Muscle-targeting complexes and their use for treating dystrophinopathies - Patents.com

Abstract

Aspects of the present disclosure relate to a complex comprising a muscle targeting agent covalently linked to a molecular payload. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, the molecular payload promotes the expression or activity of a functional dystrophin protein. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide, for example an oligonucleotide that causes exon skipping in mRNA expressed from a mutant DMD allele.

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 220030, entitled "MUSCLE-TARGETING COMPLEXES AND USES THEREOF FOR TREATING DYSTROPHINOPATHIES," filed July 9, 2021, the entire contents of which are incorporated herein by reference.

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

[0003] Electronic Sequence Listing Reference The contents of the electronic sequence listing (D082470066WO00-SEQ-COB.xml, size: 1,203,807 bytes, created July 7, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0004] Background of the Invention Dystrophinopathies are a group of distinct neuromuscular diseases caused by mutations in the gene encoding dystrophin. Dystrophinopathies include Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy. The DMD gene ("DMD") encoding dystrophin is a large gene containing 79 exons and approximately 2.6 million total base pairs. Numerous DMD mutations, such as exonic frameshifts, deletions, substitutions, and duplications, can reduce the expression of functional dystrophin, resulting in dystrophinopathies. Several drugs targeting human DMD exons have been approved by the U.S. Food and Drug Administration (FDA), including casimersen, viltolarsen, golodirsen, and eteplirsen. Among these, eteplirsen targets exon 51. Summary of the Invention

[0005] Summary of the Invention According to some aspects, the present disclosure provides complexes that target muscle cells for the purpose of delivering molecular payloads to those cells, as well as molecular payloads that can be used therein. In some embodiments, the complexes provided herein are particularly useful for delivering molecular payloads that increase or restore the expression or activity of functional dystrophin proteins. In some embodiments, the complexes comprise oligonucleotide-based molecular payloads that promote the expression of functional dystrophin proteins through in-frame exon skipping or suppression of stop codons, such as by promoting skipping of DMD exon 51. In some embodiments, the molecular payloads provided herein are useful for facilitating exon skipping in DMD sequences, such as skipping of DMD exon 51. Thus, in some embodiments, the complexes provided herein comprise a muscle-targeting agent (e.g., a muscle-targeting antibody) that specifically binds to a receptor on the surface of muscle cells for the purpose of delivering the molecular payload to muscle cells. In some embodiments, the complexes are internalized into cells via receptor-mediated internalization, after which the molecular payload can be released into the cells and perform its function. For example, a conjugate engineered to deliver an oligonucleotide can release the oligonucleotide so that it can promote the expression of functional dystrophin protein in muscle cells (e.g., via an exon skipping mechanism, such as by facilitating skipping of DMD exon 51). In some embodiments, the oligonucleotide is released by endosomal cleavage of a covalent linker connecting the oligonucleotide and the muscle-targeting agent of the conjugate. The conjugates and molecular payloads provided herein can be used to treat subjects with a mutant DMD gene, such as a mutant DMD gene suitable for skipping exon 51.

[0006] According to some aspects, provided herein is a conjugate comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured to induce skipping of exon 51 in DMD pre-mRNA, wherein the oligonucleotide comprises a region of complementarity that is complementary to at least 8 contiguous nucleotides of any one of SEQ ID NOs: 160-383.

[0007] In some embodiments, the anti-TfR1 antibody is (i) heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 33, heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 34, heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 35, light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 36, light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 37, and light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 32; (ii) CDR-H1 of SEQ ID NO: 7, CDR-H2 of SEQ ID NO: 8, CDR-H3 of SEQ ID NO: 9, CDR-L1 of SEQ ID NO: 10, CDR-L2 of SEQ ID NO: 11, and CDR-L3 of SEQ ID NO: 6; (iii) CDR-H1 of SEQ ID NO: 7, CDR-H2 of SEQ ID NO: 20, CDR-H3 of SEQ ID NO: 9, CDR-L1 of SEQ ID NO: 10, CDR-L2 of SEQ ID NO: 11, and CDR-L3 of SEQ ID NO: 6; (iv) CDR-H1 of SEQ ID NO: 7, CDR-H2 of SEQ ID NO: 24, CDR-H3 of SEQ ID NO: 9, CDR-L1 of SEQ ID NO: 10, CDR-L2 of SEQ ID NO: 11, and CDR-L3 of SEQ ID NO: 6; (v) CDR-H1 of SEQ ID NO: 51, CDR-H2 of SEQ ID NO: 52, CDR-H3 of SEQ ID NO: 53, CDR-L1 of SEQ ID NO: 54, CDR-L2 of SEQ ID NO: 55, and CDR-L3 of SEQ ID NO: 50; (vi) CDR-H1 of SEQ ID NO: 64, CDR-H2 of SEQ ID NO: 52, CDR-H3 of SEQ ID NO: 53, CDR-L1 of SEQ ID NO: 54, CDR-L2 of SEQ ID NO: 55, and CDR-L3 of SEQ ID NO: 50; or (vii) CDR-H1 of SEQ ID NO: 67, CDR-H2 of SEQ ID NO: 52, CDR-H3 of SEQ ID NO: 53, CDR-L1 of SEQ ID NO: 54, CDR-L2 of SEQ ID NO: 55, and CDR-L3 of SEQ ID NO: 50 Includes:

[0008] In some embodiments, the anti-TfR1 antibody is (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 75; (ii) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 69; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 70; (iii) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 71; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 70; (iv) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 72; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 70; (v) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 73; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 74; (vi) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 73; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 75; (vii) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 76; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 74; (viii) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 77; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 78; (ix) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 79; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 80; or (x) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 77; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 80; Includes:

[0009] In some embodiments, the anti-TfR1 antibody is (i) 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; (ii) 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; (iii) 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; (iv) 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; (v) 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; (vi) 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; (vii) 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; (viii) 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; (ix) 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; or (x) 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; Includes:

[0010] In some embodiments, the anti-TfR1 antibody may be a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an scFv, an Fv fragment, or a full-length IgG.

[0011] In some embodiments, the anti-TfR1 antibody is a Fab fragment.

[0012] In some embodiments, the anti-TfR1 antibody is (i) 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; (ii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 97; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (iii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 98; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (iv) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 99; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (v) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89; (vi) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90; (vii) 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: 89; (viii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 93; (ix) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 103; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; or (x) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; Includes:

[0013] In some embodiments, the anti-TfR1 antibody is (i) 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; (ii) 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; (iii) 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; (iv) 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; (v) 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; (vi) 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; (vii) 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; (viii) 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; (ix) 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; or (x) 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. Includes:

[0014] In some embodiments, the anti-TfR1 antibody does not specifically bind to the transferrin binding site of transferrin receptor 1 and / or the anti-TfR1 antibody does not inhibit the binding of transferrin to transferrin receptor 1.

[0015] In some embodiments, the oligonucleotide comprises a region of complementarity to at least four consecutive nucleotides of a splicing feature of the DMD pre-mRNA.

[0016] In some embodiments, the splicing feature is an exonic splicing enhancer (ESE) in exon 51 of the DMD pre-mRNA, and optionally, the ESE comprises the sequence of any one of SEQ ID NOs: 860-894.

[0017] In some embodiments, the splicing feature is a branch point, splice donor site, or splice acceptor site, and optionally, the splicing feature is located across the junction of exon 50 and intron 50, within intron 50, across the junction of intron 50 and exon 51, across the junction of exon 51 and intron 51, within intron 51, or across the junction of intron 51 and exon 52 of the DMD pre-mRNA, and further optionally, the splicing feature comprises the sequence of any one of SEQ ID NOs: 855-859 and 895-898.

[0018] In some embodiments, the oligonucleotide comprises a sequence complementary to any one of SEQ ID NOs: 160-383, or any one of SEQ ID NOs: 384-831, wherein 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.

[0019] In some embodiments, the oligonucleotide comprises one or more phosphorodiamidate morpholinos, and optionally, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

[0020] In some embodiments, the anti-TfR1 antibody is covalently linked to the oligonucleotide via a cleavable linker, optionally, the cleavable linker comprises a valine-citrulline sequence.

[0021] In some embodiments, the anti-TfR1 antibody is covalently linked to the oligonucleotide via conjugation to a lysine or cysteine ​​residue of the antibody.

[0022] According to some aspects, provided herein are oligonucleotides targeting DMD, wherein the oligonucleotide comprises a region of complementarity to any one of SEQ ID NOs: 160-383, and optionally, the region of complementarity comprises at least 15 contiguous nucleosides complementary to any one of SEQ ID NOs: 160-383.

[0023] In some embodiments, the oligonucleotide comprises at least 15 consecutive nucleosides of any one of SEQ ID NOs: 384-831, and optionally, the oligonucleotide comprises the sequence of any one of SEQ ID NOs: 384-831, wherein each thymine base (T) is independently and optionally substituted with a uracil base (U), and each U is independently and optionally substituted with a T.

[0024] According to some aspects, provided herein is a method of delivering an oligonucleotide to a cell, the method comprising contacting the cell with a complex disclosed herein or an oligonucleotide disclosed herein.

[0025] According to some aspects, provided herein is a method of promoting dystrophin protein expression or activity in a cell, the method comprising contacting the cell with a conjugate disclosed herein or an oligonucleotide disclosed herein in an amount effective to promote internalization of the oligonucleotide into the cell, optionally wherein the cell is a muscle cell.

[0026] In some embodiments, the subject has a DMD gene suitable for exon 51 skipping.

[0027] In some embodiments, the DMD protein is a truncated DMD protein. [Brief explanation of the drawings]

[0028] [Figure 1]FIG. 1 shows data demonstrating that a conjugate containing an anti-TfR1 Fab (3M12 VH4 / Vκ3) conjugated to a DMD exon skipping oligonucleotide enhanced exon skipping compared to naked DMD exon skipping oligo in myotubes from Duchenne muscular dystrophy patients. DETAILED DESCRIPTION OF THE INVENTION

[0029] Detailed Description of the Invention Aspects of the present disclosure relate to the recognition that, although certain molecular payloads (e.g., oligonucleotides, peptides, small molecules) can have beneficial effects on muscle cells, effectively targeting such cells has proven difficult. Accordingly, as described herein, the present disclosure provides a complex comprising a muscle targeting agent covalently linked to a molecular payload to overcome such challenges. In some embodiments, the complex is particularly useful for delivering a molecular payload that modulates (e.g., promotes) the expression or activity of a dystrophin protein (e.g., a truncated dystrophin protein) or DMD (e.g., a mutant DMD allele). In some embodiments, the complex provided herein may comprise an oligonucleotide that promotes the expression and activity of a dystrophin protein or DMD, such as by promoting in-frame exon skipping and / or suppression of a premature stop codon. For example, the complex may comprise an oligonucleotide that induces the skipping of an exon(s) of a DMD RNA (e.g., a pre-mRNA), such as an oligonucleotide that induces the skipping of exon 51. In some embodiments, synthetic nucleic acid payloads (eg, DNA or RNA payloads) can be used that express one or more proteins that promote the normal expression and activity of dystrophin protein or DMD.

[0030] Duchenne muscular dystrophy is an X-linked muscular disorder caused by one or more mutations in the DMD gene located on Xp21. Dystrophin protein typically forms the dystrophin-associated glycoprotein complex (DGC) in sarcolemma, which connects the sarcolemma structure to the extracellular matrix and protects the sarcolemma from contraction-induced damage. In patients with Duchenne muscular dystrophy, dystrophin protein is generally absent, and muscle fibers typically become damaged due to mechanical overstrain. Mutations in the DMD gene are associated with two types of muscular dystrophies: Duchenne muscular dystrophy and Becker muscular dystrophy, depending on whether the translational reading frame is lost or maintained. Becker muscular dystrophy is a clinically milder form of Duchenne muscular dystrophy and is characterized by features similar to Duchenne muscular dystrophy. In some embodiments, exon skipping induced by oligonucleotides (e.g., oligonucleotides delivered using the conjugates provided herein) can be used to restore the reading frame of the mutant DMD allele, resulting in the production of a truncated dystrophin protein that is sufficiently functional to improve muscle function. In some embodiments, such exon skipping converts the Duchenne muscular dystrophy phenotype to the milder Becker muscular dystrophy phenotype.

[0031] Further aspects of the disclosure, including explanations of defined terms, are provided below.

[0032] 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 a subject).

[0033] 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 (more 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 when such number exceeds 100% of a workable value).

[0034] 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, except that 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, an 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, an antibody comprises a constant region, for example, an Fc region. An immunoglobulin constant region refers to a heavy or light chain constant region. Human IgG heavy and light chain constant region amino acid sequences and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an 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 an antibody described herein can comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, an antibody described herein comprises a human gamma 1 CH1 domain, CH2 domain, and / or (for example, and), a CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any sequence known in the art.Non-limiting examples of human constant region sequences are described in the art; see, e.g., 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 at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules comprise a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues linked by a peptide bond and is used to link one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Furthermore, an antibody may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of one or more other proteins or peptides with the antibody or antibody portion. 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, biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0035] Branch point: As used herein, the term "branch point" or "branch site" refers to a nucleic acid sequence motif within an intron of a gene or pre-mRNA that is involved in splicing pre-mRNA to mRNA (i.e., removing the intron from the pre-mRNA) and can be referred to as a splicing feature. Branch points are typically located 18 to 40 nucleotides from the 3' end of the intron and contain an adenine, but are otherwise relatively unconstrained in sequence. Common branch point sequence motifs are YNYYRAY, YTRAC, and YNYTRAY, where Y is a pyrimidine, N is any nucleotide, R is any purine, and A is an adenine. During splicing, the pre-mRNA is cleaved at the 5' end of the intron and then binds to the downstream branch point region via a transesterification bond between a guanine and an adenine from the 5' end and the branch point, respectively, forming a looped lariat structure.

[0036] CDR: As used herein, the term "CDR" refers to a complementarity-determining region within an antibody variable sequence. A typical antibody molecule contains a heavy chain variable region (VH) and a light chain variable region (VL), which are typically involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity-determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). Each VH and VL typically consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methodologies known in the art, such as 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) 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 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 CDRs means that the two antibodies have the same amino acid sequence of their CDRs when determined by the same method, for example, the IMGT definition.

[0037] 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 have been defined differently according to various systems. The system described by Kabat (Kabat et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any antibody variable region, 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, whose boundaries overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Still other CDR boundary definitions may not strictly adhere to one of the above systems, but may still overlap with the Kabat CDRs, and may be shortened or extended in light of predictions or experimental findings that a particular residue or group of residues, or even an entire CDR, does not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are shown in Table 1. [Table 1]

[0038] 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 the sequence of one or more of the CDR regions of its VH and / or (for example, and) VL have been replaced with CDR sequences from another species, such as an antibody having murine heavy and light chain variable regions but in which one or more of the murine CDRs (for example, CDR3) have been replaced with human CDR sequences.

[0039] 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 human constant regions.

[0040] Complementary: As used herein, the term "complementary" refers to the ability for precise pairing between two nucleosides or two pairs of nucleosides.In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in binding between two nucleosides or two pairs of nucleosides.For example, if the base of an oligonucleotide at a certain position can hydrogen bond with the base of a target nucleic acid (for example, mRNA) at the corresponding position, then the bases are considered to be complementary to each other at that position.Base pairing may include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (for example, Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U, or T. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U, or T.

[0041] 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, and can be found, for example, in 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, FMA Usubel, 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.

[0042] Covalently linked: As used herein, the term "covalently linked" refers to the characteristic of two or more molecules being linked together via at least one covalent bond. In some embodiments, two molecules may be covalently linked together by a single bond (e.g., a disulfide bond or disulfide bridge) that acts as an intermolecular linker. However, in some embodiments, two or more molecules may be covalently linked together via a molecule that acts as a linker that connects two or more molecules together through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker.

[0043] 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 is capable of specifically binding to more than one antigen of the same type or class (e.g., multiple homologous, paralogous, or orthologous 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) is capable of binding 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.

[0044] DMD: As used herein, the term "DMD" refers to the gene encoding dystrophin protein, which is an important component of the dystrophin-glycoprotein complex that bridges the internal cytoskeleton of muscle cells, especially muscle fibers, with the extracellular matrix. Deletions, duplications, and point mutations in DMD can cause dystrophinopathies such as Duchenne muscular dystrophy, Becker muscular dystrophy, or cardiomyopathy. Alternative promoter usage and alternative splicing result in a large number of different transcriptional variants and protein isoforms for this gene. In some embodiments, the dystrophin gene (DMD or DMD gene) can be a human (gene ID: 1756), non-human primate (e.g., gene ID: 465559), or rodent gene (e.g., gene ID: 13405, gene ID: 24907). Furthermore, multiple human transcript variants (e.g., annotated under GenBank RefSeq accession numbers NM_000109.3, NM_004006.2, NM_004009.3, NM_004010.3, and NM_004011.3) encoding different protein isoforms have been characterized.

