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

Abstract

Aspects of the disclosure relate to oligonucleotides designed to target DUX4 RNA, and targeting conjugates for delivering the oligonucleotides to cells (e.g., muscle cells), and their uses, particularly for treating diseases (e.g., FSHD), where the conjugates include an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured to reduce expression or activity of DUX4.

Description

[Technical field]

[0001] Related Applications This application is a joint venture under 35 U.S.C. § 119(e) with U.S. Provisional Application No. 63 / 278,882, entitled “MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”, filed on November 1, 2021; U.S. Provisional Application No. 63 / 278,993, entitled “TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”, filed on November 12, 2021; U.S. Provisional Application No. 63 / 278,993, entitled “MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”, filed on February 22, 2022; No. 63 / 312,617, filed Feb. 22, 2022, entitled “TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; and U.S. Provisional Application No. 63 / 312,633, filed Feb. 22, 2022, entitled “TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY,” the contents of each of which are incorporated by reference in their entirety.

[0002] FIELD OF THEINVENTION This application relates to targeted complexes for delivering molecular payloads (eg, oligonucleotides) to cells and their uses, particularly for the treatment of disease.

[0003] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (D082470074WO00-SEQ-CBD.xml; size: 467,675 bytes; and creation date November 3, 2022) are incorporated herein by reference in their entirety. [Background technology]

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

[0005] Summary of the Invention In some aspects, the present disclosure provides oligonucleotides designed to target DUX4 RNA. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA that are useful for reducing the level of DUX4 mRNA and / or protein associated with features of facioscapulohumeral muscular dystrophy (FSHD), including muscle atrophy, inflammation, and reduced differentiation potential and oxidative stress. In some embodiments, the oligonucleotides provided herein target the 3'UTR of DUX4 RNA. In some embodiments, the oligonucleotides provided herein are designed to direct the degradation of DUX4 RNA. In some embodiments, the oligonucleotides are designed to block the translation of DUX4 RNA to produce DUX4 protein. In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desired binding affinity properties. In some embodiments, the oligonucleotides are designed to have desired toxicity and / or immunogenicity profiles.

[0006] According to some aspects, the present disclosure provides a complex for targeting muscle cells (e.g., primary myoblast cells) for delivery of a molecular payload (e.g., a DUX4-targeting oligonucleotide described herein) to the muscle cells. In some embodiments, the complexes provided herein are particularly useful for delivery of a molecular payload that inhibits DUX4 expression or activity, e.g., in subjects having or suspected of having facioscapulohumeral muscular dystrophy (FSHD). Accordingly, in some embodiments, the complexes provided herein include muscle-targeting agents (e.g., muscle targeting antibodies) that specifically bind to receptors on the muscle cell surface for delivery of the molecular payload to the muscle cells. In some embodiments, the complexes are taken up into the cells via receptor-mediated internalization, whereupon the molecular payload is released to perform a function inside the cells. 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 and the muscle-targeting agent of the complex.

[0007] Some embodiments of the present disclosure provide a conjugate comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured to reduce expression or activity of DUX4, wherein the anti-TfR1 antibody comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), light chain complementarity determining region 3 (CDR-L3) of any of the anti-TfR1 antibodies listed in Tables 2-7, and wherein the oligonucleotide comprises an antisense strand comprising a region of complementarity to a DUX4 sequence as represented by SEQ ID NO: 160 or SEQ ID NO: 365.

[0008] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL) of any of the anti-TfR1 antibodies listed in Table 3. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76 and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75. 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. In some embodiments, the anti-TfR1 antibody is a Fab, optionally wherein the Fab comprises the heavy and light chains of any of the anti-TfR1 Fabs listed in Table 5. In some embodiments, the Fab comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101 and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90. In some embodiments, the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90.

[0009] In some embodiments, the oligonucleotide is 20-30 nucleotides in length. In some embodiments, the oligonucleotide comprises a region of complementarity of at least 15 consecutive nucleotides to the DUX4 sequence as represented by SEQ ID NO: 160 or SEQ ID NO: 365. In some embodiments, the oligonucleotide comprises a region of complementarity of at least 15 consecutive nucleotides to the DUX4 sequence as represented by any one of SEQ ID NO: 161-168 or 213-288. In some embodiments, the oligonucleotide comprises at least 15 consecutive nucleotides of any one of SEQ ID NO: 169-176 or 289-364, wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by a T. In some embodiments, the oligonucleotide does not comprise the nucleotide sequence of SEQ ID NO: 151. In some embodiments, the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NO: 169-176 or 289-364.

[0010] In some embodiments, the oligonucleotide further comprises a sense strand that hybridizes to the antisense strand to form a double-stranded siRNA.

[0011] In some embodiments, the oligonucleotide comprises at least one modified internucleoside linkage. In some embodiments, the oligonucleotide comprises one or more modified nucleosides. In some embodiments, the one or more modified nucleosides are 2'-modified nucleosides. In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0012] In some embodiments, the antibody and the oligonucleotide are covalently linked via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a valine-citrulline sequence.

[0013] Another aspect of the present disclosure provides a method of reducing DUX4 expression in muscle cells, comprising contacting the muscle cells with a complex described herein in an amount effective to promote internalization of an oligonucleotide into the muscle cells. In some embodiments, the cell is in vitro. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human.

[0014] Further provided herein is a method for treating facioscapulohumeral muscular dystrophy (FSHD), comprising administering an effective amount of a complex described herein to a subject in need thereof, wherein the subject has abnormal production of DUX4 protein. In some embodiments, the subject has one or more deletions of D4Z4 repeats in chromosome 4. In some embodiments, the subject has 10 or fewer D4Z4 repeats. In some embodiments, the subject has 9, 8, 7, 6, 5, 4, 3, 2, or 1 D4Z4 repeat. In some embodiments, the subject has no D4Z4 repeats.

[0015] Further provided herein is an oligonucleotide comprising the nucleotide sequence of any one of SEQ ID NOs: 169-176 or 289-364. In some embodiments, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). [Brief description of the drawings]

[0016] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figure 1 shows that a conjugate containing anti-TfR Fab 3M12 VH4 / Vk3 conjugated to a DUX4-targeting oligonucleotide (SEQ ID NO: 151) inhibited the DUX4 transcriptome in C6 (AB1080)-immortalized FSHD1 cells, as indicated by decreased mRNA expression of MDB3L2, TRIM43, and ZSCAN4. The conjugate showed superior activity in inhibiting the DUX4 transcriptome compared to unconjugated DUX4-targeting oligonucleotide.

[0017] [Diagram 2] Figure 2A_2B shows dose response curves for gene knockdown. Figure 2A shows MBD3L2 knockdown in C6 (AB1080) immortalized FSHD1 cells treated with a conjugate containing anti-TfR Fab 3M12 VH4 / Vk3 conjugated to DUX4-targeting oligonucleotide (SEQ ID NO: 151). Figure 2B shows MBD3L2, TRIM43, and ZSCAN4 knockdown in myotubes of FSHD patients treated with a conjugate containing anti-TfR Fab 3M12 VH4 / Vk3 conjugated to DUX4-targeting oligonucleotide (SEQ ID NO: 151). Figure 2B encompasses the MBD3L2 data shown in Figure 2A.

[0018] [Diagram 3] FIG. 3 shows plasma levels of DUX4-targeted oligonucleotide (SEQ ID NO: 151) in non-human primates over time following administration of 30 mg / kg unconjugated ("naked") oligonucleotide, or 3, 10, or 30 mg / kg of an equivalent oligonucleotide comprising anti-TfR1 Fab 3M12 VH4 / Vk3 covalently linked to the DUX4-targeted oligonucleotide ("Fab-oligonucleotide conjugate").

[0019] [Figure 4] FIG. 4 shows tissue levels of DUX4-targeted oligonucleotide (SEQ ID NO: 151) measured in non-human primate muscle tissue samples two weeks after administration of 30 mg / kg unconjugated ("naked") oligonucleotide or 3, 10, or 30 mg / kg of an oligonucleotide equivalent to a conjugate comprising anti-TfR1 Fab 3M12 VH4 / Vk3 covalently linked to a DUX4-targeted oligonucleotide ("Fab-oligonucleotide conjugate").

[0020] [Diagram 5]FIG. 5 shows tissue levels of DUX4-targeted oligonucleotide (SEQ ID NO: 151) measured in non-human primate muscle tissue samples taken one week by biopsy (left five bars) or two weeks by necropsy (right five bars) from administration of 30 mg / kg unconjugated oligonucleotide ("oligo") or 3, 10, or 30 mg / kg of an oligonucleotide equivalent to a conjugate comprising anti-TfR1 Fab 3M12 VH4 / Vk3 covalently linked to a DUX4-targeted oligonucleotide ("conjugate").

[0021] [Figure 6] FIG. 6 shows that conjugates containing anti-TfR Fab 3M12 VH4 / Vk3 conjugated to DUX4-targeting oligonucleotides (#8, #1, or #2 in Table 8, corresponding to SEQ ID NOs: 176, 169, and 170, respectively) and a control DUX4-targeting oligonucleotide (corresponding to SEQ ID NO: 151) reduced the expression levels of DUX4 transcriptome markers (MBD3L2, TRIM43, ZSCAN4), indicating that the conjugates reduced DUX4 expression levels in FSHD patient cells in vitro. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Detailed Description of the Invention In some aspects, the present disclosure provides oligonucleotides designed to target DUX4 RNA. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA that are useful for reducing the level of DUX4 mRNA and / or protein associated with features of facioscapulohumeral muscular dystrophy (FSHD), including muscle atrophy, inflammation, and reduced differentiation potential and oxidative stress. In some embodiments, the oligonucleotides provided herein target the 3'UTR of DUX4 RNA. In some embodiments, the oligonucleotides provided herein are designed to direct the degradation of DUX4 RNA. In some embodiments, the oligonucleotides are designed to block the translation of DUX4 RNA to produce DUX4 protein. In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desired binding affinity properties. In some embodiments, the oligonucleotides are designed to have desired toxicity and / or immunogenicity profiles.

[0023] In some aspects, the present disclosure provides a complex comprising a muscle targeting agent covalently linked to a DUX4 targeting oligonucleotide for effective delivery of oligonucleotide to muscle cells.In some embodiments, the complex is particularly useful for delivering a molecular payload that inhibits the expression or activity of a target gene in muscle cells, for example in a subject that has or is suspected of having a rare muscle disease.For example, in some embodiments, a complex for targeting DUX4 is provided for treating a subject with FSHD.In some embodiments, the complex provided herein comprises an oligonucleotide that inhibits the expression of DUX4 in a subject that has one or more deletions of D4Z4 repeats on chromosome 4.

[0024] Further aspects of the disclosure, including descriptions of defined terms, are provided below.

[0025] I. Definition Administering: As used herein, the term "administering" or "administration" means providing a conjugate to a subject in a physiologically and / or (for example and) pharmacologically useful manner (for example, to treat a disease in the subject).

[0026] Approximately: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a broad range of values ​​that fall within plus or minus (greater or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value, unless otherwise stated or clear from the context (except where such number would exceed 100% of a feasible value).

[0027] Antibody: As used herein, the term "antibody" refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope, that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody, or a nanobody derived from a shark antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody comprises a framework with human germline sequences. In another embodiment, the antibody comprises a heavy chain constant region selected from the group consisting of the constant regions of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. In some embodiments, the antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or (for example and) a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody comprises a constant region, for example an Fc region. An immunoglobulin constant region refers to the constant region of a heavy or light chain. The constant region amino acid sequences of human IgG heavy and light chains and 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 comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, the antibody described herein comprises a human gamma 1 CH1 domain, CH2 domain, and / or (for example and) CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any known in the art.Non-limiting examples of human constant region sequences are described in the art, see, for example, U.S. Patent No. 5,693,780 and Kabat EA et al., (1991), supra. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, for example, via glycosylation, phosphorylation, sumoylation, and / or (for 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 (for example and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the antibody is a construct comprising a linker polypeptide or a polypeptide comprising one or more antigen-binding fragments of the present disclosure linked to an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues linked together by peptide bonds and is used to link one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Still further, an antibody may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of the antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0028] CDR: As used herein, the term "CDR" refers to a complementarity determining region within an antibody variable sequence. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are mostly involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). Each VH and VL is typically composed of three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methodologies known in the art, such as by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (for example and) the contact definition, all of which are well known in the art.For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark) http: / / 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. al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDRs may refer to CDRs defined by any method known in the art.Two antibodies having the same CDR means that the two antibodies have the same amino acid sequence of that CDR when determined by the same method, for example, the IMGT definition.

[0029] There are three CDRs in each of the heavy and light chain variable regions, designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" as used herein refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Sub-portions of the CDRs are sometimes designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but may still overlap with the Kabat CDRs, and may be shortened or extended in light of predictions or experimental findings that specific residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are provided in Table 1. Table 1. CDR definitions [Table 1] 1 IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark), imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999) 2 Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242 3 Chothia et al., J.Mol.Biol.196:901-917(1987))

[0030] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species but in which one or more sequences of the CDR regions of its VH and / or VL are replaced with CDR sequences of another species, such as an antibody having murine heavy and light chain variable regions but in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0031] Chimeric antibody: The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to a human constant region.

[0032] Complementary: As used herein, the term "complementary" refers to the ability for precise pairing between two nucleotides or two sets of nucleotides. In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in binding between two nucleosides or two sets of nucleosides. For example, if a base of an oligonucleotide at a position can hydrogen bond with a base of a target nucleic acid (e.g., mRNA) at a corresponding position, then the bases are considered to be complementary to each other at that position. Base pairing may include both 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 pairing, an adenosine-based base (A) is complementary to a thymidine-based base (T) or a uracil-based base (U), a cytosine-based base (C) is complementary to a guanosine-based base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize to and is considered complementary to any A, C, U, or T. Inosine (I) is also considered in the art to be a universal base and is considered complementary to any A, C, U, or T.

[0033] Conservative amino acid substitution: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, references that summarize such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include those made to amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0034] Covalently linked: As used herein, the term "covalently linked" refers to the characteristic of two or more molecules being linked together through at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond (e.g., a disulfide bond or a disulfide bridge) that acts as an intermolecular linker. However, in some embodiments, two or more molecules can be covalently linked together through a molecule that acts as a linker that connects two or more molecules together through multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker.

[0035] Cross-reacting: As used herein, and in the context of targeting agents (e.g., antibodies), the term "cross-reacting" refers to the property of an agent that can specifically bind to more than one antigen of a similar type or class (e.g., multiple homologues, paralogs, or orthologs of antigens) with similar affinity or avidity. For example, in some embodiments, an antibody that cross-reacts to a similar type or class of human and non-human primate antigens (e.g., human transferrin receptor and non-human primate transferrin receptor) can bind to a human antigen and a non-human primate antigen with similar affinity or avidity. In some embodiments, an antibody cross-reacts to a similar type or class of human antigen and rodent antigen. In some embodiments, an antibody cross-reacts to a similar type or class of rodent antigen and non-human primate antigen. In some embodiments, an antibody cross-reacts to a similar type or class of human antigen, non-human primate antigen, and rodent antigen.

[0036] DUX4: As used herein, the term "DUX4" refers to the gene encoding double homeobox 4, a protein that is generally expressed during fetal development and in adult male testes. In some embodiments, DUX4 may be a human (Gene ID: 100288687), a non-human primate (e.g., Gene ID: 750891, Gene ID: 100405864), or a rodent (e.g., Gene ID: 306226). In humans, expression of the DUX4 gene outside fetal development and testis is associated with facioscapulohumeral muscular dystrophy. In addition, multiple human transcript variants that code for different protein isoforms (e.g., as annotated under GenBank RefSeq accession numbers: NM_001293798.2, NM_001306068.2, NM_001363820.1) have been characterized.

[0037] Facioscapulohumeral muscular dystrophy (FSHD): As used herein, the term "facioscapulohumeral muscular dystrophy (FSHD)" refers to a genetic disease caused by mutations in the DUX4 or SMCHD1 genes, characterized by reduced muscle mass and muscle atrophy, mainly in the muscles of the face, scapula, and upper arm. Two types of the disease, type 1 and type 2, have been described. Type 1 is associated with a deletion in the D4Z4 repeat region on variant 4qA of chromosome 4 allele, which contains the DUX4 gene. Type 2 is associated with a mutation in the SMCHD1 gene. Both types 1 and 2 FSHD are characterized by abnormal production of DUX4 protein outside the testes after fetal development. Facioscapulohumeral muscular dystrophy, the genetic basis of the disease, and associated symptoms have been described in the art (see, e.g., Campbell, AE, et al., "Facioscapulohumeral dystrophy: Activating an early embryonic transcriptional program in human skeletal muscle" Human Mol Genet. (2018); and Tawil, R., "Facioscapulohumeral muscular dystrophy" Handbook Clin. Neurol. (2018), 148:541-548). FSHD1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry #158900. FSHD2 is associated with OMIM Entry #158901.

[0038] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of the CDR sequence can be determined by different systems, the meaning of the framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain, and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework region on the light chain and the heavy chain into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. The framework region that does not specify a specific subregion as FR1, FR2, FR3, or FR4, when referred to by others, represents the combined FR(s) in the variable region of a naturally occurring single immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that contain framework regions. Acceptor sequences for human heavy and light chains are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0039] Human antibody: The term "human antibody" as used herein 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, for example, in the CDRs, particularly CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0040] Humanized antibody: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but at least a portion of the VH and / or (e.g., and) VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-TfR1 receptor antibodies and antigen-binding portions are provided. Such antibodies may be produced by obtaining a mouse anti-TfR1 antibody using existing hybridoma technology, followed by humanization using in vitro genetic engineering (such as that disclosed in PCT Publication No. WO 2005 / 123126 A2 to Kasaian et al.).

[0041] Internalizing cell surface receptor: As used herein, the term "internalizing cell surface receptor" refers to a cell surface receptor that is internalized by a cell upon, for example, an external stimulus (for example, a ligand binds to the receptor). In some embodiments, the internalizing cell surface receptor is internalized by endocytosis. In some embodiments, the internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalizing cell surface receptor is internalized by a clathrin-independent pathway, such as phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or (for example and) an extracellular domain, which may optionally further comprise a ligand-binding domain. In some embodiments, the cell surface receptor becomes internalized by a cell after ligand binding. In some embodiments, the ligand may be a muscle-targeting agent or a muscle-targeting antibody. In some embodiments, the internalizing cell surface receptor is a transferrin receptor.

[0042] Isolated antibody: "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (for example, an isolated antibody that specifically binds to the transferrin receptor is substantially free of antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to the transferrin receptor complex may have cross-reactivity to other antigens, such as transferrin receptor molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or (for example and) chemicals.

[0043] Kabat numbering: The terms "Kabat numbering", "Kabat definition" and "Kabat labeling" are used interchangeably herein. These terms, while recognized in the art, refer to a system of numbering amino acid residues that are more variable (i.e., more variable) than other amino acid residues in the variable regions of the heavy and light chains of an antibody or its antigen-binding portion (Kabat et al. (1971) Ann. NY Acad, Sci. 190: 382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable region extends from amino acid position 31 to 35 for CDR1, from amino acid position 50 to 65 for CDR2, and from amino acid position 95 to 102 for CDR3. In the light chain variable region, the hypervariable region spans amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

[0044] Molecular payload: As used herein, the term "molecular payload" refers to a molecule or species that functions to modulate a biological outcome. In some embodiments, the molecular payload is linked or otherwise associated with a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, a protein, a peptide, a nucleic acid, or an oligonucleotide. In some embodiments, the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide that includes a strand having a region of complementarity with a target gene.

