modified versions (e.g., MS7 - MS9) of SEQ ID NOs: 205 - 235 listed in Table 8. In some embodiments, the sense strand of the oligonucleotides described herein is selected from the modified versions (e.g., MS7 - MS9) of SEQ ID NOs: 220, 222, 231, 217, and 221 listed in Table 9. For example, in some embodiments, the sense strand is selected from MS7 - 220, MS8 - 221, MS7 - 222, MS8 - 231, MS9 - 217, and MS7 - 217.

[0328] In some embodiments, the oligonucleotides described herein may include an antisense strand containing any one of the antisense strand structures described herein (e.g., MAS1 - MAS4 regardless of its nucleobase sequence) and a sense strand containing any one of the sense strand structures described herein (e.g., MS1 - MS6 regardless of its nucleobase sequence). In some embodiments, the oligonucleotides described herein may include an antisense strand containing any one of the antisense strand structures described herein (e.g., MAS5, MAS6, MAS7, VP - MAS5, VP - MAS6, and VP - MAS7 regardless of its nucleobase sequence) and a sense strand containing any one of the sense strand structures described herein (e.g., MS7 - MS9 regardless of its nucleobase sequence).

[0329] In some embodiments, the oligonucleotides described herein include an antisense strand having the following structure (5' to 3'; MAS3): fN*fN*mNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS3) comprising the following structure: mN*mN*mNmNfNmNmNmNfNfNfNmNmNfNmNmNmNmNfNmNmN wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0330] In some embodiments, the oligonucleotides described herein comprise an antisense strand (5' to 3'; MAS4) having the following structure: fN*fN*mNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS4) having the following structure: mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmN wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0331] In some embodiments, the oligonucleotides described herein comprise an antisense strand (5' to 3'; MAS4) having the following structure: fN*fN*mNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS6) having the following structure: mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNfNmNmNfNmNmN comprising, wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0332] In some embodiments, the oligonucleotides described herein comprise an antisense strand (5' to 3'; MAS2) having the following structure: fN*fN*mNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS5) having the following structure: mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNfNfNmNmNmNmN comprising, wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0333] In some embodiments, the oligonucleotides described herein comprise an antisense strand (5' to 3'; MAS2) having the following structure: fN*fN*mNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS4) having the following structure: mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmNmNmN comprising, wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0334] In some embodiments, the oligonucleotides described herein include an antisense strand (5' to 3'; MAS1) having the following structure: fN*fN*mNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmN*fN*mN and a sense strand (5' to 3'; MS1) having the following structure: mN*mN*fNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfN wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0335] In some embodiments, the oligonucleotides described herein include an antisense strand (5' to 3'; MAS4) having the following structure: fN*fN*mNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS2) having the following structure: mN*mN*mNmNfNmNfNmNfNfNfNmNmNmNmNfNmNmNmNmNmN wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0336] In some embodiments, the oligonucleotides described herein include an antisense strand (5' to 3'; MAS5) having the following structure: mN*fN*mNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS7) comprising the following structure: mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmN*mN*mN comprising, wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0337] In some embodiments, the oligonucleotides described herein include an antisense strand (5' to 3'; MAS6) having the following structure: mN*fN*mNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS7) comprising the following structure: mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmN*mN*mN comprising, wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0338] In some embodiments, the oligonucleotides described herein include an antisense strand (5' to 3'; MAS7): mN*fN*mNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS8): mN*mN*mNmNfNmNmNmNfNfNfNmNmNfNmNmNmNmNfN*mN*mN comprising, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0339] In some embodiments, the oligonucleotides described herein comprise an antisense strand (5' to 3'; MAS6) having the following structure: mN*fN*mNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS9) having the following structure:

[0340] mN*mN*mNmNfNmNfNmNfNfNfNmNmNmNmNfNmNmNmN*mN*mN comprising, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. In some embodiments, the oligonucleotides described herein comprise an antisense strand (5' to 3'; VP-MAS5) having the following structure: VP-mN*fN*mNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS7) having the following structure: mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmN*mN*mN comprising, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0341] In some embodiments, the oligonucleotides described herein include an antisense strand (5' to 3'; VP-MAS6) having the following structure: VP-mN*fN*mNmNmNmNfNmNfNfNmNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS7) having the following structure: mN*mN*mNmNmNmNfNmNfNfNfNmNmNmNmNmNmNmNmN*mN*mN wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0342] In some embodiments, the oligonucleotides described herein include an antisense strand (5' to 3'; VP-MAS7) having the following structure: VP-mN*fN*mNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS8): mN*mN*mNmNfNmNmNmNfNfNfNmNmNfNmNmNmNmNfN*mN*mN wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0343] In some embodiments, the oligonucleotides described herein include an antisense strand (5' to 3'; VP-MAS6) having the following structure: VP-mN*fN*mNmNmNmNfNmNfNfNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN and a sense strand (5' to 3'; MS9) comprising the following structure:

[0344] mN*mN*mNmNfNmNfNmNfNfNfNmNmNmNmNfNmNmNmN*mN*mN comprising, wherein, "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. In some embodiments, the oligonucleotides described herein are siRNAs selected from the siRNAs listed in Table 8. In some embodiments, the oligonucleotides described herein are siRNAs selected from the siRNAs listed in Table 9.

[0345] In some embodiments, any one of the oligonucleotides described herein (e.g., an siRNA selected from the siRNAs listed in Table 8) can be in the form of a salt (e.g., sodium, potassium, magnesium salts). In some embodiments, any one of the oligonucleotides described herein (e.g., an siRNA selected from the siRNAs listed in Table 9) can be in the form of a salt (e.g., sodium, potassium, magnesium salts).

[0346] In some embodiments, any one of the 5' or 3' nucleosides (e.g., the terminal nucleoside) of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8) is optionally conjugated to an amine group via a spacer. In some embodiments, any one of the 5' or 3' nucleosides (e.g., the terminal nucleoside) of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 9) is optionally conjugated to an amine group via a spacer. In some embodiments, the spacer comprises an aliphatic moiety. In some embodiments, the spacer comprises a polyethylene glycol moiety. In some embodiments, a phosphodiester linkage is present between the spacer of the oligonucleotide and the 5' or 3' nucleoside. In some embodiments, any one of the 5' or 3' nucleosides (e.g., the terminal nucleoside) of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8) is conjugated to a spacer, wherein the spacer is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)2NR A -, -NR A S(O)2-, or a combination thereof; each R Ais, independently, hydrogen, or a substituted or unsubstituted alkyl. In some embodiments, any 5' or 3' nucleoside (e.g., the terminal nucleoside) of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 9) is conjugated to a spacer, and the spacer is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)2NR A -, -NR A S(O)2-, or a combination thereof; each R A is, independently, hydrogen, or a substituted or unsubstituted alkyl. In one embodiment, 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.

[0347] In some embodiments, any one 5' or 3' nucleoside of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8, sense or antisense strand) is of the formula -NH2-(CH2) n- (wherein n is an integer from 1 to 12) is conjugated to a compound represented by. In some embodiments, the 5' or 3' nucleoside of any one of the oligonucleotides described herein (for example, the oligonucleotides listed in Table 9, sense or antisense strand) is of the formula -NH2-(CH2) n - (wherein n is an integer from 1 to 12) is conjugated to a compound represented by. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, n is 6. In some embodiments, the phosphodiester linkage is of the formula NH2-(CH2) n - between a compound of and the 5' or 3' nucleoside of an oligonucleotide (for example, the oligonucleotides listed in Table 8, sense or antisense strand), where n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In certain embodiments, n is 6.

[0348] In some embodiments, the 5' nucleoside of the sense strand of any one of the oligonucleotides described herein (for example, the oligonucleotides listed in Table 8) is of the formula NH2-(CH2) n - conjugated to a compound of, where n is an integer from 1 to 12 (for example, 6).