[0045] DMD allele: As used herein, the term "DMD allele" refers to any one of the alternative forms (for example, wild type or mutant) of the DMD gene. In some embodiments, the DMD allele can encode dystrophin that maintains its normal and typical function. In some embodiments, the DMD allele can contain one or more mutations that cause muscular dystrophy. Common mutations that cause Duchenne muscular dystrophy include frameshift, deletion, substitution and duplication mutations in one or more of the 79 exons present in the dystrophin allele, for example, exon 8, exon 23, exon 41, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53 or exon 55. Further examples of DMD mutations are disclosed, for example, in Flanigan KM et al., Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009 Dec;30(12):1657-66, the entire contents of which are incorporated herein by reference.

[0046] Dystrophinopathy: As used herein, the term "dystrophinopathy" refers to a muscle disease resulting from one or more mutant DMD alleles. Dystrophinopathy includes a spectrum of conditions (ranging from mild to severe), such as Duchenne muscular dystrophy, Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy (DCM). In some embodiments, at one end of the spectrum, dystrophinopathy is phenotypically associated with asymptomatic increases in serum concentrations of creatine phosphokinase (CK) and / or muscle spasms accompanied by (for example) myoglobinuria. In some embodiments, at the other end of the spectrum, dystrophinopathy is phenotypically associated with progressive muscle diseases generally classified as Duchenne or Becker muscular dystrophy when skeletal muscle is primarily affected, or DMD-associated dilated cardiomyopathy (DCM) when the heart is primarily affected. Symptoms of Duchenne muscular dystrophy include muscle loss or degeneration, decreased muscle function, pseudohypertrophy of the tongue and calf muscles, a higher risk of neurological abnormalities, and a shortened lifespan. Duchenne muscular dystrophy is associated with Online Mendelian Inheritance in Man (OMIM) Entry #310200. Becker muscular dystrophy is associated with OMIM Entry #300376. Dilated cardiomyopathy is associated with OMIM Entry X#302045.

[0047] Exonic splicing enhancer (ESE): As used herein, the term "exonic splicing enhancer" or "ESE" refers to a nucleic acid sequence motif within an exon of a gene, pre-mRNA, or mRNA that directs or enhances splicing of the pre-mRNA into mRNA, as described, for example, in Blencowe et al., Trends Biochem Sci 25, 106-10. (2000), incorporated herein by reference. ESEs are sometimes referred to as splicing features. ESEs may direct or enhance splicing, for example, to remove one or more introns and / or one or more exons from a gene transcript. ESE motifs are typically 6 to 8 nucleobases in length. SR proteins (e.g., proteins encoded by genes SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, SRSF12, TRA2A, or TRA2B) bind to ESEs via their RNA recognition motif regions to promote splicing. ESE motifs can be identified by several methods, such as those described in Cartegni et al., Nucleic Acids Research, 2003, Vol. 31, No. 13, 3568-3571, which is incorporated herein by reference.

[0048] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Because the exact definition of the CDR sequence can be determined by various systems, the meaning of the framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain, and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains 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. When a framework region is referred to by others without specifying the specific subregion as FR1, FR2, FR3, or FR4, it refers to the combined FR(s) in the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR(s) refers to two or more of the four subregions containing framework regions. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0049] 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, amino acid residues in the CDRs, particularly CDR3, 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, have been grafted onto human framework sequences.

[0050] 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 in which at least a portion of the VH and / or (e.g., and) VL sequences have been altered to be more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced onto non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-TfR1 antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining a murine anti-TfR1 monoclonal antibody using existing hybridoma technology, followed by humanization using in vitro genetic engineering (such as that disclosed in WO 2005 / 123126 to Kasaian et al.).

[0051] 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 an external stimulus (e.g., a ligand binding to the receptor). In some embodiments, the internalizing cell surface receptor is internalized by endocytosis. In some embodiments, the internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalizing cell surface receptor is internalized by a clathrin-independent pathway, such as phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or (e.g., and) an extracellular domain, which optionally further 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.

[0052] Isolated antibody: As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the transferrin receptor is substantially free of antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to 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 (by way of example and) chemicals.

[0053] Kabat numbering: The terms "Kabat numbering," "Kabat definition," and "Kabat labeling" are used interchangeably herein. These terms, recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions 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, USDapartment of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable regions span amino acid positions 31-35 for CDR1, positions 50-65 for CDR2, and positions 95-102 for CDR3. In the light chain variable region, the hypervariable region spans amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

[0054] 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, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region of complementarity to a target gene.

[0055] 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, e.g., 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 can be internalized into the muscle cell through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, protein, peptide, nucleic acid (e.g., an aptamer), or antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0056] Muscle-targeting antibody: As used herein, the term "muscle-targeting antibody" refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, the muscle-targeting antibody specifically binds to an antigen on muscle cells that facilitates internalization of the muscle-targeting antibody (and any attached molecular payload) into the muscle cells. In some embodiments, the muscle-targeting antibody specifically binds to an internalizing cell surface receptor present on muscle cells. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0057] 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, phosphorodiamidates, 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 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, which may be in an Rp or Sp stereochemical configuration.

[0058] 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., an antibody expressed using a recombinant expression vector transfected into a host cell (as described in more detail in this disclosure), an antibody 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), or an antibody isolated from a human immunoglobulin gene transgenic animal (e.g., a mouse) (see, e.g., Taylor, LD, et al. (See, e.g., 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 would 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, which can be generated using techniques well known in the art, such as, but not limited to, techniques using human Ig phage libraries (e.g., those disclosed in WO 2005 / 007699 to Jermutus et al.).

[0059] Region of Complementarity: As used herein, the term "region of complementarity" refers to a nucleotide sequence (e.g., a nucleotide sequence of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., a nucleotide sequence of a target nucleic acid) such that the two nucleotide sequences are capable of annealing to each other under physiological conditions (e.g., in a cell). In some embodiments, the region of complementarity is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the region of complementarity is only partially complementary (e.g., at least 80%, 90%, 95%, or 99% complementary) to the cognate nucleotide sequence of the target nucleic acid. In some embodiments, the region of complementarity contains one, two, three, or four mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0060] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner in a binding assay or other binding context, with a degree of affinity or avidity that can be used to distinguish the binding partner from an appropriate control. With respect to an antibody, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen, compared to an appropriate reference antigen, or an antigen that can be used to distinguish the specific antigen from other antigens, with a degree of affinity or avidity (e.g., that allows preferential targeting to certain cells (e.g., muscle cells) through binding to the antigen, as described herein). In some embodiments, the antibody binds to the target with at least about 10 -4 M, 10 -5 M, 10 -6 M, 10-7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less 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.

[0061] Splice acceptor site: As used herein, the term "splice acceptor site" or "splice acceptor" refers to a nucleic acid sequence motif spanning the 3' end of an intron or intron / exon junction of a gene or pre-mRNA that is involved in splicing pre-mRNA to mRNA (i.e., removing the intron from the pre-mRNA), and can be referred to as a splicing feature. A splice acceptor site contains a terminal AG sequence at the 3' end of the intron, which is usually preceded (in the 5' direction) by a pyrimidine-rich region (C / U). Upstream of the splice acceptor site is the branch point. Formation of a lariat loop intermediate structure by a transesterification reaction between the branch point and the splice donor site releases the 3'-OH of the 5' exon, which then reacts with the first nucleotide of the 3' exon, thereby joining the exons and releasing the intron lariat. The AG sequence at the 3' end of the intron at the splice acceptor site is known to be important for proper splicing, as altering one of these nucleotides results in inhibition of splicing. Rarely, alternative splice acceptor sites have AC at the 3' end of the intron rather than the more common AG. Common splice acceptor site motifs are [Y-rich region]-NCAGG or Y- x It has a sequence of NYAGG or a sequence similar thereto, where N represents a pyrimidine, N represents any nucleotide, and x is a number from 4 to 20. The cleavage site is followed by AG, which represents the 3'-terminal nucleotide of the excised intron.

[0062] Splice donor site: As used herein, the term "splice donor site" or "splice donor" refers to a nucleic acid sequence motif spanning the 5' end of an intron or exon / intron junction of a gene or pre-mRNA that is involved in splicing pre-mRNA to mRNA (i.e., removing the intron from the pre-mRNA) and can be referred to as a splicing feature. The splice donor site contains a terminal GU sequence at the 5' end of the intron within a larger, largely unconstrained sequence. During splicing, the 2'-OH of the nucleotide within the branch point initiates a transesterification reaction via nucleophilic attack on the 5' G of the intron within the splice donor site. This causes the G to be cleaved from the pre-mRNA and instead bind to the branch point nucleotide, forming a loop lariat structure. The 3' nucleotide of the upstream exon then binds to the splice acceptor site, joining the exon and excising the intron. A typical splice donor site has the sequence GGGURAGU or AGGURNG or a sequence similar thereto, where R represents a purine and N represents any nucleotide. The cleavage site precedes the first GU (i.e., GG / GURAGU or AG / GURNG), which represents the 5'-terminal nucleotide of the excised intron.

[0063] 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, e.g., a human patient, having or suspected of having a disease. In some embodiments, the subject is a human patient having or suspected of having a disease caused by a mutated DMD gene sequence, e.g., a mutation in an exon of the DMD gene sequence. In some embodiments, the subject has a dystrophinopathy, e.g., Duchenne muscular dystrophy. In some embodiments, the subject is a patient with a DMD gene mutation suitable for skipping exon 51.

[0064] Transferrin receptor: As used herein, the term "transferrin receptor" (also known as TFRC, CD71, p90, or TFR1) refers to an internalized cell surface receptor that binds to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may originate from humans (NCBI Gene ID 7037), non-human primates (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodents (e.g., NCBI Gene ID 22042). In addition, multiple human transcript variants encoding different isoforms of the receptor have been characterized (e.g., those annotated with GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0065] 2'-modified nucleoside: As used herein, the terms "2'-modified nucleoside" and "2'-modified ribonucleoside" are used interchangeably and refer to a nucleoside having a sugar moiety modified at the 2'-position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, in which the 2' and 4' positions of the sugar are bridged (e.g., by a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, in which the 2' position of the sugar moiety is substituted. Non-limiting examples of 2'-modified nucleosides include the following: 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acids (LNA, methylene-bridged nucleic acids), ethylene-bridged nucleic acids (ENA), and (S)-constrained ethyl-bridged nucleic acids (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] Although these examples are shown using a phosphate group, any internucleoside linkage is contemplated between the 2'-modified nucleosides.

[0066] II. Complex Provided herein are conjugates comprising a targeting agent, for example, an antibody covalently linked to a molecular payload. In some embodiments, the conjugate comprises a muscle-targeting antibody covalently linked to an oligonucleotide. The conjugate may comprise an antibody that specifically binds to a single antigen site, or an antibody that binds to at least two antigen sites, which may be present on the same antigen or different antigens.

[0067] 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 in the complex is responsible for modulating the gene, protein, and / or (for example, and) nucleic acid. The molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and / or (for example, and) nucleic acid in a cell.

[0068] In some embodiments, the complex comprises a muscle targeting agent, e.g., an anti-transferrin receptor antibody, covalently linked to a molecular payload, e.g., an antisense oligonucleotide, that targets DMD and promotes exon skipping in a transcript encoded from a mutant DMD allele. In some embodiments, the complex targets DMD pre-mRNA and promotes exon 51 skipping in the DMD pre-mRNA.

[0069] 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 upon binding to the antigen on the muscle cell, e.g., via endocytosis. It should be understood that various types of muscle-targeting agents can be used in accordance with the present disclosure. It should be understood that any muscle target (e.g., muscle surface proteins) can be targeted by any type of muscle-targeting agent described herein. For example, 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). The muscle-targeting agent may comprise or consist of a small molecule. Exemplary muscle-targeting agents are described in further detail herein; however, it should be understood that the exemplary muscle-targeting agents provided herein are not intended to be limiting.

[0070] Some aspects of the present disclosure provide muscle targeting agents that specifically bind to antigens on muscles, such as skeletal muscle, smooth muscle, or cardiac muscle. In some embodiments, any of the muscle targeting agents provided herein bind (e.g., specifically bind) to antigens on skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells.

[0071] Interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins) can achieve both tissue localization and selective uptake into muscle cells. In some embodiments, molecules that are substrates 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 internalized, e.g., via clathrin-mediated endocytosis.

[0072] The use of muscle-targeting agents can be useful for concentrating molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, muscle-targeting agents concentrate the bound molecular payload in muscle cells compared to other cell types within a subject. In some embodiments, muscle-targeting agents concentrate the bound molecular payload in muscle cells (e.g., skeletal muscle cells, smooth muscle cells, or cardiomyocytes) to an amount at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than the amount in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or adipocytes). In some embodiments, the toxicity in a subject of the molecular payload when conjugated to a muscle-targeting agent is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when delivered to a subject.

[0073] 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. Of course, while transporter-based approaches provide a direct pathway for cell entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0074] i. Muscle targeting antibody In some embodiments, the muscle targeting agent is an antibody. Generally, the high specificity of antibodies for their target antigens allows for the possibility of selectively targeting muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells). This specificity may also limit off-target toxicity. Examples of antibodies capable of targeting surface antigens of muscle cells have been reported and are within the scope of the present disclosure. For example, antibodies 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.

[0075] 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 internalized cell surface receptor that transports transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to the transferrin receptor. Accordingly, aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to the transferrin receptor. In some embodiments, the binding proteins that bind to the transferrin receptor are internalized into muscle cells along with any attached molecular payload. As used herein, antibodies that bind to the transferrin receptor may be referred to interchangeably 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.

[0076] It will be appreciated that anti-TfR1 antibodies can be produced, synthesized, and / or (for example, and) derivatized using several known methodologies, for example, library design using phage display. Exemplary methodologies have been described in the art and are incorporated by reference (Diez, P. et al., "High-throughput phage-display screening in array format," Enzyme and microbial technology, 2015, 79, 34-41; Christoph MH and Stanley, JR, "Antibody Phage Display: Technique and Applications," J Invest Dermatol. 2014, 134:2; Engleman, Edgar (Ed.), "Human Hybridomas and Monoclonal Antibodies," 1985, Springer). In other embodiments, the anti-TfR1 antibody has 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 December 4, 1979, entitled "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Pat. No. 8,409,573, filed June 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 December 19, 2014, entitled "Low affinity blood brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed December 24, 2014, entitled "Novel anti-Transferrin receptor antibody that passes through the blood-brain barrier"; Schneider C. et al. al. “Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9.” J Biol Chem.1982,257:14,8516-8522. Lee et al. “Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse” 2000, J (See Pharmacol. Exp. Ther., 292:1048-1052).

[0077] 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 the transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR1 antibodies provided herein bind to the human transferrin receptor. In some embodiments, the anti-TfR1 antibodies described herein bind to an amino acid segment of the human or non-human primate transferrin receptor, such as those 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 set forth in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor.

[0078] In some embodiments, an anti-TfR1 antibody described herein (e.g., anti-TfR clone 8 in Table 2 below) binds to an epitope of TfR1, wherein the epitope comprises residues from amino acids 214-241 and / or amino acids 354-381 of SEQ ID NO: 105. In some embodiments, an anti-TfR1 antibody described herein binds to an epitope comprising residues from amino acids 214-241 and 354-381 of SEQ ID NO: 105. In some embodiments, an anti-TfR1 antibody described herein binds to an epitope comprising one or more of residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1 set forth 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.

[0079] In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope of TfR1 comprising residues of 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 comprising residues of 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 comprising one or more of residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as set forth in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and its variants in Table 2 below) bind to an epitope comprising residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as set forth in SEQ ID NO: 105.

[0080] 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: (SEQ ID NO: 105)

[0081] An example of a primate non-human transferrin receptor amino acid sequence, corresponding to the NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Macaca mulatta), is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKI QVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNV LKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTM DTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 106)

[0082] An example of a primate non-human transferrin receptor amino acid sequence corresponding to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: (SEQ ID NO: 107)

[0083] 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)

[0084] In some embodiments, an anti-TfR1 antibody binds to an amino acid segment of the receptor as follows:FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and does not inhibit the binding interaction between the transferrin receptor and transferrin and / or (by way of example and not limitation) human hemochromatosis protein (also known as HFE). In some embodiments, the anti-TfR1 receptor antibodies described herein do not bind to the epitope of SEQ ID NO: 109.

[0085] 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 in any form or entity, for example, in a recombinant or naturally occurring form or entity. Hybridomas are screened using standard methods, for example, ELISA screening, to find at least one hybridoma producing an antibody that targets a specific antigen. Antibodies may also be produced through screening of protein expression libraries (for example, phage display libraries) that express antibodies. Phage display library design may also be used in some embodiments (see, e.g., U.S. Pat. No. 5,223,409, filed March 1, 1991, entitled "Directed evolution of novel binding proteins"; WO 1992 / 18619, filed April 10, 1992, entitled "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, filed May 1, 1991, entitled "Recombinant library screening methods"; WO 1992 / 20791, filed May 15, 1992, entitled "Methods for producing members of specific binding pairs"; and WO 1992 / 15679, filed February 28, 1992, entitled "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 antibody is obtained from the non-human animal, it may then optionally be 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).

[0086] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or 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 WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0087] In some embodiments, the anti-TfR1 antibodies of the present disclosure comprise 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 comprise a constant region comprising the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (for example, IgG2a and IgG2b) of immunoglobulin molecules. Non-limiting examples of human constant regions are described in the art; see, e.g., Kabat EA et al., (1991), supra.

