Muscle-targeting complexes and uses thereof

Abstract

To provide a method for treating muscle diseases.SOLUTION: Provided is a method for treating a subject diagnosed with a muscle disease associated with a gain-of-function disease allele, the method including administering to the subject a conjugate comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit expression or activity of the disease allele, and the muscle targeting agent specifically binding to an internalized cell surface receptor on the subject's muscle cells.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] Related Applications This application is related to U.S. Provisional Application No. 62 / 714,010, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF," filed August 2, 2018; U.S. Provisional Application No. 62 / 779,173, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF," filed December 13, 2018; U.S. Provisional Application No. 62 / 855,781, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF," filed May 31, 2019; U.S. Provisional Application No. 62 / 858,925, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF," filed June 7, 2019; and U.S. Provisional Application No. 62 / 858,925, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF," filed June 10, 2019. This application claims the benefit of the filing date of U.S. Provisional Application No. 62 / 859,694, filed on May 1, 2006, entitled "THEREOF," the entire contents of each 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 for the treatment of disease.

[0003] Reference to sequence listing This application has been filed in electronic format with a Sequence Listing, which is provided as a file entitled D082470006WO00-SEQ.txt, created on July 31, 2019, and is 56 kilobytes in size. The information in the electronic Sequence Listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Background of the Invention Muscle diseases are often associated with muscle weakness and / or dysfunction, leading to life-threatening complications. Many examples of such diseases have been characterized, including muscular dystrophies (e.g., Duchenne, facioscapulohumeral, myotonic, and oculopharyngeal), Pompe disease, centronuclear myopathy, familial hypertrophic cardiomyopathy, Laing distal myopathy, fibrodysplasia ossificans progressiva, Friedreich's ataxia, myofibrillar myopathy, and various other forms. These diseases are generally inherited but can arise spontaneously. They are often congenital but can also arise later in life. Many rare muscle diseases are single-gene disorders associated with gain-of-function or loss-of-function mutations that may have dominant or recessive phenotypes. For example, activating mutations have been identified in genes encoding ion channels, structural proteins, metabolic proteins, and signaling proteins that contribute to muscle diseases. Despite advances in understanding the genetic etiology of muscle diseases, effective treatment options remain limited. Summary of the Invention

[0005] Summary of the Invention According to some aspects, the present disclosure provides a complex that targets muscle cells for the purpose of delivering a molecular payload to those cells. In some embodiments, the complexes of the present disclosure facilitate muscle-specific delivery of a molecular payload that targets a muscle disease allele. For example, in some embodiments, the complexes provided herein are particularly useful for delivering a molecular payload that modulates the expression or activity of a gene in a subject having or suspected of having a muscle disease associated with that gene (e.g., a gene / disease in Table 1). In some embodiments, the complexes provided herein include a muscle targeting agent (e.g., a muscle-targeting antibody) that specifically binds to a receptor on the surface of muscle cells for the purpose of delivering the molecular payload to muscle cells. In some embodiments, the complex is internalized into the cell via receptor-mediated internalization (e.g., a transferrin receptor), whereupon the molecular payload can be released inside the cell to perform its function. For example, a complex modified to deliver an oligonucleotide can release the oligonucleotide so that the oligonucleotide can modulate the expression or activity of a muscle disease allele. 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.

[0006] In some embodiments, methods are provided for treating a subject diagnosed with a muscle disease associated with a disease allele (e.g., a gain-of-function disease allele). In some embodiments, the methods involve administering to the subject a complex comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit expression or activity of the disease allele. In some embodiments, the muscle targeting agent specifically binds to an internalized cell surface receptor on the subject's muscle cells. In some embodiments, the muscle disease may be hereditary and exhibit increasing severity in successive family generations of the subject. In some embodiments, the subject has been diagnosed with the muscle disease based on genetic analysis of the disease allele. In some embodiments, the subject exhibits progressive muscle weakness and / or sarcopenia prior to administration. In some embodiments, the subject exhibits myotonia prior to administration.

[0007] According to some aspects, methods are provided for treating a subject diagnosed with a muscle disease (e.g., associated with a gain-of-function disease allele). In some embodiments, the method comprises administering to the subject a conjugate comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit expression or activity of the disease allele. In some embodiments, the muscle targeting agent specifically binds to an internalized cell surface receptor on the subject's muscle cells.

[0008] In some embodiments, the muscle disease is hereditary. In some embodiments, the muscle disease exhibits increasing severity in successive family generations of the subject. In some embodiments, the subject has been diagnosed with the muscle disease based on genetic analysis of disease alleles. In some embodiments, the subject exhibits progressive muscle weakness and / or sarcopenia prior to administration. In some embodiments, the subject exhibits myotonia, e.g., measurable by electromyography, prior to administration.

[0009] In some embodiments, the muscle-targeting agent is a muscle-targeting antibody. In some embodiments, the muscle-targeting antibody specifically binds to an extracellular epitope of the transferrin receptor. In some embodiments, the extracellular epitope of the transferrin receptor comprises an epitope of the apical domain of the transferrin receptor. In some embodiments, the muscle-targeting antibody specifically binds to an epitope of a sequence ranging from C89 to F760 of SEQ ID NOs: 1-3. In some embodiments, the equilibrium dissociation constant (Kd) for binding of the muscle-targeting antibody to the transferrin receptor is 10 -11 M to 10 -6 In some embodiments, the muscle-targeting antibody competes with an antibody listed in Table 2 for specific binding to an epitope of the transferrin receptor.

[0010] In some embodiments, the muscle-targeting antibody has a 10 -6 In some embodiments, the Kd is less than or equal to 10 -11 M~10 -6 It is in the range of M.

[0011] In some embodiments, the muscle-targeting antibody does not specifically bind to the transferrin-binding site of the transferrin receptor and / or the muscle-targeting antibody does not inhibit the binding of transferrin to the transferrin receptor. In some embodiments, the muscle-targeting antibody cross-reacts with two or more extracellular epitopes of human, non-human primate, and rodent transferrin receptors. In some embodiments, the method is configured to promote transferrin receptor-mediated internalization of a molecular payload into muscle cells.

[0012] In some embodiments, the muscle-targeting antibody is a chimeric antibody, and optionally the chimeric antibody is a humanized monoclonal antibody. In some embodiments, the muscle-targeting antibody is in the form of an ScFv, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an Fv fragment.

[0013] In some embodiments, the molecular payload is an oligonucleotide. In some embodiments, the oligonucleotide comprises a region complementary to a gene listed in Table 1 or an mRNA encoded therefrom. In some embodiments, the oligonucleotide is a gapmer oligonucleotide, a mixmer oligonucleotide, an antisense oligonucleotide, an RNAi oligonucleotide, a messenger RNA (mRNA), or a guide sequence.

[0014] In some embodiments, the conjugate is administered to the subject by intramuscular parenteral administration. In some embodiments, the conjugate is administered to the subject by intravenous administration. In some embodiments, the conjugate is administered to the subject by subcutaneous administration of the conjugate.

[0015] In some aspects, a conjugate is provided that includes a muscle targeting agent linked to a single-stranded oligonucleotide, in some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on a muscle cell, and wherein the oligonucleotide includes a region complementary to a muscle disease gene.

[0016] In some embodiments, a composition is provided that includes a plurality of conjugates, each conjugate including a muscle targeting agent covalently linked to two, at least three, or more (e.g., two to six) oligonucleotides. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on a subject's muscle cells, and each oligonucleotide includes a region complementary to a muscle disease gene.

[0017] In some aspects, conjugates are provided that include a muscle targeting agent covalently linked to a molecular payload configured to modulate the expression or activity of a muscle disease gene encoding a non-secreted compound that functions in muscle cells. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle cells.

[0018] In some embodiments, the muscle-targeting agent is a muscle-targeting antibody. In some embodiments, the muscle-targeting antibody specifically binds to an extracellular epitope of the transferrin receptor. In some embodiments, the extracellular epitope of the transferrin receptor comprises an epitope of the apical domain of the transferrin receptor. In some embodiments, the muscle-targeting antibody specifically binds to an epitope of a sequence within amino acids C89 to F760 of SEQ ID NOs: 1-3. In some embodiments, the equilibrium dissociation constant (Kd) for binding of the muscle-targeting antibody to the transferrin receptor is 10 -11 M to 10 -6 In some embodiments, the muscle-targeting antibody competes with an antibody listed in Table 2 for specific binding to an epitope of the transferrin receptor. In some embodiments, the muscle-targeting antibody competes with an antibody listed in Table 2 for specific binding to an epitope of the transferrin receptor. -6 In some embodiments, the Kd is less than or equal to 10 -11 M~10 -6 It is in the range of M.

[0019] In some embodiments, the muscle-targeting antibody does not specifically bind to the transferrin-binding site of the transferrin receptor and / or wherein the muscle-targeting antibody does not inhibit the binding of transferrin to the transferrin receptor. In some embodiments, the muscle-targeting antibody cross-reacts with two or more extracellular epitopes of human, non-human primate, and rodent transferrin receptors.

[0020] In some embodiments, the conjugate is configured to promote transferrin receptor-mediated internalization of the molecular payload into muscle cells. In some embodiments, the muscle-targeting antibody is a chimeric antibody. In some embodiments, the chimeric antibody is a humanized monoclonal antibody.

[0021] In some embodiments, the muscle-targeting antibody is in the form of an ScFv, a Fab fragment, a Fab' fragment, a F(ab')2 fragment, or an Fv fragment.

[0022] In some embodiments, the molecular payload is an oligonucleotide, hi some embodiments, the oligonucleotide comprises a region of complementarity to a muscle disease gene having a gain-of-function disease allele.

[0023] In some embodiments, the molecular payload is a polypeptide. In some embodiments, the polypeptide is an E3 ubiquitin ligase inhibitor peptide.

[0024] In some embodiments, the oligonucleotide comprises at least one modified internucleotide linkage. In some embodiments, at least one modified internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises phosphorothioate linkages in the Rp stereochemical configuration and / or in the Sp stereochemical configuration. In some embodiments, the oligonucleotide comprises phosphorothioate linkages that are all in the Rp stereochemical configuration or all in the Sp stereochemical configuration.

[0025] In some embodiments, the oligonucleotide comprises one or more modified nucleotides. In some embodiments, the one or more modified nucleotides are 2'-modified nucleotides.

[0026] In some embodiments, the oligonucleotide is a gapmer oligonucleotide that directs RNAse H-mediated cleavage of an mRNA transcript encoded by a cellular myopathy gene. In some embodiments, the gapmer oligonucleotide comprises a core of 5-15 deoxyribonucleotides flanked on either side by 2-8 modified nucleotides.

[0027] In some embodiments, the flanking modified nucleotides are 2'-modified nucleotides. In some embodiments, the oligonucleotide is a mixmer oligonucleotide.