[0349] In some embodiments, the 5' nucleoside of the sense strand of any one of the oligonucleotides described herein (for example, the oligonucleotides listed in Table 9) is of the formula NH2-(CH2) n - conjugated to a compound of, where n is an integer from 1 to 12 (for example, 6).

[0350] In some embodiments, the 3' nucleoside of the sense strand of any one of the oligonucleotides described herein (for example, the oligonucleotides listed in Table 8) is of the formula NH2-(CH2) n- is conjugated to a compound, where n is an integer from 1 to 12 (for example, 6).

[0351] In some embodiments, the 3'-nucleoside of the sense strand of any one of the oligonucleotides described herein (for example, the oligonucleotides listed in Table 9) is of the formula NH2-(CH2) n - is conjugated to a compound, where n is an integer from 1 to 12 (for example, 6).

[0352] In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5'-phosphate of the oligonucleotide (e.g., the 5'-phosphate of the sense or antisense strand). In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5'-phosphate of the sense strand of an oligonucleotide (e.g., the oligonucleotides listed in Table 8). In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5'-phosphate of the sense strand of an oligonucleotide (e.g., the oligonucleotides listed in Table 9). In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5'-phosphate of the antisense strand of an oligonucleotide (e.g., the oligonucleotides listed in Table 8). In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5'-phosphate of the antisense strand of an oligonucleotide (e.g., the oligonucleotides listed in Table 9). In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3'-phosphate of the oligonucleotide (e.g., the 3'-phosphate of the sense or antisense strand). In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3'-phosphate of the sense strand of an oligonucleotide (e.g., the oligonucleotides listed in Table 8).In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3'-phosphate of the sense strand of an oligonucleotide (e.g., the oligonucleotides listed in Table 9). In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3'-phosphate of the antisense strand of an oligonucleotide (e.g., the oligonucleotides listed in Table 8). In some embodiments, a compound of the formula NH2-(CH2)6- is conjugated to an oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3'-phosphate of the antisense strand of an oligonucleotide (e.g., the oligonucleotides listed in Table 9). In some embodiments, the oligonucleotide is conjugated to a targeting agent, e.g., a muscle targeting agent such as an anti-TfR1 antibody, via, e.g., an amine group.

[0353] In some embodiments, the oligonucleotides described herein (e.g., siRNA molecules listed in Table 8 or Table 9) have a reduced off-target effect as compared to, e.g., other known siRNAs targeting DUX4.

[0354] C. Linker The conjugates described herein generally include a linker that covalently links any one of the anti-TfR1 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 covalently links the anti-TfR1 antibody to the molecular payload. However, in some embodiments, the linker may covalently link any one of the anti-TfR1 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 typically stable in vitro and in vivo and may be stable in a particular cellular environment. In addition, typically, the linker does not negatively affect the functional properties of either the anti-TfR antibody or the molecular payload. Examples and methods of linker synthesis are known in the art (see, for example, Kline, T. et al. "Methods to Make Homogenous Antibody Drug Conjugates." Pharmaceutical Research, 2015, 32:11, 3480-3493.; Jain, N. et al. "Current ADC Linker Chemistry" Pharm Res. 2015, 32:11, 3526-3540.; McCombs, J.R. and Owen, S.C. "Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry" AAPS J. 2015, 17:2, 339-351.).

[0355] The linker will typically contain two different reactive species that enable attachment to both the anti-TfR1 antibody and the molecular payload. In some embodiments, the two different reactive species may be nucleophiles and / or electrophiles. In some embodiments, the linker contains two different electrophiles or nucleophiles that are specific for two different nucleophiles or electrophiles. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody via conjugation to a lysine residue or cysteine residue of the anti-TfR1 antibody. In some embodiments, the linker is covalently linked to a cysteine residue of the anti-TfR1 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 covalently linked to a cysteine residue of the anti-TfR1 antibody or a thiol-functionalized molecular payload via a 3-arylpropionitrile functional group. In some embodiments, the linker is covalently linked to a lysine residue of the anti-TfR1 antibody. In some embodiments, the linker is independently covalently linked to the anti-TfR1 antibody and / or (by way of example, and) the molecular payload via an amine bond, a carbamate bond, a hydrazide, a triazole, a thioether, and / or a disulfide bond.