[0088] In some embodiments, agents that bind to the transferrin receptor, e.g., anti-TfR1 antibodies, can target muscle cells and / or mediate transport of agents across the blood-brain barrier (e.g., and). The transferrin receptor is an internalized cell surface receptor that transports transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor-binding proteins capable of binding to the transferrin receptor. An antibody that binds, e.g., specifically binds, to the transferrin receptor may be internalized into the cell upon binding to the transferrin receptor, e.g., through receptor-mediated endocytosis.

[0089] In some aspects, provided herein are humanized antibodies that bind to 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 to an 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 the 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, such as those 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 set forth 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.

[0090] 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., indicated by Kd) of 1 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 bind to human TfR1 and cynomolgus TfR1 (e.g., 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 The anti-TfR1 antibodies do not bind to mouse TfR1 (with a Kd of M or smaller). 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-beta2-microglobulin binding to TfR1.

[0091] Non-limiting examples of anti-TfR1 antibodies are shown in Table 2. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0092] In some embodiments, an anti-TfR1 antibody of the disclosure is a humanized variant of any one of the anti-TfR1 antibodies shown 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 of any one of the shown 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.

[0093] Exemplary amino acid sequences of anti-TfR1 antibodies described herein are shown in Table 3. [Table 3-1] [Table 3-2]

[0094] In some embodiments, an 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 shown in Table 3, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid mutations compared to the respective VH provided in Table 3. Alternatively, or in addition (e.g., in addition), an 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 one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid mutations compared to the respective VL provided in Table 3. In some embodiments, the VH of the anti-TfR1 antibody is a humanized VH and / or the VL of the anti-TfR1 antibody is a humanized VL.

[0095] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH that includes CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR1 antibodies shown in Table 3 and that includes an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) in the framework regions compared to the respective VHs shown in Table 3. Alternatively, or in addition (e.g., in addition), an anti-TfR1 antibody of the present disclosure comprises a VL that includes CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR1 antibodies shown in Table 3 and that includes an amino acid sequence that is at least 70% identical (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) in the framework regions compared to the respective VLs shown in Table 3. In some embodiments, the VH of the anti-TfR1 antibody is a humanized VH and / or the VL of the anti-TfR1 antibody is a humanized VL.

[0096] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:69 and a humanized VL comprising the amino acid sequence of SEQ ID NO:70.

[0097] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:71 and a humanized VL comprising the amino acid sequence of SEQ ID NO:70.

[0098] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:72 and a humanized VL comprising the amino acid sequence of SEQ ID NO:70.

[0099] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73 and a humanized VL comprising the amino acid sequence of SEQ ID NO:74.

[0100] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73 and a humanized VL comprising the amino acid sequence of SEQ ID NO:75.

[0101] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a humanized VL comprising the amino acid sequence of SEQ ID NO:74.

[0102] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a humanized VL comprising the amino acid sequence of SEQ ID NO:75.

[0103] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77 and a humanized VL comprising the amino acid sequence of SEQ ID NO:78.

[0104] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:79 and a humanized VL comprising the amino acid sequence of SEQ ID NO:80.

[0105] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77 and a humanized VL comprising the amino acid sequence of SEQ ID NO:80.

[0106] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 154 and a humanized VL comprising the amino acid sequence of SEQ ID NO: 155.

[0107] In some embodiments, the anti-TfR1 antibodies described herein are full-length IgGs, which may include heavy and light chain constant regions from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be derived from any suitable source, e.g., human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is derived from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is provided below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81)

[0108] In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein comprises a mutated human IgG1 constant region. For example, the introduction of LALA mutations in the CH2 domain of human IgG1 (a mutant derived from mAb b12 in which the lower hinge residues Leu234 and Leu235 are mutated to replace Ala234 and Ala235) is known to reduce Fcγ receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The mutated human IgG1 constant region is shown below (mutations are shown in bold and underlined):

number

[0109] 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 shown below. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)

[0110] Heavy and light chain constant regions of other antibodies are well known in the art and are provided, for example, in the IMGT database (www.imgt.org) or at www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0111] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region that contains 25 or fewer amino acid mutations (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 mutations) compared to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region set forth in SEQ ID NO: 81. In some embodiments, the anti-TfR1 antibodies described herein comprise any one of the VHs listed in Table 3 or any variant thereof, and a heavy chain comprising the heavy chain constant region set forth in SEQ ID NO:82.

[0112] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region that contains 25 or fewer amino acid mutations (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 mutations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region as set forth in SEQ ID NO: 83.

[0113] Examples of IgG heavy and light chain amino acid sequences of the described anti-TfR1 antibodies are shown in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0114] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain containing 25 or fewer amino acid mutations (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 mutation) compared to the heavy chain set forth in any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively, or in addition (for example, additionally), an anti-TfR1 antibody of the present disclosure includes a light chain that contains 25 or fewer amino acid mutations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0115] 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 the amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively, or in addition (by way of example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157.

[0116] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:84 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0117] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:86 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0118] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:87 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0119] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0120] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0121] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0122] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0123] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0124] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:94 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0125] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0126] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:156 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0127] 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 conventional methods (e.g., recombinantly or by digesting the heavy chain constant region of a full-length IgG with an enzyme such as papain). For example, F(ab')2 fragments can be generated by pepsin or papain digestion of antibody molecules, and Fab fragments can be generated by reducing the disulfide bridges of F(ab')2 fragments. In some embodiments, the heavy chain constant region on the Fab fragment of an anti-TfR1 antibody described herein comprises the amino acid sequence of: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96).

[0128] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region that contains 25 or fewer amino acid mutations (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 mutations) compared to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of a VH or any variant thereof listed in Table 3, and a heavy chain constant region set forth in SEQ ID NO:96.

[0129] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region that contains 25 or fewer amino acid mutations (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 mutations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of a VL or any variant thereof listed in Table 3, and a light chain constant region as set forth in SEQ ID NO: 83.

[0130] Examples of Fab heavy and light chain amino acid sequences of the described anti-TfR1 antibodies are shown in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3]

[0131] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain containing 25 or fewer amino acid mutations (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 mutation) compared to the heavy chain set forth in any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively, or in addition (for example, additionally), an anti-TfR1 antibody of the present disclosure includes a light chain that contains 25 or fewer amino acid mutations (for example, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to the light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0132] 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 in addition (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 the amino acid sequence of any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively, or in addition (by way of example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157.

[0133] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:97 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0134] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:98 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0135] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0136] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0137] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0138] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0139] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0140] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0141] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 103 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0142] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 95.

[0143] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:158 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0144] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:159 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0145] 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 conjugates disclosed herein. Examples of known anti-TfR1 antibodies (including relevant references and binding epitopes) are listed in Table 6. In some embodiments, the anti-TfR1 antibody comprises the complementarity-determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any of the anti-TfR1 antibodies provided herein, such as those listed in Table 6. [Table 6-1] [Table 6-2] [Table 6-3]

[0146] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises 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 comprises CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, an anti-TfR1 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-TfR1 antibodies selected from Table 6.

[0147] In some embodiments, the anti-TfR1 antibodies of the present disclosure include any antibody comprising a heavy chain variable domain and / or (by way of example and not limitation) a light chain variable domain of any anti-TfR1 antibody, such as any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the anti-TfR1 antibodies of the present disclosure include any antibody, such as any one of the anti-TfR1 antibodies selected from Table 6, comprising a heavy chain variable pair and a light chain variable pair of any of the anti-TfR1 antibodies.

[0148] Aspects of the present disclosure provide anti-TfR1 antibodies having heavy chain variable (VH) and / or (e.g., and) light chain variable (VL) domain amino acid sequences homologous to any of those described herein. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable sequence or a light chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy chain variable sequence and / or any light chain variable sequence of any anti-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 (e.g., and) light chain variable amino acid sequences do not vary within 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%) may occur within the heavy chain variable and / or (e.g., and) light chain variable sequences excluding any of the CDR sequences provided herein. In some embodiments, any of the anti-TfR1 antibodies provided herein comprise heavy chain 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.

[0149] An example of a transferrin receptor antibody that can be used in accordance with the present disclosure is described in WO 2016 / 081643, which is incorporated herein by reference. The amino acid sequence of this antibody is shown in Table 7. [Table 7-1] [Table 7-2]

[0150] 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 in addition (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.

[0151] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 that comprises three or fewer amino acid mutations (e.g., three, two, or one or fewer amino acid mutations) compared to the CDR-L3 shown in Table 7. In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 that contains one amino acid mutation compared to the CDR-L3 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 present disclosure comprises 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).

[0152] In some embodiments, anti-TfR1 antibodies of the disclosure comprise heavy chain CDRs that, collectively, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs set forth in Table 7. Alternatively, or in addition (e.g., in addition), anti-TfR1 antibodies of the disclosure comprise light chain CDRs that, collectively, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the light chain CDRs set forth in Table 7.

[0153] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 124. Alternatively, or in addition (for example, in addition), an anti-TfR1 antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 125.

[0154] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 128. Alternatively, or in addition (for example, in addition), an anti-TfR1 antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 129.

[0155] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH that contains 25 or fewer amino acid mutations (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 mutations) compared to the VH set forth in SEQ ID NO: 128. Alternatively, or in addition (e.g., in addition), an anti-TfR1 antibody of the present disclosure comprises a VL that contains 15 or fewer amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 129.

[0156] In some embodiments, the anti-TfR1 antibodies of the present disclosure are full-length IgG1 antibodies, which may comprise heavy and light chain constant regions from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein may comprise 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 derived from any suitable source, e.g., human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is derived from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is as follows: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81)

[0157] 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 shown below. RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)

[0158] 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 in addition (for example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0159] 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 in addition (for example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0160] 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 in addition (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 in addition (for example, in addition), the anti-TfR1 Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0161] The anti-TfR1 antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv, etc.), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the anti-TfR1 antibodies described herein are scFvs. In some embodiments, the anti-TfR1 antibodies described herein are scFv-Fabs (e.g., scFvs fused to a portion of a constant region). In some embodiments, the anti-TfR1 antibodies described herein are scFvs fused to a constant region (e.g., the human IgG1 constant region set forth in SEQ ID NO: 81).

[0162] 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., transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one, two, 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., in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., 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 fixation, Fc receptor binding, and / or (e.g., and) antigen-dependent cellular cytotoxicity.

[0163] In some embodiments, one, two, 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), e.g., as described 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 association of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate conjugation of a linker.

[0164] In some embodiments, one, two, 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 WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0165] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant region or FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. See, for example, WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, as well as U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for examples of mutations that may alter (e.g., increase or decrease) the half-life of an antibody in vivo.

[0166] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-TfR1 antibody in vivo. In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the 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 antibodies 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 a "YTE mutant," has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody 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.

[0167] In some embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function of the anti-TfR1 antibody. The effector ligand with altered affinity can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further 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 in 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).

[0168] In some embodiments, one or more amino acid residues in the constant region of an 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 an antibody described herein are altered to thereby alter the antibody's ability to fix complement. This approach is described in further detail in WO 94 / 29351. In some embodiments, the Fc region of an 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 antibody's affinity for Fcγ receptors. This approach is described in further detail in WO 00 / 42072.

[0169] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein can 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 understood 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 the ability to specifically bind 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.

[0170] 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, which is known to occur in native IgG4 mAbs, the antibodies provided herein may contain the stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Thus, any of the antibodies may include the stabilizing "Adair" mutation.

[0171] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or 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 WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0172] In some embodiments, any one of the anti-TfR1 antibodies described herein can include a signal peptide (e.g., an N-terminal signal peptide) on the heavy and / or (e.g., and) light chain sequence. In some embodiments, the anti-TfR1 antibodies described herein include any one of the VH and VL sequences, any one of the IgG heavy and light chain sequences, or any one of the F(ab') heavy and light chain sequences described herein, and further include a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide includes the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0173] In some embodiments, the antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also referred to as pyroglutamic acid formation (pyroGlu), may occur in antibodies at N-terminal glutamic acid (Glu) and / or glutamine (Gln) residues during production. Thus, it should be understood that an antibody identified as having a sequence containing an N-terminal glutamate or glutamine residue encompasses antibodies that have undergone pyroglutamate formation due to 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.

[0174] b. Other muscle-targeting antibodies In some embodiments, the muscle-targeting antibody specifically binds to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin IIb, or CD63. In some embodiments, the muscle-targeting antibody 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 alpha7beta1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle-targeting antibody specifically binds to a skeletal muscle protein. Exemplary skeletal muscle proteins include, but are not limited to, alpha-sarcoglycan, beta-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha7, integrin alpha7beta1, integrin beta1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I; in some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a smooth muscle protein. Exemplary smooth muscle proteins include, but are not limited to, alpha-smooth muscle actin, VE-cadherin, caldesmon / CALD1, calponin 1, desmin, histamine H2R, motilin R / GPR38, transgelin / TAGLN, and vimentin, however, it will be understood that antibodies to additional targets are within the scope of this disclosure and that the exemplary list of targets provided herein is not intended to be limiting.

[0175] c. Antibody characteristics / 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., a transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one, two, 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., in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., in the hinge region), numbered according to the Kabat numbering system (e.g., EU index of Kabat)) of a muscle-targeting antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or (e.g., and) antigen-dependent cellular cytotoxicity.

[0176] In some embodiments, one, two, 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), e.g., as described 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 association of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate conjugation of a linker.

[0177] In some embodiments, one, two, 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 WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0178] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an IgG constant region or FcRn-binding fragment thereof (preferably, an Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. See, for example, WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, as well as U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for examples of mutations that may alter (e.g., increase or decrease) the half-life of an antibody in vivo.

[0179] In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the 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 antibodies 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 a "YTE mutant," has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody 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.

[0180] 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 can be, for example, an Fc receptor or the C1 component of complement. This approach is described in further 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, e.g., 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 in 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).

[0181] In some embodiments, one or more amino acid residues in the constant region of a muscle-targeting 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 an antibody described herein are altered to thereby alter the antibody's ability to fix complement. This approach is described in further detail in WO 94 / 29351. In some embodiments, the Fc region of an 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 antibody's affinity for Fcγ receptors. This approach is described in further detail in WO 00 / 42072.

[0182] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein can 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 understood 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 the ability to specifically bind 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.

[0183] 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, which is known to occur in native IgG4 mAbs, the antibodies provided herein may contain the stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Thus, any of the antibodies may include the stabilizing "Adair" mutation.

[0184] As provided herein, the antibodies of the present disclosure may optionally comprise a constant region or a portion thereof. For example, a VL domain may be attached at its C-terminus to a light chain constant region like Cκ or Cλ. Similarly, a VH domain or a portion thereof may be attached to all or a portion of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any isotype subclass. The antibody may comprise any suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of the present disclosure may comprise VH and VL domains, or antigen-binding portions thereof, combined with any suitable constant region.

[0185] ii. Muscle-targeting peptides Some aspects of the present 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 are described in Vines e., et al., A., "Cell-penetrating and cell-targeting peptides in drug delivery," Biochim Biophys Acta 2008, 1786:126-38; Jarver P., et al., "In vivo biodistribution and efficacy of peptide mediated delivery," Trends Pharmacol Sci 2010;31:528-35; Samoylova 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 for a desired tissue, e.g., muscle, can be achieved. Skeletal muscle targeting has been explored, and a wide range of molecular payloads can be delivered. These approaches, which lack many of the practical disadvantages of large antibodies or viral particles, may have high selectivity for muscle tissue. Thus, in some embodiments, the muscle-targeting agent is a muscle-targeting peptide 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.

[0186] In some embodiments, muscle-targeting peptides can bind to internalized cell surface receptors (e.g., transferrin receptors) that are overexpressed or relatively highly expressed in muscle cells compared to certain other cells. In some embodiments, muscle-targeting peptides can target (e.g., bind to) transferrin receptors. In some embodiments, peptides that target transferrin receptors can include segments of naturally occurring ligands, e.g., transferrin. In some embodiments, peptides that target transferrin receptors are as described in U.S. Patent No. 6,743,893, filed 11 / 30 / 2000, entitled "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 May 20, 2011, entitled "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY."

[0187] As mentioned above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides have been identified using phage display libraries that display surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 943) bound to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. Thus, in some embodiments, the muscle-targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 943). This peptide exhibited improved specificity for binding to cardiac and skeletal muscle tissue after intravenous injection into mice, with reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have been identified using phage display. For example, in the context of treating Duchenne muscular dystrophy, 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. Herein, a 12 amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO: 944) was identified, and this muscle-targeting peptide showed improved binding to C2C12 cells compared to the ASSLNIA (SEQ ID NO: 943) peptide.

[0188] Additional methods for identifying peptides selective for muscle (e.g., skeletal muscle) over other cell types include 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. Nonspecific cell binders were selected by preincubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After repeated selection, the 12-amino acid peptide TARGEHKEEELI (SEQ ID NO: 945) emerged most frequently. Thus, in some embodiments, the muscle-targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 945).