[0028] In some embodiments, the mixmer oligonucleotide comprises two or more different 2'-modified nucleotides. In some embodiments, the oligonucleotide is an RNAi oligonucleotide that promotes RNAi-mediated cleavage of an mRNA transcript encoded by a muscle disease gene.

[0029] In some embodiments, the oligonucleotide is a double-stranded oligonucleotide of 19 to 25 nucleotides in length. In some embodiments, the RNAi oligonucleotide comprises at least one 2'-modified nucleotide. In some embodiments, each 2'-modified nucleotide is selected from the group consisting of 2'-O-methyl, 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-MOE), and 2',4'-bridged nucleotides.

[0030] In some embodiments, one or more modified nucleotides are bridged nucleotides. In some embodiments, at least one 2'-modified nucleotide is a 2',4'-bridged nucleotide selected from 2',4'-constrained 2'-O-ethyl (cEt) and locked nucleic acid (LNA) nucleotides.

[0031] In some embodiments, the oligonucleotide comprises a guide sequence for a genome-editing nuclease.

[0032] In some embodiments, the oligonucleotide is a phosphorodiamidite morpholino oligomer. In some embodiments, the muscle-targeting agent is covalently linked to the molecular payload via a cleavable linker.

[0033] In some embodiments, the cleavable linker is selected from a protease-sensitive linker, a pH-sensitive linker, and a glutathione-sensitive linker. In some embodiments, the cleavable linker is a protease-sensitive linker. In some embodiments, the protease-sensitive linker comprises a sequence cleavable by a lysosomal protease and / or an endosomal protease. In some embodiments, the protease-sensitive linker comprises a valine-citrulline dipeptide sequence. In some embodiments, the linker is a pH-sensitive linker that is cleaved at a pH in the range of 4 to 6.

[0034] In some embodiments, the muscle-targeting agent is covalently linked to the molecular payload via a non-cleavable linker. In some embodiments, the non-cleavable linker is an alkane linker.

[0035] In some embodiments, the muscle-targeting antibody comprises an unnatural amino acid to which an oligonucleotide is covalently linked. In some embodiments, the muscle-targeting antibody is covalently linked to the oligonucleotide via conjugation to a lysine or cysteine ​​residue of the antibody. In some embodiments, the muscle-targeting antibody is conjugated to a cysteine ​​via a maleimide-containing linker, optionally wherein the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group.

[0036] In some embodiments, the muscle-targeting antibody is a glycosylated antibody comprising at least one sugar moiety to which an oligonucleotide is covalently linked. In some embodiments, the sugar moiety is a branched mannose. In some embodiments, the muscle-targeting antibody is a glycosylated antibody comprising 1 to 4 sugar moieties, each of which is covalently linked to a separate oligonucleotide.

[0037] In some embodiments, the muscle-targeting antibody is a fully glycosylated antibody. In some embodiments, the muscle-targeting antibody is a partially glycosylated antibody. In some embodiments, the partially glycosylated antibody is produced via chemical or enzymatic means. In some embodiments, the partially glycosylated antibody is produced in cells that are deficient in an enzyme in the N- or O-glycosylation pathway.

[0038] According to some aspects, methods for delivering a molecular payload to a transferrin receptor-expressing cell are provided. In some embodiments, the method comprises contacting the cell with a complex provided herein.

[0039] According to some aspects, methods for inhibiting the expression or activity of a muscle disease gene in a cell are provided. In some embodiments, the method comprises contacting the cell with a conjugate provided herein in an amount effective to promote internalization of the molecular payload into the cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in a subject. In some embodiments, the subject is human.

[0040] According to some aspects, methods of treating a subject with a muscle disease are provided. In some embodiments, the method comprises administering to the subject an effective amount of a conjugate provided herein. In some embodiments, the muscle disease is a disease listed in Table 1. In some embodiments, the muscle disease is a disease selected from adult Pompe disease, centronuclear myopathy (CNM), Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), inclusion body myopathy 2, Laing distal myopathy, myofibrillar myopathy, myotonia congenita (autosomal dominant, Thomsen's disease), myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and congenital myotonia. [Brief explanation of the drawings]

[0041] Brief description of the drawings [Figure 1] Figure 1 depicts a non-limiting schematic diagram showing the effect of transfecting Hepa1-6 cells with antisense oligonucleotides targeting DMPK (DTX-P-060) on the expression levels of DMPK compared to vehicle transfection;

[0042] [Figure 2A-B] FIG. 2A depicts a non-limiting schematic diagram showing an HIL-HPLC trace obtained during purification of a muscle-targeting complex comprising an anti-transferrin receptor antibody covalently linked to a DMPK antisense oligonucleotide.

[0043] FIG. 2B depicts a non-limiting image of an SDS-PAGE analysis of the muscle-targeting complex.

[0044] [Figure 3] FIG. 3 depicts a non-limiting schematic diagram showing the ability of a muscle-targeting conjugate (DTX-C-008) containing DTX-P-060 to reduce the expression level of DMPK.

[0045] [Figure 4A] 4A-4E depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate (DTX-C-008) containing DTX-P-060 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle experiments (N=3 C57B1 / 6 WT mice). [Figure 4B-C] Figures 4B-4C depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate (DTX-C-008) containing DTX-P-060 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle experiments (N = 3 C57B1 / 6 WT mice). [Figure 4D-E]Figures 4D-4E depict non-limiting schematic diagrams showing the ability of a muscle-targeting complex (DTX-C-008) containing DTX-P-060 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle experiments (N = 3 C57B1 / 6 WT mice).

[0046] [Figure 5A-B] Figures 5A-5B depict non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing DTX-P-060 (DTX-C-008). Muscle-targeting complexes containing DTX-P-060 (DTX-C-008) do not reduce DMPK expression levels in mouse brain or spleen tissues in vivo compared to vehicle experiments. (N = 3 C57B1 / 6 WT mice)

[0047] [Figure 6A-B] 6A-6F depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate (DTX-C-008) containing DTX-P-060 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle experiments (N=5 C57B1 / 6 WT mice). [Figure 6C-D] Figures 6C-6D depict non-limiting schematic diagrams showing the ability of a muscle-targeting complex (DTX-C-008) containing DTX-P-060 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle experiments (N = 5 C57B1 / 6 WT mice). [Figure 6E-F] Figures 6E-6F depict non-limiting schematic diagrams showing the ability of a muscle-targeting complex (DTX-C-008) containing DTX-P-060 to reduce DMPK expression levels in mouse muscle tissue in vivo compared to vehicle experiments (N = 5 C57B1 / 6 WT mice).

[0048] [Figure 7A-B]7A-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate containing DTX-P-060 (DTX-C-012) to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle experiments and compared to naked DMPK ASO (DTX-P-060). (N=3 male cynomolgus monkeys) [Figure 7C-D] 7C-7D depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate containing DTX-P-060 (DTX-C-012) to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle experiments and compared to naked DMPK ASO (DTX-P-060). (N=3 male cynomolgus monkeys) [Figure 7E-F] 7E-7F depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate containing DTX-P-060 (DTX-C-012) to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle experiments and compared to naked DMPK ASO (DTX-P-060). (N=3 male cynomolgus monkeys) [Figure 7G-H] 7G-7H depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate containing DTX-P-060 (DTX-C-012) to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle experiments and compared to naked DMPK ASO (DTX-P-060). (N=3 male cynomolgus monkeys) [Figure 7I-J] 7I-7J depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate containing DTX-P-060 (DTX-C-012) to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle experiments and compared to naked DMPK ASO (DTX-P-060). (N=3 male cynomolgus monkeys) [Figure 7K-L]Figures 7K-7L depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate containing DTX-P-060 (DTX-C-012) to reduce DMPK expression levels in cynomolgus monkey muscle tissue in vivo compared to vehicle experiments and compared to naked DMPK ASO (DTX-P-060). (N=3 male cynomolgus monkeys)

[0049] [Figure 8A-B] 8A-8B depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate containing DTX-P-060 (DTX-C-012) to reduce DMPK expression levels in cynomolgus monkey smooth muscle tissue in vivo compared to vehicle experiments and compared to naked DMPK ASO (DTX-P-060). (N=3 male cynomolgus monkeys)

[0050] [Figure 9A-B] Figures 9A-9D depict non-limiting schematic diagrams showing the tissue selectivity of a muscle-targeting complex (DTX-C-012) containing DTX-P-060. A muscle-targeting complex containing DMPK-ASO does not reduce DMPK expression levels in liver, kidney, brain, or spleen tissues of cynomolgus monkeys in vivo compared to vehicle experiments. (N = 3 male cynomolgus monkeys) [Figure 9C-D] Figures 9C-9D depict non-limiting schematic diagrams showing the tissue selectivity of a muscle-targeting complex (DTX-C-012) containing DTX-P-060. A muscle-targeting complex containing DMPK-ASO does not reduce DMPK expression levels in liver, kidney, brain, or spleen tissues of cynomolgus monkeys in vivo compared to vehicle experiments. (N = 3 male cynomolgus monkeys)

[0051] [Figure 10] Figure 10 shows normalized DMPK mRNA tissue expression levels across several tissue types in cynomolgus monkeys. (N=3 male cynomolgus monkeys)

[0052] [Figure 11A-B]11A-11B depict non-limiting schematic diagrams showing the ability of a muscle-targeting conjugate containing DTX-P-060 (DTX-C-008) to reduce expression levels of DMPK in mouse muscle tissue in vivo for up to 28 days after dosing with DTX-C-008, compared to vehicle experiments and compared to naked DMPK ASO (DTX-P-060).

[0053] [Figure 12] Figure 12 shows that a single dose of a muscle-targeting conjugate (DTX-C-012) containing DTX-P-060 is safe and tolerated in cynomolgus monkeys. (N=3 male cynomolgus monkeys) DETAILED DESCRIPTION OF THE INVENTION

[0054] Detailed Description of the Invention Aspects of the present disclosure relate to the recognition that while certain molecular payloads (e.g., oligonucleotides, peptides, small molecules) can have beneficial effects on muscle cells, effectively targeting such cells can be challenging. As described herein, the present disclosure provides conjugates comprising a muscle targeting agent covalently linked to a molecular payload to overcome such challenges. In some embodiments, the conjugates are particularly useful for delivering molecular payloads that modulate target gene expression or activity in muscle cells, e.g., in subjects with or suspected of having a muscle disease. For example, in some embodiments, the conjugates are useful for treating subjects with rare muscle diseases, including Pompe disease, centronuclear myopathy, fibrodysplasia ossificans progressiva, Friedreich's ataxia, or Duchenne muscular dystrophy. In some embodiments, different molecular payloads may be used in such conjugates depending on the disease being treated. For example, if the underlying mutation confers a splicing defect, an oligonucleotide or other payload may be used to correct the splicing defect (e.g., an oligonucleotide that inhibits exon skipping or promotes alternative splicing). If the underlying mutation results in a gain-of-function allele, oligonucleotides (e.g., RNAi, PMO, ASO-gapmer) may be used to inhibit the expression or activity of the allele. In some embodiments, for example, if the mutation results in a loss-of-function allele, the payload may include, for example, an expression construct to express a wild-type version of the allele. In some embodiments, the payload may include machinery (e.g., a guide nucleic acid, an expression construct encoding a gene editing enzyme) to correct the underlying defect, for example, by gene editing.