[0045] Muscle targeting agent: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on a muscle cell may be a membrane protein, such as an integral membrane protein or a peripheral membrane protein. Typically, a muscle targeting agent specifically binds to an antigen on a muscle cell that facilitates internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle and is capable of being internalized into the muscle cell through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0046] 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 a muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an antigen on a muscle cell that facilitates internalization of the muscle-targeting antibody (and any attached molecular payload) into the muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an internalizing cell surface receptor present on a muscle cell. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0047] Oligonucleotide: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, gapmers, mixmers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), and the like. Oligonucleotides may be single-stranded or double-stranded. In some embodiments, oligonucleotides may contain one or more modified nucleosides (e.g., 2'-O-methyl sugar modifications, purine modifications, or pyrimidine modifications). In some embodiments, oligonucleotides may contain one or more modified internucleoside linkages. In some embodiments, oligonucleotides may contain one or more phosphorothioate linkages that may be in the stereochemical configuration of Rp or Sp.

[0048] Recombinant antibody: The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (as described in more detail in this disclosure), antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from a human immunoglobulin gene transgenic animal (e.g., a mouse) (see, e.g., Taylor, LD, et al., J. Immunology ..., LD, et al., J. Immunology 29:128-145; Hoogenboom, LD, et al., J. Immunology 29:128-145; Hoogenboom, LD, et al., J. Immunology 29:128-145; Hoogenboom, LD, et al., J. Immunology 29:128-145; Hoogenboom, LD, et al., J. Immunology 29:128-145; Hoogenboom, LD, et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur within the germline repertoire of human antibodies in vivo.One aspect of the present disclosure provides fully human antibodies capable of binding to the human transferrin receptor that can be generated using techniques well known in the art, including, but not limited to, techniques using human Ig phage libraries (e.g., those disclosed in PCT Publication No. WO 2005 / 007699 A2 to Jermutus et al.).

[0049] Region of complementarity: As used herein, the term "region of complementarity" refers to a nucleotide sequence (e.g., of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., of a target nucleic acid) so that the two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the region of complementarity is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the region of complementarity is partially complementary to the cognate nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95%, or 99% complementary). In some embodiments, the region of complementarity contains 1, 2, 3, or 4 mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0050] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that allows the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context. The term "specific binding" with respect to an antibody refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or avidity that allows the antibody to distinguish the specific antigen from other antigens (e.g., to a degree that allows preferential targeting to certain cells (e.g., muscle cells) through binding to the antigen, as described herein) compared to an appropriate reference antigen(s). In some embodiments, an antibody has at least about 10% affinity to bind to a target. -4 M, 10 -5 M, 10-6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less than this K D In some embodiments, the antibody specifically binds to a transferrin receptor, e.g., an epitope in the apical domain of the transferrin receptor.

[0051] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate animal 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.

[0052] 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 to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may be of human (NCBI Gene ID 7037), non-human primate (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodent (e.g., NCBI Gene ID 22042). In addition, multiple human transcript variants encoding different isoforms of the receptor have been characterized (e.g., as annotated under GenBank RefSeq Accession Nos. NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0053] 2'-modified nucleoside: As used herein, the terms "2'-modified nucleoside" and "2'-modified ribonucleoside" are used interchangeably to refer to a nucleoside having a sugar moiety modified at the 2' position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, in which the 2' and 4' positions of the sugar are bridged (e.g., via a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, in which the 2' position of the sugar moiety is substituted (e.g., via a methylene, ethylene, or (S)-constrained ethyl bridge). Non-limiting examples of 2'-modified nucleosides include the following: 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA, methylene bridged nucleic acid), ethylene bridged nucleic acid (ENA), and (S)-constrained ethyl bridged nucleic acid (cEt). In some embodiments, the 2'-modified nucleosides described herein are high affinity modified nucleosides, and oligonucleotides comprising the 2'-modified nucleosides have increased affinity for target sequences compared to unmodified oligonucleotides. Examples of structures of 2'-modified nucleosides are provided below: [ka]

[0054] II. Complex Further provided herein is a conjugate comprising an antibody covalently linked to a targeting agent, for example a molecular payload. In some embodiments, the conjugate comprises a muscle-targeting antibody covalently linked to an oligonucleotide. The conjugate may comprise an antibody that specifically binds to a single antigen site, or an antibody that binds to at least two antigen sites, which may be present on the same antigen or on different antigens.

[0055] The complex may be used to modulate the activity or function of at least one gene, protein, and / or (for example, and) nucleic acid. In some embodiments, the molecular payload present with the complex is responsible for the modulation of gene, protein, and / or (for example, and) nucleic acid. The molecular payload may be a small molecule, a protein, a nucleic acid, an oligonucleotide, or any molecular entity that can modulate the activity or function of gene, protein, and / or (for example, and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets DUX4 in muscle cells or CNS cells.

[0056] In some embodiments, the conjugate comprises a muscle targeting agent (e.g., an anti-TfR1 antibody) covalently linked to a molecular payload (e.g., an antisense oligonucleotide targeting DUX4).

[0057] A. Muscle-targeting Agents Some aspects of the present disclosure provide muscle targeting agents, e.g., muscle targeting agents for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents are capable of binding to muscle cells and delivering the associated molecular payload to muscle cells, e.g., via specific binding to an antigen on the muscle cell. In some embodiments, the molecular payload is attached (e.g., covalently attached) to the muscle targeting agent and is internalized into the muscle cell, e.g., via endocytosis, when the muscle targeting agent binds to the antigen on the muscle cell. It should be understood that various types of muscle targeting agents may be used according to the present disclosure, and that any muscle target (e.g., muscle surface protein) may be targeted by any type of muscle targeting agent described herein. For example, the muscle targeting agent may include or consist of a small molecule, 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 muscle-targeting agents are described in further detail herein; however, it should be understood that the exemplary muscle-targeting agents provided herein are not intended to be limiting.

[0058] Some aspects of the disclosure provide muscle targeting agents that specifically bind to antigens on muscles, such as skeletal, smooth, or cardiac muscles. In some embodiments, any of the muscle targeting agents provided herein bind (e.g., specifically bind) to antigens on skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells.

[0059] Through interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, molecules that are substrates for muscle uptake transporters are useful for delivering molecular payloads into muscle tissue. Binding to muscle surface recognition elements, followed by endocytosis, can allow even macromolecules, such as antibodies, to enter muscle cells. As another example, molecular payloads conjugated to transferrin or anti-TfR1 antibodies can be taken up by muscle cells via binding to the transferrin receptor and then endocytosed, e.g., via clathrin-mediated endocytosis.

[0060] The use of muscle targeting agents can be useful to concentrate molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells compared to other cell types in a subject. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells (e.g., skeletal muscle cells, smooth muscle cells, or cardiomyocytes) at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than the amount in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or adipocytes). In some embodiments, the toxicity of the molecular payload in a 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 delivered to a subject when conjugated to a muscle targeting agent.

[0061] In some embodiments, a muscle recognition element (e.g., a muscle cell antigen) may be required to achieve muscle selectivity. As one example, the muscle targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, the muscle targeting agent may be an antibody that enters muscle cells via transporter-mediated endocytosis. As another example, the muscle targeting agent may be a ligand that binds to a cell surface receptor on muscle cells. It should be understood that transporter-based approaches provide a direct pathway for cell entry, whereas receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0062] i. Muscle targeting antibody In some embodiments, the muscle targeting agent is an antibody. In general, the high specificity of antibodies to their target antigens provides the potential to selectively target muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiomyocytes). This specificity may also limit off-target toxicity. Examples of antibodies capable of targeting surface antigens of muscle cells have been reported and are within the scope of this disclosure. For example, antibodies that target the surface of muscle cells are described in Arahata K., et al., "Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide" Nature 1988;333:861-3; Song KS, et al., "Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins" J Biol Chem 1996;271:15160-5; and Weisbart RH et al., "Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb" Mol Immunol. 2003 Mar,39(13):78309; the entire contents of each of which are incorporated herein by reference.

[0063] a. Anti-transferrin receptor (TfR) antibody Some aspects of the present disclosure are based on the recognition that agents that bind to the transferrin receptor, e.g., anti-transferrin receptor antibodies, can target muscle cells. The transferrin receptor is an internalizing cell surface receptor that transports transferrin across the cell membrane to participate in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to the transferrin receptor. Accordingly, aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to the transferrin receptor. In some embodiments, the binding proteins that bind to the transferrin receptor are internalized into muscle cells along with any molecular payloads attached. As used herein, antibodies that bind to the transferrin receptor may be interchangeably referred to as transferrin receptor antibodies, anti-transferrin receptor antibodies, or anti-TfR1 antibodies. Antibodies that bind, e.g., specifically bind, to the transferrin receptor may be internalized into cells upon binding to the transferrin receptor, e.g., through receptor-mediated endocytosis.

[0064] It should be understood that anti-TfR1 antibodies may be produced, synthesized, and / or (for example and) derivatized using several known methodologies, for example library design using phage display. Exemplary methodologies have been characterized in the art and are incorporated by reference (Diez, P. et al. "High-throughput phage-display screening in array format", Enzyme and microbial technology, 2015, 79, 34-41.; Christoph MH and Stanley, JR "Antibody Phage Display: Technique and Applications" J Invest Dermatol. 2014, 134:2.; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies." 1985, Springer). In other embodiments, anti-TfR1 antibodies have been previously characterized or disclosed.Antibodies that specifically bind to the transferrin receptor are known in the art (see, e.g., U.S. Pat. No. 4,364,934, filed Dec. 4, 1979, entitled "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Pat. No. 8,409,573, filed Jun. 14, 2006, entitled "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Pat. No. 9,708,406, filed May 20, 2014, entitled "Anti-transferrin receptor antibodies and methods of use"; U.S. Pat. No. 9,611,323, filed Dec. 19, 2014, entitled "Low affinity blood brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed Dec. 24, 2014, entitled "Novel anti-Transferrin receptor antibody that passes through blood-brain barrier"; Schneider C. et al., "Structural features of of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9."J Biol Chem.1982,257:14,8516-8522.;Lee et al."Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse"2000,J (See Pharmacol. Exp. Ther., 292:1048-1052).

[0065] 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 epitope that becomes exposed to the antibody. In some embodiments, the anti-TfR1 antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR1 antibodies provided herein bind to human transferrin receptor. In some embodiments, the anti-TfR1 antibodies described herein bind to an amino acid segment 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 an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor as represented by SEQ ID NO: 105 that is not in the apical domain of the transferrin receptor.

[0066] In some embodiments, the anti-TfR1 antibodies described herein (e.g., anti-TfR clone 8 in Table 2 below) bind to an epitope in TfR1, where the epitope includes residues at amino acids 214-241 and / or amino acids 354-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope including residues at amino acids 214-241 and amino acids 354-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope including one or more of residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1 as represented in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope comprising residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1 as set forth in SEQ ID NO:105.

[0067] In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope in TfR1, where the epitope includes residues at amino acids 258-291 and / or amino acids 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope including residues at amino acids 258-291 and amino acids 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope including one or more of residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as represented in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and its variants in Table 2 below) bind to an epitope including residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as represented by SEQ ID NO:105.

[0068] An example of a human transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows: (Sequence number 105).

[0069] An example of a primate non-human transferrin receptor amino acid sequence, which corresponds to the NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Macaca mulatta), is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKI QVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNV LKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTM DTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO:106)

[0070] An example of a non-human primate transferrin receptor amino acid sequence corresponding to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: (Sequence number 107).

[0071] An example of a mouse transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_001344227.1 (transferrin receptor protein 1, mus musculus) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMCLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYV KIQVKSsIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHHLGTGDPYTPGPFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKN VLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCCEDADYPYLGTR LDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATEWTIQGVANALSGDIWNIDNEF (query number 108)

[0072] In some embodiments, the anti-TfR1 antibodies bind to the following amino acid segment of the receptor: FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and do not inhibit the binding interaction between the transferrin receptor and transferrin and / or (by way of example and) human hemochromatosis protein (also known as HFE). In some embodiments, the anti-TfR1 antibodies described herein do not bind to the epitope of SEQ ID NO: 109.

[0073] Suitable methodologies may be used to obtain and / or (for example and) produce antibodies, antibody fragments, or antigen-binding agents, for example through the use of recombinant DNA protocols. In some embodiments, antibodies may also be produced through the generation of hybridomas (see, for example, Kohler, G and Milstein, C. "Continuous cultures of fused cells secreting antibody of predefined specificity" Nature, 1975, 256:495-497). The antigen of interest may be used as an immunogen, of any type or entity, for example recombinant or naturally occurring type or entity. Hybridomas are screened using standard methods, for example ELISA screening, to find at least one hybridoma that produces an antibody that targets a specific antigen. Antibodies may also be produced through screening of protein expression libraries (for example phage display libraries) that express antibodies. Phage display library design may also be used in some embodiments (see, e.g., U.S. 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"; and WO 1992 / 15679, filed 2 / 28 / 1992, "Improved epitope displaying phage"). In some embodiments, the antigen of interest may be used to immunize a non-human animal, e.g., a rodent or goat.In some embodiments, once the antibodies are obtained from the non-human animal, they may then be optionally modified using a number of methodologies, for example using recombinant DNA techniques. Additional examples of antibody production and methodologies are also known in the art (see, for example, Harlow et al., "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988).

[0074] In some embodiments, the antibody is modified, for example, via glycosylation, phosphorylation, sumoylation, and / or (for example, and) methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (for example, and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules are present. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell, optionally deficient in an enzyme (e.g., glycosyltransferase) in the N- or O-glycosylation pathway. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0075] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VL domain and / or (for example and) a VH domain of any one of the anti-TfR1 antibodies selected from any one of Tables 2-7, and comprises a constant region comprising the amino acid sequence of the constant region of an immunoglobulin molecule of an IgG, IgE, IgM, IgD, IgA, or IgY, of any class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or of any subclass (for example, IgG2a and IgG2b). Non-limiting examples of human constant regions are described in the art, see, for example, Kabat EA et al., (1991), supra.

[0076] In some embodiments, agents that bind to transferrin receptor, e.g., anti-TfR1 antibodies, can target muscle cells and / or mediate transport of agents (e.g., to CNS cells) across the blood-brain barrier (e.g., and). Transferrin receptors are internalizing cell surface receptors that transport transferrin across cell membranes to participate in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins that can bind to transferrin receptors. Antibodies that bind, e.g., specifically bind, to transferrin receptors may be internalized into cells upon binding to transferrin receptors, e.g., through receptor-mediated endocytosis.

[0077] Provided herein are, in some aspects, humanized antibodies that bind to the transferrin receptor with high specificity and affinity. In some embodiments, the humanized anti-TfR1 antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor or epitope that becomes exposed to the antibody. In some embodiments, the humanized anti-TfR1 antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the humanized anti-TfR1 antibodies provided herein bind to human transferrin receptor. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to an amino acid segment of the human or non-human primate transferrin receptor as provided in SEQ ID NOs: 105-108. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor as represented in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to TfR1 but not to TfR2.

[0078] 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 -13In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 (e.g., human or non-human primate TfR1) with a binding affinity (e.g., as indicated by Kd) of 10 M or less. 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 not transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR1 antibodies described herein selectively bind to human TfR1 and cyno TfR1 (e.g., 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less) but do not bind to mouse TfR1. The affinity and binding kinetics of the anti-TfR1 antibodies can be tested using any suitable method, including but not limited to biosensor technology (e.g., OCTET or BIACORE). In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit transferrin binding to TfR1. In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit HFE-beta-2-microglobulin binding to TfR1.

[0079] Non-limiting examples of anti-TfR1 antibodies are provided in Table 2. Table 2. Examples of anti-TfR1 antibodies [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] * The positions of the mutations are according to the Kabat numbering of each VH sequence containing the mutation.

[0080] In some embodiments, an anti-TfR1 antibody of the disclosure is a humanized variant of any one of the anti-TfR1 antibodies provided in Table 2. In some embodiments, an anti-TfR1 antibody of the disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 in any one of the anti-TfR1 antibodies provided in Table 2, and comprises a humanized heavy chain variable region and / or (by way of example and) a humanized light chain variable region.

[0081] Examples of amino acid sequences of humanized anti-TfR1 antibodies described herein are provided in Table 3. Table 3. Variable regions of anti-TfR1 antibodies [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] * The positions of the mutations are according to the Kabat numbering of each VH sequence containing the mutation. ** The CDRs according to the Kabat numbering system are in bold.

[0082] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework regions when compared to the respective VHs provided in Table 3. Alternatively or additionally (e.g., in addition), an anti-TfR1 antibody of the present disclosure comprises a VL comprising a CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework regions when compared to the respective VLs provided in Table 3.

[0083] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VH comprising CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises 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 amino acid sequence in the framework region compared to the respective VH provided in Table 3. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibody of the present disclosure comprises a VL comprising CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises 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 amino acid sequence in the framework region compared to the respective VL provided in Table 3.

[0084] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:69 and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0085] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:71 and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0086] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:72 and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0087] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73 and a VL comprising the amino acid sequence of SEQ ID NO:74.

[0088] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73 and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0089] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:74.

[0090] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0091] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77 and a VL comprising the amino acid sequence of SEQ ID NO:78.

[0092] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:79 and a VL comprising the amino acid sequence of SEQ ID NO:80.

[0093] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77 and a VL comprising the amino acid sequence of SEQ ID NO:80.

[0094] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:154 and a VL comprising the amino acid sequence of SEQ ID NO:155.

[0095] In some embodiments, the anti-TfR1 antibodies described herein are full-length IgG, which may include heavy and light chain constant regions from a human antibody. In some embodiments, any heavy chain of an anti-TfR1 antibody as described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, e.g., human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 81).

[0096] In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein comprises a mutant human IgG1 constant region. For example, the introduction of LALA mutations in the CH2 domain of human IgG1 (a mutant derived from mAb b12 mutated to replace the lower hinge residues Leu234 Leu235 with Ala234 and Ala235) is known to reduce Fcγ receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The mutant human IgG1 constant region is provided below (mutations in bold and underlined): [ka] (SEQ ID NO:82)

[0097] In some embodiments, the light chain of any of the anti-TfR1 antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 83).

[0098] Heavy and light chain constant regions of other antibodies are well known in the art and include, for example, those provided in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0099] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising a VH as listed in Table 3 or any one of its variants, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising a VH as listed in Table 3 or any one of its variants, and a heavy chain constant region that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising a VH as listed in Table 3 or any one of their variants, and a heavy chain constant region as represented by SEQ ID NO: 81. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising a VH as listed in Table 3 or any one of their variants, and a heavy chain constant region as represented by SEQ ID NO: 82.

[0100] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and a light chain constant region at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and a light chain constant region containing 25 or less amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and a light chain constant region represented by SEQ ID NO: 83.

[0101] Examples of IgG heavy and light chain amino acid sequences of the described anti-TfR1 antibodies are provided in Table 4 below. Table 4. Heavy and light chain sequences of example anti-TfR1 IgGs [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4] [Table 4-5] * The positions of the mutations are according to the Kabat numbering of each VH sequence containing the mutation. ** The CDRs according to the Kabat numbering system are in bold; the VH / VL sequences are underlined.

[0102] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain that contains no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) when compared to a heavy chain as represented by any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a light chain that contains no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a light chain as represented by any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0103] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or additionally (e.g., in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or additionally (by way of example, in addition), the anti-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.