[0189] The muscle-targeting agent may be an amino acid-containing molecule or peptide. The muscle-targeting peptide may correspond to the sequence of a protein that preferentially binds to a protein receptor found on muscle cells. In some embodiments, the muscle-targeting peptide has a strong hydrophobic amino acid profile (e.g., valine) so that the peptide preferentially targets muscle cells. In some embodiments, the muscle-targeting peptide has not been previously characterized or disclosed. These peptides may be conceived, produced, synthesized, and / or derivatized (e.g., and) using any of several methodologies, e.g., phage-displayed peptide libraries, one-bead-one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. Exemplary methodologies have been characterized in the art and are incorporated by reference (Gray, B. P. and Brown, K. C., "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides," Chem Rev. 2014, 114:2, 1020-1081; Samoylova, T. I. and Smith, B. F., "Elucidation of muscle-binding peptides by phage display screening." Muscle Nerve, 1999, 22:4, 460-6).In some embodiments, muscle-targeting peptides have been previously disclosed (see, e.g., Writer MJ et al. "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display." J. Drug Targeting. 2004;12:185; Cai, D. "BDNF-mediated enhancement of inflammation and injury in the aging heart." Physiol Genomics. 2006,24:3,191-7; Zhang, L. "Molecular profiling of heart endothelial cells." Circulation, 2005,112:11,1601-11; McGuire, MJ et al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004,342:1,171-82). Exemplary muscle-targeting peptides include the following group of amino acid sequences: CQAQGQLVC (SEQ ID NO: 946), CSERSMNFC (SEQ ID NO: 947), CPKTRRVPC (SEQ ID NO: 948), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 949), ASSLNIA (SEQ ID NO: 943), CMQHSMRVC (SEQ ID NO: 950), and DDTRHWG (SEQ ID NO: 951). In some embodiments, muscle-targeting peptides may contain 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 contain naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art.In some embodiments, the muscle-targeting peptide may be linear; in other embodiments, the muscle-targeting peptide may be cyclic (e.g., bicyclic) (see, e.g., Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

[0190] 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, which binds to an internalized cell surface receptor expressed by muscle cells. Thus, in some embodiments, the muscle-targeting agent is transferrin or a derivative thereof that binds to the transferrin receptor. Alternatively, the muscle-targeting ligand may 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 containing cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolenic acid, linoleic acid, myristic acid, sterol, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and alkoxy acids.

[0191] 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 previously been 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 are described in the art and are incorporated by reference (Yan, A.C. and Levy, M., "Aptamers and aptamer targeted delivery," RNA biology, 2009, 6:3, 316-20; Germer, K. et al., "RNA aptamers and their therapeutic and diagnostic applications," Int. J. Biochem. Mol. Biol. 2013; 4:27-40). In some embodiments, 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.

[0192] 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. 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, for example, a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0193] In some embodiments, the muscle-targeting agent is any muscle-targeting agent described herein (e.g., an antibody, nucleic acid, small molecule, peptide, aptamer, lipid, or sugar moiety) that targets the SLC superfamily of transporters. In some embodiments, the muscle-targeting agent is a substrate of the SLC superfamily of transporters. SLC transporters are either equilibrium-type or use a 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, thereby providing opportunities for muscle targeting.

[0194] In some embodiments, the muscle-targeting agent is a substrate for the equilibrative nucleoside transporter 2 (ENT2) transporter. Compared with other transporters, ENT2 has one of the highest mRNA expression levels in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs, such as the brain, heart, placenta, thymus, pancreas, prostate, and kidney, but is particularly abundant in skeletal muscle. Human ENT2 facilitates the uptake of its substrates according to their concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. The hENT2 transporter has low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except for inosine. Therefore, 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 is 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.

[0195] In some embodiments, the muscle-targeting agent is a substrate of the organic cation / carnitine transporter (OCTN2), a sodium ion-dependent, high-affinity carnitine transporter. In some embodiments, the muscle-targeting agent is carnitine, mildronate, acetylcarnitine, or a 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).

[0196] A muscle-targeting agent may be a protein that exists in at least one soluble form and 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 N-terminal signaling, and / or (for example, and) may lack C-terminal anchor domains. In some embodiments, the hemojuvelin may be annotated with GenBank RefSeq accession numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It will be appreciated that the hemojuvelin may originate from a human, a non-human primate, or a rodent.

[0197] B. Molecular Payload Some aspects of the present disclosure provide molecular payloads for modulating biological outcomes, e.g., transcription of DNA sequences, splicing and processing of RNA sequences, protein expression, or protein activity. In some embodiments, the molecular payload is linked or otherwise associated with a muscle targeting agent. In some embodiments, such molecular payloads are capable of targeting muscle cells, e.g., via specific binding to nucleic acids or proteins in muscle cells upon delivery to the muscle cells by the associated muscle targeting agent. It should be understood that various types of molecular payloads may be used in accordance with the present disclosure. For example, the molecular payload may comprise or consist of an oligonucleotide (e.g., an antisense oligonucleotide), a peptide (e.g., a peptide that binds to a nucleic acid or protein in a disease-associated muscle cell), a protein (e.g., a protein that binds to a nucleic acid or protein in a disease-associated muscle cell), or a small molecule (e.g., a small molecule that modulates the function of a nucleic acid or protein in a disease-associated muscle cell). In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region of complementarity to a mutant DMD allele. Exemplary molecular payloads are described in further detail herein; however, it will be understood that the exemplary molecular payloads provided herein are not intended to be limiting.

[0198] i. Oligonucleotides Aspects of the present disclosure relate to, for example, oligonucleotides configured to regulate (e.g., increase) the expression of dystrophin from a DMD allele. In some embodiments, the oligonucleotides provided herein are configured to alter the splicing of DMD pre-mRNA to promote the expression of a dystrophin protein (e.g., a functional truncated dystrophin protein). In some embodiments, the oligonucleotides provided herein are configured to promote the skipping of one or more exons in DMD, for example, in a mutant DMD allele, to restore the reading frame. In some embodiments, the oligonucleotide enables expression of a functional dystrophin protein (see, e.g., Kinali M, Arechevala-Gomeza V, Feng L, et al. Local restoration of dystrophin expression with the morpholino oligomer AVI-4658 in Duchenne muscular dystrophy: a single-blind, placebo-controlled, dose-escalation, proof-of-concept study. Lancet Neurol. 2009;8(10):918-928, and Watanabe N, Nagata T, Satou Y, et al. NS-065 / NCNP-01: an antisense oligonucleotide for potential treatment of exon 53 skipping in Duchenne muscular dystrophy. Mol Ther Nucleic Acids. 2018;13:442-449). In some embodiments, the provided oligonucleotides are configured to promote skipping of exon 51 to generate a shorter but functional version of dystrophin (e.g., containing an in-frame deletion).In some embodiments, oligonucleotides that promote skipping of exon 51 are provided (e.g., oligonucleotides that promote skipping of DMD exons 3-50, 4-50, 5-50, 6-50, 9-50, 10-50, 11-50, 13-50, 14-50, 15-50, 16-50, 17-50, 19-50, 21-50, 23-50, 24-50, 25-50, 26-50, 27-50, 28-50, 29-50, 30-50, 31-50, 32-50, 33-50, 34-50, 35-50, 36-50, 37-50, 38-50, 39-50, 40-50, 41-50, 42-50, 43-50, 44-50, 45-50, 46-50, 47-50, 48-50, 49-50, 50-50, 51-50, 52-50, 53-50, 54-50, 55-50, 56-50, 57-50, 58-50, 59-50, 60-50, 61-50, 62-50, 63-50, 64-50, 65-50, 66-50, 67-50, 68-50, 69-50, 70-50, 71-50, 72-50, 73-50, 74-50, 75-50, 76-50, 77 A significant number of patients may be suitable for exon 51 skipping, such as those with deletions in exon 50, 32-50, 33-50, 34-50, 35-50, 36-50, 37-50, 38-50, 39-50, 40-50, 41-50, 42-50, 43-50, 45-50, 47-50, 48-50, 49-50, 50, 52, 52-58, 52-61, 52-63, 52-64, 52-66, 52-76, or 52-77.

[0199] Table 8 shows non-limiting examples of oligonucleotide sequences useful for targeting DMD, for example, exon skipping, and targeting sequences within DMD. In some embodiments, the oligonucleotides can include any of the antisense sequences shown in Table 8 or sequences complementary to the target sequences shown in Table 8. [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4] [Table 8-5] [Table 8-6]

Table 8-7

Table 8-8

Table 8-9

[0200] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a region of the DMD sequence. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a region of DMD RNA (e.g., the Dp427m transcript of SEQ ID NO: 130). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region of complementarity to DMD RNA (e.g., the Dp427m transcript of SEQ ID NO: 130). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region of complementarity to an exon of DMD RNA (e.g., SEQ ID NO: 131, 838, or 854). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region of complementarity to an intron of DMD RNA (e.g., SEQ ID NO: 834 or 846). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) contain a region of complementarity to a portion of the DMD sequence (e.g., a sequence provided by any one of SEQ ID NOs: 832, 844, 833, 845, 835, 836, 847-852, 837, 853, 839-843). Exemplary DMD sequences are shown below. Each of the DMD sequences shown below contains thymine nucleotides (T), although it should be understood that each sequence can represent a DNA or RNA sequence in which any or all of the Ts are replaced with uracil nucleotides (U).

[0201] Homo sapiens dystrophin (DMD), transcript variant Dp427m, mRNA (NCBI Reference Sequence: NM_004006.2)

[0202] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 50 (nucleotide positions 7445-7553 in NCBI Reference Sequence: NM_004006.2, and nucleotide positions 1524527-1524635 in NCBI Reference Sequence: NG_012232.1) AGGAAGTTAGAAGATCTGAGCTCTGAGTGGAAGGCGGTAAACCGTTTACTTCAAGAGCTGAGGGCAAAGCAGCCTGACCTAGCTCCTGGACTGACCACTATTGGAGCCT (SEQ ID NO: 131)

[0203] Homo sapiens dystrophin (DMD) exon 50 / intron 50 junction (nucleotide positions 1524606 to 1524665 of NCBI reference sequence: NG_012232.1) TAGCTCCTGGACTGACCACTATTGGAGCCTGTAAGTATACTGGATCCCATTCTCTTTGGC (SEQ ID NO: 832)

[0204] Homo sapiens dystrophin (DMD), exon 50 / intron 50 junction target sequence 1 (nucleotide positions 1524626 to 1524677 of NCBI reference sequence: NG_012232.1) ATTGGAGCCTGTAAGTATACTGGATCCCATTCTCTTTGGCTCTAGCTATTTG (SEQ ID NO: 833)

[0205] Homo sapiens dystrophin (DMD), intron 50 (nucleotide positions 1524636 to 1570417 of NCBI Reference Sequence: NG_012232.1)

[0206] Homo sapiens dystrophin (DMD), intron 50 target sequence 1 (nucleotide positions 1524636 to 1524685 of NCBI reference sequence: NG_012232.1) GTAAGTATACTGGATCCCATTCTCTTTGGCTCTAGCTATTTGTTCAAAAG (SEQ ID NO: 835)

[0207] Homo sapiens dystrophin (DMD), intron 50 target sequence 2 (nucleotide positions 1570168 to 1570417 of NCBI reference sequence: NG_012232.1) CGTTTTTTAAAAAATTGTTAAATGTATATTAATGAAAAGGTTGAATCTTTTCATTTTCTACCATGTATTGCTAAACAAAGTATCCACATTGTTAGAAAAAGATATATAATGTCATGAATAAGAGTTTGG CTCAAATTGTTACTCTTCAATTAAATTTGACTTATTGTTATTGAAATTGGCTCTTTAGCTTGTGTTTCTAATTTTTCTTTTTCTTCTTTTTTCCTTTTTGCAAAAACCCAAAATATTTTAG (SEQ ID NO: 836)

[0208] Homo sapiens dystrophin (DMD) intron 50 / exon 51 junction (nucleotide positions 1570388 to 1570447 of NCBI reference sequence: NG_012232.1) TCCTTTTTGCAAAAACCCAAAATATTTTAGCTCCTACTCAGACTGTTACTCTGGTGACAC (SEQ ID NO: 837)

[0209] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 51 (nucleotide positions 7554-7786 in NCBI Reference Sequence: NM_004006.2, and nucleotide positions 1570418-1570650 in NCBI Reference Sequence: NG_012232.1) CTCCTACTCAGACTGTTACTCTGGTGACACAACCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATGTTGGAGGTACCTGCTCTGGCAGATTTCAACCGGGCTTGGACAGAACTTACCGACTGGCTTTCTCTGCTTGATCAAGTTATAAAATCACAGAGGGTGATGGTGGGTGACCTTGAGGATATCAACGAGATGATCATCAAGCAGAAG (SEQ ID NO: 838)

[0210] Homo sapiens dystrophin (DMD), exon 51 target sequence 1 (nucleotide positions 1570442 to 1570487 of NCBI reference sequence: NG_012232.1) TGACACAACCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGA (SEQ ID NO: 839)

[0211] Homo sapiens dystrophin (DMD), exon 51 target sequence 2 (nucleotide positions 1570455 to 1570498 of NCBI reference sequence: NG_012232.1) GGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTT (SEQ ID NO: 840)

[0212] Homo sapiens dystrophin (DMD), exon 51 target sequence 3 (nucleotide positions 1570465 to 1570506 of NCBI reference sequence: NG_012232.1) GAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATG (SEQ ID NO: 841)

[0213] Homo sapiens dystrophin (DMD), exon 51 target sequence 4 (nucleotide positions 1570442 to 1570506 of NCBI reference sequence: NG_012232.1) TGACACAACCTGTGGTTACTAAGGAAACTGCCATCTCCAAACTAGAAATGCCATCTTCCTTGATG (SEQ ID NO: 842)

[0214] Homo sapiens dystrophin (DMD), exon 51 target sequence 5 (nucleotide positions 1570518 to 1570567 of NCBI reference sequence: NG_012232.1) TGCTCTGGCAGATTTCAACCGGGCTTGGACAGAACTTACCGACTGGCTTT (SEQ ID NO: 843)

[0215] Homo sapiens dystrophin (DMD) exon 51 / intron 51 junction (nucleotide positions 1570621 to 1570680 of NCBI reference sequence: NG_012232.1) GATATCAACGAGATGATCATCAAGCAGAAGGTATGAGAAAAAATGATAAAAGTTGGCAGA (SEQ ID NO: 844)

[0216] Homo sapiens dystrophin (DMD), exon 51 / intron 51 junction target 1 (nucleotide positions 1570623 to 1570674 of NCBI reference sequence: NG_012232.1) TATCAACGAGATGATCATCAAGCAGAAGGTATGAGAAAAAATGATAAAAGTT (SEQ ID NO: 845)

[0217] Homo sapiens dystrophin (DMD), intron 51 (nucleotide positions 1570651 to 1614861 of NCBI Reference Sequence: NG_012232.1)

[0218] Homo sapiens dystrophin (DMD), intron 51 target sequence 1 (nucleotide positions 1570651 to 1570700 of NCBI reference sequence: NG_012232.1) GTATGAGAAAAAATGATAAAAGTTGGCAGAAGTTTTTCTTTAAAATGAAG (SEQ ID NO: 847)

[0219] Homo sapiens dystrophin (DMD), intron 51 target sequence 2 (nucleotide positions 1570651 to 1570693 of NCBI reference sequence: NG_012232.1) GTATGAGAAAAAATGATAAAAGTTGGCAGAAGTTTTTCTTTAA (SEQ ID NO: 848)

[0220] Homo sapiens dystrophin (DMD), intron 51 target sequence 3 (nucleotide positions 1570703 to 1570765 of NCBI reference sequence: NG_012232.1) TTTCCACCAATCACTTTACTCTCCTAGACCATTTCCCACCAGTTCTTAGGCAACTGTTTCTCT (SEQ ID NO: 849)

[0221] Homo sapiens dystrophin (DMD), intron 51 target sequence 4 (nucleotide positions 1614751 to 1614804 of NCBI reference sequence: NG_012232.1) TATTTCTAAAAGTGTTTTGGCTGGTCTCACAATTGTACTTTACTTTGTATTATG (SEQ ID NO: 850)

[0222] Homo sapiens dystrophin (DMD), intron 51 target sequence 5 (nucleotide positions 1614793 to 1614847 of NCBI reference sequence: NG_012232.1) CTTTGTATTATGTAAAAGGAATACACAACGCTGAAGAACCCTGATACTAAGGGAT (SEQ ID NO: 851)

[0223] Homo sapiens dystrophin (DMD), intron 51 target sequence 6 (nucleotide positions 1614612 to 1614861 of NCBI reference sequence: NG_012232.1) CGGAATGTCTCCATTTGAGCCTTTAAATGAAGAAAATCTATAGTCAAGATTTTCATTTGAAATATTTTTGATATCTAAGAATGAAACATATTTCCTGTTAAATTGTTTTCTATAAACCCTTATACAGTA ACATCTTTTTTATTTCTAAAAGTGTTTTGGCTGGTCTCACAATTGTACTTTACTTTGTATTATGTAAAAGGAATACACAACGCTGAAGAACCCTGATACTAAGGGATATTTGTCTTACAG (SEQ ID NO: 852)

[0224] Homo sapiens dystrophin (DMD) intron 51 / exon 52 junction (nucleotide positions 1614832 to 1614891 of NCBI reference sequence: NG_012232.1) CCTGATACTAAGGGATATTTGTTCTTACAGGCAACAATGCAGGATTTGGAACAGAGGCGT (SEQ ID NO: 853)

[0225] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 52 (nucleotide positions 7787-7904 in NCBI Reference Sequence: NM_004006.2, and nucleotide positions 1614862-1614979 in NCBI Reference Sequence: NG_012232.1) GCAACAATGCAGGATTTGGAACAGAGGCGTCCCCAGTTGGAAGAACTCATTACCGCTGCCCAAAATTTGAAAAACAAGACCAGCAATCAAGAGGCTAGAACAATCATTACGGATCGAA (SEQ ID NO: 854)

[0226] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target splicing features of the DMD sequence (e.g., DMD pre-mRNA). In some embodiments, the splicing feature of the DMD sequence is an exonic splicing enhancer (ESE), branch point, splice donor site, or splice acceptor site of the DMD sequence. In some embodiments, the ESE is within exon 51 of the DMD sequence (e.g., DMD pre-mRNA). In some embodiments, the branch point is in intron 50 or intron 51 of the DMD sequence (e.g., DMD pre-mRNA). In some embodiments, the splice donor site is located across the junction of exon 50 and intron 50, within intron 50, across the junction of exon 51 and intron 51, or within intron 51 of the DMD sequence (e.g., DMD pre-mRNA). In some embodiments, the splice acceptor site is present within intron 50, spanning the junction of intron 50 and exon 51, within intron 51, or spanning the junction of intron 51 and exon 52 of the DMD sequence (e.g., DMD pre-mRNA). In some embodiments, oligonucleotides useful for targeting DMD promote skipping of exon 51, such as by targeting a splicing feature (e.g., an ESE, branch point, splice donor site, or splice acceptor site) of the DMD sequence (e.g., DMD pre-mRNA). Examples of ESEs, branch points, splice donor sites, and splice acceptor sites are shown in Table 9.