[0055] Further aspects of the disclosure, including a description of defined terms, are provided below.

[0056] I. Definition Administering: As used herein, the terms "administering" or "administration" mean providing a conjugate to a subject in a physiologically and / or pharmacologically useful manner (e.g., treating a disease in a subject).

[0057] about: As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In some embodiments, the term "approximately" or "about" refers to a broad range of values ​​that fall within plus or minus (greater or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value, unless otherwise stated or clear from the context (except when such number exceeds 100% of a feasible value).

[0058] antibody: As used herein, the term "antibody" refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope, that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a 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 a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises a constant region, e.g., 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, CH2, and / or 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, for example, via glycosylation, phosphorylation, sumoylation, and / or 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 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 and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0059] CDR: As used herein, the term "CDR" refers to a complementarity-determining region within an antibody variable sequence. 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 particular residues or groups of residues, or even entire CDRs, do not significantly affect antigen binding. While the methods used herein may utilize CDRs defined according to any of these systems, preferred embodiments use CDRs defined by Kabat or Chothia.

[0060] CDR-grafted antibodies: The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species but in which the sequences of one or more of the CDR regions of its VH and / or VL have been replaced with CDR sequences from another species, such as an antibody having murine heavy and light chain variable regions but in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0061] Chimeric antibodies: The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0062] Complementary: As used herein, the term "complementary" refers to the ability for precise pairing between two nucleotides or two pairs of nucleotides.In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in binding between two nucleotides or two pairs of nucleotides.For example, if the base of an oligonucleotide at a certain position can hydrogen bond with the base of 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 and be considered complementary to 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.

[0063] Conservative amino acid substitutions: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, 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 within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0064] 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.

[0065] Cross-reacting: As used herein, and in the context of targeting agents (e.g., antibodies), the term "cross-reacting" refers to the property of an agent that can specifically bind to more than one antigen of the same type or class (e.g., multiple 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.

[0066] Disease allele: As used herein, the term "disease allele" refers to any one of alternative forms (e.g., mutant forms) of a gene in which the allele correlates with and / or directly or indirectly contributes to or causes a disease. A disease allele may contain genetic alterations compared to a wild-type (non-disease) allele, including, but not limited to, insertions (e.g., disease-associated repeats described below), deletions, missense mutations, nonsense mutations, and splice site mutations. In some embodiments, a disease allele has a loss-of-function mutation. In some embodiments, a disease allele has a gain-of-function mutation. In some embodiments, a disease allele encodes an activating mutation (e.g., encodes a constitutively active protein). In some embodiments, a disease allele is a recessive allele with a recessive phenotype. In some embodiments, a disease allele is a dominant allele with a dominant phenotype.

[0067] Disease-associated repeats: As used herein, the term "disease-associated repeat" refers to a repetitive nucleotide sequence at a genomic location where several units of the repetitive nucleotide sequence correlate with and / or directly or indirectly contribute to or cause a genetic disease. Each repeat unit of the disease-associated repeat may be 2, 3, 4, 5, or more nucleotides in length. For example, in some embodiments, the disease-associated repeat is a dinucleotide repeat. In some embodiments, the disease-associated repeat is a trinucleotide repeat. In some embodiments, the disease-associated repeat is a tetranucleotide repeat. In some embodiments, the disease-associated repeat is a pentanucleotide repeat. In some embodiments, the disease-associated repeat comprises a CAG repeat, a CTG repeat, a CUG repeat, a CGG repeat, a CCTG repeat, or the nucleotide complement of any thereof. In some embodiments, the disease-associated repeat is in a non-coding portion of a gene. However, in some embodiments, the disease-associated repeat is in a coding region of a gene. In some embodiments, the disease-associated repeat is expanded from a normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, the disease-associated repeat is in RNA (e.g., an RNA transcript). In some embodiments, the disease-associated repeats are in DNA (e.g., chromosomes, plasmids). In some embodiments, the disease-associated repeats are expanded in chromosomes of germline cells. In some embodiments, the disease-associated repeats are expanded in chromosomes of somatic cells. In some embodiments, the disease-associated repeats are expanded into multiple repeat units associated with congenital onset. In some embodiments, the disease-associated repeats are expanded into multiple repeat units associated with childhood onset of the disease. In some embodiments, the disease-associated repeats are expanded into multiple repeat units associated with adult onset of the disease.

[0068] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Because the precise definition of a CDR sequence can be determined by various systems, the meaning of a 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 framework regions that do not specify a specific subregion as FR1, FR2, FR3, or FR4 are referred to by others, they represent the combined FRs 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.

[0069] Human antibodies: The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include, for example, in the CDRs, particularly CDR3, amino acid residues 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.

[0070] Humanized antibodies: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., a mouse), but in which at least a portion of the VH and / or VL sequences have been altered to be more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced onto non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen-binding portions are provided. Such antibodies may be produced by obtaining a murine anti-transferrin receptor monoclonal antibody using conventional hybridoma technology, followed by humanization using in vitro genetic engineering (such as that disclosed in PCT Publication No. WO 2005 / 123126 A2 to Kasaian et al.).

[0071] Internalizing cell surface receptors: As used herein, the term "internalizing cell surface receptor" refers to a cell surface receptor that is internalized by a cell upon an external stimulus (e.g., a ligand binding to the receptor). In some embodiments, the internalizing cell surface receptor is internalized by endocytosis. In some embodiments, the internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalizing cell surface receptor is internalized by a clathrin-independent pathway, such as phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or 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.

[0072] Isolated antibodies: An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (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 chemicals.

[0073] 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., more 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 region spans 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.

[0074] 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 to 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 complementary to a target gene.

[0075] Muscle disease genes: As used herein, the term "muscle disease gene" refers to a gene having at least one disease allele that correlates with and / or directly or indirectly contributes to or causes a muscle disease. In some embodiments, the muscle disease is a rare disease, e.g., as defined by the Genetic and Rare Diseases Information Center (GARD), a program of the National Center for Advancing Translational Sciences (NCATS). In some embodiments, the muscle disease is a rare disease characterized as affecting fewer than 200,000 people. In some embodiments, the muscle disease is a monogenic disease. In some embodiments, the muscle disease gene is a gene listed in Table 1.

[0076] Muscle-targeting agents: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on a muscle cell may be a membrane protein, 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, facilitating internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle and is capable of being internalized into the muscle cell through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, protein, peptide, nucleic acid (e.g., an aptamer), or antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0077] Muscle targeting antibodies: As used herein, the term "muscle-targeting antibody" refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, 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.

[0078] Oligonucleotides: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, gapmers, mixmers, phosphorodiamidite 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 nucleotides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, oligonucleotides may contain one or more modified internucleotide linkages. In some embodiments, oligonucleotides may contain one or more phosphorothioate linkages, which may be in an Rp or Sp stereochemical configuration.

[0079] Recombinant antibodies: The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, created, or isolated by recombinant means, e.g., 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 may not naturally occur within the germline repertoire of human antibodies in vivo.One aspect of the present disclosure provides fully human antibodies capable of binding to the human transferrin receptor that can be produced using techniques well known in the art, including, but not limited to, techniques using human Ig phage libraries (e.g., those disclosed in PCT Publication No. WO 2005 / 007699 A2 to Jermutus et al.).

[0080] Regions of complementarity: As used herein, the term "region of complementarity" refers to a nucleotide sequence (e.g., a nucleotide sequence of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., a nucleotide sequence of a target nucleic acid) such that the two nucleotide sequences are capable of annealing to each other under physiological conditions (e.g., in a cell). In some embodiments, the region of complementarity is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the region of complementarity is 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.

[0081] Specific binding to: As used herein, the term "specifically binds" 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 "specifically binds" refers to the ability of an antibody to bind to a specific antigen, relative 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.

[0082] 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 a disease or suspected of having a disease. In some embodiments, the subject is a human patient having a muscle disease (e.g., any of the diseases provided in Table 1) or suspected of having FOP.

[0083] 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).

[0084] II. Complex Provided herein are conjugates comprising a targeting agent, e.g., 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 antigenic site, or an antibody that binds to at least two antigenic sites, which may be on the same antigen or different antigens. The conjugate may be used to modulate the activity or function of at least one gene, protein, and / or nucleic acid. In some embodiments, the molecular payload present with the conjugate is responsible for modulating the gene, protein, and / or 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 nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets a muscle disease allele in muscle cells.

[0085] In some embodiments, the conjugate comprises a muscle targeting agent (e.g., an anti-transferrin receptor antibody) covalently linked to a molecular payload (e.g., an antisense oligonucleotide that targets a muscle disease allele).

[0086] In some embodiments, the conjugates are useful for treating muscle diseases in which the molecular payload affects the activity of a corresponding gene provided in Table 1. For example, depending on the disease, the molecular payload may modulate (e.g., decrease, increase) the transcription or expression of the gene, modulate the expression of a protein encoded by the gene, or modulate the activity of the encoded protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region of complementarity to a target gene provided in Table 1.

[0087] Table 1 - List of muscle diseases and corresponding genes [Table 1-1] [Table 1-2] [Table 1-3]

[0088] 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 cells. In some embodiments, the molecular payload is attached (e.g., covalently attached) to the muscle-targeting agent, and is internalized into the muscle cells upon binding to the antigen on the muscle cells, e.g., via endocytosis. It should be understood that various types of muscle-targeting agents may be used in accordance with the present disclosure. 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). 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.

[0089] 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 cardiac muscle cells.

[0090] 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 enable even macromolecules, such as antibodies, to enter muscle cells. As another example, molecular payloads conjugated to transferrin or anti-transferrin receptor antibodies can be taken up by muscle cells via binding to the transferrin receptor and then endocytosed, e.g., via clathrin-mediated endocytosis.

[0091] 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) 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.

[0092] In some embodiments, a muscle recognition element (e.g., a muscle cell antigen) may be required to achieve muscle selectivity. As one example, the muscle targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, the muscle targeting agent may be an antibody that enters muscle cells via transporter-mediated endocytosis. As another example, the muscle targeting agent may be a ligand that binds to a cell surface receptor on muscle cells. It should be understood that transporter-based approaches provide a direct pathway for cell entry, whereas receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0093] Muscle cells encompassed by this disclosure include, but are not limited to, skeletal muscle cells, smooth muscle cells, cardiac muscle cells, myoblasts, and myocytes.