[0104] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:84 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0105] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:86 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0106] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:87 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0107] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0108] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0109] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0110] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0111] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0112] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:94 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0113] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0114] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:156 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0115] 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). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by routine methods (e.g., recombinantly or by digesting the heavy chain constant region of a full-length IgG using an enzyme such as papain). For example, F(ab')2 fragments can be produced by pepsin or papain digestion of an antibody molecule, and Fab fragments can be generated by reducing disulfide bridges of F(ab')2 fragments. In some embodiments, the heavy chain constant region on the Fab fragment of the anti-TfR1 antibody described herein comprises the amino acid sequence of: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96).

[0116] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising a VH or any one of variants 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: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising a VH or any one of variants thereof as listed in Table 3, and a heavy chain constant region that contains 25 or less amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less amino acid variations) compared to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising a VH or any one of variants thereof as listed in Table 3, and a heavy chain constant region as represented by SEQ ID NO: 96.

[0117] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and a light chain constant region at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and a light chain constant region containing 25 or less amino acid variations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less amino acid variations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising a VL or any one of variants thereof as listed in Table 3, and a light chain constant region represented by SEQ ID NO: 83.

[0118] Examples of amino acid sequences of the Fab heavy and light chains of the described anti-TfR1 antibodies are provided in Table 5 below. Table 5. Heavy and light chain sequences of example anti-TfR1 Fabs [Table 5-1] [Table 5-2] [Table 5-3] * The positions of the mutations are according to the Kabat numbering of each VH sequence containing the mutation. ** The CDRs according to the Kabat numbering system are in bold; the VH / VL sequences are underlined.

[0119] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain 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) when compared to a heavy chain as set forth in any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a light chain that contains no more than 25 amino acid variations (for example, no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to a light chain as represented by any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0120] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively or additionally (e.g., in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively or additionally (by way of example, in addition), the anti-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.

[0121] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:97 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0122] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:98 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0123] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0124] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0125] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0126] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0127] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0128] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0129] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:103 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0130] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0131] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:158 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0132] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:159 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0133] Other known anti-TfR1 antibodies Any other suitable anti-TfR1 antibody known in the art can be used as a muscle targeting agent in the complex 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 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, for example, the anti-TfR1 antibodies listed in Table 6. Table 6 - List of anti-TfR1 antibody clones with relevant references and binding epitope information [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0134] In some embodiments, an anti-TfR1 antibody of the disclosure includes one or more of the CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, an anti-TfR1 antibody includes CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, an anti-TfR1 antibody includes 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.

[0135] In some embodiments, the anti-TfR1 antibodies of the disclosure include any antibody that includes a heavy chain variable domain and / or (by way of example and) a light chain variable domain of any anti-TfR antibody, such as any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the anti-TfR1 antibodies of the disclosure include any antibody that includes a 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.

[0136] Aspects of the disclosure provide anti-TfR1 antibodies having heavy chain variable (VH) and / or (by way of example and) light chain variable (VL) domain amino acid sequences homologous to any of the anti-TfR1 antibodies described herein. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable sequence 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 in any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) can occur within the heavy and / or (by way of example and) light chain variable sequences that excludes any of the CDR sequences provided herein. In some embodiments, any of the anti-TfR1 antibodies provided herein comprise heavy and light chain variable sequences that comprise framework sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequences of any anti-TfR1 antibody, such as any one of the anti-TfR1 antibodies selected from Table 6.

[0137] An example of a transferrin receptor antibody that may be used in accordance with the present disclosure is described in International Application Publication WO 2016 / 081643, which is incorporated herein by reference. The amino acid sequence of this antibody is provided in Table 7. Table 7. Heavy and light chain CDRs of examples of known anti-TfR1 antibodies [Table 7-1] [Table 7-2]

[0138] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-H1, CDR-H2, and CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

[0139] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 that contains 3 or fewer amino acid variations (e.g., 3, 2, or 1 or fewer amino acid variations) compared to a CDR-L3 as shown in Table 7. In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 that contains 1 amino acid variation compared to a CDR-L3 as shown in Table 7. In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system). In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7, and comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system).

[0140] In some embodiments, anti-TfR1 antibodies of the disclosure comprise heavy chain CDRs that, together, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs as shown in Table 7. Alternatively or additionally (e.g., in addition), anti-TfR1 antibodies of the disclosure comprise light chain CDRs that, together, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the light chain CDRs as shown in Table 7.

[0141] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 124. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 125.

[0142] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 128. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 129.

[0143] In some embodiments, the anti-TfR1 antibodies of the present disclosure include a VH 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) when compared to the VH as set forth in SEQ ID NO: 128. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibodies of the present disclosure include a VL that contains 15 or fewer amino acid variations (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid variations) when compared to the VL as set forth in SEQ ID NO: 129.

[0144] In some embodiments, the anti-TfR1 antibodies of the present disclosure are full-length IgG1 antibodies that may include heavy and light chain constant regions from a human antibody. In some embodiments, any heavy chain of an anti-TfR1 antibody as described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, e.g., human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 81).

[0145] In some embodiments, the light chain of any of the anti-TfR1 antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 83).

[0146] 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 (for example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0147] In some embodiments, the anti-TfR1 antibodies described herein are fully human antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 134. Alternatively or additionally (for example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0148] 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 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 136. Alternatively or additionally (for example, in addition), the anti-TfR1 Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-TfR1 Fab described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 137. Alternatively or additionally (for example, in addition), the anti-TfR1 Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0149] The anti-TfR1 antibodies described herein may be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as 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 with a portion of a constant region). In some embodiments, the anti-TfR1 antibodies described herein are scFv fused with a constant region (e.g., human IgG1 constant region as represented by SEQ ID NO:81).

[0150] In some embodiments, conservative mutations can be introduced into an antibody sequence (e.g., CDR or framework sequences) at positions where the residues are unlikely to be involved in interactions with a target antigen (e.g., transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) of an 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 cellular cytotoxicity to cells.

[0151] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased) as described, e.g., in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0152] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publications Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0153] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. See, for example, International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that will alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0154] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-TfR1 antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (e.g., and) in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al., (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, termed "YTE mutant", has been shown to exhibit a 4-fold increased half-life when compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.

[0155] In some embodiments, one or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the anti-TfR1 antibody. The effector ligand with altered affinity to itself can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of a constant region domain (through point mutation or other means) can reduce binding of circulating antibodies to Fc receptors, thereby increasing tumor localization. See, for example, U.S. Pat. Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0156] In some embodiments, one or more amino acid residues in the constant region of the anti-TfR1 antibody described herein may be replaced with a different amino acid residue, such that the antibody may have altered C1q binding and / or (for example and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are altered, thereby altering the antibody's ability to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or (for example and) increase the affinity of the antibody to Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0157] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein may be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be appreciated by one of skill in the art, any variant, CDR-grafted, chimeric, humanized, or composite antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and will retain specific binding ability to the transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it was derived.

[0158] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibody. For example, to avoid potential complications due to Fab-arm exchange that is known to occur in native IgG4 mAbs, the antibodies provided herein may contain a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include a stabilizing "Adair" mutation.

[0159] In some embodiments, the antibody is modified, for example, via glycosylation, phosphorylation, sumoylation, and / or (for example, and) methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (for example, and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules are present. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell, optionally deficient in an enzyme (e.g., glycosyltransferase) in the N- or O-glycosylation pathway. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0160] 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 and / or (e.g., and) light chain sequences. In some embodiments, the anti-TfR1 antibodies described herein include any one of the VH and VL sequences, any one of the IgG heavy and light chain sequences, or any one of the 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).

[0161] In some embodiments, the antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also referred to as pyroglutamic acid formation (pyro-Glu), may occur in antibodies at the N-terminal glutamic acid (Glu) and / or glutamine (Gln) residues during production. Thus, an antibody identified as having a sequence that includes an N-terminal glutamic acid or glutamine residue should be understood to encompass an antibody that has undergone pyroglutamic acid formation resulting from post-translational modification. In some embodiments, pyroglutamic acid formation occurs in the heavy chain sequence. In some embodiments, pyroglutamic acid formation occurs in the light chain sequence.

[0162] b. Other muscle-targeting antibodies In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin IIb, or CD63. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a myogenic precursor protein. Exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha7, integrin alpha7 beta1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a skeletal muscle protein. Exemplary skeletal muscle proteins include, without limitation, alpha-sarcoglycan, beta-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha 7, integrin alpha 7 beta 1, integrin beta 1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a smooth muscle protein. Exemplary smooth muscle proteins include, without limitation, alpha-smooth muscle actin, VE-cadherin, caldesmon / CALD1, calponin 1, desmin, histamine H2 R, motilin R / GPR38, Transgelin / TAGLN, and vimentin, however, it should be understood that antibodies to additional targets are within the scope of the present disclosure and that the exemplary list of targets provided herein is not intended to be limiting.

[0163] c. Antibody Features / Modifications In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where the residue is unlikely to be involved in interactions with the target antigen (e.g., the transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region) of a muscle-targeting antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or (e.g., and) antigen-dependent cellular cytotoxicity to cells.

[0164] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased) as described, e.g., in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0165] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publications Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0166] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. See, for example, International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that will alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0167] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (e.g., and) in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al., (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, termed "YTE mutant", has been shown to exhibit a 4-fold increased half-life when compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.

[0168] In some embodiments, one or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the anti-transferrin receptor antibody. The effector ligand with altered affinity to itself can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce binding of circulating antibodies to Fc receptors, thereby increasing tumor localization. See, for example, U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0169] In some embodiments, one or more amino acid residues in the constant region of the muscle-targeting antibody described herein may be replaced with a different amino acid residue, such that the antibody may have altered Clq binding and / or (for example and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are altered, thereby altering the antibody's ability to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or (for example and) increase the affinity of the antibody to Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0170] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein may be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be appreciated by one of skill in the art, any variant, CDR-grafted, chimeric, humanized, or composite antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and will retain specific binding ability to the transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it was derived.

[0171] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibody. For example, to avoid potential complications due to Fab-arm exchange that is known to occur in native IgG4 mAbs, the antibodies provided herein may contain a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include a stabilizing "Adair" mutation.

[0172] As provided herein, the antibodies of the present 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 a heavy chain, such as IgA, IgD, IgE, IgG, and IgM, and any subclass of isotype. The antibody may include a suitable constant region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, an antibody within the scope of the present disclosure may include a VH and a VL domain, or an antigen-binding portion thereof, in combination with any suitable constant region.

[0173] ii. Muscle-targeting peptides Some aspects of the disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences (e.g., peptide sequences between 5 and 20 amino acids in length) that bind to specific cell types have been described. For example, cell-targeting peptides can be found in the following publications: Vines e.,et al.,A., "Cell-penetrating and cell-targeting peptides in drug delivery" Biochim Biophys Acta 2008,1786:126-38; Jarver P.,et al., "In vivo biodistribution and efficacy of peptide mediated delivery" Trends Pharmacol Sci 2010;31:528-35; Samoylova TI,et al., "Elucidation of muscle-binding peptides by phage display screening" Muscle Nerve 1999;22:460-6; ​​U.S. Patent No. 6,329,501, issued December 11, 2001, entitled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE"; and Samoylov AM,et al., "Recognition of cell-specific binding of phage display derived peptides using an acoustic wave sensor." Biomol Eng 2002;18:269-72; the entire contents of each of which are incorporated herein by reference. By designing peptides to interact with specific cell surface antigens (e.g., receptors), selectivity to desired tissues (e.g., muscle) can be obtained. Skeletal muscle targeting has been investigated, and a wide range of molecular payloads can be delivered. These approaches, which do not have many of the practical disadvantages of large antibodies or viral particles, may have high selectivity to muscle tissue. Consequently, in some embodiments, the muscle targeting agent is a muscle targeting peptide that is 4 to 50 amino acids in length.In some embodiments, the muscle-targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Muscle-targeting peptides can be generated using any of several methods, such as phage display.

[0174] In some embodiments, the muscle targeting peptide may bind to an internalizing cell surface receptor (e.g., transferrin receptor) that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells. In some embodiments, the muscle targeting peptide may target (e.g., bind to) the transferrin receptor. In some embodiments, the transferrin receptor targeting peptide may include a segment of a naturally occurring ligand, e.g., transferrin. In some embodiments, the transferrin receptor targeting peptide is as described in U.S. Patent No. 6,743,893, 11 / 30 / 2000 application, "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR." In some embodiments, the transferrin receptor targeting peptide is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells." BMC Cancer. 2011 Aug 18;11:359. In some embodiments, the transferrin receptor targeting peptide is as described in U.S. Patent No. 8,399,653, filed 5 / 20 / 2011, "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY."

[0175] As discussed above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides have been identified using phage display libraries that display surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 184) binds to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. As a result, in some embodiments, the muscle targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 184). This peptide showed improved specificity for binding to cardiac and skeletal muscle tissues after intravenous injection into mice, with reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have also been identified using phage display. For example, in the context of treatment for 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 hereby incorporated by reference. Therein, a 12 amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO:185) was identified, and this muscle-targeting peptide showed improved binding to C2C12 cells compared to the ASSLNIA (SEQ ID NO:184) peptide.

[0176] 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 contents of which are incorporated herein by reference. Non-specific cell binders were selected by pre-incubating a random 12-mer (-12mer) peptide phage display library with a mixture of non-muscle cell types. After undergoing rounds of selection, the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 177) emerged most frequently. Consequently, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 177).

[0177] The muscle targeting agent may be a molecule or peptide containing amino acids. The muscle targeting peptide may correspond to a sequence of a protein that preferentially binds to a protein receptor found on muscle cells. In some embodiments, the muscle targeting peptide contains a high propensity of hydrophobic amino acids (e.g., valine) such that the peptide may preferentially target muscle cells. In some embodiments, the muscle targeting peptide has not been previously characterized or disclosed. These peptides may be conceived, produced, synthesized, and / or (for example and) derivatized using any of several methodologies, for example, phage-displayed peptide libraries, one-bead one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. Exemplary methodologies are characterized in the art and are incorporated by reference (Gray, BP and Brown, KC "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides" Chem Rev. 2014, 114:2, 1020-1081.; Samoylova, TI and Smith, BF "Elucidation of muscle-binding peptides by phage display screening." Muscle Nerve, 1999, 22:4.460-6).In some embodiments, muscle-targeting peptides have been previously disclosed (see, e.g., Writer MJ et al. "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display." J. Drug Targeting. 2004;12:185; Cai, D. "BDNF-mediated enhancement of inflammation and injury in the aging heart." Physiol Genomics. 2006,24:3,191-7.; Zhang, L. "Molecular profiling of heart endothelial cells." Circulation, 2005,112:11,1601-11.; McGuire, MJ et al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004,342:1,171-82). Exemplary muscle-targeting peptides include the amino acid sequences of the following group: CQAQGQLVC (SEQ ID NO: 178), CSERSMNFC (SEQ ID NO: 179), CPKTRRVPC (SEQ ID NO: 180), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 181), ASSLNIA (SEQ ID NO: 184), CMQHSMRVC (SEQ ID NO: 182), and DDTRHWG (SEQ ID NO: 183). In some embodiments, muscle-targeting peptides may include about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. Muscle-targeting peptides may include naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring amino acids, or modified amino acids.Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the muscle-targeting peptides may be linear; in other embodiments, the muscle-targeting peptides may be cyclic (e.g., bicyclic) (see, e.g., Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

[0178] iii. Muscle-targeted receptor ligands The muscle targeting agent may be a ligand, e.g., a ligand that binds to a receptor protein. The muscle targeting ligand may be a protein, e.g., transferrin, that binds to an internalized cell surface receptor expressed by muscle cells. Accordingly, in some embodiments, the muscle targeting agent is transferrin or a derivative thereof that binds to the transferrin receptor. The muscle targeting ligand may alternatively be a small molecule, e.g., a lipophilic small molecule that preferentially targets muscle cells over other cell types. Exemplary lipophilic small molecules that may target muscle cells include compounds that include cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linoleic acid, linoleic acid, myristic acid, sterol, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and alkoxy acids.

[0179] iv. Muscle-targeting aptamers The muscle targeting agent may be an aptamer, e.g., an RNA aptamer, that preferentially targets muscle cells over other cell types. In some embodiments, the muscle targeting aptamer has not been previously characterized or disclosed. These aptamers may be conceived, produced, synthesized, and / or (e.g., and) derivatized using any of several methodologies, e.g., Systematic Evolution of Ligands by Exponential Enrichment. Exemplary methodologies have been characterized in the art and are incorporated by reference (Yan, AC and Levy, M. "Aptamers and aptamer targeted delivery" RNA biology, 2009, 6:3, 316-20.; Germer, K. et al. "RNA aptamers and their therapeutic and diagnostic applications." Int. J. Biochem. Mol. Biol. 2013; 4:27-40). In some embodiments, muscle-targeting aptamers have been previously disclosed (see, e.g., Phillippou, S. et al. "Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers." Mol Ther Nucleic Acids. 2018, 10:199-214.; Thiel, W H et al. "Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation." Mol Ther. 2016, 24:4, 779-87). Exemplary muscle-targeting aptamers include the A01B RNA aptamer and RNA Apt 14. In some embodiments, the aptamer is a nucleic acid-based aptamer, an oligonucleotide aptamer, or a peptide aptamer. In some embodiments, the aptamer may be about 5-15 kDa, about 5-10 kDa, about 10-15 kDa, about 1-5 Da, about 1-3 kDa, or smaller.

[0180] v. Other muscle-targeting agents One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use a substrate of a muscle transporter protein, such as a transporter protein expressed on the sarcolemma. In some embodiments, the muscle targeting agent is a substrate of an influx transporter specific to muscle tissue. In some embodiments, the influx transporter is specific to skeletal muscle tissue. The two major classes of transporters expressed on the sarcolemma of skeletal muscle are (1) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue, and (2) the solute carrier (SLC) superfamily, which can facilitate the influx of substrates into skeletal muscle. In some embodiments, the muscle targeting agent is a substrate that binds to the ABC or SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a naturally occurring substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-naturally occurring substrate, such as a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0181] 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 equilibria or use the proton or sodium ion gradient created across the membrane to drive transport of the substrate. Exemplary SLC transporters with high expression in skeletal muscle include, without limitation, the SATT transporter (ASCT1; SLC1A4), the GLUT4 transporter (SLC2A4), the GLUT7 transporter (GLUT7; SLC2A7), the ATRC2 transporter (CAT-2; SLC7A2), the LAT3 transporter (KIAA0245; SLC7A6), the PHT1 transporter (PTR4; SLC15A4), the OATP-J transporter (OATP5A1; SLC21A15), the OCT3 transporter (EMT; SLC22A3), the OCTN2 transporter (FLJ46769; SLC22A5), the ENT transporters (ENT1; SLC29A1 and ENT2; SLC29A2), the PAT2 transporter (SLC36A2), and the SAT2 transporter (KIAA1382; SLC38A2). These transporters may facilitate the entry of substrates into skeletal muscle, providing opportunities for muscle targeting.

[0182] In some embodiments, the muscle targeting agent is a substrate for equilibrative nucleoside transporter 2 (ENT2) transporter. Compared to other transporters, ENT2 has one of the highest mRNA expression in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs, such as brain, heart, placenta, thymus, pancreas, prostate, and kidney, but is particularly abundant in skeletal muscle. Human ENT2 facilitates the uptake of its substrates depending on their concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. hENT2 transporter has low affinity for all nucleosides except inosine (adenosine, guanosine, uridine, thymidine, and cytidine). Consequently, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and clofarabine. In some embodiments, any of the muscle targeting agents provided herein are associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular payload.