[0227] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target an exon splicing enhancer (ESE) in the DMD sequence. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target an ESE in DMD exon 51 (e.g., an ESE listed in Table 9).

[0228] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) comprise a region of complementarity to a target sequence comprising one or more complete or partial ESEs of a DMD transcript (e.g., one or more complete or partial ESEs listed in Table 9). In some embodiments, an oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs of DMD exon 51. In some embodiments, an oligonucleotide comprises a region of complementarity to a target sequence comprising one or more complete or partial ESEs set forth in SEQ ID NOs: 860-894. In some embodiments, an oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., four, five, six, seven, or eight) consecutive nucleotides of an ESE set forth in any one of SEQ ID NOs: 860-894. In some embodiments, an oligonucleotide comprises at least four (e.g., four, five, six, seven, or eight) consecutive nucleotides of an ESE antisense sequence set forth in any one of SEQ ID NOs: 904-938.

[0229] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least six (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) of DMD exon 51. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least six (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) nucleotides of one or more ESEs (e.g., 2, 3, 4, or more adjacent ESEs) set forth in SEQ ID NOs: 860-894. In some embodiments, the oligonucleotide comprises at least six (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) nucleotides of one or more ESE antisense sequences (e.g., antisense sequences of two, three, four, or more adjacent ESEs) set forth in SEQ ID NOs: 904-938.

[0230] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 18-35 nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE set forth in any one of SEQ ID NOs: 860-894. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 20-30 (e.g., 20, 25, 30) nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE set forth in any one of SEQ ID NOs: 860-894. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 20-25 (i.e., 20, 21, 22, 23, 24, or 25) nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE set forth in any one of SEQ ID NOs: 860-894. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of an ESE set forth in any one of SEQ ID NOs: 860-894.

[0231] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a branch point in the DMD sequence. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a branch point in DMD intron 50 or intron 51 (e.g., a branch point listed in Table 9).

[0232] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) comprise a region of complementarity to a target sequence comprising a full or partial branchpoint of the DMD transcript (e.g., a full or partial branchpoint listed in Table 9). In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising a full or partial branchpoint of DMD intron 50 or intron 51. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising a full or partial branchpoint set forth in any one of SEQ ID NOs: 856-858, 896, and 897. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., four, five, six, or seven) consecutive nucleotides of the branchpoint set forth in any one of SEQ ID NOs: 856-858, 896, and 897. In some embodiments, the oligonucleotide comprises at least four (eg, four, five, six, or seven) consecutive nucleotides of the branchpoint antisense sequence set forth in any one of SEQ ID NOs: 900-902, 940, and 941.

[0233] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 18 to 35 nucleotides in length and contain a region of complementarity to a target sequence that includes at least four (e.g., 4, 5, 6, or 7) consecutive nucleotides of the branch point set forth in any one of SEQ ID NOs: 856-858, 896, and 897. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 20 to 30 (e.g., 20, 25, 30) nucleotides in length and contain a region of complementarity to a target sequence that includes at least four (e.g., 4, 5, 6, or 7) consecutive nucleotides of the branch point set forth in any one of SEQ ID NOs: 856-858, 896, and 897. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 20-25 (i.e., 20, 21, 22, 23, 24, or 25) nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, or 7) consecutive nucleotides of the branch point set forth in any one of SEQ ID NOs: 856-858, 896, and 897. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, or 7) consecutive nucleotides of the branch point set forth in any one of SEQ ID NOs: 856-858, 896, and 897.

[0234] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a splice donor site in the DMD sequence. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a splice donor site spanning the junction of exon 50 and intron 50, within intron 50, spanning the junction of exon 51 and intron 51, or within intron 51 (e.g., a splice donor site listed in Table 9).

[0235] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) comprise a region of complementarity to a target sequence comprising a complete or partial splice donor site of a DMD transcript (e.g., a complete or partial splice donor site listed in Table 9). In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising a complete or partial splice donor site spanning the junction of exon 50 and intron 50 of DMD, spanning the junction of exon 51 and intron 51 in intron 50, or within intron 51. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising a complete or partial splice donor site set forth in SEQ ID NO: 855 or 895. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., four, five, six, or seven) consecutive nucleotides of the splice donor site set forth in SEQ ID NO: 855 or 895. In some embodiments, the oligonucleotide comprises at least four (eg, four, five, six, or seven) consecutive nucleotides of the splice donor site antisense sequence set forth in SEQ ID NO:899 or 939.

[0236] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 18-35 nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, or 7) consecutive nucleotides of the splice donor site set forth in SEQ ID NO: 855 or 895. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 20-30 (e.g., 20, 25, 30) nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, or 7) consecutive nucleotides of the splice donor site set forth in SEQ ID NO: 855 or 895. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 20-25 (i.e., 20, 21, 22, 23, 24, or 25) nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, or 7) consecutive nucleotides of the splice donor site set forth in SEQ ID NO: 855 or 895. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, or 7) consecutive nucleotides of the splice donor site set forth in SEQ ID NO: 855 or 895.

[0237] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a splice acceptor site in the DMD sequence. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a splice acceptor site within intron 50, spanning the junction of intron 50 and exon 51, within intron 51, or spanning the junction of intron 51 and exon 52 (e.g., a splice acceptor site listed in Table 9).

[0238] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) comprise a region of complementarity to a target sequence comprising a complete or partial splice acceptor site of a DMD transcript (e.g., a complete or partial splice acceptor site listed in Table 9). In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising a complete or partial splice acceptor site within intron 50, spanning the junction of intron 50 and exon 51, within intron 51, or spanning the junction of intron 51 and exon 52 of DMD. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising a complete or partial splice acceptor site set forth in SEQ ID NO: 859 or 898. In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, 8, or 9) consecutive nucleotides of the splice acceptor site set forth in SEQ ID NO: 859 or 898. In some embodiments, the oligonucleotide comprises at least four (eg, four, five, six, seven, eight, or nine) consecutive nucleotides of the splice acceptor site antisense sequence set forth in SEQ ID NO:903 or 942.

[0239] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 18-35 nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, 8, or 9) consecutive nucleotides of the splice acceptor site set forth in SEQ ID NO: 859 or 898. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 20-30 (e.g., 20, 25, 30) nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, 8, or 9) consecutive nucleotides of the splice acceptor site set forth in SEQ ID NO: 859 or 898. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping, such as skipping of exon 51) are 20-25 (i.e., 20, 21, 22, 23, 24, or 25) nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, or 8) consecutive nucleotides of the splice acceptor site set forth in SEQ ID NO: 859 or 898. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 30 nucleotides in length and contain a region of complementarity to a target sequence comprising at least four (e.g., 4, 5, 6, 7, 8, or 9) consecutive nucleotides of the splice acceptor site set forth in SEQ ID NO: 859 or 898.

[0240] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region of complementarity to an exon-intron junction of DMD RNA (e.g., any one of the exon / intron junctions provided by SEQ ID NOs: 832, 833, 837, 844, 845, and 853). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region of complementarity to at least 10 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more) consecutive nucleosides of an exon-intron junction of DMD RNA (e.g., any one of the exon / intron junctions provided by SEQ ID NOs: 832, 833, 837, 844, 845, and 853). In some embodiments, oligonucleotides useful for targeting DMD (eg, for exon skipping) are complementary to any one of SEQ ID NOs: 832, 833, 837, 844, 845, and 853.

[0241] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region of complementarity to a target sequence of DMD RNA (e.g., a target sequence provided by any one of SEQ ID NOS: 833, 835-837, 845, 847-853, and 839-843). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region of complementarity to at least 10 (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive nucleosides of a target sequence of DMD RNA (e.g., a target sequence provided by any one of SEQ ID NOS: 833, 835-837, 845, 847-853, and 839-843). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are complementary to any one of SEQ ID NOs: 833, 835-837, 845, 847-853, and 839-843. [Table 9-1] [Table 9-2]

[0242] In some embodiments, any one of the oligonucleotides useful for targeting DMD (eg, for exon skipping) is a phosphorodiamidate morpholino oligomer (PMO).

[0243] In some embodiments, the oligonucleotide may have a region of complementarity to a mutant DMD allele, e.g., a DMD allele with at least one mutation in any of exons 1-79 of DMD in humans that leads to a frameshift and improper RNA splicing / processing.

[0244] In some embodiments, any one of the oligonucleotides may be in a salt form, for example, as a sodium, potassium, or magnesium salt.

[0245] 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)-, or a combination thereof; each R A are independently hydrogen or substituted or unsubstituted alkyl. In certain embodiments, the spacer is a substituted or unsubstituted alkylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )-, or -C(=O)N(R A)2, or a combination thereof.

[0246] 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 and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to the oligonucleotide by reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide.

[0247] In some embodiments, the oligonucleotide is conjugated to a targeting agent, e.g., a muscle-targeting agent such as an anti-TfR1 antibody, e.g., via an amine group.

[0248] a. Oligonucleotide size / sequence Oligonucleotides may be of various different lengths, for example, depending on the format. In some embodiments, oligonucleotides are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length. In some embodiments, oligonucleotides are 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 21 to 23 nucleotides in length, 20 to 25 nucleotides in length, etc.

[0249] In some embodiments, for purposes of this disclosure, the nucleic acid sequence of an oligonucleotide is "complementary" to a target nucleic acid if it can specifically hybridize 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) modulates 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 the oligonucleotide has a sufficient degree of complementarity to its target nucleic acid so that it does not hybridize to non-target sequences under conditions where it is desired to avoid non-specific binding, e.g., physiological conditions. Thus, in some embodiments, the oligonucleotide can 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 can specifically hybridize with target nucleic acid or does not need to be 100% complementary to the sequence of the target that is specific to target nucleic acid.In certain embodiments, oligonucleotide contains one or more mismatched nucleobases with target nucleic acid.In certain embodiments, such mismatch reduces target-related activity, but reduces non-target-related activity to a greater extent (i.e., increases selectivity for target nucleic acid and reduces off-target effect).

[0250] In some embodiments, the oligonucleotide comprises a region of complementarity to the target nucleic acid that ranges from 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, 15 to 20, 20 to 25, or 5 to 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 8 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain 1, 2, or 3 base mismatches compared to a portion of consecutive nucleotides in 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.

[0251] In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary) to the target sequence of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8). In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary) to the target sequence of any one of the oligonucleotides provided by SEQ ID NOs: 384-831. In some embodiments, such target sequences are 100% complementary to the oligonucleotides listed in Table 8. In some embodiments, such target sequences are 100% complementary to the oligonucleotides provided by SEQ ID NOs: 384-831. In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary) to the target sequences provided herein (e.g., the target sequences listed in Table 8). In some embodiments, the oligonucleotide is complementary (eg, at least 85%, at least 90%, at least 95%, or 100% complementary) to any one of SEQ ID NOs: 160-383.

[0252] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region of complementarity to a target sequence of DMD RNA (e.g., a target sequence provided by any one of SEQ ID NOS: 160-383). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region of complementarity to at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive nucleosides of a target sequence of DMD RNA (e.g., a target sequence provided by any one of SEQ ID NOS: 160-383). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are complementary to any one of SEQ ID NOS: 160-383.

[0253] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a sequence comprising at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive nucleobases of a DMD-targeting sequence provided herein (e.g., an antisense sequence listed in Table 8). In some embodiments, the oligonucleotide comprises a sequence comprising at least 8 (e.g., 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more) consecutive nucleobases of any one of SEQ ID NOs: 384-831. In some embodiments, the oligonucleotide comprises the sequence of any one of SEQ ID NOs: 384-831.

[0254] In some embodiments, it is understood that methylation of the nucleobase uracil at the C5 position 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.

[0255] In some embodiments, any one or more of the thymine bases (T) in any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 8) may independently and optionally be a uracil base (U), and / or any one or more of the U's in the oligonucleotides provided herein may independently and optionally be a T. In some embodiments, any one or more of the thymine bases (T) in any one of the oligonucleotides provided by SEQ ID NOs: 608-831, or in the oligonucleotides complementary to any one of SEQ ID NOs: 160-383, may optionally be a uracil base (U), and / or any one or more of the U's in the oligonucleotide may optionally be a T. In some embodiments, any one or more of the uracil bases (U) in any one of the oligonucleotides provided by SEQ ID NOs: 384-607, or in the oligonucleotides complementary to any one of SEQ ID NOs: 160-383, may optionally be a thymine base (T), and / or any one or more of the T's in the oligonucleotide may optionally be a U.

[0256] b. Oligonucleotide Modifications: The oligonucleotides described herein may be modified, including, for example, modified sugar moieties, modified internucleoside linkages, modified nucleotides or nucleosides, and / or (for example, and) combinations thereof.In addition, in some embodiments, 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 in cells; minimize TLR stimulation; or avoid pattern recognition receptors.Any of the modified chemical properties or formats of the oligonucleotides described herein can be combined with each other.For example, 1, 2, 3, 4, 5, or more different types of modifications can be included in the same oligonucleotide.

[0257] In some embodiments, specific nucleotide or nucleoside modifications can be used to make the oligonucleotides they are incorporated into more resistant to nuclease digestion than natural oligodeoxynucleotide or oligoribonucleotide molecules, and these modified oligonucleotides remain intact for longer periods than unmodified oligonucleotides.Specific examples of modified oligonucleotides include those that contain modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short-chain alkyl or cycloalkyl intersugar linkages, or modified internucleoside linkages, such as short-chain heteroatom or heterocyclic intersugar linkages.Therefore, the oligonucleotides of the present disclosure can be stabilized against nucleic acid degradation by modification, for example, by incorporating nucleotide or nucleoside modifications.

[0258] In some embodiments, the oligonucleotide can be up to 50 nucleotides in length or up to 100 nucleotides in length, and 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45, or more nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The oligonucleotide can be 8 to 30 nucleotides in length, and 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, or 2 to 30 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The oligonucleotide can be 8 to 15 nucleotides in length, and 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, or 2 to 14 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. Optionally, the oligonucleotide can have all but 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides / nucleosides modified. Oligonucleotide modifications are further described herein.

[0259] C. modified nucleoside In some embodiments, the oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of the sugar. In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleoside. In some embodiments, all of the nucleosides on the oligonucleotide are 2'-modified nucleosides.

[0260] 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'-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-methylacetamido (2'-O-NMA) modified nucleosides.

[0261] In some embodiments, the oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleosides, wherein the ribose ring in the nucleoside comprises a bridging moiety connecting two atoms in the ring (e.g., 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 Publication No. 2008 / 043753, entitled "RNA Antagonist Compounds For The Modulation Of PCSK9," published April 17, 2008, the contents of which are incorporated herein by reference in their entirety. Examples of ENAs are provided in International Publication No. 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.

[0262] In some embodiments, the oligonucleotide is selected from the group consisting of 6-Modified Bicyclic Nucleic Acid Analogs, 6-Modified Bicyclic Nucleic Acid Analogs, and 6-Modified Bicyclic Nucleic Acid Analogs. No. 7,314,923, issued January 1, 2008, entitled "Novel Nucleoside And Oligonucleotide Analogues," U.S. Pat. No. 7,816,333, issued October 19, 2010, entitled "Oligonucleotide Analogues And Methods Utilizing The Same," and U.S. Patent Application Publication No. 2011 / 0009471, now U.S. Pat. No. 8,957,201, issued February 17, 2015, entitled "Oligonucleotide Analogues And Methods Utilizing The Same," the entire contents of each of which are incorporated herein by reference for all purposes.