[0094] i. Muscle targeting antibody In some embodiments, the muscle targeting agent is an antibody. Generally, the high specificity of antibodies for their target antigens offers the potential to selectively target muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and / or 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.

[0095] anti-transferrin receptor 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. Consequently, 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 also be referred to as anti-transferrin receptor 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.

[0096] It should be understood that anti-transferrin receptor antibodies may be produced, synthesized, and / or derivatized using several known methodologies, e.g., library design using phage display. Exemplary methodologies have been characterized in the art and are incorporated by reference (Diez, P. et al. "High-throughput phage-display screening in array format," Enzyme and microbial technology, 2015, 79, 34-41; Christoph MH and Stanley, JR "Antibody Phage Display: Technique and Applications," J Invest Dermatol. 2014, 134:2; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies," 1985, Springer). In other embodiments, the anti-transferrin 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, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing the same"; U.S. Pat. No. 8,409,573, filed June 14, 2006, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Pat. No. 9,708,406, filed May 20, 2014, "Anti-transferrin receptor antibodies and methods of use"; U.S. Pat. No. 9,611,323, filed December 19, 2014, "Low affinity blood-brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed December 24, 2014, "Novel anti-Transferrin receptor antibody that passes through the blood-brain barrier"; Schneider C. et al., "Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9.”J Biol Chem.1982,257:14,8516-8522.;Lee et al.“Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse”2000,J Pharmacol. Exp. Ther., 292:1048-1052).

[0097] Any suitable anti-transferrin receptor antibody may be used in the conjugates disclosed herein. Examples of anti-transferrin receptor antibodies, including associated reference and binding epitopes, are listed in Table 2. In some embodiments, the anti-transferrin receptor antibody comprises any of the complementarity determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of the anti-transferrin receptor antibodies provided herein, e.g., the anti-transferrin receptor antibodies listed in Table 2.

[0098] Table 2 - List of anti-transferrin receptor antibody clones, including associated reference epitope and binding epitope information [Table 2-1] [Table 2-2] [Table 2-3]

[0099] In some embodiments, the muscle-targeting agent is an anti-transferrin receptor antibody. In some embodiments, the anti-transferrin receptor antibody specifically binds to a transferrin protein having an amino acid sequence as disclosed herein. In some embodiments, the anti-transferrin receptor antibody may specifically bind to any extracellular epitope of the transferrin receptor or epitope that becomes exposed to the antibody, including the apical domain, the transferrin-binding domain, and the protease-like domain. In some embodiments, the anti-transferrin receptor antibody binds to an amino acid segment of the human or non-human primate transferrin receptor as provided in SEQ ID NOS: 1-3 ranging from amino acids C89 to F760. In some embodiments, the anti-transferrin receptor antibody binds to 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 As used herein, an anti-transferrin receptor antibody specifically binds to the transferrin receptor with a binding affinity of 10 M or less. -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 It may be possible to compete with other anti-transferrin receptor antibodies (e.g., OKT9, 8D3) that bind at or below M.

[0100] 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: 1).

[0101] 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: (SEQ ID NO: 2).

[0102] 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: 3).

[0103] 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: (SEQ ID NO: 4). In some embodiments, the anti-transferrin receptor antibody is an amino acid segment of the receptor as follows: FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 5) and does not inhibit the binding interaction between transferrin receptor and transferrin and / or human hemochromatosis protein (also known as HFE).

[0104] Suitable methodologies may be used to obtain and / or 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, "Directed evolution of novel binding proteins"; WO 1992 / 18619, filed April 10, 1992, "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, filed May 1, 1991, "Recombinant library screening methods"; WO 1992 / 20791, filed May 15, 1992, "Methods for producing members of specific binding pairs"; and WO 1992 / 15679, filed February 28, 1992, "Improved epitope displaying phage"). In some embodiments, the antigen of interest may be used to immunize a non-human animal, e.g., a rodent or goat.In some embodiments, once the 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).

[0105] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or 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 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 International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0106] Some aspects of the present disclosure provide proteins that bind to transferrin receptors (e.g., the extracellular portion of the transferrin receptor). In some embodiments, the anti-transferrin receptor antibodies provided herein specifically bind to transferrin receptors (e.g., the human transferrin receptor). 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. In some embodiments, the anti-transferrin receptor antibodies provided herein specifically bind to transferrin receptors from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-transferrin receptor antibodies provided herein bind to the human transferrin receptor. In some embodiments, the anti-transferrin receptor antibodies provided herein specifically bind to the apical domain of the human transferrin receptor. In some embodiments, the anti-transferrin receptor antibodies provided herein specifically bind to the apical domain of the human transferrin receptor.

[0107] In some embodiments, an anti-transferrin receptor antibody of the present disclosure includes one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, an anti-transferrin receptor antibody includes CDR-H1, CDR-H2, and CDR-H3 as provided in any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, an anti-transferrin receptor antibody includes CDR-L1, CDR-L2, and CDR-L3 as provided in any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, an anti-transferrin antibody includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided in any one of the anti-transferrin receptor antibodies selected from Table 2. The present disclosure also encompasses any nucleic acid sequence encoding a molecule comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, antibody heavy and light chain CDR3 domains may play a particularly important role in the binding specificity / affinity of an antibody to an antigen. Consequently, an anti-transferrin receptor antibody of the present disclosure may comprise at least the heavy and / or light chain CDR3 of any one of the anti-transferrin receptor antibodies selected from Table 2.

[0108] In some examples, any of the anti-transferrin receptor antibodies of the present disclosure have one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 sequences from one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the position of one or more CDRs along the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and / or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) regions of an antibody described herein may vary by one, two, three, four, five, or six amino acid positions, so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived). For example, in some embodiments, the positions defining the CDRs of any of the antibodies described herein may vary by one, two, three, four, five, or six amino acid positions compared to the CDR positions of any one of the antibodies described herein by shifting the N-terminal and / or C-terminal boundaries of the CDRs, so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).In another embodiment, the length of one or more CDRs along the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) and / or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) regions of an antibody described herein can vary by 1, 2, 3, 4, 5, or more amino acids (e.g., shorter or longer), so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).

[0109] Consequently, in some embodiments, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3 described herein may be shorter by 1, 2, 3, 4, 5, or more amino acids than one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 2), so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived). In some embodiments, CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3 described herein may be shorter by 1, 2, 3, 4, 5, or more amino acids than one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 2), so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3 described herein can be extended by a difference of 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 2), so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).The carboxy portion of CDR-H2 and / or CDR-H3 can be extended by a difference of 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 2), so long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived). In some embodiments, the amino portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 2), as long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived). In some embodiments, the carboxy portion of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3 described herein can be shortened by 1, 2, 3, 4, 5, or more amino acids compared to one or more of the CDRs described herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 2), as long as immunospecific binding to a transferrin receptor (e.g., a human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding of the original antibody from which it is derived).Either method can be used to determine whether immunospecific binding to the transferrin receptor (e.g., the human transferrin receptor) is maintained, using, for example, binding assays and conditions described in the art.

[0110] In some examples, any of the anti-transferrin receptor antibodies of the present disclosure have one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any one of the anti-transferrin receptor antibodies selected from Table 2. For example, an antibody may include one or more CDR sequence(s) from any of the anti-transferrin receptor antibodies selected from Table 2 containing up to 5, 4, 3, 2, or 1 amino acid residue variations compared to the corresponding CDR region in any one of the CDRs provided herein (e.g., a CDR from any of the anti-transferrin receptor antibodies selected from Table 2), so long as immunospecific binding to transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% maintained relative to the binding of the original antibody from which it was derived). In some embodiments, any amino acid variations in any of the CDRs provided herein may be conservative variations. Conservative variations can be introduced into CDRs at positions where the residues are unlikely to be involved in interactions with the transferrin receptor protein (e.g., human transferrin receptor protein), for example, as determined based on a crystal structure. Some aspects of the present disclosure provide anti-transferrin receptor antibodies comprising one or more of the heavy chain variable (VH) and / or light chain variable (VL) domains provided herein. In some embodiments, any of the VH domains provided herein include one or more of the CDR-H sequences (e.g., CDR-H1, CDR-H2, and CDR-H3) provided herein (e.g., any of the CDR-H sequences provided in any one of the anti-transferrin receptor antibodies selected from Table 2). In some embodiments, any of the VL domains provided herein include one or more of the CDR-L sequences (e.g., CDR-L1, CDR-L2, and CDR-L3) provided herein (e.g., any of the CDR-L sequences provided in any one of the anti-transferrin receptor antibodies selected from Table 2).

[0111] In some embodiments, anti-transferrin receptor antibodies of the present disclosure include any antibody that includes the heavy chain variable domain and / or light chain variable domain of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, anti-transferrin receptor antibodies of the present disclosure include any antibody that includes the heavy chain variable and light chain variable pair of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2.

[0112] Aspects of the present disclosure provide anti-transferrin receptor antibodies having heavy chain variable (VH) and / or light chain variable (VL) domain amino acid sequences homologous to any of the anti-transferrin receptor antibodies described herein. In some embodiments, the anti-transferrin receptor 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-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the homologous heavy chain variable and / or light chain variable amino acid sequence does 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 light chain variable sequence that excludes any of the CDR sequences provided herein. In some embodiments, any of the anti-transferrin receptor 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-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2.

[0113] In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain that includes any of the CDR-L domains (CDR-L1, CDR-L2, and CDR-L3) of any of the anti-transferrin receptor antibodies selected from Table 2, or a CDR-L domain variant provided herein. In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., a human transferrin receptor) comprises a light chain variable VL domain that includes CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, an anti-transferrin receptor antibody comprises a light chain variable (VL) region sequence that includes one, two, three, or four of the framework regions of the light chain variable region sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the anti-transferrin receptor antibody comprises one, two, three, or four framework regions of a light chain variable region sequence that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to one, two, three, or four framework regions of the light chain variable region sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the light chain variable framework region derived from said amino acid sequence consists of said amino acid sequence apart from the presence of up to 10 amino acid substitutions, deletions, and / or insertions, preferably up to 10 amino acid substitutions. In some embodiments, the light chain variable framework region derived from said amino acid sequence consists of said amino acid sequence with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues substituted for amino acids found in analogous positions in the light chain variable framework region of a corresponding non-human primate animal or human.

[0114] In some embodiments, an anti-transferrin receptor antibody that specifically binds to transferrin receptor comprises CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the antibody further comprises one, two, three, or all four VL framework regions derived from the VL of a human or primate antibody. The primate or human light chain framework regions of the selected antibodies for use with the light chain CDR sequences described herein can have, for example, at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%) identity to the light chain framework regions of the non-human parent antibody. The selected primate or human antibody can have the same or substantially the same number of amino acids in its light chain complementarity determining regions as the amino acids in the light chain complementarity determining regions of any of the antibodies provided herein (e.g., any of the anti-transferrin receptor antibodies selected from Table 2). In some embodiments, the amino acid residues of the primate or human light chain framework region are from a naturally occurring primate or human antibody light chain framework region that has at least 75% identity, at least 80% identity, at least 85% identity, at least 90% identity, at least 95% identity, at least 98% identity, at least 99% (or more) identity to the light chain framework region of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable kappa subfamily. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable lambda subfamily.