[0183] In some embodiments, the muscle targeting agent is a substrate for the organic cation / carnitine transporter (OCTN2), a sodium ion-dependent high affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, mildronate, acetylcarnitine, or any derivative thereof that binds to OCTN2. In some embodiments, carnitine, mildronate, acetylcarnitine, or a derivative thereof is covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0184] A muscle targeting agent may be a protein, which is a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, hemojuvelin may be full-length or a fragment or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack the N-terminal signaling and / or may lack (by way of example) the C-terminal anchoring domain. In some embodiments, the hemojuvelin may be annotated under GenBank RefSeq Accession Nos. NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It is to be understood that the hemojuvelin may be of human, non-human primate, or rodent origin.

[0185] B. Molecular Payload Some aspects of the present disclosure provide molecular payloads, for example oligonucleotides designed to target DUX4 RNA to modulate the expression or activity of DUX4. In some embodiments, modulating the expression of DUX4 includes reducing the levels of DUX4 RNA and / or (for example and) protein. In some embodiments, the DUX4 targeting oligonucleotide is linked to or otherwise associated with a muscle targeting agent described herein. In some embodiments, such oligonucleotides can target DUX4 in muscle cells via specific binding to the DUX4 sequence in muscle cells upon delivery to the muscle cells by the muscle targeting agent to which it is linked, for example. It should be understood that various types of muscle targeting agents may be used in accordance with the present disclosure. In some embodiments, the oligonucleotide comprises a strand having a region of complementarity to the DUX4 sequence. Exemplary oligonucleotides targeting DUX4 RNA are described in further detail herein, however, it should be understood that the exemplary molecular payloads provided herein are not intended to be limiting.

[0186] i. Oligonucleotides In some embodiments, the oligonucleotide may be designed to cause degradation of mRNA (for example, the oligonucleotide may be a gapmer, siRNA, ribozyme, or aptamer that causes degradation). In some embodiments, the oligonucleotide may be designed to block translation of mRNA. In some embodiments, the oligonucleotide may be designed to block both mRNA degradation and translation. In some embodiments, the oligonucleotide may be designed to cause reduced expression of DUX4 RNA. In some embodiments, the oligonucleotide may be designed to cause reduced expression of DUX4 protein. Other examples of oligonucleotides are provided herein. It should be understood that in some embodiments, oligonucleotides of one format (for example, antisense oligonucleotides) may be suitably adapted to another format (for example, siRNA oligonucleotides) by incorporating functional sequences (for example, antisense strand sequences) from one format into the other format.

[0187] Any suitable oligonucleotide may be used as a molecular payload, as described herein. Examples of oligonucleotides useful for targeting DUX4 are described in U.S. Patent No. 9,988,628, published on February 2, 2017, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. Patent No. 9,469,851, published on October 30, 2014, entitled "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4"; U.S. Patent Application Publication No. 20120225034, published on September 6, 2012, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; PCT Patent Application Publication No. WO 2013 / 023363, published on August 15, 2013, entitled "MORPHOLINO TARGETING DUX4 FOR TREATING FSHD"; No. 2013 / 120038; 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 contents of each of which are incorporated herein in their entirety. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA, or another oligonucleotide that hybridizes to the target DUX4 gene or mRNA.

[0188] In some embodiments, the oligonucleotide may have a region of complementarity to a sequence as represented as follows: human DUX4 corresponding to NCBI sequence NM_001293798.1 (SEQ ID NO: 186), NCBI sequence: NM_001293798.2 (SEQ ID NO: 187), and / or (by way of example and) NM_001306068.3 (SEQ ID NO: 188), and / or mouse DUX4 corresponding to NCBI sequence NM_001081954.1 (SEQ ID NO: 189), as shown below. In some embodiments, the oligonucleotide may have a region of complementarity to hypomethylated, shortened D4Z4 repeats as per Daxinger, et al., "Genetic and Epigenetic Contributors to FSHD," Lim JW, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1;24(17):4817-4828, published in Curr Opin Genet Dev in 2015 (the entire contents of each of which are incorporated herein).

[0189]

[0190] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence depicted as follows, which is an example of a human DUX4 gene sequence (NM_001293798.2) (SEQ ID NO: 187):

[0191] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence depicted as follows, which is an example of a human DUX4 gene sequence (NM_001306068.3) (SEQ ID NO: 188):

[0192]

[0193]

[0194] In some embodiments, the oligonucleotide may have regions of complementarity to multiple DUX4 gene sequences, for example selected from human, mouse, and non-human species.

[0195] In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence that comprises a region of complementarity of at least 12 contiguous nucleotides (e.g., 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, at least 26, or more contiguous nucleotides) to a DUX4 sequence represented by any one of SEQ ID NOs: 186-189.

[0196] In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence that includes a region of complementarity to a DUX4 sequence corresponding to nucleotides 1519-1553 in SEQ ID NO: 187. In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence that includes a region of complementarity of at least 12 contiguous nucleotides (e.g., 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, at least 26, or more contiguous nucleotides) to a DUX4 sequence corresponding to nucleotides 1519-1553 in SEQ ID NO: 187. In some embodiments, the DUX4-targeting oligonucleotides described herein are 15-30 nucleotides in length (e.g., 15-30, 18-28, 20-26, 22-27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and include a nucleotide sequence that includes a region of complementarity of at least 15 contiguous nucleotides (e.g., 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, at least 26, or more contiguous nucleotides) to a DUX4 sequence corresponding to nucleotides 1519-1553 in SEQ ID NO:187.

[0197] In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence that comprises a region complementary to the DUX4 sequence as set forth in SEQ ID NO: 160: CCTGGATGATTAGTTCAGAGATATATTAAAATGCC (SEQ ID NO: 160). In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence that comprises a region complementary to the DUX4 sequence set forth in SEQ ID NO: 160 of at least 12 consecutive nucleotides (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides). In some embodiments, the DUX4-targeting oligonucleotides described herein are 15-30 nucleotides in length (e.g., 15-30, 18-28, 20-26, 22-27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and include a nucleotide sequence that includes a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) to the DUX4 sequence represented by SEQ ID NO:160.

[0198] Non-limiting examples of DUX4-targeting oligonucleotides are provided in Table 8. Table 8. Non-limiting examples of DUX4-targeting oligonucleotides† [Table 8]

[0199] In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a region of complementarity to at least 15 contiguous nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more contiguous nucleotides) of any one of SEQ ID NOs: 161-168. In some embodiments, the DUX4 targeting oligonucleotides described herein are 15-30 nucleotides in length (e.g., 15-20, 20-30, 22-27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and include a nucleotide sequence that includes a region of complementarity of at least 15 contiguous nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more contiguous nucleotides) to any one of SEQ ID NOs: 161-168. In some embodiments, the DUX4 targeting oligonucleotides described herein do not include a region of complementarity of 25 nucleotides to the DUX4 target sequence of AGTTCAGAGATATATTAAAATGCCC (SEQ ID NO: 150).

[0200] In some embodiments, the DUX4 targeting oligonucleotide described herein comprises at least 15 consecutive nucleosides (e.g., 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, at least 26, or more consecutive nucleosides) of any one of the nucleotide sequences of SEQ ID NOs: 169-176, where each thymine base (T) may be independently and optionally replaced with a uracil base (U), and each U may be independently and optionally replaced with a T. In some embodiments, the DUX4 targeting oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the DUX4 targeting oligonucleotide described herein does not comprise the nucleotide sequence GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 151).

[0201] In some embodiments, the DUX4-targeting oligonucleotides described herein comprise the nucleotide sequence of any one of SEQ ID NOs: 169-176, wherein each thymine base (T) is independently and optionally replaced with a uracil base (U), and each U is independently and optionally replaced with a T.

[0202] In some embodiments, any one of the DUX4-targeting oligonucleotides described herein is a phosphorodiamidate morpholino oligomer (PMO).

[0203] In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence that includes a region of complementarity to the DUX4 sequence corresponding to nucleotides 1474-1574 in SEQ ID NO: 187. In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence that includes a region of complementarity of at least 12 contiguous nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more contiguous nucleotides) to the DUX4 sequence corresponding to nucleotides 1474-1574 in SEQ ID NO: 187. In some embodiments, the DUX4-targeting oligonucleotides described herein are 15-30 nucleotides in length (e.g., 15-30, 18-28, 20-26, 22-27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and include a nucleotide sequence that includes a region of complementarity of at least 15 contiguous nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more contiguous nucleotides) to a DUX4 sequence corresponding to nucleotides 1474-1574 in SEQ ID NO:187.

[0204] In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence that comprises a region complementary to the DUX4 sequence as set forth in SEQ ID NO: 365: CACCTTCCGACGCTGTCTAGGCAAACCTGGATTAGAGTTACATCTCCTGGATGATTAGTTCAGAGATATATTAAAATGCCCCCTCCCTGTGGATCCTATAG (SEQ ID NO: 365). In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence that comprises a region complementary to the DUX4 sequence set forth in SEQ ID NO: 365 of at least 12 consecutive nucleotides (e.g., at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides). In some embodiments, the DUX4-targeting oligonucleotides described herein are 15-30 nucleotides in length (e.g., 15-30, 18-28, 20-26, 22-27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and include a nucleotide sequence that includes a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) to the DUX4 sequence represented by SEQ ID NO:365.

[0205] Non-limiting examples of DUX4-targeting oligonucleotides are provided in Table 9. Table 9. Non-limiting examples of DUX4-targeting oligonucleotides† [Table 9-1] [Table 9-2]

[0206] In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a region of complementarity to at least 15 contiguous nucleotides (e.g., 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, or more contiguous nucleotides) of any one of SEQ ID NOs:213-288. In some embodiments, the DUX4 targeting oligonucleotides described herein are 15-30 nucleotides in length (e.g., 15-20, 20-30, 22-27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) and include a nucleotide sequence that includes a region of complementarity of at least 15 contiguous nucleotides (e.g., 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, or more contiguous nucleotides) to any one of SEQ ID NOs: 213-288. In some embodiments, the DUX4 targeting oligonucleotides described herein do not include a region of complementarity of 25 nucleotides to the DUX4 target sequence of AGTTCAGAGATATATTAAAATGCCC (SEQ ID NO: 150).

[0207] In some embodiments, the DUX4 targeting oligonucleotide described herein comprises at least 15 consecutive nucleosides (e.g., 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, or more consecutive nucleosides) of any one of the nucleotide sequences of SEQ ID NOs: 289-364, where each thymine base (T) may be independently and optionally replaced with a uracil base (U), and each U may be independently and optionally replaced with a T. In some embodiments, the DUX4 targeting oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the DUX4 targeting oligonucleotide described herein does not comprise the nucleotide sequence GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 151).

[0208] In some embodiments, the DUX4-targeting oligonucleotides described herein comprise the nucleotide sequence of any one of SEQ ID NOs: 289-364, wherein each thymine base (T) is independently and optionally replaced with a uracil base (U), and each U is independently and optionally replaced with a T.

[0209] In some embodiments, any one of the DUX4-targeting oligonucleotides described herein is a phosphorodiamidate morpholino oligomer (PMO).

[0210] In some embodiments, any one of the oligonucleotides may be in a salt form, such as a sodium salt, a potassium salt, or a magnesium salt.

[0211] In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any one of the oligonucleotides described herein is conjugated to an amine group, optionally via a spacer. In some embodiments, the spacer comprises an aliphatic moiety. In some embodiments, the spacer comprises a polyethylene glycol moiety. In some embodiments, a phosphodiester linkage is present between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any one of the oligonucleotides described herein is conjugated to a spacer, which is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)NR A -, -NR A S(O)2-, or a combination thereof; each R A are independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, the spacer is a substituted or unsubstituted alkylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )-, or -C(=O)N(R A )2, or a combination of these.

[0212] In some embodiments, the 5' or 3' nucleoside of any one of the oligonucleotides described herein has the formula -NH2-(CH2) n -, where n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the phosphodiester linkage is conjugated to a compound of the formula NH2-(CH2) n - is present between the compound represented by the formula NH2-(CH2)6- and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the compound represented by the formula NH2-(CH2)6- is conjugated to the oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide.

[0213] In some embodiments, the oligonucleotide is conjugated, e.g., via an amine group, to a targeting agent, e.g., a muscle targeting agent such as an anti-TfR antibody.

[0214] a. Oligonucleotide size / sequence Oligonucleotides may be of a variety of different lengths, depending, for example, on the format. In some embodiments, the oligonucleotides are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides in length or longer. In some embodiments, the oligonucleotides are 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 21-23 nucleotides in length, 15-20 nucleotides in length, 20-25 nucleotides in length, 20-30 nucleotides in length, etc.

[0215] In some embodiments, the nucleic acid sequence of an oligonucleotide for the purposes of this disclosure is "complementary" to a target nucleic acid when it is specifically hybridizable to the target nucleic acid. In some embodiments, an oligonucleotide that hybridizes to a target nucleic acid (e.g., an mRNA or pre-mRNA molecule) results in modulation of the activity or expression of the target (e.g., reduced mRNA translation, altered pre-mRNA splicing, exon skipping, target mRNA degradation, etc.). In some embodiments, the nucleic acid sequence of an oligonucleotide has a sufficient degree of complementarity to its target nucleic acid such that it does not hybridize to non-target sequences under conditions where it is desired to avoid non-specific binding (e.g., under physiological conditions). Thus, in some embodiments, an 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 a target nucleic acid. In some embodiments, complementary nucleotide sequence does not need to be 100% complementary to the sequence of the target that can specifically hybridize to target nucleic acid or be specific to target nucleic acid.In some embodiments, oligonucleotide comprises one or more mismatched nucleobases compared to target nucleic acid.In some embodiments, the activity of target is reduced by such mismatch, but the activity of non-target is reduced by a greater amount (i.e., the selectivity for target nucleic acid is increased and off-target effect is reduced).

[0216] In some embodiments, the oligonucleotide comprises a region of complementarity to a target nucleic acid that ranges from 8-15, 8-30, 8-40, or 10-50, or 5-50, 15-20, 20-25, or 5-40 nucleotides in length. In some embodiments, the region of complementarity of the oligonucleotide to the target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 12 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain 1, 2, or 3 base mismatches compared to the contiguous nucleotides of a portion 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.

[0217] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 consecutive nucleotides of a sequence comprising any one of SEQ ID NOs: 169-176 or 289-364. In some embodiments, the oligonucleotide comprises a sequence comprising any one of SEQ ID NOs: 169-176 or 289-364. In some embodiments, the oligonucleotide comprises a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity with at least 12 (e.g., 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, at least 26) consecutive nucleotides of any one of SEQ ID NOs: 169-176 or 289-364. In some embodiments, the oligonucleotide targeting DUX4 does not comprise the sequence GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 151).

[0218] In some embodiments, the oligonucleotide comprises a region of complementarity to a nucleotide sequence represented by any one of SEQ ID NOs: 161-168 or 213-288. In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides (e.g., consecutive nucleotides) that are complementary to a nucleotide sequence represented by any one of SEQ ID NOs: 161-168 or 213-288. In some embodiments, the oligonucleotide comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementary to at least 12 or at least 15 consecutive nucleotides of any one of SEQ ID NOs: 161-168 or 213-288. In some embodiments, oligonucleotides targeting DUX4 do not contain the 25 nucleotide region of complementarity to the DUX4 target sequence of AGTTCAGAGATATATTAAAATGCCC (SEQ ID NO: 150).

[0219] 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 or Table 9). In some embodiments, such target sequence is 100% complementary to the oligonucleotide sequence listed in Table 8 or Table 9.

[0220] It should be understood that in some embodiments, methylation at the C5 position of the nucleobase uracil forms thymine. Thus, in some embodiments, a nucleotide or nucleoside having a C5 methylated uracil (or 5-methyl-uracil) may be equivalently identified as a thymine nucleotide or nucleoside.

[0221] In some embodiments, any one or more of the thymine bases (T) in any one of the oligonucleotides provided herein (the oligonucleotides listed in Table 8 or Table 9) may independently and optionally be a uracil base (U), and / or any one or more of the U's may independently and optionally be a T.

[0222] b. Oligonucleotide Modification: The oligonucleotides described herein may be modified, including, for example, modified sugar moieties, modified internucleoside linkages, modified nucleotides or nucleosides, and / or combinations thereof.In addition, in some embodiments, the oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immunostimulatory; are nuclease resistant; have improved cellular uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; have improved endosomal exit into cells; minimize TLR stimulation; or avoid pattern recognition receptors.Any of the modified chemistries or formats of the oligonucleotides described herein may be combined with each other.For example, 1, 2, 3, 4, 5, or more different types of modifications may be included in the same oligonucleotide.

[0223] In some embodiments, specific nucleotide or nucleoside modifications may be used that make the oligonucleotides that incorporate the modifications more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides remain intact longer than unmodified oligonucleotides.Specific examples of modified oligonucleotides include those that include modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages.As a result, the oligonucleotides of the present disclosure can be stabilized against nucleic acid degradation by modifications, such as the incorporation of nucleotide or nucleoside modifications.

[0224] In some embodiments, the oligonucleotides can be up to 50 or up to 100 nucleotides in length, where 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30, 2-40, 2-45, or more nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The oligonucleotides can be 8-30 nucleotides in length, where 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The oligonucleotide may be an oligonucleotide 8-15 nucleotides in length, with 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14 nucleotides of the oligonucleotide being modified nucleotides / nucleosides. Optionally, the oligonucleotide may have any nucleotide or nucleoside modified except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides / nucleosides. Oligonucleotide modifications are further described herein.

[0225] C modified nucleosides In some embodiments, the oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of the sugar. In some embodiments, the oligonucleotides comprise at least one 2'-modified nucleoside. In some embodiments, all of the nucleosides in the oligonucleotide are 2'-modified nucleosides.

[0226] In some embodiments, the oligonucleotides described herein include one or more non-bicyclic 2'-modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA) modified nucleosides.

[0227] In some embodiments, the oligonucleotides described herein include one or more 2'-4' bicyclic nucleosides in which the ribose ring contains a bridging moiety connecting two atoms in the ring (e.g., connecting the 2'-O atom to the 4'-C atom via a methylene (LNA) bridge, an ethylene (ENA) bridge, or an (S)-constrained ethyl (cEt) bridge). Examples of LNAs are described in International Patent Application Publication WO / 2008 / 043753, published April 17, 2008, entitled "RNA Antagonist Compounds For The Modulation Of PCSK9," the contents of which are incorporated herein by reference in their entirety. Examples of ENAs are provided in International Patent Publication No. WO 2005 / 042777, published May 12, 2005, entitled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006; and Horie et al., Nucleic Acids Symp. Ser(Oxf), 49:171-172, 2005, the disclosures of which are incorporated herein by reference in their entireties. Examples of cEt are provided in US Patents 7,101,993; 7,399,845 and 7,569,686, each of which is incorporated herein by reference in its entirety.

[0228] In some embodiments, the oligonucleotide comprises a modified nucleoside disclosed in one of the following U.S. patents or patent application publications: U.S. Patent 7,399,845, issued July 15, 2008, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,741,457, issued June 22, 2010, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 8,022,193, issued September 20, 2011, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,569,686, issued August 4, 2009, entitled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,335,765, issued February 26, 2008, entitled "Novel Nucleoside And Oligonucleotide Analogs"; No. 7,314,923, issued Jan. 1, 2008, entitled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent 7,816,333, issued Oct. 19, 2010, entitled "Oligonucleotide Analogues And Methods Utilizing The Same" and U.S. Publication No. 2011 / 0009471, now U.S. Patent 8,957,201, issued Feb. 17, 2015, entitled "Oligonucleotide Analogues And Methods Utilizing The Same," the contents of each of which are incorporated by reference in their entirety for all purposes.