[0263] In some embodiments, an oligonucleotide comprises at least one modified nucleoside that confers 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 collectively confers 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.

[0264] Oligonucleotides may contain a mixture of different types of nucleosides. For example, oligonucleotides may contain a mixture of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro-modified nucleosides. Oligonucleotides may contain a mixture of deoxyribonucleosides or ribonucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain a mixture of 2'-fluoro-modified nucleosides and 2'-O-Me-modified nucleosides. Oligonucleotides may contain a mixture of 2'-4' bicyclic nucleosides and 2'MOE, 2'-fluoro, or 2'-O-Me-modified nucleosides. 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).

[0265] Oligonucleotides can contain different types of alternating nucleosides.For example, oligonucleotides can contain alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides.Oligonucleotides can contain alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides.Oligonucleotides can contain alternating 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides.Oligonucleotides can 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 (eg, 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (eg, LNA, ENA, cEt).

[0266] In some embodiments, the oligonucleotides described herein comprise a 5'-vinylphosphonate modification, one or more abasic residues, and / or one or more inverted abasic residues.

[0267] d. Internucleoside linkage / backbone In some embodiments, oligonucleotides may contain phosphorothioate or other modified internucleoside linkages. In some embodiments, oligonucleotides include phosphorothioate internucleoside linkages. In some embodiments, oligonucleotides include phosphorothioate internucleoside linkages between at least two nucleosides. In some embodiments, oligonucleotides include phosphorothioate internucleoside linkages between all nucleosides. For example, in some embodiments, oligonucleotides include modified internucleoside linkages at the first, second, and / or (for example, and) third internucleoside linkages at the 5' or 3' end of the nucleotide sequence.

[0268] Phosphorus-containing linkages that may be used include, but are not limited to, normal 3'-5' linkages, 2'-5' linked analogs of these, phosphorothioates, 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, as well as those of reverse polarity (where adjacent pairs of nucleoside units are 3'-5' to 5'-3' or 2' -5' to 5'-2') and the like; U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,6 See Nos. 76; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0269] In some embodiments, oligonucleotides may have heteroatom backbones such as methylene (methylimino) or MMI backbones, amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374), morpholino backbones (see Summerton and Weller, U.S. Pat. No. 5,034,506), or peptide nucleic acid (PNA) backbones (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone and the nucleotides are linked directly or indirectly to aza nitrogen atoms of the polyamide backbone; see Nielsen et al., Science 1991, 254, 1497).

[0270] e. stereospecific oligonucleotides In some embodiments, the internucleotide phosphorus atom 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 can 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 December; 40(12):5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided, comprising nucleoside units linked to each other by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages. In some embodiments, such phosphorothioate oligonucleotides with substantially chiral pure intersugar linkages are prepared by enzymatic synthesis or chemical synthesis, for example, as described in U.S. Patent No. 5,587,261, issued December 12, 1996, the contents of which are incorporated herein by reference in their entirety. In some embodiments, chiral-controlled oligonucleotide provides selective cleavage pattern of target nucleic acid.For example, in some embodiments, chiral-controlled oligonucleotide provides single cleavage site in the complementary sequence of nucleic acid, for example, as described in U.S. Patent Application Publication No. 20170037399, entitled "CHIRAL DESIGN," published on February 2, 2017 (the contents of which are incorporated herein by reference in their entirety).

[0271] f. morpholino In some embodiments, the oligonucleotide may be a morpholino-based compound.Morpholino-based oligomeric compounds are described in Dwaine A.Braasch and David R.Corey, Biochemistry, 2002, 41(14), 4503-4510; Genesis, volume 30, issue 3, 2001; Heasman, J., Dev.Biol., 2002, 243, 209-214; Nasevicius et al., Nat.Genet., 2000, 26, 216-220; Lacerra et al., Proc.Natl.Acad.Sci., 2000, 97, 9591-9596; and U.S. Patent No. 5,034,506, issued July 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (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 herein by reference in their entireties).

[0272] 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 an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimic that has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an oligonucleotide is replaced with an amide-containing backbone, such as an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atoms 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 teachings on PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.

[0273] h.Mixmer In some embodiments, the oligonucleotides described herein may be mixmers or include mixmer sequence patterns. Generally, mixmers are oligonucleotides that contain both naturally occurring nucleosides and non-naturally occurring nucleosides, or oligonucleotides that contain two different types of non-naturally occurring nucleosides, typically in a staggered pattern. Mixmers generally have higher binding affinity than unmodified oligonucleotides and specifically bind to target molecules, for example, they can be used to block binding sites on target molecules. Generally, mixmers do not allow RNase to be recruited to target molecules, and therefore do not promote the cleavage of target molecules. Such oligonucleotides that are not capable of recruiting RNase H have been described, see, for example, WO 2007 / 112754 or WO 2007 / 112753.

[0274] In some embodiments, a mixmer comprises or consists of a repeating pattern of nucleoside analogs and naturally occurring nucleosides, or a repeating pattern of one type of nucleoside analog and another type of nucleoside analog. However, a mixmer need not comprise a repeating pattern, but instead can comprise any arrangement of modified nucleosides and naturally occurring nucleosides, or any arrangement of one type of modified nucleoside and another type of modified nucleoside. Illustratively, the repeating pattern may be such that every third or third nucleoside is a modified nucleoside, such as LNA, with the remaining nucleosides being naturally occurring nucleosides, such as DNA, or 2'-substituted nucleoside analogs, such as 2'MOE or 2'-fluoro analogs, or any other modified nucleoside described herein. It is recognized that repeating patterns of modified nucleosides, such as LNA units, may be combined with modified nucleosides at fixed positions, for example at the 5' or 3' termini.

[0275] In some embodiments, a mixmer does not contain a region of six or more, five or more, four or more, or three or more consecutive naturally occurring nucleosides, such as DNA nucleosides. In some embodiments, a mixmer contains at least a region consisting of at least two consecutive modified nucleosides, such as at least two consecutive LNAs. In some embodiments, a mixmer contains at least a region consisting of at least three consecutive modified nucleosides, such as at least three consecutive LNAs.

[0276] In some embodiments, the mixmer does not contain a region of 8 or more, 7 or more, 6 or more, 5 or more, 4 or more, or 3 or more consecutive nucleoside analogs such as LNA. In some embodiments, the LNA units may be replaced with other nucleoside analogs, such as those mentioned herein.

[0277] Mixmers may be designed to contain a mixture of affinity-enhancing modified nucleosides, such as, in non-limiting examples, LNA nucleosides and 2'-O-Me nucleosides. In some embodiments, a mixmer contains modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, or more nucleosides.

[0278] 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 Publication Nos. US20060128646, US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. Patent No. 7,687,617.

[0279] In some embodiments, a mixmer comprises one or more morpholino nucleosides. For example, in some embodiments, a mixmer may comprise morpholino nucleosides mixed (e.g., alternating) with one or more other nucleosides (e.g., DNA, RNA nucleosides) or modified nucleosides (e.g., LNA, 2'-O-Me nucleosides).

[0280] In some embodiments, mixmers are useful for splicing correction or exon skipping, as reported, for example, 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), and 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).

[0281] i. Multimers 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 can be increased beyond the available linking sites on the targeting agent (e.g., available thiol sites on an antibody) or otherwise tailored to achieve a specific payload. The oligonucleotides in the multimer can be the same or different (e.g., targeting different genes, or different sites on the same gene, or their products).

[0282] 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 to 5, 2 to 10, or 4 to 20 oligonucleotides linked together.

[0283] In some embodiments, a multimer comprises two or more oligonucleotides linked end-to-end (in a linear configuration). 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, a basic linker). In some embodiments, a multimer comprises the 5' end of one oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, a multimer comprises the 3' end of one oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, a multimer comprises the 5' end of one oligonucleotide linked to the 5' end of another oligonucleotide. Still further, in some embodiments, a multimer may comprise a branched structure comprising multiple oligonucleotides linked together by a branched linker.

[0284] Further examples of multimers that may be used in the conjugates provided herein are disclosed, for example, in U.S. Patent Application No. 2015 / 0315588, published November 5, 2015, entitled "Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers," U.S. Patent Application No. 2015 / 0247141, published September 3, 2015, entitled "Multimeric Oligonucleotide Compounds," U.S. Patent Application No. 2011 / 0158937, published June 30, 2011, entitled "Immunostimulatory Oligonucleotide Multimers," and U.S. Patent No. 5,693,773, published December 2, 1997, entitled "Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers Targeting Nucleic Acids Comprising Mixed Sequences of Purines and Pyrimidines," the contents of each of which are incorporated herein by reference in their entirety.

[0285] C. Linker The conjugates described herein generally include a linker covalently linking 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 can be a single bond, such as a disulfide bond or disulfide bridge, that covalently links the anti-TfR1 antibody to the molecular payload. However, in some embodiments, the linker can covalently link any one of the anti-TfR1 antibodies described herein to the molecular payload via 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. The linker is typically stable in vitro and in vivo and can be stable in a particular cellular environment. In addition, the linker typically does not negatively affect the functional properties of either the anti-TfR1 antibody or the molecular payload. Examples and methods of linker synthesis are known in the art (see, for example, Kline, T. et al., "Methods to Make Homogenous Antibody Drug Conjugates." Pharmaceutical Research, 2015, 32:11, 3480-3493; Jain, N. et al., "Current ADC Linker Chemistry." Pharm Res. 2015, 32:11, 3526-3540; McCombs, JR and Owen, SC, "Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry." AAPS J. 2015, 17:2, 339-351).

[0286] The linker typically comprises 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 comprises two different electrophiles or nucleophiles 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, optionally comprising a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some embodiments, the linker is covalently linked to a cysteine ​​residue of the anti-TfR1 antibody or a thiol-functionalized molecular payload via a 3-arylpropionitrile functional group. In some embodiments, the linker is covalently linked to a lysine residue of the anti-TfR1 antibody. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or molecular payload (e.g., and) independently via an amide bond, a carbamate bond, a hydrazide bond, a triazole bond, a thioether bond, and / or a disulfide bond.

[0287] i. Cleavable linker The cleavable linker may be a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker, which are typically cleavable only intracellularly and are preferably stable in an extracellular environment, e.g., outside of a muscle cell.

[0288] 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 or alanine-citrulline sequence. In some embodiments, the protease-sensitive linker may be cleaved by lysosomal proteases, such as cathepsin B, and / or endosomal proteases (e.g., α- and β-amino acids).

[0289] A pH-sensitive linker is a covalent linkage that is readily degraded in high or low pH environments. In some embodiments, the pH-sensitive linker may be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved in an endosome or lysosome.

[0290] In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with glutathione species inside a cell. In some embodiments, the disulfide moiety further comprises at least one amino acid, for example, a cysteine ​​residue.

[0291] 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, prior to conjugation, the linker comprises the following structure: [ka]

[0292] In some embodiments, after conjugation, the linker comprises the following structure: [ka]

[0293] In some embodiments, prior to conjugation, the linker comprises the following structure: [ka] (wherein n is any number from 0 to 10.) In some embodiments, n is 3.

[0294] In some embodiments, the linker comprises the following structure: [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.

[0295] In some embodiments, the linker comprises the following structure: [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.

[0296] ii. Non-cleavable linker In some embodiments, a non-cleavable linker may be used. Generally, a non-cleavable linker cannot be easily degraded in a cellular or physiological environment. In some embodiments, the 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, the 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 is carried out to link an anti-TfR1 antibody comprising an LPXT sequence to (G) n It can be used to covalently link to molecular payloads containing sequences (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.).

[0297] 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.

[0298] 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 via a phosphate or phosphorothioate group, for example, a phosphate at the end of the oligonucleotide backbone. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody via a lysine or cysteine ​​residue present on the anti-TfR1 antibody.

[0299] In some embodiments, the linker, or a portion thereof, is covalently linked to the anti-TfR1 antibody and / or (for example, and) a molecular payload via a cycloaddition reaction between an azide and an alkyne to form a triazole, and the azide or alkyne may be located on the anti-TfR1 antibody, the molecular payload, or the linker. In some embodiments, the alkyne may be a cyclic alkyne, 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 No. WO2011136645, entitled "Fused Cyclooctyne Compounds and Their Use in Metal-free Click Reactions," published November 3, 2011. In some embodiments, the azide may be an azide-containing sugar or carbohydrate molecule. In some embodiments, the azide can 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 Publication No. 2016170186, 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 and alkyne can be located on the anti-TfR1 antibody, the molecular payload, or the linker) is as described in WO 2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or WO 2016170186, 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."

[0300] 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.

[0301] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (for example, and) the molecular payload via a Diels-Alder reaction between a dienophile and a diene / hetero-diene, although 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 via other pericyclic reactions, such as an ene reaction. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (for example, and) the molecular payload via 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 via a condensation reaction to form an oxime, hydrazone, or semicarbazide group present between the linker and the anti-TfR1 antibody and / or (for example, and) the molecular payload.

[0302] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (for example, and) molecular payload by a conjugate addition reaction between a nucleophile, such as an amine group or a hydroxyl group, and an electrophile, such as a carboxylic acid, carbonate, or aldehyde. In some embodiments, prior to the reaction between the linker and the anti-TfR1 antibody or molecular payload, the nucleophile may be present on the linker, and the electrophile may be present on the anti-TfR1 antibody or molecular payload. In some embodiments, prior to the reaction between the linker and the anti-TfR1 antibody or molecular payload, the electrophile may be present on the linker, and the nucleophile may be present on the anti-TfR1 antibody or molecular payload. In some embodiments, the electrophile may be an azide, pentafluorophenyl, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimidyl ester, sulfosuccinimidyl ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center, and / or activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, optionally substituted alkyne, optionally substituted aryl, optionally substituted heterocyclyl, hydroxyl group, amino group, alkylamino group, anilide group, and / or thiol group.

[0303] 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 the following structure: [ka] (wherein n is any number from 0 to 10.) In some embodiments, n is 3.

[0304] In some embodiments, a linker comprising the structure of Formula (A) is covalently linked (e.g., optionally via an additional chemical moiety) to a molecular payload (e.g., an oligonucleotide). In some embodiments, a linker comprising the structure of Formula (A) is covalently linked to an oligonucleotide, e.g., via nucleophilic substitution with an amine-L1-oligonucleotide to form a carbamate bond, resulting in a compound comprising the following structure: [ka] (wherein n is any number from 0 to 10.) In some embodiments, n is 3.

[0305] In some embodiments, the compound of Formula (B) is further covalently linked to an additional moiety via a triazole, which 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 the following structure: [ka] (wherein m is any number from 0 to 10.) In some embodiments, m is 4.

[0306] In some embodiments, the azide of a compound of structure (B) forms a triazole via a click reaction with an alkyne of a compound of structure (C) to form a compound comprising the following structure: [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 m is 4.

[0307] In some embodiments, the compound of structure (D) is further covalently linked to a lysine of an anti-TfR1 antibody to form a conjugate comprising the following structure: [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 (by way of example, and) m is 4. It will be appreciated 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.

[0308] In some embodiments, the compound of Formula (C) is further covalently linked to a lysine of an anti-TfR1 antibody to form a compound comprising the following structure: [ka] (wherein m is 0 to 15 (for example, 4).) It will be appreciated that the amide shown adjacent to the anti-TfR1 antibody in formula (F) results from reaction with an amine of the anti-TfR1 antibody, such as a lysine epsilon amine.

[0309] In some embodiments, the azide of a compound of structure (B) forms a triazole via a click reaction with an alkyne of a compound of structure (F) to form a conjugate comprising the following structure: [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 (by way of example, and) m is 4. It will be appreciated 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.

[0310] In some embodiments, the azide of a compound of structure (A) forms a triazole via a click reaction with an alkyne of a compound of structure (F) to form a compound comprising the following structure: [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 (by way of example, and) m is 4. In some embodiments, the oligonucleotide is covalently attached to a compound comprising the structure of formula (G), thereby forming a conjugate comprising the structure of formula (E). It will be appreciated that the amide shown adjacent to the anti-TfR1 antibody in formula (G) results from reaction with an amine of the anti-TfR1 antibody, such as a lysine epsilon amine.

[0311] In some embodiments, in any one of the conjugates described herein, the anti-TfR1 antibody is covalently linked to a molecular payload (e.g., an oligonucleotide) via a lysine on the anti-TfR1 antibody via a linker comprising the following structure: [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.

[0312] In some embodiments, in any one of the conjugates described herein, the anti-TfR1 antibody is covalently linked to a molecular payload (e.g., an oligonucleotide) via a lysine on the anti-TfR1 antibody via a linker comprising the following structure: [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.

[0313] In some embodiments, in formulas (B), (D), (E), and (I), L is, in some embodiments, a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, —O—, —N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)NR A -, -NR A S(O)-, or a combination thereof; R A is independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L is [ka] (wherein L2 is [ka] or [ka] where a represents a moiety directly attached to the carbamate moiety of formulas (B), (D), (E), and (I), and b represents a moiety covalently linked to the oligonucleotide (directly or via an additional chemical moiety).