[0356] i. Cleavable linker The cleavable linker may be a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. These linkers are typically cleavable only intracellularly and are preferably stable in the extracellular environment, for example, extracellular to muscle cells.

[0357] Protease-sensitive linkers are cleavable by protease enzyme activity. These linkers typically contain peptide sequences and may be 2 to 10 amino acids in length, about 2 to 5 amino acids, about 5 to 10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids. In some embodiments, the peptide sequence may contain 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 contains a valine-citrulline sequence or an alanine-citrulline sequence. In some embodiments, the protease-sensitive linker can be cleaved by proteases in lysosomes, such as cathepsin B, and / or proteases in endosomes.

[0358] pH-sensitive linkers are covalent linkages that are readily degraded in high or low pH environments. 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 contains a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved within endosomes or lysosomes.

[0359] In some embodiments, glutathione-sensitive linkers contain a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with glutathione species inside the cell. In some embodiments, the disulfide moiety further contains at least 1 amino acid, such as a cysteine residue.

[0360] In some embodiments, the linker comprises a valine-citrulline sequence (as described, for example, in U.S. Patent 6,214,345, which is incorporated herein by reference). In some embodiments, prior to conjugation, the linker has the structure:

Chem.

[0361] In some embodiments, after conjugation, the linker has the structure:

Chem.

[0362] In some embodiments, prior to conjugation, the linker has the formula (A):

Chem.

[0363] In some embodiments, the linker has the formula (H):

Chem.

[0364] In some embodiments, the linker has the formula (I):

Chem.

[0365] ii. Non-cleavable linker In some embodiments, a non-cleavable linker may be used. Generally, a non-cleavable linker cannot be readily degraded in a cellular or physiological environment. In some embodiments, a non-cleavable linker contains an optionally substituted alkyl group, where the substitution may include halogen, hydroxyl group, oxygen species, and other common substitutions. In some embodiments, the linker may include an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, heteroarylene, a peptide sequence containing at least one non-natural amino acid, a truncated glycan, a sugar(s) that cannot be enzymatically degraded, azide, alkyne-azide, a peptide sequence containing an LPXT sequence, thioether, biotin, biphenyl, a polyethylene glycol of repeating units or an equivalent compound, an acid ester, an acid amide, a sulfamide, and / or an alkoxy-amine linker. In some embodiments, sortase-mediated ligation can 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).

[0366] In some embodiments, the linker is a substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted cycloalkylene, optionally substituted cycloalkenylene, optionally substituted arylene, optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; imino, optionally substituted nitrogen species, optionally substituted oxygen species O, optionally substituted sulfur species, or poly(alkylene oxide), for example, polyethylene oxide or polypropylene oxide. In some embodiments, the linker may be an uncleavable N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS) linker.

[0367] iii. Linker conjugation In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (for example, and) the molecular payload via a phosphate, thioether, ether, carbon-carbon, carbamate, or amide bond. In some embodiments, the linker is covalently linked to the oligonucleotide through a phosphate or phosphorothioate group, for example, the terminal phosphate of the oligonucleotide backbone. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody through a lysine or cysteine residue present on the anti-TfR1 antibody.