[0229] In some embodiments, an oligonucleotide comprises at least one modified nucleoside that results in an increase in the Tm of the oligonucleotide in the range of 1° C., 2° C., 3° ​​C., 4° C., or 5° C., compared to an oligonucleotide that does not have at least one modified nucleoside. An oligonucleotide may have multiple modified nucleosides that, in total, result in an increase in the Tm of the oligonucleotide in the range of 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., or more, compared to an oligonucleotide that does not have the modified nucleoside.

[0230] An oligonucleotide may contain a mix of different types of nucleosides. For example, an oligonucleotide may contain a mix of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. An oligonucleotide may contain a mix of deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may contain a mix of 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may contain a mix of bridged nucleosides and 2'-fluoro or 2'-O-methyl modified nucleosides. An oligonucleotide may contain a mix of non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). The oligonucleotides may contain a mixture of 2'-fluoro and 2'-O-Me modified nucleosides. The oligonucleotides may contain a mixture of 2'-4' bicyclic nucleosides and 2'MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. The oligonucleotides may contain a mixture 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).

[0231] The oligonucleotide may contain different types of alternating nucleosides. For example, the oligonucleotide may contain alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. The oligonucleotide may contain alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. The oligonucleotide may contain alternating 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. The oligonucleotide may contain alternating bridged nucleosides and 2'-fluoro or 2'-O-methyl modified nucleosides. The oligonucleotide may contain alternating non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). The oligonucleotides may contain alternating 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. The oligonucleotides may contain alternating non-bicyclic 2' modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0232] In some embodiments, the oligonucleotides described herein contain a 5'-vinylphosphonate modification, one or more abasic residues, and / or one or more inverted abasic residues.

[0233] d. Internucleoside linkage / backbone In some embodiments, the oligonucleotide may contain phosphorothioate or other modified internucleoside linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between at least two nucleosides. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleosides. For example, in some embodiments, the oligonucleotide comprises modified internucleoside linkages at the first, second, and / or (for example, and) third internucleoside linkages at the 5'-end or 3'-end of the nucleotide sequence.

[0234] Phosphorus-containing linkages that may be used include, but are not limited to, regular 3'-5' linkages, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3' alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, as well as those of opposite polarity (where adjacent pairs of nucleoside units are 3'-5' to 5'-3' linked analogs thereof). 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,322,132; 5,321,132; 5,322,132; 5,322,132; 5,322,132; 5,322,132; 5,322,132; 5,322,132; 5,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; ,399,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,306; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; and No. 5,625,050.

[0235] In some embodiments, oligonucleotides may have heteroatom backbones such as methylene (methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleic acid (PNA) backbones (in which the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone and the nucleotides are attached directly or indirectly to aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

[0236] e. Stereospecific oligonucleotides In some embodiments, the phosphorus atom between the nucleotides of the oligonucleotide is chiral, and the properties of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atom. In some embodiments, suitable methods may be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms.Chem Soc Rev.2011 Dec;40(12):5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided that include nucleoside units linked together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages. In some embodiments, such phosphorothioate oligonucleotides with substantially chirally pure intersugar linkages are prepared by enzymatic synthesis or chemical synthesis, for example, as described in U.S. Patent No. 5,587,261, issued Dec. 12, 1996, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the chiral controlled oligonucleotide provides a selective cleavage pattern of the target nucleic acid. For example, in some embodiments, the chiral controlled oligonucleotide provides a single cleavage site within the complementary sequence of the nucleic acid, for example, as described in U.S. Patent Application Publication No. 20170037399 A1, entitled "CHIRAL DESIGN," published on February 2, 2017, the contents of which are incorporated herein by reference in their entirety.

[0237] F. Morpholino In some embodiments, the oligonucleotide may be a morpholino-based compound.Morpholino-based oligomeric compounds are described in Dwaine A.Braasch and David R.Corey,Biochemistry,2002,41(14),4503-4510;Genesis,volume 30,issue 3,2001;Heasman,J.,Dev.Biol.,2002,243,209-214;Nasevicius et al.,Nat.Genet.,2000,26,216-220;Lacerra et al.,Proc.Natl.Acad.Sci.,2000,97,9591-9596;and U.S. Patent No. 5,034,506 issued on July 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated by reference in their entireties herein).

[0238] g. Peptide nucleic acid (PNA) In some embodiments, both the sugar and the internucleoside linkage (backbone) of the nucleotide unit of the oligonucleotide are replaced with novel groups. In some embodiments, the base unit is maintained for hybridization with the appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimic, has been shown to have excellent hybridization properties and is called peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide-containing backbone, such as an aminoethylglycine backbone. The nucleobase is retained and is directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative publications reporting the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is incorporated herein by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0239] h. gapmer In some embodiments, the oligonucleotides described herein are gapmers. Gapmer oligonucleotides generally have the formula 5'-XYZ-3' with X and Z as flanking regions surrounding the gap region Y. In some embodiments, the flanking region X of the formula 5'-XYZ-3' is also referred to as the X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, the flanking region Z of the formula 5'-XYZ-3' is also referred to as the Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, the gap region Y of the formula 5'-XYZ-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.

[0240] In some embodiments, the Y region is a region of contiguous stretches of nucleotides, e.g., 6 or more DNA nucleotides, that are capable of recruiting RNases, such as RNase H. In some embodiments, the gapmer binds to a target nucleic acid at which point RNases can be recruited and then the target nucleic acid can be cleaved. In some embodiments, the Y region is flanked on both the 5' and 3' ends by regions X and Z that contain high affinity modified nucleosides, e.g., 1-6 high affinity modified nucleosides. Examples of high affinity modified nucleosides include, but are not limited to, 2'-modified nucleosides (e.g., 2'-MOE, 2'O-Me, 2'-F), or 2'-4' bicyclic nucleosides (e.g., LNA, cEt, ENA). In some embodiments, the flanking sequences X and Z may be 1-20 nucleotides, 1-8 nucleotides, or 1-5 nucleotides in length. The flanking sequences X and Z may be of similar or dissimilar lengths. In some embodiments, the gap segment Y can be a nucleotide sequence between 5 and 20 nucleotides, between 5 and 15 nucleotides, between 5 and 12 nucleotides, or between 6 and 10 nucleotides in length.

[0241] In some embodiments, the gap region of a gapmer oligonucleotide may contain, in addition to DNA nucleosides, modified nucleotides known to be permissive for efficient RNase H action, such as C4'-substituted nucleosides, acyclic nucleosides, and arabino-configured nucleosides. In some embodiments, the gap region contains one or more unmodified internucleosides. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least 2, at least 3, at least 4, at least 5, or more nucleotides. In some embodiments, the gap region and the two flanking regions each independently contain modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least 2, at least 3, at least 4, at least 5, or more nucleotides.

[0242] Gapmers may be produced using any suitable method. Representative U.S. patents, U.S. patent publications, and PCT publications which 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; 5,6 No. 23,065; No. 5,652,355; No. 5,652,356; No. 5,700,922; No. 5,898,031; No. 7,015,315; No. 7,1 No. 01,993; No. 7,399,845; No. 7.432,250; No. 7,569,686; No. 7,683,036; No. 7,750,131; No. 8,5 No. 80,756; No. 9,045,754; No. 9,428.534; No. 9,695,418; No. 10,017,764; No. 10,260,069; No. 9.428,534; No. 8,580,756; U.S. Patent Publication Nos. US20050074801, US20090221685; US200902869 69, US20100197762, and US20110112170; PCT Publication Nos. W02004069991; W02005023825; W02008049085 and W02009090182; and European Patent No. EP 2,149,605, each of which is incorporated herein by reference in its entirety.

[0243] In some embodiments, gapmers are 10-40 nucleosides in length. For example, gapmers can be 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35, or 35-40 nucleosides in length. In some embodiments, the gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleosides in length.

[0244] In some embodiments, the gap region Y in the gapmer is 5-20 nucleosides in length. For example, the gap region Y may be 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 nucleosides in length. In some embodiments, the gap region Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, each nucleoside in the gap region Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides in the gap region Y are 2'-deoxyribonucleosides. In some embodiments, one or more of the nucleosides in the gap region Y are modified nucleosides (e.g., 2'-modified nucleosides, such as those described herein). In some embodiments, one or more cytosines in gap region Y are optionally 5-methylcytosines. In some embodiments, each cytosine in gap region Y is a 5-methylcytosine.

[0245] In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are independently 1 to 20 nucleosides in length. For example, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) may 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'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) 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'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are of the same length. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are of different lengths. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is longer than the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is shorter than the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula).

[0246] 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, 3-1 1-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1- 13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4 -11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4,1-19-1、1-18-2、2-18-1、1-17-3、3-17-1、2-17-2、1-16-4、4-16-1、2-16-3、3-16-2、1-15-5、2-15-4、4-15-2、3-15-3、1-14-6、6-14-1、2-14-5、5-14-2、3-14-4、4-14-3、1-13-7、7-13-1、2-13-6、6-13-2、3-13-5、5-13-3、4-13-4、1-12-8、8-12-1、2-12-7、7-12-2、3-12-6、6-12-3、4-12-5、5-12-4、2-11-8、8-11-2、3-11-7、7-11-3、4-11-6、6-11-4、5-11-5、1-20-1、1-19-2、2-19-1、1-18-3、3-18-1、2-18-2、1-17-4、4-17-1、2-17-3、3-17-2、1-16-5、2-16-4、4-16-2、3-16-3、1-15-6、6-15-1、2-15-5、5-15-2、3-15-4、4-15-3、1-14-7、7-14-1、2-14-6、6-14-2、3-14-5、5-14-3、4-14-4、1-13-8、8-13-1、2-13-7、7-13-2、3-13-6、6-13-3、4-13-5、5-13-4、2-12-8、8-12-2、3-12-7、7-12-3、4-12-6、6-12-4、5-12-5、3-11-8、8-11-3、4-11-7、7-11-4、5-11-6、6-11-5、1-21-1、1-20-2、2-20-1、1-20-3、3-19-1、2-19-2、1-18-4、4-18-1、2-18-3、3-18-2、1-17-5、2-17-4、4-17-2、3-17-3、1-16-6、6-16-1、2-16-5、5-16-2、3-16-4、4-16-3、1-15-7、7-15-1、2-15-6、6-15-2、3-15-5、5-15-3、4-15-4、1-14-8、8-14-1、2-14-7、7-14-2、3-14-6、6-14-3、4-14-5、5-14-4、2-13-8、8-13-2、3-13-7、7-13-3、4-13-6、6-13-4、5-13-5、1-12-10、10-12-1、2-12-9、9-12-2、3-12-8、8-12-3、4-12-7、7-12-4、5-12-6、6-12-5、4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1 -15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6 5'-XYZ-3'. The numbers indicate the number of nucleosides in the X, Y, and Z regions of the 5'-XYZ-3' gapmer.

[0247] In some embodiments, one or more nucleosides in the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) or the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) are modified nucleosides (e.g., high affinity modified nucleosides). In some embodiments, the modified nucleoside (e.g., high affinity modified nucleoside) is a 2'-modified nucleoside. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside or a non-bicyclic 2' modified nucleoside. In some embodiments, the high affinity modified nucleoside is a 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2'-modified nucleoside (e.g., 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-ODMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA)).

[0248] In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) are high affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is a high affinity modified nucleoside. In some embodiments, one or more nucleosides in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are high affinity modified nucleosides. In some embodiments, each nucleoside in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a high affinity modified nucleoside. In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) are high affinity modified nucleosides and one or more nucleosides in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are high affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is a high affinity modified nucleoside and each nucleoside in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a high affinity modified nucleoside.

[0249] In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) comprises the same high affinity nucleosides as the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) 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 of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) may comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XY-Z-3' formula) is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt).

[0250] In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where each nucleoside in X and Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me) and each nucleoside in Y is a 2'-deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where each nucleoside in X and Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt) and each nucleoside in Y is a 2'-deoxyribonucleoside. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) comprises a different high affinity nucleoside than the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) may comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) may comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) may comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) may comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0251] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where each nucleoside in X is a non-bicyclic 2'-modified nucleoside (e.g., 2'MOE or 2'-O-Me), each nucleoside in Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside in Y is a 2'-deoxyribonucleoside. In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where each nucleoside in X is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), each nucleoside in Z is a non-bicyclic 2' modified nucleoside (e.g., 2'MOE or 2'-O-Me), and each nucleoside in Y is a 2'-deoxyribonucleoside.

[0252] In some embodiments, the 5' wing region of a gapmer (Z in the 5'-XYZ-3' formula) comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-OMe) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0253] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where at least one, but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 (the most 5' position is position 1) in X is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), where the remainder of the nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and where each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where at least one, but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 (the most 5' position is position 1) in Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), where the remainder of the nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and where each nucleoside in Y is a 2' deoxyribonucleoside.In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where at least one, but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 in X and positions 1, 2, 3, 4, 5, or 6 in Z are 5'-XYZ-3'. , 4, 5, 6, or 7 (the 5'-most position is position 1), and at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) positions are non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), where the remainder of the nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and where each nucleoside in Y is a 2' deoxyribonucleoside.

[0254] Non-limiting examples of gapmer configurations having a mix of 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 the gapmer (X in the 5'-XYZ-3' formula) and / or the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) 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;BB B-(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;LEL E-(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-E BEB;EKEK-(D)n-EKEK;ELEL-(D)n-ELEL;AABB-(D)n-BBAA;BBAA-(D)n-AABB;AAKK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;AABB-(D)n-BBAA;AAKK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;BBB-(D)n-BBA;KKK-(BBA; D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE; E;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;ABBB-(D)n-BBBA;ACC-KK-KKA; ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK-(D)n-KKKE;ELLL-(D)n-LLLE;ABBB-(D)n-BBBA;AKKK-(D)n-KKKA;ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK -(D)n-CCKE;ELLL-(D)n-LLLE;ABBB-(D)n-BBBAA;ACC-(D)n-CCKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;ECC-(D)n-CCKEE;ELLL-(D)n-LLLEE;ACC-(D)n-LLLAA; BBB-(D)n-BBBAA;ACC-(D)n-CCKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;CCK-(D)n-CCKEE;ELLL-(D)n-LLLEE;AABBB-(D)n-BBB;AACCC-(D)n-CCKKK" ;AALLL-(D)n-LLL;EEBBB-(D)n-BBB;EEKKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBB;AACKK-(D)n-KKK;AALLL-(D)n-LLL; KKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBBA;AACKK-(D)n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKK-(D)n-KKKE;EELLL-(D)n-LLLE;AABBB-(D)n-BBBA;AAKKK-(D)n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE;ABBAABB-(D)n-BB;AKKAAK K-(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-EEEEKEKE;K-(D)n-EEEEKEKEE;K-(D)n- EEKEK;EK-(D)n-EEEEKEKE;EK-(D)n-EEEKEK;EEK-(D)n-KEEKE;EK-(D)n-EEKEK;EK-(D)n-KEEK;EEK-(D)n-EEEEKEK;EK-(D)n-KEEEKEE;EK-(D)n -EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK; where "A" represents a 2'-modified nucleoside; "B" represents a 2'-4' bicyclic nucleoside; "K" represents a constrained ethyl nucleoside (cEt); "L" represents an LNA nucleoside; "E" represents a 2'-MOE modified ribonucleoside; "D" represents a 2'-deoxyribonucleoside; "n" represents the length of the gap segment (Y in the 5'-XYZ-3' configuration) and is an integer between 1 and 20;

[0255] In some embodiments, any one of the gapmers described herein comprises one or more modified nucleoside linkages (e.g., phosphorothioate linkages) in each of the X, Y, and Z regions. In some embodiments, each internucleoside linkage in any one of the gapmers described herein is a phosphorothioate linkage. In some embodiments, each of the X, Y, and Z regions independently comprises a mix of phosphorothioate and phosphodiester linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate linkage, 5' wing region X comprises a mix of phosphorothioate and phosphodiester linkages, and 3' wing region Z comprises a mix of phosphorothioate and phosphodiester linkages.

[0256] i.Mixmar In some embodiments, the oligonucleotides described herein may be mixmers or contain mixmer sequence patterns. In general, mixmers are oligonucleotides that contain both naturally occurring and non-naturally occurring nucleosides, or contain two different types of non-naturally occurring nucleosides, typically in an alternating pattern. Mixmers generally have higher binding affinity than unmodified oligonucleotides, and may be used to specifically bind to target molecules, for example to block binding sites on target molecules. In general, mixmers do not recruit RNase to target molecules, and thus do not promote cleavage of target molecules. Such oligonucleotides that are unable to recruit RNase H have been described, see, for example, WO2007 / 112754 or WO2007 / 112753.

[0257] In some embodiments, the mixmer comprises or consists of a repeating pattern of nucleoside analogs and naturally occurring nucleosides, or one type of nucleoside analog and another type of nucleoside analog. However, the mixmer need not comprise a repeating pattern, but may instead comprise any arrangement of modified nucleosides and naturally occurring nucleosides, or one type of modified nucleosides and another type of modified nucleosides. The repeating pattern may illustratively be a pattern in which every second or every third nucleoside is a modified nucleoside, such as LNA, and the remaining nucleosides are naturally occurring nucleosides, such as DNA, or 2'-substituted nucleoside analogs, such as 2'-MOE analogs or 2' fluoro analogs, or any other modified nucleosides described herein. It is recognized that repeating turns of modified nucleosides, such as LNA units, may be combined with modified nucleosides at a fixed position, for example at the 5' or 3' end.

[0258] In some embodiments, the mixmer does not contain a region of more than 5, more than 4, more than 3, or more than 2 consecutive naturally occurring nucleosides, such as DNA nucleosides. In some embodiments, the mixmer contains at least a region consisting of at least two consecutive modified nucleosides, such as at least two consecutive LNAs. In some embodiments, the mixmer contains at least a region consisting of at least three consecutive modified nucleoside units, such as at least three consecutive LNAs.

[0259] In some embodiments, the mixmer does not contain a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleoside analogues, such as LNA. In some embodiments, the LNA units may be replaced with other nucleoside analogues, such as those mentioned herein.

[0260] Mixmers may be designed to contain a mixture of affinity-enhancing modified nucleosides, such as, in non-limiting examples, LNA and 2'-O-Me nucleosides. In some embodiments, mixmers contain modified internucleoside linkages, such as phosphorothioate internucleoside linkages or other linkages, between at least 2, at least 3, at least 4, at least 5, or more nucleosides.

[0261] Mixmers may be produced using any suitable method. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of mixmers include U.S. Patent Publications US20060128646, US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. Patent No. 7,687,617.

[0262] In some embodiments, a mixmer comprises one or more morpholino nucleosides. For example, in some embodiments, a mixmer may comprise a morpholino nucleoside mixed (e.g., in an alternating manner) with one or more other nucleosides (e.g., DNA nucleosides, RNA nucleosides) or modified nucleosides (e.g., LNA nucleosides, 2'-O-Me nucleosides). In some embodiments, mixmers are useful for splice correction or exon skipping, e.g., as reported in Touznik A., et al., LNA / DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1 SMA fibroblasts Scientific Reports, volume 7, Article number: 3672 (2017); Chen S. et al., Synthesis of a Morpholino Nucleic Acid (MNA)-Uridine Phosphoramidite, and Exon Skipping Using MNA / 2'-O-Methyl Mixmer Antisense Oligonucleotide, Molecules 2016, 21, 1582 (the contents of each of which are incorporated herein by reference).