[0115] In some embodiments, any of the anti-transferrin receptor antibodies provided herein comprises a light chain variable domain that further comprises a light chain constant region. In some embodiments, the light chain constant region is a kappa or lambda light chain constant region. In some embodiments, the kappa or lambda light chain constant region is from a mammal, e.g., a human, a monkey, a rat, or a mouse. In some embodiments, the light chain constant region is a human kappa light chain constant region. In some embodiments, the light chain constant region is a human lambda light chain constant region. It should be understood that any of the light chain constant regions provided herein may be a variant of any of the light chain constant regions provided herein. In some embodiments, the light chain constant region comprises an amino acid sequence at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to any of the light chain constant regions of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2.

[0116] In some embodiments, the anti-transferrin receptor antibody is any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2.

[0117] In some embodiments, the anti-transferrin receptor antibody comprises a VL domain comprising the amino acid sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2, wherein the constant region comprises the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some embodiments, the anti-transferrin receptor antibody comprises a VL domain, or any variant of the VL domain, and a VH domain, or any variant of the VH domain, wherein the VL and VH domains, or variants thereof, are from the same antibody clone, and wherein the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b) of an immunoglobulin molecule. Non-limiting examples of human constant regions have been described in the art; see, e.g., Kabat EA et al. (1991), supra.

[0118] In some embodiments, the antibodies of the present disclosure have relatively high affinity, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or lower K DFor example, an anti-transferrin receptor antibody may bind to a transferrin receptor protein (e.g., human transferrin receptor) with an affinity between 5 pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM. The present disclosure also encompasses antibodies that compete with any of the antibodies described herein for binding to a transferrin receptor protein (e.g., human transferrin receptor) and have an affinity of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of anti-transferrin receptor antibodies can be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE).

[0119] In some embodiments, the antibodies of the present disclosure have relatively high affinity, e.g., 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M or lower K D For example, an anti-transferrin receptor antibody may bind to a transferrin receptor protein (e.g., human transferrin receptor) with an affinity between 5 pM and 500 nM, e.g., between 50 pM and 100 nM, e.g., between 500 pM and 50 nM. The present disclosure also encompasses antibodies that compete with any of the antibodies described herein for binding to a transferrin receptor protein (e.g., human transferrin receptor) and have an affinity of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of anti-transferrin receptor antibodies can be tested using any suitable method, including, but not limited to, biosensor technology (e.g., OCTET or BIACORE).

[0120] In some embodiments, the muscle-targeting agent is an anti-transferrin receptor antibody (e.g., an antibody and variants thereof as described in International Application Publication WO 2016 / 081643, which is incorporated herein by reference).

[0121] The heavy and light chain CDRs of antibodies according to various definition systems are provided in Table 1.1. Various definition systems, such as the Kabat definition, the Chothia definition, and / or the Contact definition, are described. See, for example, (e.g., Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDapartment of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. (1997) J. Mol. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs). Table 1.1. Heavy and light chain CDRs of mouse anti-transferrin receptor antibodies [Table 3]

[0122] Heavy chain variable domain (VH) and light chain variable domain sequences are also provided:

[0123] VH QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSS (SEQ ID NO: 33)

[0124] VL DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELK (SEQ ID NO: 34)

[0125] In some embodiments, an anti-transferrin receptor antibody of the disclosure comprises the same CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1 Alternatively or additionally, an anti-transferrin receptor antibody of the disclosure comprises the same CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1.

[0126] In some embodiments, an anti-transferrin receptor antibody of the present disclosure comprises CDR-H1, CDR-H2, and CDR-H3 that collectively contain no more than five amino acid variations (e.g., no more than five, four, three, two, or one amino acid variations) when compared to CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1. "Combined" means that the total number of amino acid variations in all three heavy chain CDRs is within a defined range. Alternatively or in addition, an anti-transferrin receptor antibody of the present disclosure may comprise CDR-L1, CDR-L2, and CDR-L3 that collectively contain no more than five amino acid variations (e.g., no more than five, four, three, two, or one amino acid variations) when compared to CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1.

[0127] In some embodiments, anti-transferrin receptor antibodies of the present disclosure comprise CDR-H1, CDR-H2, and CDR-H3, at least one of which contains no more than three amino acid variations (e.g., no more than three, two, or one amino acid variations) when compared to the corresponding heavy chain CDR as shown in Table 1.1. Alternatively or additionally, anti-transferrin receptor antibodies of the present disclosure may comprise CDR-L1, CDR-L2, and CDR-L3, at least one of which contains no more than three amino acid variations (e.g., no more than three, two, or one amino acid variations) when compared to the corresponding light chain CDR as shown in Table 1.1.

[0128] In some embodiments, an anti-transferrin receptor antibody of the present disclosure comprises a CDR-L3 that contains no more than three amino acid variations (e.g., no more than three, two, or one amino acid variations) when compared to a CDR-L3 as shown in Table 1.1. In some embodiments, a transferrin receptor antibody of the present disclosure comprises a CDR-L3 that contains no more than one amino acid variation when compared to a CDR-L3 as shown in Table 1.1. In some embodiments, an anti-transferrin receptor antibody of the present disclosure comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 31, according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 32, according to the Contact definition system). In some embodiments, an anti-transferrin receptor antibody of the disclosure comprises the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 as shown in Table 1.1, and comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 31, according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 32, according to the Contact definition system).

[0129] In some embodiments, anti-transferrin receptor antibodies of the present disclosure comprise heavy chain CDRs that, taken together, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs as shown in Table 1.1. Alternatively or in addition, anti-transferrin receptor antibodies of the present disclosure comprise light chain CDRs that, taken together, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the light chain CDRs as shown in Table 1.1.

[0130] In some embodiments, an anti-transferrin receptor antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 33. Alternatively or additionally, an anti-transferrin receptor antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 34.

[0131] In some embodiments, an anti-transferrin receptor antibody of the disclosure comprises a VH that contains no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) when compared to the VH as set forth in SEQ ID NO: 33. Alternatively or in addition, an anti-transferrin receptor antibody of the disclosure comprises a VL that contains no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) when compared to the VL as set forth in SEQ ID NO: 34.

[0132] In some embodiments, an anti-transferrin receptor antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH as set forth in SEQ ID NO: 33. Alternatively or in addition, an anti-transferrin receptor antibody of the present disclosure comprises a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL as set forth in SEQ ID NO: 34.

[0133] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure are humanized antibodies (e.g., humanized variants of antibodies). In some embodiments, the anti-transferrin receptor antibodies of the present disclosure comprise the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as set forth in Table 1.1, and comprise a humanized heavy chain variable region and / or a humanized light chain variable region.

[0134] Humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the recipient's complementarity-determining regions (CDRs) are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the imported CDR or framework sequences, but may also include residues incorporated to further refine and optimize antibody performance. In general, humanized antibodies will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. A humanized antibody will optimally also comprise at least a portion of an immunoglobulin (typically a human immunoglobulin) constant region or domain (Fc). The antibody may have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (1, 2, 3, 4, 5, 6) that are altered with respect to the original antibody, also referred to as one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also be affinity matured.

[0135] In some embodiments, humanization is achieved by grafting CDRs (e.g., as shown in Table 1.1) into IGKV1-NL1*01 and IGHV1-3*01 human variable domains. In some embodiments, the anti-transferrin receptor antibodies of the disclosure are humanized variants comprising one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 (when compared to the VL as set forth in SEQ ID NO:34), and / or one or more amino acid substitutions at positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 (when compared to the VH as set forth in SEQ ID NO:33). In some embodiments, the anti-transferrin receptor antibodies of the disclosure are humanized variants that include amino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and 70 (when compared to the VL as set forth in SEQ ID NO: 34), and / or amino acid substitutions at all of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 66, 75, 81, 83, 87, and 108 (when compared to the VH as set forth in SEQ ID NO: 33).

[0136] In some embodiments, the anti-transferrin receptor antibody of the disclosure is a humanized antibody and contains residues at positions 43 and 48 of the VL as set forth in SEQ ID NO: 34. Alternatively, or in addition, the anti-transferrin receptor antibody of the disclosure is a humanized antibody and contains residues at positions 48, 67, 69, 71, and 73 of the VH as set forth in SEQ ID NO: 33.

[0137] The VH and VL amino acid sequences of examples of humanized antibodies that may be used in accordance with the present disclosure are provided below:

[0138] Humanized VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSS (SEQ ID NO: 35)

[0139] Humanized VL DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELK (SEQ ID NO: 36)

[0140] In some embodiments, an anti-transferrin receptor antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 35. Alternatively or in addition, an anti-transferrin receptor antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 36.

[0141] In some embodiments, an anti-transferrin receptor antibody of the disclosure comprises a VH that contains no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) when compared to the VH as set forth in SEQ ID NO: 35. Alternatively or in addition, an anti-transferrin receptor antibody of the disclosure comprises a VL that contains no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) when compared to the VL as set forth in SEQ ID NO: 36.

[0142] In some embodiments, an anti-transferrin receptor antibody of the present disclosure comprises a VH comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to a VH as set forth in SEQ ID NO: 35. Alternatively or in addition, an anti-transferrin receptor antibody of the present disclosure comprises a VL comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to a VL as set forth in SEQ ID NO: 36.

[0143] In some embodiments, the anti-transferrin receptor antibodies of the disclosure are humanized variants that include an amino acid substitution at one or more of positions 43 and 48 (when compared to the VL as set forth in SEQ ID NO: 34), and / or an amino acid substitution at one or more of positions 48, 67, 69, 71, and 73 (when compared to the VH as set forth in SEQ ID NO: 33). In some embodiments, the anti-transferrin receptor antibodies of the disclosure are humanized variants that include an S43A and / or V48L mutation (when compared to the VL as set forth in SEQ ID NO: 34), and / or one or more of an A67V, L69I, V71R, and K73T mutation (when compared to the VH as set forth in SEQ ID NO: 33).

[0144] In some embodiments, the anti-transferrin receptor antibodies of the disclosure are humanized variants that include amino acid substitutions at one or more of positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 (when compared to the VL as set forth in SEQ ID NO: 34), and / or amino acid substitutions at one or more of positions 1, 5, 7, 11, 12, 20, 38, 40, 44, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 (when compared to the VH as set forth in SEQ ID NO: 33).