[0368] In some embodiments, the linker, or a portion thereof, is covalently linked to the anti-TfR1 antibody and / or (by way of example, and) the molecular payload by a cycloaddition reaction between an azide and an alkyne that forms a triazole, where the azide or alkyne may be located on the anti-TfR1 antibody, the molecular payload, or the linker. In some embodiments, the alkyne may be a cyclic alkyne, such as cyclooctyne. In some embodiments, the alkyne may be bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctyne is as described in International Patent Application Publication WO2011136645, published November 3, 2011, entitled "Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions". 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 sugar or carbohydrate molecule containing an azide is as described in International Patent Application Publication WO2016170186, published 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 that forms a triazole (where the azide and alkyne may be located on an anti-TfR antibody, a molecular payload, or a linker) is as described in International Patent Application Publication WO2014065661, published May 1, 2014, titled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or International Patent Application Publication WO2016170186, published October 27, 2016, titled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase".

[0369] In some embodiments, the linker further comprises a spacer, such as a polyethylene glycol spacer or an acyl / carbamoyl sulfamide spacer, such as a HydraSpace™ spacer. In some embodiments, the spacer is as described in Verkade, J.M.M. et al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates", Antibodies, 2018, 7, 12.

[0370] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (by way of example, and) the molecular payload by a Diels-Alder reaction between the dienophile and the diene / hetero-diene, where the dienophile or diene / hetero-diene may be located on the anti-TfR1 antibody, the molecular payload, or the linker. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (by way of example, and) the molecular payload by other pericyclic reactions such as an ene reaction. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (by way of example, and) the molecular payload by an amide, thioamide, or sulfonamide bond-forming reaction. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (by way of example, and) the molecular payload by a condensation reaction that forms an oxime, hydrazone, or semicarbazide group present between the linker and the anti-TfR1 antibody and / or (by way of example, and) the molecular payload.

[0371] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) the molecular payload by a conjugate addition reaction between a nucleophile (e.g., an amine or hydroxyl group) and an electrophile (e.g., a carboxylic acid, carbonate, or aldehyde). In some embodiments, prior to the reaction between the linker and the anti-TfR1 antibody or molecular payload, the nucleophile may be present on the linker and the electrophile may be present on the anti-TfR1 antibody or molecular payload. In some embodiments, prior to the reaction between the linker and the anti-TfR1 antibody or molecular payload, the electrophile may be present on the linker and the nucleophile may be present on the anti-TfR1 antibody or molecular payload. In some embodiments, the electrophile may be an azide, pentafluorophenyl, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimidyl ester, sulfosuccinimidyl ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center, and / or activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, optionally substituted alkyne, optionally substituted aryl, optionally substituted heterocyclyl, hydroxyl group, amino group, alkylamino group, anilide group, and / or thiol group.

[0372] In some embodiments, the linker comprises a valine-citrulline sequence covalently linked to a reactive chemical moiety (e.g., an azide moiety or a BCN moiety for click chemistry). In some embodiments, a linker comprising a valine-citrulline sequence covalently linked to a reactive chemical moiety (e.g., an azide moiety for click chemistry) has the formula (A):

Chemical formula

[0373] In some embodiments, a linker comprising the structure of formula (A) is covalently linked (e.g., optionally via an additional chemical moiety) to a molecular payload (e.g., an oligonucleotide). In some embodiments, the molecular payload is a double-stranded siRNA oligonucleotide and the linker comprising the structure of formula (A) is covalently linked (e.g., optionally via an additional chemical moiety) to the sense strand of the siRNA oligonucleotide (e.g., at the 5′-end). In some embodiments, the molecular payload is a double-stranded siRNA oligonucleotide and the linker comprising the structure of formula (A) is covalently linked (e.g., optionally via an additional chemical moiety) to the sense strand of the siRNA oligonucleotide (e.g., at the 3′-end). In some embodiments, the linker comprising the structure of formula (A) is covalently linked to an oligonucleotide, for example, through nucleophilic substitution by an amine-L1-oligonucleotide that forms a carbamate bond, to give a compound having the structure of formula (B):

Chemical formula

Claims

1. A complex comprising a muscle targeting agent covalently linked to an oligonucleotide targeting double homeobox 4 (DUX4) RNA, wherein the oligonucleotide comprises an antisense strand 18 to 25 nucleotides in length and has complementarity to a target sequence as set forth in SEQ ID NOs: 200, 191, 189, 186, 190, 174 - 185, 187, 188, 192 - 199, and 201 - 235, and wherein the complementary region is at least 16 consecutive nucleosides in length, said complex.