[0263] j.RNA interference (RNAi) In some embodiments, the oligonucleotides provided herein may be in the form of small interfering RNA (siRNA) (also known as short interfering RNA or silencing RNA). SiRNAs are a class of double-stranded RNA molecules, typically about 20-25 base pairs in length, that target nucleic acids (e.g., mRNA) for degradation via the RNA interference (RNAi) pathway in cells. The specificity of an siRNA molecule may be determined by the binding of the antisense strand molecule to its target RNA. Effective siRNA molecules are generally less than 30-35 base pairs in length to avoid triggering non-specific RNA interference pathways in cells via the interferon response, although longer siRNAs may also be effective. In some embodiments, the siRNA molecules are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more base pairs in length. In some embodiments, the siRNA molecules are 8-30 base pairs in length, 10-15 base pairs in length, 10-20 base pairs in length, 15-25 base pairs in length, 19-21 base pairs in length, 21-23 base pairs in length.

[0264] After selecting a suitable target RNA sequence, siRNA molecules that contain nucleotide sequences that are complementary to all or part of the target sequence, i.e., antisense sequences, can be designed and prepared using suitable methods (see, for example, PCT Publication No. WO2004 / 016735; and US Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791). siRNA molecules can be double-stranded (i.e., dsRNA molecules that contain an antisense strand and a complementary sense strand that hybridizes to form dsRNA) or single-stranded (i.e., ssRNA molecules that contain only an antisense strand). siRNA molecules can contain a double-stranded, asymmetric double-stranded, hairpin, or asymmetric hairpin secondary structure with self-complementary sense and antisense strands.

[0265] In some embodiments, the antisense strand of the siRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleosides in length. In some embodiments, the antisense strand is 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 19-21 nucleotides in length, 21-23 nucleotides in length.

[0266] In some embodiments, the sense strand of the siRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, or more nucleotides in length. In some embodiments, the sense strand is 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 19-21 nucleotides in length, 21-23 nucleotides in length.

[0267] In some embodiments, the siRNA molecule comprises an antisense strand that comprises a region of complementarity to a target region in a target mRNA.In some embodiments, the region of complementarity is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the target region in the target mRNA.In some embodiments, the target region is a region of consecutive nucleotides in the target mRNA.In some embodiments, the complementary nucleotide sequence does not need to be 100% complementary to the sequence of the target in order to be specifically hybridizable or specific to the target RNA sequence.

[0268] In some embodiments, the siRNA molecule comprises an antisense strand that includes a region of complementarity to a target RNA sequence, the region of complementarity being in the range of 8-15, 8-30, 8-40, or 10-50, or 5-50, or 5-40 nucleotides in length. In some embodiments, the region of complementarity is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25 or more consecutive nucleotides of the target RNA sequence.In some embodiments, the siRNA molecule comprises a nucleotide sequence that contains no more than 1, 2, 3, 4 or 5 bases mismatched compared to a portion of the consecutive nucleotides of the target RNA sequence.In some embodiments, the siRNA molecule comprises a nucleotide sequence that has up to 3 mismatches over 15 bases or up to 2 mismatches over 10 bases.

[0269] In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary (for example, at least 85%, at least 90%, at least 95%, or 100%) to the target RNA sequence of the oligonucleotide provided herein. In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to the oligonucleotide provided herein. In some embodiments, the siRNA molecule comprises an antisense strand comprising at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, or more consecutive nucleotides of the oligonucleotide provided herein.

[0270] Double-stranded siRNA may comprise sense and antisense RNA strands of the same or different length.Double-stranded siRNA molecules can also be assembled from a single oligonucleotide of stem-loop structure (wherein the self-complementary sense and antisense regions of siRNA molecules are linked using nucleic acid-based or non-nucleic acid-based linker(s)), as well as from circular single-stranded RNA with two or more loop structures and a stem that comprises self-complementary sense and antisense strands (wherein the circular RNA can be processed either in vivo or in vitro to generate active siRNA molecules that can mediate RNAi).Therefore, small hairpin RNA (shRNA) molecules are also contemplated herein.These molecules comprise specific antisense sequences in addition to reverse-complementary (sense) sequences, typically separated by spacer or loop sequences. Cleavage of the spacer or loop provides the single-stranded RNA molecule and its reverse complement (optionally with additional processing steps that may result in the addition or removal of one, two, three or more nucleotides from the 3'-end and / or (by way of example and) the 5'-end of one or both strands) such that they can anneal to form a dsRNA molecule. The spacer may be of sufficient length to allow the antisense and sense sequences to anneal to form a double-stranded structure (or stem) prior to cleavage of the spacer (and optionally with subsequent processing steps that may result in the addition or removal of one, two, three, four or more nucleotides from the 3'-end and / or (by way of example and) the 5'-end of one or both strands). The spacer sequence may be an unrelated nucleotide sequence located between two complementary nucleotide sequence regions that, once annealed to the double-stranded nucleic acid, will comprise the shRNA.

[0271] The overall length of siRNA molecule can vary from about 14 nucleotides to about 100 nucleotides depending on the type of siRNA molecule designed.Generally, these nucleotides between about 14 and about 50 are complementary to RNA target sequence, i.e., constitute the specific antisense sequence of siRNA molecule.For example, when siRNA is double-stranded siRNA or single-stranded siRNA, the length can vary from about 14 nucleotides to about 50 nucleotides, while when siRNA is shRNA or circular molecule, the length can vary from about 40 nucleotides to about 100 nucleotides.

[0272] The siRNA molecule may comprise a 3' overhang at one end of the molecule. The other end may be blunt or may also have an overhang (5' or 3'). When the siRNA molecule comprises an overhang at both ends of the molecule, the length of the overhang may be the same or different. In one embodiment, the siRNA molecule of the present disclosure comprises a 3' overhang of about 1 to about 3 nucleotides on both ends of the molecule. In some embodiments, the siRNA molecule comprises a 3' overhang of about 1 to about 3 nucleotides on the sense strand. In some embodiments, the siRNA molecule comprises a 3' overhang of about 1 to about 3 nucleotides on the antisense strand. In some embodiments, the siRNA molecule comprises a 3' overhang of about 1 to about 3 nucleotides on both the sense and antisense strands.

[0273] 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 comprises a modified sugar moiety (e.g., 2'-modified nucleotides). In some embodiments, the siRNA molecule comprises one or more 2' modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O--NMA). In some embodiments, each nucleotide of the siRNA molecule is a modified nucleotide (such as a 2'-modified nucleotide). In some embodiments, the siRNA molecule comprises one or more phosphorodiamidate morpholinos. In some embodiments, each nucleotide of the siRNA molecule is a phosphorodiamidate morpholino.

[0274] In some embodiments, the siRNA molecule contains phosphorothioate or other modified internucleotidic linkages. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the siRNA molecule comprises modified internucleotidic linkages at the 5'-end or 3'-end of the siRNA molecule, at the first, second, and / or (for example and) third internucleoside linkages.

[0275] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage.In some embodiments, the phosphorus-containing linkage that may be used includes, but is not limited to, phosphorothioates with normal 3'-5' linkage, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, their 2'-5' linked analogs, and those with adjacent pairs of nucleoside units 3'-5' vs. 5'-3' or 2'-5' vs. 5'-2'. '; U.S. Patents Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399 ,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,306; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; and No. 5,625,050.

[0276] Any of the modified chemistries or formats of the siRNA molecules described herein can be combined with each other, for example, one, two, three, four, five, or more different types of modifications can be included within the same siRNA molecule.

[0277] In some embodiments, the antisense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the antisense strand comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleotide linkages. In some embodiments, the modified nucleotide comprises a modified sugar moiety (e.g., 2'-modified nucleotides). In some embodiments, the antisense strand comprises one or more 2' modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAE0E), or 2'-ON-methylacetamide (2'-O-NMA). In some embodiments, each nucleotide of the antisense strand is a modified nucleotide (such as a 2'-modified nucleotide). In some embodiments, the antisense strand comprises one or more phosphorodiamidate morpholinos. In some embodiments, the antisense strand is a phosphorodiamidate morpholino oligomer (PMO).

[0278] In some embodiments, the antisense strand contains phosphorothioate or other modified internucleotidic linkages. In some embodiments, the antisense strand comprises phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the antisense strand comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the antisense strand comprises modified internucleotidic linkages at the 5'-end or 3'-end of the siRNA molecule, at the first, second, and / or (for example and) third internucleoside linkages. In some embodiments, the modified internucleotidic linkages are phosphorus-containing linkages.In some embodiments, phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates having normal 3'-5' linkages, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boranophosphates, 2'-5' linked analogs thereof, as well as nucleoside linkages in which adjacent pairs of nucleoside units are 3'-5' to 5'-3' or 2'-5' to 5'-2'. '; U.S. Patents Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399 ,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,306; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; and No. 5,625,050.

[0279] Any of the antisense strand modified chemistries or formats described herein can be combined with each other, for example, one, two, three, four, five, or more different types of modifications can be included within the same antisense strand.

[0280] In some embodiments, the sense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the sense strand comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleotide linkages. In some embodiments, the modified nucleotides comprise modified sugar moieties (e.g., 2'-modified nucleotides). In some embodiments, the sense strand comprises one or more 2' modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAE0E), or 2'-ON-methylacetamide (2'-O-NMA). In some embodiments, each nucleotide of the sense strand is a modified nucleotide (such as a 2'-modified nucleotide). In some embodiments, the sense strand comprises one or more phosphorodiamidate morpholinos. In some embodiments, the antisense strand is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the sense strand contains phosphorothioate or other modified internucleotidic linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the sense strand comprises modified internucleotidic linkages at the 5'-end or 3'-end of the siRNA molecule, at the first, second, and / or (for example and) third internucleoside linkages.

[0281] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage.In some embodiments, the phosphorus-containing linkage that may be used includes, but is not limited to, phosphorothioates with normal 3'-5' linkage, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, their 2'-5' linked analogs, and those with adjacent pairs of nucleoside units 3'-5' vs. 5'-3' or 2'-5' vs. 5'-2'. '; U.S. Patents Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399 ,676; No. 5,405,939; No. 5,453,496; No. 5,455,233; No. 5,466,677; No. 5,476,925; No. 5,519,126; No. 5,536,821; No. 5,541,306; No. 5,550,111; No. 5,563,253; No. 5,571,799; No. 5,587,361; and No. 5,625,050.

[0282] Any of the sense strand modification chemistries or formats described herein can be combined with each other, for example, one, two, three, four, five, or more different types of modifications can be included within the same sense strand.

[0283] In some embodiments, the antisense or sense strand of the siRNA molecule comprises a modification that enhances or reduces RNA-induced silencing complex (RISC) loading. In some embodiments, the antisense strand of the siRNA molecule comprises a modification that enhances RISC loading. In some embodiments, the sense strand of the siRNA molecule comprises a modification that reduces RISC loading and also 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 herein 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 oriented RNA-induced silencing complex (RISC) loading of the modified strand. In some embodiments, the antisense strand of the siRNA molecule contains a 2'-OMe-phosphorodithioate modification, which increases RISC loading as described in Wu et al., (2014) Nat Commun 5:3459, which is incorporated herein by reference in its entirety.

[0284] In some embodiments, the sense strand of the siRNA molecule contains a 5'-morpholino, which reduces RISC loading of the sense strand to improve antisense strand selection and RNAi activity as described in Kumar et al., (2019) Chem Commun (Camb) 55(35): 5139-5142, which is incorporated herein by reference in its entirety. In some embodiments, the sense strand of the siRNA molecule is modified with a locked nucleic acid (LNA), a synthetic RNA-like high affinity nucleotide analog, which reduces RISC loading of the sense strand to further enhance incorporation of the antisense strand into RISC as described in Elman et al., (2005) Nucleic Acids Res. 33(1): 439-447, which is incorporated herein 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 RISC loading of the sense strand and improves the silencing power of the antisense strand, as described in Snead et al., (2013) Mol Ther Nucleic Acids 2(7):e103 (incorporated herein in its entirety by reference).In some embodiments, the sense strand of the siRNA molecule comprises a 5-nitroindole modification, which reduces the RNAi power of the sense strand and reduces off-target effects, as described in Zhang et al., (2012) Chembiochem 13(13):1940-1945 (incorporated herein in its entirety by reference). In some embodiments, the sense strand contains a 2'-O' methyl (2'-O-Me) modification, which reduces RISC loading and off-target effects of the sense strand as described in Zheng et al., FASEB (2013) 27(10):4017-4026, which is incorporated herein by reference in its entirety.In some embodiments, the sense strand of the siRNA molecule is fully substituted with morpholino, 2'-MOE, or 2'-O-Me residues and is not recognized by RISC as described in Kole et al., (2012) Nature reviews. Drug Discovery 11(2):125-140, which is incorporated herein by reference in its entirety. In some embodiments, the antisense strand of the siRNA molecule comprises a 2'-MOE modification and the sense strand comprises a 2'-O-Me modification (see, e.g., Song et al., (2017) Mol Ther Nucleic Acids 9:242-250). In some embodiments, at least one (e.g., at least 2, at least 3, at least 4, at least 5, at least 10) siRNA molecule is linked (e.g., covalently) to a muscle targeting agent. In some embodiments, the muscle targeting agent may comprise or consist of a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide). In some embodiments, the muscle targeting agent is an antibody. In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody (e.g., any one of the anti-TfR antibodies provided herein). 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 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 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 linked internally to the antisense strand of the siRNA molecule.

[0285] k. MicroRNA (miRNA) In some embodiments, the oligonucleotide may be a microRNA (miRNA). MicroRNAs (referred to as "miRNAs") are a class of small non-coding RNAs that control gene expression by binding to complementary sites on target RNA transcripts. Typically, miRNAs are generated from large RNA precursors (termed pri-miRNAs) that are processed in the nucleus into approximately 70 nucleotide pre-miRNAs that fold into an incomplete stem-loop structure. These pre-miRNAs typically undergo an additional processing step in the cytoplasm, where mature miRNAs, 18-25 nucleotides in length, are excised from one side of the pre-miRNA hairpin by the RNase III enzyme Dicer.

[0286] As used herein, miRNA includes pri-miRNA, pre-miRNA, mature miRNA, or fragments of their variants that retain the biological activity of mature miRNA. In one embodiment, the size range of miRNA can be from 21 nucleotides to 170 nucleotides. In one embodiment, the size range of miRNA is from 70 nucleotides to 170 nucleotides in length. In another embodiment, mature miRNAs of 21 nucleotides to 25 nucleotides in length can be used.

[0287] l Aptamer In some embodiments, the oligonucleotides provided herein may be in the form of aptamers. In general, in the context of molecular payloads, aptamers are any nucleic acids that specifically bind to targets, such as small molecules, proteins, nucleic acids, etc., in cells. In some embodiments, the aptamers are DNA aptamers or RNA aptamers. In some embodiments, the nucleic acid aptamers are single-stranded DNA or RNA (ssDNA or ssRNA). It should be understood that single-stranded nucleic acid aptamers may form helical structures and / or (for example and) loop structures. The nucleic acids forming nucleic acid aptamers may include naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (for example, alkylene) or polyether-linkers (for example, PEG linkers) inserted between one or more nucleotides, modified nucleotides with hydrocarbon linkers or PEG linkers inserted between one or more nucleotides, or combinations thereof. Exemplary publications and patents describing aptamers and methods of producing aptamers include, by way of example, Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Patent Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; ​​5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438, and PCT application WO 99 / 31275, each of which is incorporated herein by reference.

[0288] m. multimer In some embodiments, the molecular payload may comprise a multimer (e.g., a concatemer) of two or more oligonucleotides connected by a linker. Thus, in some embodiments, the oligonucleotide loading of the complex / conjugate can be increased beyond the available linking sites on the targeting agent (e.g., the available thiol sites on an antibody) or can be tuned differently to achieve a specific payload loading content. The oligonucleotides in the multimer can be the same or different (e.g., targeting different genes, or different sites on the same gene, or its products).

[0289] In some embodiments, a multimer comprises two or more oligonucleotides linked together by a cleavable linker. However, in some embodiments, a multimer comprises two or more oligonucleotides linked together by a non-cleavable linker. In some embodiments, a multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides linked together. In some embodiments, a multimer comprises 2-5, 2-10, or 4-20 oligonucleotides linked together.

[0290] In some embodiments, a multimer comprises two or more oligonucleotides linked end-to-end (in a linear arrangement). In some embodiments, a multimer comprises two or more oligonucleotides linked end-to-end via an oligonucleotide-based linker (e.g., a poly-dT linker, which is an abasic linker). In some embodiments, a multimer comprises a 5' end of one oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, a multimer comprises a 3' end of another oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, a multimer comprises a 5' end of another oligonucleotide linked to the 5' end of another oligonucleotide. Still, in some embodiments, a multimer may comprise a branched structure comprising multiple oligonucleotides linked together by a branched linker.

[0291] Further examples of multimers that may be used in the conjugates provided herein include, for example, U.S. Patent Application No. 2015 / 0315588 A1, published November 5, 2015, and entitled Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers; U.S. Patent Application No. 2015 / 0247141 A1, published September 3, 2015, and entitled Multimeric Oligonucleotide Compounds; U.S. Patent Application No. US 2011 / 0158937 A1, published June 30, 2011, and entitled Immunostimulatory Oligonucleotide Multimers; and U.S. Patent Application No. 2011 / 0158937 A1, published December 2, 1997, and entitled Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers Targeting Nucleic Acids Comprising Mixed Sequences Of Purines And No. 5,693,773, entitled "Polymers of Novel Pyrimidines," the contents of each of which are incorporated herein by reference in their entireties.

[0292] 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 certain cellular environment. In addition, the linker typically does not negatively affect the functional properties of either the anti-TfR, 1 antibody or the molecular payload. Examples and methods of linker synthesis are known in the art (see, e.g., Kline, T. et al., "Methods to Make Homogenous Antibody Drug Conjugates." Pharmaceutical Research, 2015, 32:11, 3480-3493.; Jain, N. et al., "Current ADC Linker Chemistry" Pharm Res. 2015, 32:11, 3526-3540.; McCombs, JR and Owen, SC, "Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry" AAPS J. 2015, 17:2, 339-351.).

[0293] The linker will typically contain two different reactive species that can be attached 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 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 the maleimide-containing linker optionally comprises a maleimidocaproyl group or a 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 covalently linked to the anti-TfR1 antibody and / or (by way of example and) the molecular payload, independently, via an amide bond, a carbamate bond, a hydrazide, a triazole, a thioether, and / or a disulfide bond.

[0294] 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 only cleavable intracellularly and are preferably stable in the extracellular environment, e.g., outside of muscle cells or CNS cells.

[0295] Protease-sensitive linkers are cleavable by protease enzyme activity. These linkers typically comprise peptide sequences and may be 2-10 amino acids, about 2-5 amino acids, about 5-10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length. In some embodiments, the peptide sequence may comprise naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the protease-sensitive linker comprises a valine-citrulline sequence or an alanine-citrulline sequence. In some embodiments, the protease-sensitive linker may be cleaved by lysosomal proteases, such as cathepsin B, and / or endosomal proteases.

[0296] A pH-sensitive linker is a covalent linkage that is easily degraded in a high or low pH environment. In some embodiments, the pH-sensitive linker may be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved in an endosome or lysosome.