[0145] In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a chimeric antibody that can include heavy and light chain constant regions from a human antibody. A chimeric antibody refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from a certain mammal (e.g., a non-human mammal such as a mouse, rabbit, or rat), while the constant regions are homologous to sequences in antibodies from another mammal, such as a human. In some embodiments, amino acid modifications can be made in the variable and / or constant regions.

[0146] In some embodiments, the anti-transferrin receptor antibodies described herein are chimeric antibodies that can include heavy and light chain constant regions from a human antibody. A chimeric antibody refers to an antibody having a variable region or a portion of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, the variable regions of both the light and heavy chains mimic the variable regions of antibodies from a certain mammal (e.g., a non-human mammal such as a mouse, rabbit, or rat), while the constant regions are homologous to sequences in antibodies from another mammal, such as a human. In some embodiments, amino acid modifications can be made in the variable and / or constant regions.

[0147] In some embodiments, the heavy chain of any of the anti-transferrin receptor antibodies as 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 belong to any suitable origin, e.g., human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An exemplary human IgG1 constant region is provided below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 37)

[0148] In some embodiments, the light chain of any of the anti-transferrin receptor antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 38)

[0149] 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.

[0150] Exemplary heavy and light chain amino acid sequences of the described anti-transferrin receptor antibodies are provided below:

[0151] Heavy chain (VH + human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 39)

[0152] Light chain (VL + kappa light chain) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 40)

[0153] Heavy chain (humanized VH + human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 41)

[0154] Light chain (humanized VL + kappa light chain) DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 42)

[0155] In some embodiments, the anti-transferrin receptor antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 39. Alternatively or in addition, the anti-transferrin receptor antibodies described herein comprise a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 40. In some embodiments, the anti-transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 39. Alternatively or in addition, the anti-transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 40.

[0156] In some embodiments, an anti-transferrin receptor antibody of the disclosure comprises a heavy chain that contains no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) when compared to a heavy chain as set forth in SEQ ID NO: 39. Alternatively or in addition, an anti-transferrin receptor antibody of the disclosure comprises a light chain that contains no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) when compared to a light chain as set forth in SEQ ID NO: 40.

[0157] In some embodiments, the anti-transferrin receptor antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 41. Alternatively or in addition, the anti-transferrin receptor antibodies described herein comprise a light chain comprising an amino acid sequence at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 42. In some embodiments, the anti-transferrin receptor antibodies described herein comprise a heavy chain comprising the amino acid sequence of SEQ ID NO: 41. Alternatively or in addition, the anti-transferrin receptor antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 42.

[0158] In some embodiments, an anti-transferrin receptor antibody of the present disclosure comprises a heavy chain that contains no more than 20 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) when compared to the heavy chain of a humanized antibody as set forth in SEQ ID NO: 39. Alternatively or in addition, an anti-transferrin receptor antibody of the present disclosure comprises a light chain that contains no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) when compared to the light chain of a humanized antibody as set forth in SEQ ID NO: 40.

[0159] In some embodiments, the anti-transferrin receptor antibody is an antigen-binding fragment (FAB) of an intact antibody (full-length antibody). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared via routine methods. For example, F(ab')2 fragments can be produced by pepsin digestion of the antibody molecule, and Fab fragments can be generated by reducing the disulfide bridges of the F(ab')2 fragment. Exemplary FAB amino acid sequences of the anti-transferrin receptor antibodies described herein are provided below:

[0160] Heavy chain FAB (VH + part of human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 43)

[0161] Heavy chain FAB (humanized VH + part of human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 44)

[0162] The anti-transferrin receptor antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv, etc.), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the anti-transferrin receptor antibodies described herein are scFvs. In some embodiments, the anti-transferrin receptor antibodies described herein are scFv-Fabs (e.g., scFvs fused with a portion of a constant region). In some embodiments, the anti-transferrin receptor antibodies described herein are scFvs fused with a constant region (e.g., a human IgG1 constant region as set forth in SEQ ID NO: 39).

[0163] 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, without limitation, alpha-sarcoglycan, beta-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha7, integrin alpha7beta1, integrin beta1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, the muscle-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 should 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.

[0164] c. Antibody Features / Modifications In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where the residue is unlikely to be involved in interactions with the target antigen (e.g., a transferrin receptor), as determined, for example, based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or in the CH3 domain (residues 341-447 of human IgG1), and / or 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 antigen-dependent cellular cytotoxicity.

[0165] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased), 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 assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0166] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or in the CH3 domain (residues 341-447 of human IgG1), and / or in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0167] In some embodiments, one 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, e.g., International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for mutations that may alter (e.g., increase or decrease) the half-life of an antibody in vivo.

[0168] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, the Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, 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 in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al. (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, termed 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.

[0169] 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 into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0170] In some embodiments, one or more amino acid residues in the constant region of the muscle-targeting antibody described herein can be replaced with a different amino acid residue so that the antibody can have altered Clq binding and / or reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are altered to thereby alter the antibody's ability to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or to increase the antibody's affinity for Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0171] In some embodiments, the heavy and / or 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.

[0172] 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. Consequently, any of the antibodies may include the stabilizing "Adair" mutation.

[0173] 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.

[0174] 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 5-20 amino acids in length) that bind to specific cell types have been described. For example, cell-targeting peptides can be prepared by methods 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 is being explored, and a wide range of molecular payloads can be delivered. These approaches, without many of the practical disadvantages of large antibodies or viral particles, may have high selectivity for muscle tissue. Consequently, in some embodiments, the muscle-targeting agent is a muscle-targeting peptide ranging from 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.

[0175] In some embodiments, the muscle-targeting peptide may bind to an internalized cell surface receptor (e.g., transferrin receptor) that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells. In some embodiments, the muscle-targeting peptide may target (e.g., bind to) the transferrin receptor. In some embodiments, the transferrin receptor-targeting peptide may comprise a segment of a naturally occurring ligand, e.g., transferrin. In some embodiments, the transferrin receptor-targeting peptide is as described in U.S. Patent No. 6,743,893, 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 5 / 20 / 2011, entitled "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY."

[0176] 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: 6) bound to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. Consequently, in some embodiments, a muscle-targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 6). 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 DMD, a 12-amino acid peptide was identified by a phage display library for muscle targeting. See Yoshida D., et al., "Targeting of salicylate to skin and muscle following topical injections in rats." Int J Pharm 2002;231:177-84, the entire contents of which are hereby incorporated by reference. Herein, a 12-amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO: 7) was identified, and this muscle-targeting peptide showed improved binding to C2C12 cells compared to the ASSLNIA (SEQ ID NO: 6) peptide.

[0177] 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 rounds of selection, the 12-amino acid peptide TARGEHKEEELI (SEQ ID NO: 8) emerged most frequently. Consequently, in some embodiments, the muscle-targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 8).

[0178] 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 contains a high propensity of hydrophobic amino acids (e.g., valine) so that the peptide can preferentially target muscle cells. In some embodiments, the muscle-targeting peptide has not been previously characterized or disclosed. These peptides may be conceived, produced, synthesized, and / or derivatized using any of several methodologies, such as phage-displayed peptide libraries, one-bead-one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. Exemplary methodologies are characterized in the art and 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 amino acid sequences: CQAQGQLVC (SEQ ID NO: 9), CSERSMNFC (SEQ ID NO: 10), CPKTRRVPC (SEQ ID NO: 11), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 12), ASSLNIA (SEQ ID NO: 6), CMQHSMRVC (SEQ ID NO: 13), and DDTRHWG (SEQ ID NO: 14). 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 ​​or 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, muscle-targeting peptides can be linear; in other embodiments, muscle-targeting peptides can be cyclic (e.g., bicyclic) (see, e.g., Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

[0179] iii. Muscle-targeted receptor ligands The muscle-targeting agent may be a ligand, e.g., a ligand that binds to a receptor protein. The muscle-targeting ligand may be a protein, e.g., transferrin, that binds to an internalized cell surface receptor expressed by muscle cells. Consequently, 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.

[0180] 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 derivatized using any of several methodologies, e.g., systematic evolution of ligands by exponential enrichment. Exemplary methodologies have been 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.

[0181] 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.

[0182] In some embodiments, the muscle-targeting agent is a substrate for the SLC superfamily of transporters. SLC transporters are either equilibrium or use proton or sodium ion gradients 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.

[0183] 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. Consequently, in some embodiments, the muscle-targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and clofarabine. In some embodiments, any of the muscle-targeting agents provided herein 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.

[0184] 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).

[0185] 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, a fragment, or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack the N-terminal signaling domain, and / or may lack the C-terminal anchoring domain. 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 is to be understood that the hemojuvelin may be of human, non-human primate, or rodent origin.

[0186] B. Molecular Payload Some aspects of the present disclosure provide molecular payloads, e.g., molecular payloads for modulating a biological outcome (e.g., transcription of a DNA sequence, expression of a protein, or activity of a protein). In some embodiments, the molecular payload is linked or otherwise associated with a muscle-targeting agent. In some embodiments, such molecular payloads can target muscle cells, e.g., via specific binding to a nucleic acid or protein in muscle cells upon delivery to the muscle cells by the associated muscle-targeting agent. It should be understood that various types of muscle-targeting agents 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 muscle cell associated with a disease), a protein (e.g., a protein that binds to a nucleic acid or protein in a muscle cell associated with a disease), or a small molecule (e.g., a small molecule that modulates the function of a nucleic acid or protein in a muscle cell associated with a disease). In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a region complementary to a gene provided in Table 1. Exemplary molecular payloads are described in further detail herein; however, it should be understood that the exemplary molecular payloads provided herein are not intended to be limiting.

[0187] In some embodiments, at least one (e.g., at least two, at least three, at least four, at least five, at least ten) molecular payloads (e.g., oligonucleotides) are linked to the muscle-targeting agent. In some embodiments, all molecular payloads linked to the muscle-targeting agent are the same, e.g., target the same gene. In some embodiments, all molecular payloads linked to the muscle-targeting agent are different, e.g., molecular payloads may target different portions of the same target gene, or molecular payloads may target at least two different target genes. In some embodiments, a muscle-targeting agent may be linked to some molecular payloads that are the same and some that are different.

[0188] The present disclosure also provides compositions comprising a plurality of conjugates, wherein at least 80% (e.g., 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%, or at least 99%) of the conjugates comprise a muscle-targeting agent linked to the same number of molecular payloads (e.g., oligonucleotides).

[0189] i. Oligonucleotides Any suitable oligonucleotide may be used as a molecular payload as described herein. In some embodiments, the oligonucleotide may be designed to cause degradation of mRNA (e.g., the oligonucleotide may be a gapmer, siRNA, ribozyme, or aptamer that causes degradation). In some embodiments, the oligonucleotide may be designed to block translation of mRNA (e.g., the oligonucleotide may be a mixmer, siRNA, or aptamer that blocks translation). In some embodiments, the oligonucleotide may be designed to cause degradation of mRNA to block its translation. In some embodiments, the oligonucleotide may be a guide nucleic acid (e.g., a guide RNA) to direct the activity of an enzyme (e.g., a gene editing enzyme). Other examples of oligonucleotides are provided herein. It should be understood that in some embodiments, an oligonucleotide of one format (e.g., an antisense oligonucleotide) may be suitably adapted to another format (e.g., an siRNA oligonucleotide) by incorporating a functional sequence (e.g., an antisense strand sequence) from one format into the other format.