2. The complex of claim 1, wherein the muscle targeting agent is an anti-transferrin receptor 1 (TfR1) antibody.

3. The complex of claim 1 or claim 2, wherein the oligonucleotide is an RNAi oligonucleotide.

4. The complex of any one of claims 1 - 3, wherein the antisense strand comprises any one nucleotide sequence of SEQ ID NOs: 262, 253, 251, 262, 248, 252, 236 - 247, 249, 250, 254 - 261, 263 - 266.

5. The complex of any one of claims 1 - 4, wherein the oligonucleotide further comprises a sense strand comprising at least 18 consecutive nucleosides complementary to the antisense strand, and optionally wherein the sense strand comprises 21 consecutive nucleosides complementary to the antisense strand.

6. The complex of claim 5, wherein the sense strand comprises any one nucleotide sequence of SEQ ID NOs: 231, 222, 220, 217, 221, 205 - 216, 218, 219, 223, 230, 232 - 235.

7. The complex of claim 5 or claim 6, wherein the muscle targeting agent is covalently linked to the 5' end or 3' end of the sense strand.

8. The complex of any one of claims 1 - 7, wherein the antisense strand further comprises 5'-(E)-vinylphosphonate.

9. The complex of any one of claims 1 - 8, wherein the oligonucleotide comprises one or more modified nucleosides.

10. The complex according to claim 9, wherein the at least one modified nucleoside is a 2'-modified nucleotide, and optionally, the at least one 2'-modified nucleoside is selected from 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-O-N-methylacetamide (2'-O-NMA).

11. The complex according to claim 10, wherein each 2'-modified nucleotide is 2'-O-methyl (2'-O-Me) or 2'-fluoro (2'-F).

12. The complex according to any one of claims 1 to 11, wherein the oligonucleotide comprises at least one phosphorothioate internucleoside linkage.

13. The complex according to claim 12, wherein the at least one phosphorothioate internucleoside linkage is present on the antisense strand of the oligonucleotide, and optionally, the internucleoside linkage between two nucleosides at the 3'-end of the antisense strand is a phosphorothioate internucleoside linkage, and / or the internucleoside linkage between two nucleosides at the 5'-end of the antisense strand is a phosphorothioate internucleoside linkage.

14. The complex according to claim 12 or claim 13, wherein the at least one phosphorothioate internucleoside linkage is present on the sense strand of the oligonucleotide, and optionally, the internucleoside linkage between two nucleosides at the 3'-end of the sense strand is a phosphorothioate internucleoside linkage, and / or the internucleoside linkage between two nucleosides at the 5'-end of the sense strand is a phosphorothioate internucleoside linkage.

15. The complex according to any one of claims 6 to 14, wherein the antisense strand is selected from the modified versions (e.g., MAS1 to MAS4) of SEQ ID NOs: 236 to 266 listed in Table 8, and / or the sense strand is selected from the modified versions (e.g., MS1 to MS6) of SEQ ID NOs: 205 to 235 listed in Table 8.

16. The antisense strand is selected from the modified versions of SEQ ID NOs: 262, 253, 251, 248, and 252 listed in Table 9 (examples are VP-MAS5, VP-MAS6, and VP-MAS7), and / or herein, the sense strand is selected from the modified versions of SEQ ID NOs: 231, 222, 220, 217, and 221 listed in Table 9 (examples are MS7-MS9), the complex according to any one of claims 6-14.

17. The complex according to any one of claims 1-15, wherein the oligonucleotide is a siRNA molecule selected from the siRNAs listed in Table 8.