[0297] In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by disulfide exchange reaction with glutathione species inside the cell. In some embodiments, the disulfide moiety further comprises at least one amino acid, for example a cysteine ​​residue.

[0298] In some embodiments, the linker comprises a valine-citrulline sequence (e.g., as described in U.S. Pat. No. 6,214,345, incorporated herein by reference). In some embodiments, the linker prior to conjugation comprises the following structure: [ka]

[0299] In some embodiments, the linker after conjugation comprises the following structure: [ka]

[0300] In some embodiments, the linker prior to conjugation comprises a structure represented by formula (A): [ka] wherein n is any number from 0 to 10. In some embodiments, n is 3.

[0301] In some embodiments, the linker comprises a structure represented by formula (H): [ka] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and / or (for example and) m is 4.

[0302] In some embodiments, the linker comprises a structure represented by formula (I): [ka] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and / or (for example and) m is 4.

[0303] ii. Non-cleavable linker In some embodiments, a non-cleavable linker may be used. In general, a non-cleavable linker cannot be easily degraded in a cellular or physiological environment. In some embodiments, a non-cleavable linker comprises an optionally substituted alkyl group, where the substitution may include halogen, hydroxyl group, oxygen species, and other common substitutions. In some embodiments, a linker may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one unnatural amino acid, a truncated glycan, an enzymatically non-degradable sugar(s), an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, a biotin, a biphenyl, a repeating unit of polyethylene glycol or an equivalent compound, an acid ester, an acid amide, a sulfamide, and / or an alkoxy-amine linker. In some embodiments, sortase-mediated ligation may be utilized to covalently link an anti-TfR1 antibody comprising an LPXT sequence to a molecular payload comprising 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).

[0304] In some embodiments, the linker may comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S, an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), such as polyethylene oxide or polypropylene oxide. In some embodiments, the linker may be a non-cleavable N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS) linker.

[0305] 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 group or a 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.

[0306] In some embodiments, the linker or a portion thereof is covalently linked to the anti-TfR1 antibody and / or (for example and) the molecular payload by a cycloaddition reaction between an azide and an alkyne forming a triazole, where the azide or the 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, for example, a cyclooctyne. In some embodiments, the alkyne may be a bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the 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 an azide-containing sugar or carbohydrate molecule. In some embodiments, the azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the azide-containing sugar or carbohydrate molecule is as described in International Patent Application Publication WO2016170186, published October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase."In some embodiments, the cycloaddition reaction between an azide and an alkyne to form a triazole (wherein the azide or alkyne may be located on the anti-TfR1 antibody, the molecular payload, or the linker) is as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or International Patent Application Publication WO2016170186, published on October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase."

[0307] In some embodiments, the linker comprises a spacer, e.g., a polyethylene glycol spacer or an acyl / carbamoyl sulfamide spacer, e.g., a HydraSpace™ spacer. In some embodiments, the spacer is as described in Verkade, JMM et al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates", Antibodies, 2018, 7, 12.

[0308] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (for example and) the molecular payload by a Diels-Alder reaction between a dienophile and a diene / hetero-diene, where the dienophile 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 (for example and) the molecular payload by other pericyclic reactions, such as ene reactions. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (for example and) the molecular payload by an amide, thioamide, or sulfonamide coupling reaction. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (for example and) the molecular payload by a condensation reaction forming an oxime group, a hydrazone group, or a semicarbazide group present between the linker and the anti-TfR1 antibody and / or (for example and) the molecular payload.

[0309] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (for example and) the molecular payload by a conjugate addition reaction between a nucleophile (for example, an amine group or a hydroxyl group) and an electrophile (for example, a carboxylic acid, carbonate, or aldehyde). In some embodiments, the nucleophile may be present on the linker and the electrophile may be present on the anti-TfR1 antibody or the molecular payload prior to the reaction between the linker and the anti-TfR1 antibody or the molecular payload. In some embodiments, the electrophile may be present on the linker and the nucleophile may be present on the anti-TfR1 antibody or the molecular payload prior to the reaction between the linker and the anti-TfR1 antibody or the molecular payload. In some embodiments, the electrophile may be an azide, pentafluorophenyl, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimidyl ester, sulfosuccinimidyl ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center, and / or activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilide group, and / or a thiol group.

[0310] 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, the linker comprising a valine-citrulline sequence covalently linked to a reactive chemical moiety (e.g., an azide moiety for click chemistry) comprises a structure represented by formula (A): [ka] wherein n is any number from 0 to 10. In some embodiments, n is 3.

[0311] In some embodiments, a linker comprising a structure represented by formula (A) is covalently linked (e.g., optionally via an additional chemical moiety) to a molecular payload (e.g., an oligonucleotide). In some embodiments, a linker comprising a structure represented by formula (A) is covalently linked to an oligonucleotide, e.g., via nucleophilic displacement with an amine-L1-oligonucleotide forming a carbamate bond, to produce a compound comprising a structure represented by formula (B): [ka] wherein n is any number from 0 to 10. In some embodiments, n is 3.

[0312] In some embodiments, the compound of formula (B) is further covalently linked to an additional moiety via a triazole, where the triazole is formed by a click reaction between an azide of formula (A) or formula (B) and an alkyne provided on the bicyclononyne. In some embodiments, the bicyclononyne-containing compound comprises a structure of formula (C): [ka] wherein m is any number from 0 to 10. In some embodiments, m is 4.

[0313] In some embodiments, an azide of a compound represented by structure (B) forms a triazole via a click reaction with an alkyne of a compound represented by structure (C) to form a compound comprising a structure represented by formula (D): [ka] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and m is 4.

[0314] In some embodiments, the compound represented by structure (D) is further covalently linked to a lysine of an anti-TfR1 antibody to form a conjugate comprising a structure represented by formula (E): [ka] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and / or (by way of example and) m is 4. It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0315] In some embodiments, the compound represented by structure (C) is further covalently linked to a lysine of an anti-TfR1 antibody to form a compound comprising a structure represented by formula (F): [ka] wherein m is 0 to 15 (e.g., 4). It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (F) results from reaction of an amine of the anti-TfR1 antibody with an amine such as the lysine epsilon amine.

[0316] In some embodiments, the azide of the compound represented by structure (B) forms a triazole via a click reaction with an alkyne of the compound represented by structure (F) to form a conjugate comprising a structure represented by formula (E): [ka] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and / or (by way of example and) m is 4. It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0317] In some embodiments, an azide of a compound represented by structure (A) forms a triazole via a click reaction with an alkyne of a compound represented by structure (F) to form a compound comprising a structure represented by formula (G): [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3, and / or (for example and) m is 4. In some embodiments, the oligonucleotide is covalently linked to a compound comprising a structure represented by formula (G), thereby forming a complex comprising a structure represented by formula (E). It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (G) results from the reaction of the anti-TfR1 antibody with an amine, such as a lysine epsilon amine.

[0318] In some embodiments, in any one of the conjugates described herein, the anti-TfR1 antibody is covalently linked through a lysine of the anti-TfR1 antibody to a molecular payload (e.g., an oligonucleotide) via a linker comprising a structure represented by formula (H): [ka] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and / or (for example and) m is 4.

[0319] In some embodiments, in any one of the conjugates described herein, the anti-TfR1 antibody is covalently linked through a lysine of the anti-TfR1 antibody to a molecular payload (e.g., an oligonucleotide) via a linker comprising a structure represented by formula (I): [ka] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and / or (for example and) m is 4.

[0320] In some embodiments, L in Structural Formulas (B), (D), (E), and (I) is a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(RA)-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NRA-, -NRAC(=O)-, -NRAC(=O)RA-, -C(=O)RA-, -NRAC(=O)O-, -NRAC(=O)N(RA)-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(RA)-, -S(O)2NRA-, -NRAS(O)2-, or a combination thereof, wherein each R A is independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L is [ka] where L2 is [ka] where a labels a site that is directly linked to a carbamate moiety represented by formulas (B), (D), (E), and (I); and b labels a site that is covalently linked (directly or via an additional chemical moiety) to an oligonucleotide.

[0321] In some embodiments, L1 is the following: [ka] where a labels a site that is directly linked to a carbamate moiety represented by formulas (B), (D), (E), and (I); and b labels a site that is covalently linked (directly or via an additional chemical moiety) to an oligonucleotide.

[0322] In some embodiments, L1 is [ka] It is.

[0323] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide.

[0324] In some embodiments, L1 is optional (eg, does not have to be present).

[0325] In some embodiments, any one of the conjugates described herein has a structure represented by formula (J): [ka] where n is 0 to 15 (for example, 3) and m is 0 to 15 (for example, 4). It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (J) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0326] In some embodiments, any one of the conjugates described herein has a structure represented by formula (K): [ka] wherein n is 0 to 15 (for example, 3) and m is 0 to 15 (for example, 4).

[0327] In some embodiments, the oligonucleotide is modified to contain an amine group at the 5' end, at the 3' end, or internally (e.g., as an amine-functionalized nucleobase) prior to linkage to a compound, e.g., a compound of Formula (A) or Formula (G).

[0328] Although the linker conjugates are described in the context of anti-TfR1 antibodies and oligonucleotide molecular payloads, it should be understood that the use of such linker conjugates on other muscle targeting agents, such as other muscle targeting antibodies, and / or on other molecular payloads is contemplated.

[0329] D. Examples of Antibody-Molecular Payload Conjugates Further provided herein are non-limiting examples of conjugates comprising any one of the anti-TfR1 antibodies described herein covalently linked to any one of the molecular payloads described herein (e.g., oligonucleotides). In some embodiments, the anti-TfR1 antibody (e.g., any one of the anti-TfR1 antibodies provided in Tables 2-7) is covalently linked to the molecular payload (e.g., an oligonucleotide such as the oligonucleotides provided in Tables 8 or 9) via a linker. Any of the linkers described herein may be used. In some embodiments, when the molecular payload is an oligonucleotide, the linker is linked to the 5' end of the oligonucleotide, the 3' end of the oligonucleotide, or to an internal site of the oligonucleotide. In some embodiments, the linker is linked to the anti-TfR1 antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the anti-TfR1 antibody). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is linked to the antibody (e.g., an anti-TfR1 antibody described herein) via an amine group (e.g., via a lysine in the antibody). In some embodiments, the molecular payload is a DUX4-targeting oligonucleotide (eg, a DUX4-targeting oligonucleotide listed in Table 8 or Table 9).

[0330] An example structure of a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload via a linker is provided below: [ka] wherein the linker is linked to the antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody). In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in Table 8 or Table 9).

[0331] Another example of the structure of a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload via a linker is provided below: [ka] where n is a number between 0 and 10, where m is a number between 0 and 10, where the linker is linked to the antibody via an amine group (e.g., on a lysine residue) and / or (e.g., and) where the linker is linked to the oligonucleotide (e.g., at the 5' end, the 3' end, or internally). In some embodiments, the linker is linked to the antibody via a lysine, where the linker is linked to the oligonucleotide at the 5' end, n is 3, and m is 4. In some embodiments, the molecular payload is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in Table 8 or Table 9). It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine of the anti-TfR1 antibody, such as a lysine epsilon amine.

[0332] It should be understood that antibodies can be linked to molecular payloads with various stoichiometries, a property sometimes referred to as drug-antibody ratio (DAR), where the "drug" is the molecular payload. In some embodiments, one molecular payload is linked to the antibody (DAR=1). In some embodiments, two molecular payloads are linked to the antibody (DAR=2). In some embodiments, three molecular payloads are linked to the antibody (DAR=3). In some embodiments, four molecular payloads are linked to the antibody (DAR=4). In some embodiments, a mixture of different conjugates, each having a different DAR, is provided. In some embodiments, the average DAR of the conjugates in such a mixture may range from 1-3, 1-4, 1-5 or more. The DAR may be increased by conjugating the molecular payloads to various sites on the antibody and / or by (for example and) conjugating multimers to one or more sites on the antibody. For example, a DAR of 2 may be achieved by conjugating a single molecular payload to two different sites on an antibody, or by conjugating a dimeric molecular payload to a single site on an antibody.

[0333] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody described herein (e.g., an antibody provided in Tables 2-7) covalently linked to a molecular payload. In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody described herein (e.g., an antibody provided in Tables 2-7) covalently linked to a molecular payload via a linker (e.g., a linker comprising a valine-citrulline sequence). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is linked to an antibody (e.g., an anti-TfR1 antibody described herein) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is linked to an antibody (e.g., an anti-TfR1 antibody described herein) via an amine group (e.g., via a lysine in the antibody). In some embodiments, the molecular payload is a DUX4-targeting oligonucleotide (e.g., a DUX4-targeting oligonucleotide listed in Tables 8 or 9).

[0334] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of any one of the antibodies listed in Table 2. In some embodiments, the molecular payload is a DUX4-targeting oligonucleotide (e.g., a DUX4-targeting oligonucleotide listed in Table 8 or Table 9).

[0335] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, where the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 71, or SEQ ID NO: 72, and a VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0336] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0337] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0338] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77, and a VL comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0339] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 79, and a VL comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0340] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 154, and a VL comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0341] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 86, or SEQ ID NO: 87, and a light chain comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0342] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0343] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0344] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 94, and a light chain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0345] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0346] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 156, and a light chain comprising the amino acid sequence of SEQ ID NO: 157. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0347] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99, and a light chain comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0348] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101, and a light chain comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0349] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101, and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0350] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody 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. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0351] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 or SEQ ID NO: 103, and a light chain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0352] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 158 or SEQ ID NO: 159, and a light chain comprising the amino acid sequence of SEQ ID NO: 157. In some embodiments, the molecular payload is a DUX4-targeted oligonucleotide (e.g., a DUX4-targeted oligonucleotide listed in Table 8 or Table 9).

[0353] In any of the example conjugates described herein, in some embodiments, the anti-TfR1 antibody is covalently linked to the molecular payload via a linker comprising a structure represented by formula (I): [ka] In the formula, n is 3 and m is 4.

[0354] In some embodiments, the conjugate described herein comprises an anti-TfR1 antibody covalently linked to the 5' end of a DUX4-targeting oligonucleotide (e.g., a DUX4-targeting oligonucleotide listed in Table 8 or Table 9) via a lysine in the anti-TfR1 antibody, where the anti-TfR1 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of any one of the antibodies listed in Table 2, where the conjugate has a structure represented by formula (E): [ka] where n is 3 and m is 4. It is to be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0355] In some embodiments, the conjugate described herein comprises an anti-TfR1 antibody covalently linked to the 5' end of a DUX4-targeting oligonucleotide (e.g., a DUX4-targeting oligonucleotide listed in Table 8 or Table 9) via a lysine in the anti-TfR1 antibody, where the anti-TfR1 antibody comprises a VH and VL of any one of the antibodies listed in Table 3, where the conjugate has a structure represented by formula (E): [ka] where n is 3 and m is 4. It is to be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0356] In some embodiments, the conjugate described herein comprises an anti-TfR1 antibody covalently linked to the 5' end of a DUX4-targeting oligonucleotide (e.g., a DUX4-targeting oligonucleotide listed in Table 8 or Table 9) via a lysine in the anti-TfR1 antibody, where the anti-TfR1 antibody comprises the heavy and light chains of any one of the antibodies listed in Table 4, and where the conjugate has a structure represented by formula (E): [ka] where n is 3 and m is 4. It is to be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0357] In some embodiments, the conjugate described herein comprises an anti-TfR1 Fab covalently linked via a lysine in an anti-TfR1 antibody to a 5' end of a DUX4-targeting oligonucleotide (e.g., a DUX4-targeting oligonucleotide listed in Table 8 or Table 9), wherein the anti-TfR1 Fab comprises the heavy and light chains of any one of the antibodies listed in Table 5, and wherein the conjugate has a structure represented by formula (E): [ka] where n is 3 and m is 4. It is to be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0358]

[0023] In some embodiments, L1 in any one of the example conjugates described herein is a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(RA)-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NRA-, -NRAC(=O)-, -NRAC(=O)RA-, -C(=O)RA-, -NRAC(=O)O-, -NRAC(=O)N(RA)-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(RA)-, -S(O)2NRA-, -NRAS(O)2-, or a combination thereof, wherein each R A is independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L is [ka] where L2 is [ka] where a labels a site that is directly linked to the carbamate moiety of formula (E); and b labels a site that is covalently linked (directly or via an additional chemical moiety) to the oligonucleotide.

[0359] In some embodiments, L1 is the following: [ka] where a labels the moiety that is directly linked to the carbamate moiety of formula (E); and b labels the moiety that is covalently linked (directly or via an additional chemical moiety) to the oligonucleotide.

[0360] In some embodiments, L1 is [ka] It is.

[0361] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide.

[0362] In some embodiments, L1 is optional (eg, does not have to be present).

[0363] III. Preparations The complexes provided herein may be formulated in any suitable manner. In general, the complexes provided herein are formulated in a manner suitable for pharmaceutical use. For example, the complexes may be delivered to a subject using a formulation that minimizes degradation, facilitates delivery and / or (for example and) uptake, or provides another beneficial property to the complex in the formulation. In some embodiments, provided herein is a composition comprising the complex and a pharma- ceutically acceptable carrier. Such compositions may be suitably formulated so that a sufficient amount of the complex can enter the target cell (for example, a muscle cell or a CNS cell) when administered either into the environment surrounding the target cell of a subject or into the entire body of a subject. In some embodiments, the complexes are formulated in a buffer solution, such as phosphate buffered saline solution, in a liposome, in a micellar structure, and in a capsid.

[0364] It should be understood that in some embodiments, a composition may individually include one or more components of the conjugates provided herein (e.g., a muscle targeting agent, a linker, a molecular payload, or a precursor molecule of any one of these).

[0365] In some embodiments, the complex is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the complex is formulated in a basic buffered aqueous solution (e.g., PBS). In some embodiments, the formulation as disclosed herein includes an excipient. In some embodiments, the excipient confers improved stability, improved absorption, improved solubility, and / or (e.g., and) therapeutic enhancement of the active ingredient to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or a vehicle (e.g., a buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil).

[0366] In some embodiments, the complex or a component thereof (e.g., an oligonucleotide or an antibody) is lyophilized to extend its shelf life and then brought into solution prior to use (e.g., administration to a subject).Accordingly, the excipient in a composition comprising the complex or a component thereof described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone), or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0367] In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, and subcutaneous. Typically, the route of administration is intravenous or subcutaneous.

[0368] Pharmaceutical compositions suitable for use in injections include sterile aqueous solutions (wherein they are soluble in water) or dispersions, and sterile powders for extemporaneous preparation of sterile injection solutions or dispersions. Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. In some embodiments, the formulations include isotonic agents in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. Sterile injection solutions can be prepared by incorporating the required amount of the complex with one or a combination of the above-listed ingredients in the selected solvent, and then optionally filtering and sterilizing.

[0369] In some embodiments, the composition may contain at least about 0.1% of the complex or its components, although the percentage of active ingredient(s) may be between about 1% and about 80% or more by weight or volume of the total composition.Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will be taken into account by those skilled in the art who prepare such pharmaceutical formulations.Therefore, different dosages and treatment schedules may be desired.

[0370] IV. Method of Use / Treatment The complex comprising the muscle targeting agent covalently linked to the molecular payload as described herein is effective for treating FSHD.In some embodiments, the complex is effective for treating FSHD type 1.In some embodiments, the complex is effective for treating FSHD type 2.In some embodiments, FSHD is associated with the deletion of D4Z4 repeats on chromosome 4, which contains the DUX4 gene.In some embodiments, FSHD is associated with a mutation in the SMCHD1 gene.