[0190] In some embodiments, the oligonucleotides may comprise a region of complementarity to a target gene provided in Table 1. Further non-limiting examples are provided below for selected genes in Table 1.

[0191] DMPK / DM1 In some embodiments, examples of useful oligonucleotides for targeting DMPK, e.g., for the treatment of DM1, are described in U.S. Patent Application Publication No. 20100016215A1, published January 1, 2010, entitled Compound And Method For Treating Myotonic Dystrophy; U.S. Patent Application Publication No. 20130237585A1, published July 19, 2010, entitled Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; U.S. Patent Application Publication No. 20150064181A1, published March 5, 2015, entitled "Antisense Conjugates For Decreasing Expression Of Dmpk"; and U.S. Patent Application Publication No. 20150064181A1, published March 5, 2015, entitled "Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy." U.S. Patent Application Publication No. 20150238627A1, published August 27, 2015, entitled "Myotonic Dystrophy"; Pandey, SK et al., "Identification and Characterization of Modified Antisense Oligonucleotides Targeting DMPK in Mice and Nonhuman Primates for the Treatment of Myotonic Dystrophy Type 1," J. of Pharmacol Exp Ther, 2015, 355:329-340; Langlois, M. et al., "Cytoplasmic and Nuclear Retained DMPK mRNAs Are Targets for RNA Interference in Myotonic Dystrophy Cells," J. Biological Chemistry, 2005, 280:17, 16949-16954; Jauvin, D. et al.“Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice”, Mol. Ther: Nucleic Acids, 2017, 7:465-474.; Mulders, SA et al. “Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy” PNAS, 2009, 106:33, 13915-13920.; Wheeler, TM et al., “Targeting nuclear RNA for in vivo correction of myotonic dystrophy” Nature, 2012, 488(7409):111-115.; and “Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) and U.S. Patent Application Publication No. 20160304877A1, published October 20, 2016, entitled "Methods for Producing a Novel Fluorescent Microwave Oxide-Based Microwave Oxide Expression," the entire contents of each of which are incorporated herein by reference.

[0192] Examples of oligonucleotides for facilitating DMPK gene editing include U.S. Patent Application Publication No. 20170088819A1, published March 3, 2017, entitled "Genetic Correction Of Myotonic Dystrophy Type 1"; and International Patent Application Publication No. WO18002812A1, published April 1, 2018, entitled "Materials And Methods For Treatment Of Myotonic Dystrophy Type 1 (DM1) And Other Related Disorders," the entire contents of each of which are incorporated herein by reference.

[0193] In some embodiments, the oligonucleotide may have a region of complementarity to a mutant form of DMPK, such as the mutant forms reported in Botta A. et al. "The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients." J Med Genet. 2008 Oct;45(10):639-46.; and Machuca-Tzili L. et al. "Clinical and molecular aspects of the myotonic dystrophies: a review." Muscle Nerve. 2005 Jul;32(1):1-18. (The entire contents of each of which are incorporated herein by reference).

[0194] In some embodiments, the oligonucleotides provided herein are antisense oligonucleotides targeting DMPK. In some embodiments, the oligonucleotide targeting is any one of the DMPK-targeting antisense oligonucleotides (e.g., Gapmers) described in U.S. Patent Application Publication No. US20160304877A1, entitled "Compounds and Methods for Modulation of Dystrophia Myotonica-Protein Kinase (DMPK) Expression," published on October 20, 2016, which is incorporated herein by reference. In some embodiments, the DMPK-targeting oligonucleotide targets a region of the DMPK gene sequence represented by Genbank Accession No. NM_001081560.2 or Genbank Accession No. NG_009784.1.

[0195] In some embodiments, the DMPK-targeting oligonucleotide comprises a nucleotide sequence comprising a region complementary to a target region in Genbank Accession No. NM_001081560.2 that is at least 10 contiguous nucleotides (e.g., at least 10, at least 12, at least 14, at least 16, or more contiguous nucleotides).

[0196] In some embodiments, the DMPK-targeting oligonucleotide contains a gapmer motif. "Gapmer" refers to a chimeric antisense compound in which an internal region having multiple nucleotides that support RNase H cleavage is positioned between external regions having one or more nucleotides, where the nucleotides comprising the internal region are chemically distinct from the nucleotides comprising the external region. The internal region may be referred to as a "gap segment," and the external region may be referred to as a "wing segment." In some embodiments, the DMPK-targeting oligonucleotide contains one or more modified nucleotides and / or one or more modified internucleotide linkages. In some embodiments, the internucleotide linkages are phosphorothioate linkages. In some embodiments, the oligonucleotide contains a complete phosphorothioate backbone. In some embodiments, the oligonucleotide is a DNA gapmer with a cET terminus (e.g., 3-10-3; cET-DNA-cET). In some embodiments, the DMPK-targeting oligonucleotide contains one or more 6'-(S)-CH3 bicyclic nucleotides, one or more β-D-2'-deoxyribonucleotides, and / or one or more 5-methylcytosine nucleotides.

[0197] DUX4 / FSHD In some embodiments, examples of useful oligonucleotides for targeting DUX4, e.g., for the treatment of FSHD, are described in U.S. Patent No. 9,988,628, published February 2, 2017, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. Patent No. 9,469,851, published October 30, 2014, entitled "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4"; U.S. Patent Application Publication No. 20120225034, published September 6, 2012, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY ...MORPHOLINO TARGETING DUX4 FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; and Ansseau et al., "Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy (FSHD)," Genes, 2017, 8, 93, the entire contents of each of which are incorporated herein. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA, or another nucleotide that hybridizes to the target DUX4 gene or mRNA.

[0198] In some embodiments, for example, for the treatment of FSHD, the oligonucleotide may have a region complementary to a hypomethylated, shortened D4Z4 repeat, as in Daxinger, et al., "Genetic and Epigenetic Contributors to FSHD," Lim JW, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1; 24(17): 4817-4828, published in Curr Opin Genet Dev in 2015 (the entire contents of each of which are incorporated herein).

[0199] DNM2 / CNM In some embodiments, examples of useful oligonucleotides for targeting DNM2, e.g., for the treatment of CNM, are provided in U.S. Patent Application Publication No. 20180142008, published May 24, 2018, entitled "DYNAMIN 2 INHIBITOR FOR THE TREATMENT OF DUCHENNE'S MUSCULAR DYSTROPHY," and PCT Application Publication No. WO 2018 / 100010A1, published June 7, 2018, entitled "ALLELE-SPECIFIC SILENCING THERAPY FOR DYNAMIN 2-RELATED DISEASES." For example, in some embodiments, the oligonucleotide is an RNAi, antisense nucleic acid, siRNA, or ribozyme that specifically interferes with DNM2 expression. Other examples of useful oligonucleotides for targeting DNM2 are provided in Tasfaout, et al., "Single Intramuscular Injection of AAV-shRNA Reduces DNM2 and Prevents Myotubular Myopathy in Mice," published in Mol. Ther. on April 4, 2018, and Tasfaout, et al., "Antisense oligonucleotide-mediated Dnm2 knockdown prevents and reverts myotubular myopathy in mice," Nature Communications volume 8, Article number: 15661 (2017). In some embodiments, the oligonucleotide is an shRNA or morpholino that efficiently targets DNM2 mRNA.In some embodiments, the oligonucleotide encodes wild-type DNM2 that is resistant to miR-133 activity, as described in Todaka, et al., "Overexpression of NF90-NF45 Represses Myogenic MicroRNA Biogenesis, Resulting in Development of Skeletal Muscle Atrophy and Centronuclear Muscle Fibers," published in Mol. Cell Biol. in July 2015. Further examples of oligonucleotides useful for targeting DNM2 are provided in Gibbs, et al., "Two Dynamin-2 Genes are Required for Normal Zebrafish Development," published in PLoS One in 2013, the entire contents of each of which are incorporated herein.

[0200] In some embodiments, by way of example, for the treatment of CNM, the oligonucleotide may have a region complementary to a mutation in DNM2 associated with CNM, as in Boehm et al., "Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy," published in Hum. Mutat. in 2012 (the entire contents of which are incorporated herein).

[0201] Pompe disease In some embodiments, for example, for the treatment of Pompe disease, the oligonucleotide mediates exon 2 inclusion in the GAA disease allele as in van der Wal, et al., "GAA Deficiency in Pompe Disease is Alleviated by Exon Inclusion in iPSC-Derived Skeletal Muscle Cells," Mol Ther Nucleic Acids. 2017 Jun 16; 7: 101-115 (the entire contents of which are incorporated herein). Consequently, in some embodiments, the oligonucleotide may have a region of complementarity to the GAA disease allele.

[0202] In some embodiments, for example, for the treatment of Pompe disease, oligonucleotides such as RNAi or antisense oligonucleotides are utilized to suppress expression of wild-type GYS1 in muscle cells, as reported, for example, in Clayton, et al., "Antisense Oligonucleotide-Mediated Suppression of Muscle Glycogen Synthase 1 Synthesis as an Approach for Substrate Reduction Therapy of Pompe Disease," published in Mol Ther Nucleic Acids in 2017, or in U.S. Patent Application Publication No. 2017182189, published June 29, 2017, entitled "INHIBITING OR DOWNREGULATING GLYCOGEN SYNTHASE BY CREATING PREMATURE STOP CODONS USING ANTISENSE OLIGONUCLEOTIDES," the contents of which are incorporated herein by reference. Consequently, in some embodiments, the oligonucleotide may have an antisense strand that has a region of complementarity to a sequence that is the human GYS1 sequence corresponding to RefSeq number NM_002103.4 and / or the mouse GYS1 sequence corresponding to RefSeq number NM_030678.3.