[0314] In some embodiments, L1 is [ka] where a represents the site directly linked to the carbamate moiety of formulas (B), (D), (E), and (I), and b represents the site covalently linked (directly or via an additional chemical moiety) to the oligonucleotide.

[0315] In some embodiments, L1 is [ka] is.

[0316] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide. In some embodiments, the phosphate is a phosphodiester. In some embodiments, L1 is linked to the 5' phosphorothioate of the oligonucleotide. In some embodiments, L1 is linked to the 5' phosphoramidate of the oligonucleotide. In some embodiments, L1 is linked to the 5' end of the oligonucleotide via a phosphorodiamidate bond.

[0317] In some embodiments, L1 is optional (eg, does not have to be present).

[0318] In some embodiments, any one of the conjugates described herein has the following structure: [ka] (wherein n is 0 to 15 (e.g., 3) and m is 0 to 15 (e.g., 4).) It will be appreciated 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 a lysine epsilon amine.

[0319] In some embodiments, any one of the conjugates described herein has the following structure: [ka] (In the formula, n is 0 to 15 (for example, 3), and m is 0 to 15 (for example, 4).)

[0320] In some embodiments, the oligonucleotide is modified to include an amine group at the 5' or 3' end, or internally (e.g., as an amine-functionalized nucleobase) before linking to a compound, e.g., a compound of Formula (A) or Formula (G).

[0321] Although the linker conjugates are described in relation to anti-TfR1 antibodies and oligonucleotide molecular payloads, it will be appreciated that the use of such linker conjugates for other muscle-targeting agents, such as other muscle-targeting antibodies, and / or other molecular payloads is contemplated.

[0322] 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 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 listed in Tables 2-7) is covalently linked to the molecular payload (e.g., an oligonucleotide such as the oligonucleotide listed in Table 8) 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 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 including 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8, such as provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0323] 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 covalently 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0324] 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] (wherein n is a number between 0 and 10, m is a number between 0 and 10, and the linker is linked to the antibody via an amine group (e.g., on a lysine residue) and / or (for example, and) 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, 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide provided by any one of SEQ ID NOs: 384-831, or a DMD-targeting oligonucleotide listed in Table 8 that is complementary to any one of SEQ ID NOs: 160-383). It will be appreciated 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.

[0325] It should be understood that antibodies can be linked to molecular payloads with various stoichiometries, a property that may be referred to as the drug-antibody ratio (DAR), where "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, mixtures of different conjugates, each having a different DAR, are provided. In some embodiments, the average DAR of the conjugates in such a mixture can range from 1 to 3, 1 to 4, 1 to 5, or more. The average DAR of the conjugates in a mixture need not be an integer value. The DAR may be increased by conjugating 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.

[0326] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody described herein (e.g., an antibody shown 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 shown 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 attached to the antibody (e.g., an anti-TfR1 antibody described herein) via a thiol-reactive bond (e.g., via a cysteine ​​in the antibody). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is attached 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8, such as provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0327] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein 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. In some embodiments, the molecular payload is a DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0328] 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: 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0329] 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0330] 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0331] In some embodiments, a conjugate described herein comprises an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0332] 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0333] In some embodiments, a conjugate described herein comprises an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0335] 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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: 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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: 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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: 102 and a light chain comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the molecular payload is a DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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: 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[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: 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 DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383).

[0346] In any of the exemplary conjugates described herein, in some embodiments, the anti-TfR1 antibody is covalently linked to the molecular payload via a linker comprising the following structure: [ka] (In the formula, n is 3 and m is 4.)

[0347] In some embodiments, the conjugate described herein comprises an anti-TfR1 antibody covalently linked to the 5' end of a DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8, such as provided by any one of SEQ ID NOs: 384-831, or complementary to any one of SEQ ID NOs: 160-383) via a lysine of the anti-TfR1 antibody, wherein 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, and the conjugate has the following structure: [ka] (where n is 3 and m is 4.) It will be appreciated 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.

[0348] In some embodiments, the conjugate described herein comprises an anti-TfR1 antibody covalently linked via a lysine of the anti-TfR1 antibody to the 5' end of a DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831 or complementary to any one of SEQ ID NOs: 160-383), wherein the anti-TfR1 antibody comprises the VH and VL of any one of the antibodies listed in Table 3, and the conjugate has the following structure: [ka] (where n is 3 and m is 4.) It will be appreciated 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.

[0349] In some embodiments, the conjugate described herein comprises an anti-TfR1 antibody covalently linked via a lysine of the anti-TfR1 antibody to the 5' end of a DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831 or complementary to any one of SEQ ID NOs: 160-383), wherein the anti-TfR1 antibody comprises the heavy and light chains of any one of the antibodies listed in Table 4, and the conjugate has the following structure: [ka] (where n is 3 and m is 4.) It will be appreciated 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.

[0350] In some embodiments, the conjugate described herein comprises an anti-TfR1 Fab covalently linked via a lysine of the anti-TfR1 antibody to the 5' end of a DMD-targeting oligonucleotide (e.g., a DMD-targeting oligonucleotide listed in Table 8 provided by any one of SEQ ID NOs: 384-831 or complementary to any one of SEQ ID NOs: 160-383), wherein the anti-TfR1 Fab comprises the heavy and light chains of any one of the antibodies listed in Table 5, and the conjugate has the following structure: [ka] (where n is 3 and m is 4.) It will be appreciated 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.

[0351] In some embodiments, in any one of the example conjugates described herein, L1 is [ka] (wherein L2 is [ka] or [ka] where a represents a moiety directly attached to the carbamate moiety of formulas (B), (D), (E), and (I), and b represents a moiety covalently linked to an oligonucleotide (directly or via an additional chemical moiety).

[0352] In some embodiments, L1 is [ka] where a represents the site directly linked to the carbamate moiety of formulas (B), (D), (E), and (I), and b represents the site covalently linked (directly or via an additional chemical moiety) to the oligonucleotide.

[0353] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide. In some embodiments, the phosphate is a phosphodiester. In some embodiments, L1 is linked to the 5' phosphorothioate of the oligonucleotide. In some embodiments, L1 is linked to the 5' phosphoramidate of the oligonucleotide. In some embodiments, L1 is linked to the 5' end of the oligonucleotide via a phosphorodiamidate bond.

[0354] In some embodiments, L1 is optional (eg, does not have to be present).

[0355] III. Preparations The conjugates provided herein may be formulated in any suitable manner. Generally, the conjugates provided herein are formulated in a manner suitable for pharmaceutical use. For example, the conjugates 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 conjugate in the formulation. In some embodiments, provided herein are compositions comprising a conjugate and a pharmaceutically acceptable carrier. Such compositions may be suitably formulated so that a sufficient amount of the conjugate can enter the target muscle cell when administered either into the environment surrounding the target cell of a subject or into the subject's systemic environment. In some embodiments, the conjugates are formulated in a buffer solution such as phosphate-buffered saline, in a liposome, in a micellar structure, or in a capsid.

[0356] It will be appreciated 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).

[0357] 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, a 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).

[0358] 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 before use (e.g., administration to a subject). Thus, the excipient in a composition comprising the complex or a component thereof described herein may be a cryoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0359] In some embodiments, the pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, and subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous administration.

[0360] Pharmaceutical compositions suitable for use in injections include sterile aqueous solutions (wherein the complex is soluble in water) or dispersions, and sterile powders for the extemporaneous preparation of sterile injection solutions or dispersions. Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. In some embodiments, the formulations include isotonic agents, such as sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. 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, followed by filtration sterilization as required.

[0361] In some embodiments, the composition may contain at least about 0.1% of the complex or its components, or more, although the percentage of active ingredient may be from about 1% to about 80%, or more, by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art when preparing such pharmaceutical formulations. Therefore, various dosages and treatment regimens may be desirable.

[0362] IV. Methods of Use / Treatment Conjugates comprising a muscle-targeting agent covalently linked to a molecular payload described herein are effective in treating subjects with dystrophinopathy, e.g., Duchenne muscular dystrophy. In some embodiments, the conjugate comprises a molecular payload that is an oligonucleotide, e.g., an antisense oligonucleotide, that facilitates exon skipping of pre-mRNA expressed from a mutant DMD allele.

[0363] 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 Duchenne muscular dystrophy or other dystrophinopathy. In some embodiments, the subject has a mutant DMD allele, which may optionally include at least one mutation in a DMD exon that causes a frameshift mutation and leads to improper RNA splicing / processing. In some embodiments, the subject suffers from severe dystrophinopathy symptoms, such as muscle atrophy or sarcopenia. In some embodiments, the subject has asymptomatic increases in serum levels of creatine phosphokinase (CK) and / or muscle spasms accompanied by myoglobinuria (for example, and). In some embodiments, the subject has a progressive muscular disease, such as Duchenne or Becker muscular dystrophy or DMD-related dilated cardiomyopathy (DCM). In some embodiments, the subject does not suffer from dystrophinopathy symptoms.

[0364] In some embodiments, the subject has a mutation in the DMD gene that is amenable to skipping of exon 51. In some embodiments, a conjugate comprising a muscle-targeting agent covalently linked to a molecular payload described herein is effective in treating a subject having a mutation in the DMD gene that is amenable to skipping of exon 51. In some embodiments, the conjugate comprises a molecular payload that is an oligonucleotide, e.g., an antisense oligonucleotide that facilitates skipping of exon 51 of a pre-mRNA, such as a pre-mRNA encoded from a mutant DMD gene (e.g., a mutant DMD gene that is amenable to skipping of exon 51).

[0365] Aspects of the present disclosure include methods involving administering to a subject an effective amount of a conjugate described herein. In some embodiments, an effective amount of a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent covalently linked to a molecular payload can be administered to a subject in need of treatment. In some embodiments, a pharmaceutical composition comprising a conjugate as described herein can 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, administration can be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the pharmaceutical composition can be in solid, aqueous, or liquid form. In some embodiments, the aqueous or liquid form can be sprayed or lyophilized. In some embodiments, the sprayed or lyophilized form can be reconstituted with an aqueous or liquid solution.

[0366] Compositions for intravenous administration may contain various 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 infusion, whereby a pharmaceutical formulation containing the antibody and a pharmaceutically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0367] 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, including implantable depot sources, localized delivery catheters, site-specific carriers, direct injection, or direct application of the conjugate.

[0368] In some embodiments, a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent covalently linked to a molecular payload is administered at an effective concentration to confer a therapeutic effect on 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 no more than routine experimentation. In some embodiments, the effective concentration is the maximum dose deemed safe for the patient. In some embodiments, the effective concentration will be the lowest feasible concentration that provides maximum efficacy.

[0369] Empirical considerations, such as the half-life of the conjugate in the subject, will generally help determine the concentration of pharmaceutical composition used for treatment. Dosage frequency may be empirically determined and adjusted to maximize the effectiveness of treatment.

[0370] The effectiveness of treatment may be assessed using any suitable method. In some embodiments, the effectiveness of treatment may be assessed by observing symptoms associated with dystrophinopathy, such as muscle atrophy or muscle weakness, by subject self-reported outcomes, such as measures of mobility, self-care, usual activities, pain / discomfort, and anxiety / depression, or by quality of life indicators, such as lifespan.

[0371] 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 modulate 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% compared to a control (e.g., a baseline level of gene expression prior to treatment). Additional Aspects 1. A conjugate comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to a molecular payload configured to induce skipping of exon 51 in DMD pre-mRNA, wherein the anti-TfR1 antibody is an antibody identified in any one of Tables 2-7. 2. Anti-TfR1 antibody, (i) heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 33, heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 34, heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 35, light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 36, light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 37, and light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 32; (ii) CDR-H1 of SEQ ID NO: 7, CDR-H2 of SEQ ID NO: 8, CDR-H3 of SEQ ID NO: 9, CDR-L1 of SEQ ID NO: 10, CDR-L2 of SEQ ID NO: 11, and CDR-L3 of SEQ ID NO: 6; (iii) CDR-H1 of SEQ ID NO: 7, CDR-H2 of SEQ ID NO: 20, CDR-H3 of SEQ ID NO: 9, CDR-L1 of SEQ ID NO: 10, CDR-L2 of SEQ ID NO: 11, and CDR-L3 of SEQ ID NO: 6; (iv) CDR-H1 of SEQ ID NO: 7, CDR-H2 of SEQ ID NO: 24, CDR-H3 of SEQ ID NO: 9, CDR-L1 of SEQ ID NO: 10, CDR-L2 of SEQ ID NO: 11, and CDR-L3 of SEQ ID NO: 6; (v) CDR-H1 of SEQ ID NO: 51, CDR-H2 of SEQ ID NO: 52, CDR-H3 of SEQ ID NO: 53, CDR-L1 of SEQ ID NO: 54, CDR-L2 of SEQ ID NO: 55, and CDR-L3 of SEQ ID NO: 50; (vi) CDR-H1 of SEQ ID NO: 64, CDR-H2 of SEQ ID NO: 52, CDR-H3 of SEQ ID NO: 53, CDR-L1 of SEQ ID NO: 54, CDR-L2 of SEQ ID NO: 55, and CDR-L3 of SEQ ID NO: 50; or (vii) CDR-H1 of SEQ ID NO: 67, CDR-H2 of SEQ ID NO: 52, CDR-H3 of SEQ ID NO: 53, CDR-L1 of SEQ ID NO: 54, CDR-L2 of SEQ ID NO: 55, and CDR-L3 of SEQ ID NO: 50 2. The complex of embodiment 1, comprising: 3. Anti-TfR1 antibody (i) a heavy chain variable region (VH) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 76; and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% identical to SEQ ID NO: 75; (ii) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 69; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 70; (iii) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 71; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 70; (iv) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 72; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 70; (v) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 73; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 74; (vi) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 73; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 75; (vii) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 76; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 74; (viii) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 77; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 78; (ix) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 79; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 80; or (x) a VH comprising an amino acid sequence at least 85% identical to SEQ ID NO: 77; and / or a VL comprising an amino acid sequence at least 85% identical to SEQ ID NO: 80; 3. The complex of embodiment 1 or embodiment 2, comprising: 4. Anti-TfR1 antibody (i) 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; (ii) 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; (iii) 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; (iv) 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; (v) 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; (vi) 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; (vii) 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; (viii) 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; (ix) 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; or (x) 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; 4. The conjugate of any one of aspects 1 to 3, comprising: 5. The conjugate of any one of aspects 1 to 4, wherein the anti-TfR1 antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, an Fv, or a full-length IgG. 6. The conjugate of embodiment 5, wherein the anti-TfR1 antibody is a Fab fragment. 7. Anti-TfR1 antibody (i) 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; (ii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 97; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (iii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 98; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (iv) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 99; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 85; (v) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 89; (vi) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 100; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90; (vii) 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: 89; (viii) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 93; (ix) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 103; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; or (x) a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 102; and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 95; 7. The complex of embodiment 6, comprising: 8. Anti-TfR1 antibody (i) 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; (ii) 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; (iii) 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; (iv) 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; (v) 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; (vi) 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; (vii) 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; (viii) 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; (ix) 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; or (x) 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. 8. The complex of embodiment 6 or embodiment 7, comprising: 9. The conjugate of any one of aspects 1 to 8, wherein the anti-TfR1 antibody does not specifically bind to the transferrin binding site of transferrin receptor 1 and / or the anti-TfR1 antibody does not inhibit binding of transferrin to transferrin receptor 1. 10. The conjugate of any one of aspects 1 to 9, wherein the molecular payload comprises an oligonucleotide. 11. The conjugate of aspect 10, wherein the oligonucleotide promotes antisense-mediated exon skipping in the DMD pre-RNA. 12. The conjugate of aspect 10 or 11, wherein the oligonucleotide comprises a region of complementarity to a splicing feature of the DMD pre-mRNA. 13. The complex of aspect 12, wherein the splicing feature is an exonic splicing enhancer (ESE) of the DMD pre-mRNA. 14. The complex of embodiment 13, wherein the splicing feature is in exon 51 of the DMD pre-mRNA, and optionally the ESE comprises the sequence of any one of SEQ ID NOs: 860-894. 15. The complex of embodiment 12, wherein the splicing feature is a branch point, a splice donor site, or a splice acceptor site. 16. The complex of embodiment 15, wherein the splicing feature is present across the junction of exon 50 and intron 50, within intron 50, across the junction of intron 50 and exon 51, across the junction of exon 51 and intron 51, within intron 51, or across the junction of intron 51 and exon 52 of the DMD pre-mRNA, and optionally the splicing feature comprises the sequence of any one of SEQ ID NOs: 855-8595 and 895-898. 17. The conjugate of any one of aspects 12 to 16, wherein the region of complementarity comprises at least four consecutive nucleosides complementary to the splicing feature. 18. The conjugate of any one of aspects 1 to 9, wherein the molecular payload comprises an oligonucleotide comprising a sequence complementary to any one of SEQ ID NOs: 160-383, or comprising the sequence of any one of SEQ ID NOs: 384-831, wherein each thymine base (T) is independently and optionally substituted with a uracil base (U), and each U is independently and optionally substituted with a T. 19. The conjugate according to any one of aspects 10 to 18, wherein the oligonucleotide comprises at least one modified internucleoside linkage. 20. The conjugate of embodiment 19, wherein at least one modified internucleoside linkage is a phosphorothioate linkage. 21. The conjugate according to any one of aspects 10 to 20, wherein the oligonucleotide comprises one or more modified nucleosides. 22. The conjugate of embodiment 21, wherein the one or more modified nucleosides are 2'-modified nucleosides. 23. The conjugate of any one of aspects 10 to 18, wherein the oligonucleotide comprises one or more phosphorodiamidate morpholinos, and optionally, the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO). 24. The conjugate of any one of aspects 1 to 23, wherein the anti-TfR1 antibody is covalently linked to the molecular payload via a cleavable linker. 25. The conjugate of embodiment 24, wherein the cleavable linker comprises a valine-citrulline sequence. 26. The conjugate of any one of aspects 1 to 25, wherein the anti-TfR1 antibody is covalently linked to the molecular payload via conjugation to a lysine or cysteine ​​residue of the antibody. 27. A conjugate comprising an anti-TfR1 antibody covalently linked to an oligonucleotide configured to induce skipping of exon 51 in DMD pre-mRNA, wherein the oligonucleotide comprises a region of complementarity to any one of SEQ ID NOs: 160-383. 28. The conjugate of aspect 27, wherein the anti-TfR1 antibody is an antibody identified in any one of Tables 2 to 7. 29. A conjugate comprising an anti-TfR1 antibody covalently linked to an oligonucleotide configured to induce skipping of exon 51 in DMD pre-mRNA, wherein the oligonucleotide comprises a region of complementarity to a splicing feature of DMD pre-mRNA. 30. An oligonucleotide targeted to DMD, wherein the oligonucleotide comprises a region of complementarity to any one of SEQ ID NOs: 160-383. 31. The oligonucleotide according to aspect 30, wherein the region of complementarity comprises at least 15 contiguous nucleosides complementary to any one of SEQ ID NOs: 160-383. 32. The oligonucleotide of aspect 30 or 31, wherein the oligonucleotide comprises at least 15 contiguous nucleosides of any one of SEQ ID NOs: 384-831, optionally wherein the oligonucleotide comprises the sequence of any one of SEQ ID NOs: 384-831, wherein each thymine base (T) is optionally and independently replaced with a uracil base (U), and wherein each U is optionally and independently replaced with a T. 33. A method for delivering a molecular payload to a cell, the method comprising contacting the cell with a complex according to any one of aspects 1 to 26. 34. A method for delivering an oligonucleotide to a cell, the method comprising contacting the cell with a complex according to any one of aspects 27 to 29. 35. A method for promoting expression or activity of a dystrophin protein in a cell, the method comprising contacting the cell with a complex of any one of aspects 1 to 26, in an amount effective to promote internalization of the molecular payload into the cell, optionally wherein the cell is a muscle cell. 36. A method for promoting expression or activity of a dystrophin protein in a cell, the method comprising contacting the cell with a complex of any one of aspects 27 to 29 in an amount effective to promote internalization of the oligonucleotide into the cell, wherein optionally the cell is a muscle cell. 37. The method of embodiment 35 or 36, wherein the cell is in vitro. 38. The method of embodiment 35 or 36, wherein the cell is in a subject. 39. The method of embodiment 38, wherein the subject is a human. 40. The method of embodiment 39, wherein the subject has a DMD gene suitable for skipping exon 51. 41. The method of any one of aspects 35 to 40, wherein the dystrophin protein is a truncated dystrophin protein. 42. A method of treating a subject having a mutant DMD allele associated with dystrophinopathy, the method comprising administering to the subject an effective amount of a conjugate of any one of aspects 1 to 29. 43. A method for promoting exon 51 skipping of a DMD pre-mRNA transcript in a cell, the method comprising contacting the cell with an effective amount of a complex according to any one of aspects 1 to 29. 44. A method of treating a subject having a mutant DMD allele associated with dystrophinopathy, the method comprising administering to the subject an effective amount of a conjugate of any one of aspects 1 to 29.