[0371] In some embodiments, the subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject.In some embodiments, the subject may have myotonic dystrophy.In some embodiments, the subject has elevated expression of DUX4 gene outside fetal development and testis.In some embodiments, the subject has facioscapulohumeral muscular dystrophy type 1 or type 2.In some embodiments, the subject with FSHD has a mutation in SMCHD1 gene.In some embodiments, the subject with FSHD has a deletion mutation in D4Z4 repeat region on chromosome 4.

[0372] Aspects of the present disclosure include methods involving administering to a subject an effective amount of a complex as described herein. In some embodiments, an effective amount of a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently linked to a molecular payload may be administered to a subject in need of treatment. In some embodiments, a pharmaceutical composition comprising a complex as described herein may be administered by a suitable route, which may include intravenous administration, for example as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration may be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, or intrathecal routes. In some embodiments, the pharmaceutical composition may be in a solid, aqueous, or liquid form. In some embodiments, the aqueous or liquid form may be sprayed or lyophilized. In some embodiments, the sprayed or lyophilized form may be reconstituted with an aqueous or liquid solution.

[0373] Compositions for intravenous administration may contain a variety of carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). Water-soluble antibodies for intravenous injection may be administered by drip infusion, whereby a pharmaceutical formulation containing the antibody and a pharmaceutically acceptable excipient is infused. Pharmaceutically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody, may be administered dissolved in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0374] In some embodiments, pharmaceutical compositions comprising a conjugate comprising a muscle-targeting agent covalently linked to a molecular payload are administered via site-specific or localized delivery techniques, examples of which include an implanted depot source of the conjugate, a localized delivery catheter, a site-specific carrier, direct injection, or direct application.

[0375] In some embodiments, the pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload is administered at an effective concentration that provides a therapeutic effect to the subject. The effective amount will vary depending on the severity of the disease, the specific characteristics of the subject being treated, such as age, physical condition, health, or weight, the duration of treatment, the nature of any concomitant treatments, the route of administration, and related factors, as will be appreciated by those skilled in the art. These related factors are known to those skilled in the art and can be addressed with a little routine experimentation. In some embodiments, the effective concentration is the maximum dose that is deemed safe for the patient. In some embodiments, the effective concentration will be the lowest concentration feasible that provides maximum efficacy.

[0376] Empirical considerations, such as the half-life of the conjugate in the subject, will generally contribute to determining the concentration of pharmaceutical composition used for treatment. The frequency of administration may be empirically determined and adjusted to maximize the efficacy of the treatment.

[0377] The efficacy of treatment can be assessed by any suitable method. In some embodiments, the efficacy of treatment can be assessed by evaluating the symptoms associated with FSHD, including muscle mass loss and muscle atrophy, mainly in the muscles of the face, shoulder blades, and upper arms.

[0378] In some embodiments, a pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload described herein is administered to a subject at an effective concentration sufficient to inhibit target gene activity or expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% as compared to a control (e.g., a baseline level of gene expression prior to treatment).

[0379] example Example 1. Effect of a conjugate containing an anti-TfR Fab conjugated to a DUX4-targeting oligonucleotide in immortalized myoblasts derived from FSHD patients Anti-TfR Fab 3M12 VH4 / VK3 was conjugated to a DUX4-targeting oligonucleotide (SEQ ID NO: 151) via a cleavable Val-Cit linker to achieve enhanced muscle delivery of the oligonucleotide. The oligonucleotide is a PMO and targets the polyadenylation signal of the DUX4 transcript. The activity of the conjugate was evaluated in C6 (AB1080) immortalized FSHD1 cell line, which has significant levels of surface TfR1 expression and activation of DUX4 transcriptome markers (MBD3L2, TRIM43, ZSCAN4). It has been demonstrated that receptor-mediated delivery of PMO (SEQ ID NO: 151) by anti-TfR Fab into muscle cells resulted in a ∼75% reduction in DUX4 transcriptome biomarkers at 8 nM PMO concentration, while the equivalent unconjugated PMO showed no significant biomarker reduction compared to vehicle-treated cells (Figure 1). The results show that conjugation with an anti-TfR Fab enhances delivery of oligonucleotides to muscle cells for the treatment of FSHD.

[0380] The term "unconjugated" as used in this example indicates that the oligonucleotide was not conjugated to the antibody.

[0381] In addition, the dose-response curve for the reduction of MBD3L2 mRNA is shown in Figure 2A. The half maximal concentration (IC50) value for the conjugate was 189 pM. The dose-response curve for the reduction of MBD3L2, TRIM43, and ZSCAN4 mRNA is shown in Figure 2B. The IC50 values ​​for the conjugate inhibiting MBD3L2, TRIM43, and ZSCAN4 were 200 pM, 50 pM, and 200 pM, respectively.

[0382] Experimental procedure for Example 1 Cell culture and test article treatment C6 (AB1080) immortalized FSHD myoblasts were seeded to a density of 45,000 cells / well on 96-well plates (ThermoFisher Scientific) in Skeletal Growth Media (CAT#C-23060, Promocell) with Supplementary mix (C-39365, Promocell) and 1% Penstrep (15140-122, Gibco). Growth media was replaced after 24 hours with Differentiation Media, NbActiv4 (Brainbits) and 1% Pen / Strep (Gibco). Cells were treated in technical replicates with unconjugated DUX4-targeted oligonucleotides, conjugates at a PMO concentration of 8 nM, or vehicle for 4 hours prior to washing out with 1XPBS (10010023, Gibco). Conditioned differentiation medium was immediately added back to the wells and cells were harvested 5 days later for downstream analyses.

[0383] For dose response curves for MBD3L2, TRIM43, and ZSCAN4 knockdown, C6 (AB1080) immortalized FSHD myoblasts were treated as described above but with varying concentrations of conjugate.

[0384] RNA extraction and qPCR Total RNA was extracted from cell monolayers with the RNeasy 96 Kit (Qiagen) according to the manufacturer's instructions. RNA was quantified with a Biotek Plate Reader, diluted to 50 ng per sample with Nuclease-Free Water (Qiagen), and reverse transcribed with qScript cDNA SuperMix (QuantaBio). Gene expression was analyzed by measuring the transcript levels of TRIM43 (Hs00299174_m1), MBD3L2 (Hs00544743_m1), ZSCAN4 (Hs00537549_m1), and RPL13A (Hs04194366_g1) by qPCR with specific TaqMan assays (ThermoFisher). Two-step amplification reactions and fluorescence measurements for Ct determination were performed on a QuantStudio 7 instrument (Thermo Scientific). Log fold changes in expression of transcripts of interest were measured using RPL13A as the reference gene and cells exposed to vehicle as the control group, as a 2-fold increase in expression of the transcripts of interest. ΔΔCT Calculations were performed according to the method. Data were expressed as mean ± SD.

[0385] Example 2. Pharmacokinetic characterization of antibody-oligonucleotide conjugates in non-human primates DUX4-targeted oligonucleotide (SEQ ID NO: 151) was administered intravenously to non-human primates either naked or conjugated to anti-TfR1 antibody (3M12 VH4 / Vk3 Fab). Naked oligonucleotide was administered at a dose of 30 mg / kg, and the conjugate was administered at doses of 3 mg / kg, 10 mg / kg, or 30 mg / kg oligonucleotide equivalent. Plasma levels of oligonucleotide measured over time are shown in FIG. 3. The results demonstrate that the systemic exposure of the antibody-oligonucleotide conjugate exhibits dose-dependent pharmacokinetic properties, achieving higher exposure compared to naked oligonucleotide. Plasma measurements also demonstrate that the antibody-oligonucleotide conjugate has a long serum half-life of approximately 60 hours. Furthermore, the antibody-oligonucleotide conjugate exhibits a 58-fold increase in area under the curve (AUC), C , compared to naked oligonucleotide at a dose of 30 mg / kg oligonucleotide equivalent.max These results are summarized in Table 16. Table 16. Pharmacokinetic values ​​calculated from measured plasma concentrations [Table 16]

[0386] Two weeks after administration of oligonucleotide or antibody-oligonucleotide conjugate, necropsies were performed to harvest muscle tissue from non-human primates and oligonucleotide levels were measured. In each muscle tissue tested (heart, orbicularis oris, zygomaticus major, diaphragm, trapezius, deltoid, gastrocnemius, biceps, quadriceps, and tibialis anterior), tissue oligonucleotide levels were higher at each dose of antibody-oligonucleotide conjugate (3, 10, or 30 mg / kg oligonucleotide equivalent) compared to naked oligonucleotide (30 mg / kg) (Figure 4). As a control, tissue oligonucleotide levels were also measured in tissue harvested from vehicle-treated animals, and no oligonucleotide was detected in any of the muscle tissues tested. These results demonstrate that the antibody-oligonucleotide conjugate achieved high exposure of DUX4-targeted oligonucleotide to muscle tissue, significantly higher than the naked oligonucleotide administered. At an oligonucleotide equivalent dose of 30 mg / kg, oligonucleotide concentrations in each muscle tested were 26- to 139-fold higher in animals treated with antibody-oligonucleotide conjugates compared to naked oligonucleotide.

[0387] To assess tissue accumulation of DUX4-targeted oligonucleotides over time, tissue oligonucleotide levels were measured in gastrocnemius muscle biopsies taken one week after dosing and compared to values ​​measured in autopsy samples taken two weeks after dosing. Oligonucleotide levels were significantly higher in gastrocnemius muscle biopsies taken from animals administered 3, 10, or 30 mg / kg oligonucleotide equivalents of antibody-oligonucleotide conjugate than in biopsies taken from animals administered 30 mg / kg naked oligonucleotide, and even higher in tissue taken two weeks after dosing (Figure 5). No oligonucleotide was detected in tissue samples from vehicle-treated animals. These results demonstrate that the antibody-oligonucleotide conjugate achieves higher exposure of DUX4-targeted oligonucleotides to muscle tissue compared to naked oligonucleotides, and that the conjugate continues to accumulate over time.

[0388] Example 3. Effect of a conjugate containing an anti-TfR Fab conjugated to a DUX4-targeting oligonucleotide in immortalized myoblasts derived from FSHD patients Anti-TfR Fab 3M12 VH4 / VK3 was conjugated to DUX4-targeted oligonucleotides (oligonucleotides #8, #1, or #2 as listed in Table 8, corresponding to SEQ ID NOs: 176, 169, and 170, respectively) via a cleavable Val-Cit linker to achieve enhanced muscle delivery of the oligonucleotide. A control conjugate was also produced by conjugating anti-TfR Fab 3M12 VH4 / VK3 to a control DUX4-targeted oligonucleotide (SEQ ID NO: 151) via the same cleavable Val-Cit linker. The activity of the conjugates was evaluated in the C6 (AB1080) immortalized FSHD1 cell line, which has significant levels of surface TfR1 expression and activation of transcriptomic markers of DUX4 (MBD3L2, TRIM43, ZSCAN4).

[0389] C6 (AB1080) immortalized FSHD myoblasts were seeded onto 384-well plates (ThermoFisher Scientific) to a density of 410,000 cells / well in Skeletal Growth Media (CAT#C-23060, Promocell) with Supplementary mix (C-39365, Promocell) and 1% Penstrep (15140-122, Gibco). Growth medium was replaced after 24 hours with Differentiation medium, NbActiv4 (Brainbits) and 1% Pen / Strep (Gibco). Cells were treated with conjugates at concentrations equivalent to 10 pM, 1 nM, or 100 nM of oligonucleotide for 10 days and later harvested for analysis at the end point.

[0390] As shown in Figure 6, conjugates containing anti-TfR Fab 3M12 VH4 / Vk3 conjugated to DUX4-targeting oligonucleotides (#8, #1, or #2 in Table 8, corresponding to SEQ ID NOs: 176, 169, and 170, respectively) and control conjugates reduced the expression levels of DUX4 transcriptome markers in FSHD patient cells. These results indicate that the conjugates reduced DUX4 expression levels in FSHD patient cells in vitro. Additional Aspects 1. A conjugate comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured to reduce expression or activity of DUX4, wherein the anti-TfR1 antibody comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), light chain complementarity determining region 3 (CDR-L3) of any of the anti-TfR1 antibodies listed in Tables 2-7, and wherein the oligonucleotide comprises an antisense strand comprising a region of complementarity to a DUX4 sequence as set forth in SEQ ID NO: 160 or SEQ ID NO: 365. 2. The conjugate of embodiment 1, wherein the anti-TfR1 antibody comprises the heavy chain variable region (VH) and light chain variable region (VL) of any of the anti-TfR1 antibodies listed in Table 3. 3. The conjugate of any one of embodiment 1 or embodiment 2, wherein the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76 and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75, optionally wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 75. 4. The conjugate of embodiment 1 or embodiment 2, wherein the anti-TfR1 antibody is a Fab, optionally wherein the Fab comprises the heavy and light chains of any of the anti-TfR1 Fabs listed in Table 5. 5. The conjugate of embodiment 4, wherein the Fab comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO:101 and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO:90, optionally wherein the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90. 6. The conjugate of any one of aspects 1 to 5, wherein the oligonucleotide is 20 to 30 nucleotides in length. 7. The conjugate of any one of aspects 1-6, wherein the oligonucleotide comprises a region of complementarity of at least 15 contiguous nucleotides to the DUX4 sequence as set forth in SEQ ID NO: 160 or SEQ ID NO: 365. 8. The conjugate of any one of embodiments 1-7, wherein the oligonucleotide comprises a region of complementarity of at least 15 contiguous nucleotides to a DUX4 sequence as represented in any one of SEQ ID NOs: 161-168 or 213-288. 9. The conjugate of any one of aspects 1-8, wherein the oligonucleotide comprises at least 15 contiguous nucleotides of any one of SEQ ID NOs: 169-176 or 289-364, wherein each thymine base (T) is independently and optionally replaced by a uracil base (U), and each U is independently and optionally replaced by a T. 10. The conjugate of any one of aspects 1 to 9, wherein the oligonucleotide does not comprise the nucleotide sequence of SEQ ID NO: 151. 11. The conjugate of any one of aspects 1 to 9, wherein the oligonucleotide comprises a nucleotide sequence of any one of SEQ ID NOs: 169-176 or 289-364. 12. The conjugate of any one of embodiments 1-11, wherein the oligonucleotide further comprises a sense strand that hybridizes to the antisense strand to form the double-stranded siRNA. 13. The conjugate of any one of aspects 1 to 12, wherein the oligonucleotide comprises at least one modified internucleoside linkage. 14. The conjugate of any one of aspects 1-13, wherein the oligonucleotide comprises one or more modified nucleosides, optionally wherein the one or more modified nucleosides are 2'-modified nucleosides. 15. The conjugate of any one of aspects 1 to 12, wherein the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). 16. The conjugate of any one of aspects 1 to 15, wherein the antibody and the oligonucleotide are covalently linked via a linker. 17. The conjugate of claim 16, wherein the linker is a cleavable linker, optionally wherein the linker comprises a valine-citrulline sequence. 18. A method for reducing DUX4 expression in a muscle cell, the method comprising contacting the muscle cell with a complex of any one of aspects 1-17 in an amount effective to promote internalization of the oligonucleotide into the muscle cell. 19. The method of embodiment 18, wherein the cell is in vitro. 20. The method of embodiment 18, wherein the cell is in a subject. 21. The method of embodiment 20, wherein the subject is a human. 22. A method of treating facioscapulohumeral muscular dystrophy (FSHD), the method comprising administering to a subject in need thereof an effective amount of a conjugate of any one of embodiments 1-17, wherein the subject has abnormal production of DUX4 protein. 23. The method of any one of aspects 20-22, wherein the subject has one or more deletions of the D4Z4 repeats in chromosome 4. 24. The method of embodiment 23, wherein the subject has 10 or fewer D4Z4 repeats. 25. The me...

Claims

1. A conjugate comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured to reduce the expression or activity of DUX4, wherein the anti-TfR1 antibody (i) comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 76 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 75, or (ii) comprises the VH and VL of any of the other anti-TfR1 antibodies listed in Tables 2-7, and wherein the oligonucleotide targets the 3' UTR sequence of DUX4.

2. The conjugate of claim 1 , wherein the anti-TfR1 antibody comprises the VH and VL of any of the anti-TfR1 antibodies listed in Table 3.

3. The complex described in claim 1, wherein the anti-TfR1 antibody comprises a VH consisting of the amino acid sequence of SEQ ID NO: 76 and a VL consisting of the amino acid sequence of SEQ ID NO:

75.

4. 2. The conjugate of claim 1, wherein the anti-TfR1 antibody is a Fab, optionally wherein the Fab comprises the heavy and light chains of any of the anti-TfR1 Fabs listed in Table 5.

5. The conjugate of claim 1, wherein the anti-TfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101 and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90, optionally wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO:

90.

6. A conjugate described in any one of claims 1 to 5, wherein the anti-TfR1 antibody comprises a heavy chain containing pyroglutamic acid at its N-terminus.

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

8. 6. The conjugate of any one of claims 1 to 5, wherein the oligonucleotide comprises an antisense strand comprising a region of complementarity of at least 15 consecutive nucleotides to the 3' UTR sequence of DUX4.

9. 9. The complex of claim 8, wherein the oligonucleotide further comprises a sense strand that hybridizes with the antisense strand to form a double-stranded siRNA, optionally wherein the sense strand and the antisense strand are 15 to 25 nucleotides in length, and further optionally wherein the sense strand and the antisense strand are 21 to 23 nucleotides in length.

10. 6. The conjugate of any one of claims 1 to 5, wherein the oligonucleotide does not comprise the nucleotide sequence of SEQ ID NO:

151.

11. The complex described in claim 9, wherein the sense strand and antisense strand are of different lengths, and optionally wherein the double-stranded siRNA comprises a 3' overhang of about 1 to about 3 nucleotides on the antisense strand.

12. 6. The conjugate of any one of claims 1 to 5, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

13. The conjugate of any one of claims 1 to 5, wherein the oligonucleotide comprises one or more modified nucleosides, optionally wherein one or more modified nucleosides are 2'-modified nucleosides.

14. A conjugate described in any one of claims 1 to 5, wherein the anti-TfR1 antibody and the oligonucleotide are covalently linked via a linker, optionally wherein the linker is a cleavable linker, and further optionally wherein the linker comprises a valine-citrulline sequence.

15. The conjugate of any one of claims 1 to 5, wherein the oligonucleotide is covalently linked to the anti-TfR1 antibody through a cysteine ​​residue of the anti-TfR1 antibody.

16. The conjugate of any one of claims 1 to 5, wherein the oligonucleotide is covalently linked to the anti-TfR1 antibody through a lysine residue of the anti-TfR1 antibody.

17. A complex according to any one of claims 1 to 5, wherein the oligonucleotide is in a salt form, optionally wherein the salt form is a sodium salt, a potassium salt, or a magnesium salt.

18. 6. The conjugate of any one of claims 1 to 5 for use in a method for reducing DUX4 expression in muscle cells, wherein a medicament comprising the conjugate of any one of claims 1 to 5 is formulated to promote internalization of the oligonucleotide into muscle cells.

19. 20. The conjugate of claim 18, wherein the cell is in a subject, optionally wherein the subject is a human.

20. The complex described in claim 19, wherein the subject has one or more deletions of D4Z4 repeats in chromosome 4.

21. A complex described in any one of claims 1 to 5 for use in a method for treating facioscapulohumeral muscular dystrophy (FSHD) in a subject, wherein the subject has abnormal production of DUX4 protein.

22. The complex of claim 21, wherein the subject is a human.

23. 23. The complex of claim 22, wherein the subject has one or more deletions of the D4Z4 repeat in chromosome 4.

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