18. The complex according to any one of claims 1-14 or 16, wherein the oligonucleotide is a siRNA molecule selected from the siRNAs listed in Table 9.

19. The complex according to any one of claims 2-18, wherein the anti-TfR1 antibody comprises any of the 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 the anti-TfR1 antibodies listed in Table 2.

20. The complex according to any one of claims 2-18, wherein the anti-TfR1 antibody comprises any heavy chain variable region (VH) and light chain variable region (VL) of the anti-TfR1 antibodies listed in Table 3.

21. The complex according to any one of claims 2-18, wherein the anti-TfR1 antibody is a Fab, and optionally herein, the Fab comprises any heavy and light chains of the anti-TfR1 Fabs listed in Table 5.

22. The complex according to any one of claims 2-21, wherein the anti-TfR antibody is as follows: (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 comprising the complex.

23. The complex according to claim 22, wherein the anti-TfR1 antibody comprises VH comprising the amino acid sequence of SEQ ID NO: 76 and VL comprising the amino acid sequence of SEQ ID NO:

75.

24. The complex according to claim 23, wherein the anti-TfR1 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.

25. The complex according to any one of claims 1 to 24, wherein the muscle targeting agent and the antisense oligonucleotide are covalently linked via a linker, optionally wherein the linker comprises a valine-citrulline sequence.

26. A method for reducing DUX4 expression in muscle cells, the method comprising contacting the muscle cells with an effective amount of the complex according to any one of claims 1 to 25 to promote internalization of the oligonucleotide into the muscle cells.

27. The method according to claim 26, wherein reducing DUX4 expression comprises reducing the level of DUX4 protein and / or mRNA.

28. A method for treating facioscapulohumeral muscular dystrophy (FSHD), the method comprising administering to a subject in need thereof an effective amount of the complex according to any one of claims 1 to 25, wherein the subject has abnormal production of DUX4 protein.

29. An oligonucleotide comprising a siRNA oligonucleotide selected from the siRNA oligonucleotides listed in Table 8.

30. An oligonucleotide comprising a siRNA oligonucleotide selected from the siRNA oligonucleotides listed in Table 9.

31. A method for producing a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide, comprising: (i) obtaining a compound comprising the structure of formula (B), wherein the oligonucleotide comprises the sense strand of a siRNA oligonucleotide, and optionally wherein the siRNA oligonucleotide targets DUX4 RNA; (ii) annealing the antisense strand of the siRNA oligonucleotide to the sense strand; (iii) reacting a compound comprising the structure of formula (B) with a compound comprising the structure of formula (C) to obtain a compound comprising the structure of formula (D); and (iv) covalently linking an anti-TfR1 antibody to a compound comprising the structure of formula (D) together with a muscle targeting agent to obtain a compound comprising the structure of formula (E), optionally wherein the annealing of step (ii) is performed at 30 °C, further optionally wherein the method further comprises isolating a compound comprising the structure of formula (D) after step (iii) and before step (iv); further optionally wherein the anti-TfR1 antibody is covalently linked to the 5'-end of the sense strand of a siRNA oligonucleotide targeting DUX4 RNA, said method comprising.

32. A method for producing a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide, comprising: (i) obtaining a compound comprising the structure of formula (B), wherein the oligonucleotide comprises the sense strand of a siRNA oligonucleotide, and optionally wherein the siRNA oligonucleotide targets DUX4 RNA; (ii) annealing the antisense strand of the siRNA oligonucleotide to the sense strand; (iii) covalently linking an anti-TfR1 antibody to a compound comprising the structure of formula (C) to obtain a compound comprising the structure of formula (F); and (iv) reacting a compound comprising the structure of formula (F) with a compound comprising the structure of formula (B) to obtain a compound comprising the structure of formula (E), optionally wherein the annealing of step (ii) is performed at 30 °C, further optionally wherein the anti-TfR1 antibody is covalently linked to the 5'-end of the sense strand of a siRNA oligonucleotide targeting DUX4 RNA, said method comprising.

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