[0372] example Example 1. Exon skipping activity of anti-TfR1 antibody conjugates in myotubes from Duchenne muscular dystrophy patients In this experiment, the exon skipping activity of an anti-TfR1 antibody conjugate containing an anti-TfR1 Fab (3M12 VH4 / Vκ3) covalently linked to a DMD exon 51 skipping antisense oligonucleotide (ASO) was evaluated. The DMD exon 51 skipping ASO is a 30-nucleotide phosphorodiamidate morpholino oligomer (PMO) targeting an ESE in DMD exon 51 with the sequence TGGAGGT (SEQ ID NO: 877). Immortalized human myoblasts carrying an exon 52 deletion in the DMD gene were thawed and seeded at a density of 1e6 cells / flask in Promocell skeletal muscle cell growth medium (containing 5% FBS and 1x Pen-Strep) and grown to confluence. Once confluent, the cells were trypsinized, pelleted by centrifugation, and resuspended in fresh Promocell skeletal muscle cell growth medium. The cells were counted and seeded at a density of 50,000 cells / well onto Matrigel-coated 96-well plates. Cells were allowed to recover for 24 hours. Cells were induced to differentiate into myotubes by aspirating the growth medium and replacing it with serum-free differentiation medium. Cells were then treated with 10 μM ASO of DMD exon 51 skipping oligonucleotides (not covalently linked to an antibody—"naked") or 10 μM ASO equivalent of anti-TfR1 Fab (3M12 VH4 / Vκ3) covalently linked to DMD exon 51 skipping oligonucleotides. Cells were incubated with the test compounds for 10 days, and then total RNA was harvested from the 96-well plates. 75 ng of total RNA was used for cDNA synthesis, and mutation-specific PCR was performed to assess the extent of exon 51 skipping in the cells. Mutation-specific PCR products were run on a 4% agarose gel and visualized using SYBR gold. Densitometry was used to calculate the relative amounts of skipped and non-skipped amplicons, and exon skipping was determined as the ratio of amplicons in which exon 51 was skipped divided by the total amount of amplicons present.

number

[0373] These results demonstrate that the conjugate enhanced exon skipping compared to the naked DMD exon 51 skipping oligonucleotide in patient myotubes (Figure 1). This indicates that the anti-TfR1 Fab 3M12 VH4 / Vκ3 enabled cellular internalization of the conjugate into muscle cells, resulting in the activity of the exon 51 skipping oligonucleotide in muscle cells. Similarly, an anti-TfR1 antibody (e.g., anti-TfR1 Fab 3M12 VH4 / Vκ3) can enable muscle cell internalization of a conjugate containing an anti-TfR1 antibody covalently linked to another exon skipping oligonucleotide (e.g., an exon skipping oligonucleotide provided herein, e.g., an exon 51 skipping oligonucleotide), enhancing the activity of the exon skipping oligonucleotide in muscle cells.

[0374] Example 2. Exon skipping activity of anti-TfR1 Fab-ASO conjugates in vivo in cynomolgus monkeys The anti-TfR1 Fab 3M12 VH4 / Vκ3 was covalently linked to the DMD exon 51 skipping antisense oligonucleotide (ASO) used in Example 1. The exon skipping activity of the conjugate was tested in vivo in healthy non-human primates. Naive male cynomolgus monkeys (n = 4-5 per group) were administered two doses of vehicle, 30 mg / kg naked ASO (i.e., not covalently linked to an antibody), or 122 mg / kg anti-TfR1 Fab (3M12 VH4 / Vκ3) covalently linked to the DMD exon 51 skipping oligonucleotide (equivalent to 30 mg / kg ASO) by intravenous infusion on days 1 and 8. Animals were sacrificed 2 or 4 weeks after the first dose, and tissues were harvested. Total RNA was collected from tissue samples using a Promega Maxwell® RSC instrument, and cDNA synthesis was performed using qScript cDNA SuperMix. Assessment of exon 51 skipping was performed using end-point PCR.

[0375] Exon skipping was assessed using capillary electrophoresis of PCR products, and % exon 51 skipping was calculated using the following formula:

number

[0376] Tissue ASO accumulation was also quantified using a hybridization ELISA with a probe complementary to the ASO sequence. A standard curve was generated, and ASO levels (ng / g) were derived from linear regression of the standard curve. ASOs were distributed to all tissues evaluated at higher levels after administration of the anti-TfR1 Fab VH4 / Vκ3-ASO conjugate compared to administration of naked ASO. Intravenous administration of naked ASO resulted in ASO levels near background levels in all tissues evaluated 2 and 4 weeks after the first administration. Administration of the anti-TfR1 Fab VH4 / Vκ3-ASO conjugate resulted in ASO distribution throughout the tissues evaluated in the following order: heart > diaphragm > biceps > quadriceps > gastrocnemius > tibialis anterior 2 weeks after the first administration. The duration of tissue concentration was also assessed. The concentrations of ASO in the quadriceps, biceps, and diaphragm muscles decreased by less than 50% over the evaluated period (2-4 weeks), whereas the levels of ASO in the heart, tibialis anterior, and gastrocnemius muscles remained substantially unchanged (Table 11). This indicates that the anti-TfR1 Fab 3M12 VH4 / Vκ3 enables the cellular internalization of the conjugate into muscle cells in vivo, resulting in the activity of the exon-skipping oligonucleotide in muscle cells. Similarly, an anti-TfR1 antibody (e.g., anti-TfR1 Fab 3M12 VH4 / Vκ3) can enable the internalization of a conjugate containing an anti-TfR1 antibody covalently linked to another exon-skipping oligonucleotide (e.g., the exon-skipping oligonucleotide provided herein, e.g., the exon 51-skipping oligonucleotide) into muscle cells in vivo, promoting the activity of the exon-skipping oligonucleotide in muscle cells. [Table 11]

[0377] Equivalents and Terminology The disclosure illustratively described herein may suitably be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced by either of the other two terms. The terms and expressions employed are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the present disclosure. Thus, although the present disclosure has been specifically disclosed by preferred embodiments, it should be understood that any features, modifications, and variations of the concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present disclosure.

[0378] Additionally, where features or aspects of the disclosure are described as a Markush group or other grouping of alternatives, those skilled in the art will recognize that the disclosure is thereby also described as any individual member or subgroup of members of the Markush group or other group.

[0379] Of course, in some embodiments, when describing the structure of an oligonucleotide or other nucleic acid, reference may be made to the sequences listed in the sequence listing. In such embodiments, the actual oligonucleotide or other nucleic acid may have essentially the same or similar complementary properties as the designated sequence, but may have one or more alternative nucleotides or nucleosides (e.g., RNA counterparts of DNA nucleosides, or DNA counterparts of RNA nucleosides) and / or (for example, and) one or more modified nucleotides / nucleosides and / or (for example, and) one or more modified internucleoside linkages and / or (for example, and) one or more other modifications compared to the designated sequence.

[0380] The use of the terms "a," "an," and "the" and similar referents in the context of describing the present invention (particularly in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise noted. The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually set forth herein. Unless otherwise indicated herein or clearly contradicted by context, all methods described herein can be performed in any suitable order. Unless otherwise stated herein or clearly contradicted by context, the use of any and all examples or exemplary language provided herein (e.g., "such as") is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0381] Aspects of the invention have been described herein. Variations of those aspects may become apparent to those skilled in the art upon reading the foregoing description.

[0382] The inventors anticipate that those skilled in the art will employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

**Claim 1** A complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured to induce skipping of exon 51 in dystrophin pre-mRNA, wherein the oligonucleotide comprises a complementary region complementary to at least 8 consecutive nucleotides of any one of SEQ ID NOs: 173, 176, 206, 213, 222, 160-172, 174, 175, 177-205, 207-212, 214-221, and 223-383, said complex. **Claim 2** The complex according to claim 1, wherein the anti-TfR1 antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 33, a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 34, a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 35, a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 36, a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 37, and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO:

32. **Claim 3** The complex according to claim 1 or claim 2, wherein the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 76; and a light chain variable region (VL) comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:

75. **Claim 4** The complex according to claim 1 or claim 2, 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. **Claim 5** The complex according to claim 1 or claim 2, wherein the anti-TfR1 antibody is a Fab fragment, a Fab' fragment, an F(ab')2 fragment, a scFv, an Fv, or a full-length IgG. **Claim 6** The complex according to claim 5, wherein the anti-TfR1 antibody is a Fab fragment. **Claim 7** The complex according to claim 6, wherein the anti-TfR1 antibody comprises a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 101; and a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:

90. **Claim 8** The complex according to claim 6, 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. **Claim 9** The complex according to claim 1 or claim 2, wherein the anti-TfR1 antibody comprises a heavy chain comprising an N-terminal pyroglutamic acid. **Claim 10** The complex according to claim 1 or claim 2, wherein the oligonucleotide comprises a complementary region for at least 4 consecutive nucleotides of the splicing characteristics of dystrophin pre-mRNA.

11. The complex according to claim 10, wherein the splicing characteristic is an exon splicing enhancer (ESE) in exon 51 of dystrophin pre-mRNA, and optionally the ESE comprises any one of the sequences of SEQ ID NOs: 860 to 894.

12. The complex according to claim 10, wherein the splicing characteristic is a branch point, a splice donor site or a splice acceptor site, and optionally the splicing characteristic spans across the junction of exon 50 and intron 50 of dystrophin pre-mRNA, within intron 50, across the junction of intron 50 and exon 51, across the junction of exon 51 and intron 51, within intron 51, or across the junction of intron 51 and exon 52, and further optionally the splicing characteristic comprises any one of the sequences of SEQ ID NOs: 855 to 859 and 895 to 898.

13. The complex according to claim 1 or claim 2, wherein the oligonucleotide comprises a sequence complementary to any one of SEQ ID NOs: 173, 176, 206, 213, 222, 160 to 172, 174, 175, 177 to 205, 207 to 212, 214 to 221, and 223 to 383, or comprises any one of the sequences of SEQ ID NOs: 397, 400, 430, 437, 446, 384 to 396, 398, 399, 401 to 429, 437 to 436, 438 to 445, and 447 to 831, 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 T.

14. The complex according to claim 1 or claim 2, wherein the oligonucleotide comprises one or more phosphorodiamidate morpholinos, and optionally the oligonucleotide is a phosphorodiamidate morpholino oligomer (PMO).

15. The complex according to claim 1 or claim 2, wherein the anti-TfR1 antibody is covalently linked to the oligonucleotide via a cleavable linker, and optionally the cleavable linker comprises a valine-citrulline sequence.

16. The complex according to claim 1 or claim 2, wherein the anti-TfR1 antibody is covalently linked to the oligonucleotide via a non-cleavable linker, and optionally, the non-cleavable linker contains an alkyl group which may be optionally substituted.

17. The complex according to claim 1 or claim 2, wherein the anti-TfR1 antibody is covalently linked to the oligonucleotide via a lysine residue of the antibody.

18. The complex according to claim 1 or claim 2, wherein the anti-TfR1 antibody is covalently linked to the oligonucleotide via a cysteine residue of the antibody.

19. An oligonucleotide targeting dystrophin, comprising a complementary region to any one of SEQ ID NOs: 173, 176, 206, 213, 222, 160 - 172, 174, 175, 177 - 205, 207 - 212, 214 - 221, and 223 - 383, and optionally, the complementary region contains at least 15 consecutive nucleosides complementary to any one of SEQ ID NOs: 173, 176, 206, 213, 222, 160 - 172, 174, 175, 177 - 205, 207 - 212, 214 - 221, and 223 - 383.

20. The oligonucleotide according to claim 19, wherein the oligonucleotide contains at least 15 consecutive nucleosides of any one of SEQ ID NOs: 397, 400, 430, 437, 446, 384 - 396, 398, 399, 401 - 429, 437 - 436, 438 - 445, and 447 - 831, and optionally, the oligonucleotide contains the sequence of any one of SEQ ID NOs: 621, 624, 654, 661, 670, 384 - 620, 622, 623, 625 - 653, 655 - 660, 662 - 669, and 671 - 831, where each thymine base (T) may be independently and optionally substituted with a uracil base (U), and each U may be independently and optionally substituted with T.

21. The complex according to claim 1 or claim 2 or the oligonucleotide according to claim 19 or claim 20 for use in a method of delivering an oligonucleotide to a cell, wherein the method comprises contacting the cell with the complex or the oligonucleotide.

22. The complex according to claim 1 or claim 2 or the oligonucleotide according to claim 19 or claim 20 for use in a method of promoting the expression or activity of dystrophin protein in a cell, the method comprising contacting the cell with the complex or the oligonucleotide, optionally the oligonucleotide is internalized into the cell, further optionally the cell is a muscle cell, and further optionally the muscle cell is a human muscle cell, said complex or oligonucleotide.

23. The complex according to claim 1 or claim 2 or the oligonucleotide according to claim 19 or claim 20 for use in a method of treating a subject having Duchenne muscular dystrophy, the method comprising administering the complex or the oligonucleotide to the subject, optionally the subject is human, and further optionally the subject has a mutant dystrophin allele associated with dystrophinopathy, said complex or oligonucleotide.

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