[0203] ACVR1 / FOP For example, examples of oligonucleotides useful for targeting ACVR1 for the treatment of FOP are described in U.S. Patent Application No. 2009 / 0253132, published October 8, 2009, entitled "Mutated ACVR1 for diagnosis and treatment of fibrodysplasia ossificans progressiva (FOP)"; WO 2015 / 152183, published October 8, 2015, entitled "Prophylactic agent and therapeutic agent for fibrodysplasia ossificans progressive"; Lowery, J. Wet et al., "Allele-specific RNA Interference in FOP-Silencing the FOP gene," GENE THERAPY, vol. 19, 2012, pages 701-702; Takahashi, M. et al., "Disease-causing allele-specific silencing against the ALK2 mutants, R206H and G356D, in fibrodysplasia ossificans progressiva," Gene Therapy, vol. 19, 2012, pages 701-702. Therapy(2012)19,781-785;Shi,S.et al. “Antisense-Oligonucleotide Mediated Exon Skipping in Activin-Receptor-Like Kinase 2: Inhibiting the Receptor That Is Overactive in Fibrodysplasia Ossificans Progressiva”Plos One,July 2013,Vol8:7,e69096.U.S. Patent Application No. 2017 / 0159056, published June 8, 2017, entitled "Antisense oligonucleotides and methods of use thereof"; U.S. Patent No. 8,859,752, published October 4, 2014, entitled "SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva (FOP)"; and WO2004 / 094636, published November 4, 2004, entitled "Effective siRNA knockdown constructs," the contents of each of which are incorporated herein in their entirety.

[0204] FXN / Friedreich's ataxia In some embodiments, examples of oligonucleotides useful for targeting FXN and / or otherwise compensating for frataxin deficiency, e.g., for the treatment of Friedreich's ataxia, include those described in Li, L. et al., "Activating frataxin expression by repeat-targeted nucleic acids," Nat. Comm. 2016, 7:10606; WO 2016 / 094374, published 6 / 16 / 2016, "Compositions and methods for treatment of Friedreich's ataxia."; WO 2015 / 020993, published 2 / 12 / 2015, "RNAi COMPOSITIONS AND METHODS FOR TREATMENT OF FRIEDREICH'S ATAXIA."; WO 2017 / 186815, published 11 / 2 / 2017, "Antisense oligonucleotides for enhanced expression of frataxin."; WO U.S. Patent Application No. 2008 / 018795, published 2 / 14 / 2008, "Methods and means for treating DNA repeat instability associated genetic disorders"; U.S. Patent Application No. 2018 / 0028557, published 2 / 1 / 2018, "Hybrid oligonucleotides and uses thereof"; WO 2015 / 023975, published 2 / 19 / 2015, "Compositions and methods for modulating RNA"; WO 2015 / 023939, published 2 / 19 / 2015, "Compositions and methods for modulating expression of frataxin"; U.S. Patent Application No. 2017 / 0281643, published 10 / 5 / 2017, "Compounds and methods for modulating frataxin expression"; Li L. et al., "Activating frataxin expression by repeat-targeted nucleic acids," Nature Communications, published February 4, 2016; and Li L. et al., "Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat," Nucleic Acid Ther. 2018 Feb;28(1):23-33, the entire contents of each of which are incorporated herein.

[0205] In some embodiments, the oligonucleotide payload is configured to inhibit expression of natural antisense transcripts that inhibit FXN expression (e.g., as gapmers or RNAi oligonucleotides), as disclosed, for example, in U.S. Patent No. 9,593,330, filed 6 / 9 / 2011, entitled "Treatment of frataxin (FXN) related diseases by inhibition of natural antisense transcript to FXN," the entire contents of which are incorporated herein by reference.

[0206] Examples of oligonucleotides for facilitating FXN gene editing include WO 2016 / 094845, published June 16, 2016, entitled "Compositions and methods for editing nucleic acids in cells utilizing oligonucleotides"; WO 2015 / 089354, published June 18, 2015, entitled "Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders"; WO 2015 / 139139, published September 24, 2015, entitled "CRISPR-based methods and products for increasing frataxin levels and uses thereof"; and WO 2018 / 002783, published January 4, 2018, entitled "Materials and methods for treatment of Friedreich ataxia and other related disorders," the entire contents of each of which are incorporated herein.

[0207] Examples of oligonucleotides for promoting FXN gene expression through targeting non-FXN genes, such as epigenetic regulators of FXN, include WO 2015 / 023938, published 2 / 19 / 2015, entitled "Epigenetic regulators of frataxin," the entire contents of which are incorporated herein.

[0208] In some embodiments, the oligonucleotide may have a region of complementarity to a sequence defined as the FXN gene from human (Gene ID 2395; NC_000009.12) and / or the FXN gene from mouse (Gene ID 14297; NC_000085.6). In some embodiments, the oligonucleotide may have a region complementary to a mutant form of FXN, as reported, for example, in Montermini, L. et al. "The Friedreich ataxia GAA triplet repeat: premutation and normal alleles." Hum. Molec. Genet., 1997, 6: 1261-1266; Filla, A. et al. "The relationship between trinucleotide (GAA) repeat length and clinical features in Friedreich ataxia." Am. J. Hum. Genet. 1996, 59: 554-560; Pandolfo, M. Friedreich ataxia: the clinical picture. J. Neurol. 2009, 256, 3-8 (the entire contents of each of which are incorporated herein by reference).

[0209] DMD / Dystrophinopathy Examples of useful oligonucleotides for targeting DMD are described in U.S. Patent Application Publication US20100130591A1, published May 27, 2010, entitled "MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD"; U.S. Patent Application Publication No. 8,361,979, issued January 29, 2013, entitled "MEANS AND METHOD FOR INDUCING EXON-SKIPPING"; U.S. Patent Application Publication No. 20120059042, published March 8, 2012, entitled "METHOD FOR EFFICIENT EXON (44) SKIPPING IN DUCHENNE MUSCULAR DYSTROPHY AND ASSOCIATED MEANS"; and U.S. Patent Application Publication No. 20120059042, published March 8, 2012, entitled "EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY." U.S. Patent Application Publication No. 20140329881, published November 6, 2014, entitled "ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF"; U.S. Patent Application Publication No. 8,232,384, issued July 31, 2012, entitled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA"; U.S. Patent Application Publication No. 20120022134A1, published January 26, 2012, entitled "ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING A DISPENSABLE DOG U.S. Patent Application Publication No. 20120077860, published March 29, 2012, entitled "Oligomers," and entitled "Antibody Oligomers," U.S. Patent No. 8,324,371, issued December 4, 2012, entitled "Oligomers," and U.S. Patent No. 9,078,911, issued July 14, 2015, entitled "Antibody Oligonucleotides," and entitled "Antibody Oligomers," respectively.No. 9,079,934, issued July 14, 2015, entitled "ANTISENSE NUCLEIC ACIDS"; U.S. Patent No. 9,034,838, issued May 19, 2015, entitled "MIR-31 IN DUCHENNE MUSCULAR DYSTROPHY THERAPY"; and International Patent Publication WO2017062862A3, published April 13, 2017, entitled "OLIGONUCLEOTIDE COMPOSITIONS AND METHODS THEREOF," the entire contents of each of which are incorporated herein.

[0210] Examples of oligonucleotides for facilitating DMD gene editing are described in International Patent Publication WO2018053632A1, published March 29, 2018, entitled "METHODS OF MODIFYING THE DYSTROPHIN GENE AND RESTORING DYSTROPHIN EXPRESSION AND USES THEREOF"; International Patent Publication WO2017049407A1, published March 30, 2017, entitled "MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF"; International Patent Publication WO2016161380A1, published October 6, 2016, entitled "CRISPR / CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND BECKER MUSCULAR DYSTROPHY"; and International Patent Publication WO2016161380A1, published October 6, 2016, entitled "THERAPEUTIC International Patent Publication WO2017095967, published June 8, 2017, entitled "TARGETS FOR THE CORRECTION OF THE HUMAN DYSTROPHIN GENE BY GENE EDITING AND METHODS OF USE"; International Patent Publication WO2017072590A1, published May 4, 2017, entitled "MATERIALS AND METHODS FOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY"; International Patent Publication WO2018098480A1, published May 31, 2018, entitled "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CPF1-MEDIATED GENE EDITING"; and "RNA-Guided Systems for In Vivo Gene Editing." U.S. Patent Application Publication US20170266320A1, published September 21, 2017, entitled "Editing";and U.S. Patent Application Publication No. 2013 / 0145487, published June 6, 2013, entitled "MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHY GENE AND USES THEREOF," the entire contents of each of which are incorporated herein. In some embodiments, the oligonucleotide may have regions of complementarity to DMD gene sequences of multiple species, e.g., selected from human, mouse, and non-human species;

[0211] 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.

[0212] MYH7 / hypertrophic cardiomyopathy Examples of useful oligonucleotides for targeting, e.g., MYH7, as a payload include U.S. Patent Application Publication No. 20180094262, published April 5, 2018, entitled "Inhibitors of MYH7B and Uses Thereof"; U.S. Patent Application Publication No. 20160348103, published December 1, 2016, entitled "Oligonucleotides and Methods for Treatment of Cardiomyopathy Using RNA Interference"; U.S. Patent Application Publication No. 20160237430, published August 18, 2016, entitled "Allele-specific RNA Silencing for the Treatment of Hypertrophic Cardiomyopathy"; U.S. Patent Application Publication No. 20160032286, published February 4, 2016, entitled "Inhibitors of MYH7B and Uses Thereof"; and U.S. Patent Application Publication No. 20160032286, published February 4, 2016, entitled "MicroRNA Inhibitors Comprising Locked U.S. Patent Application Publication No. 20140187603, published July 3, 2014, entitled "Dual Targeting of miR-208 and miR-499 in the Treatment of Cardiac Disorders," U.S. Patent Application Publication No. 20140179764, published June 26, 2014, entitled "Dual Targeting of miR-208 and miR-499 in the Treatment of Cardiac Disorders," and U.S. Patent Application Publication No. 20120114744, published May 10, 2012, entitled "Compositions and Methods to Treat Muscular and Cardiovascular Disorders," the entire contents of each of which are incorporated herein.

[0213] In some embodiments, the oligonucleotide may target lncRNA or mRNA, e.g., for degradation. In some embodiments, the oligonucleotide may target nucleic acids encoding proteins involved in the mismatch repair pathway, e.g., MSH2, MutL alpha, MutS beta, MutL alpha, e.g., for degradation. Non-limiting examples of proteins involved in the mismatch repair pathway (mRNAs encoding such proteins may be targeted by the oligonucleotides described herein) are described in Iyer, RR et al., "DNA triplet repeat expansion and mismatch repair," Annu Rev Biochem. 2015;84:199-226; and Schmidt MH and Pearson CE, "Disease-associated repeat instability and mismatch repair," DNA Repair (Amst). 2016 Feb;38:117-26.

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

[0215] In some embodiments, for purposes of the present disclosure, a complementary nucleic acid sequence of an oligonucleotide is specifically hybridizable to or specific for a target nucleic acid when the binding of said sequence to the target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) causing loss of activity (e.g., inhibition of translation) or loss of expression (e.g., degradation of the target mRNA) and when there is a sufficient degree of complementarity to avoid non-specific binding of said sequence to non-target sequences under conditions where avoidance of non-specific binding is desired, e.g., physiological conditions in the case of in vivo assays or therapeutic treatments and in the case of in vitro assays, and the assay is performed under suitable conditions of stringency. Thus, in some embodiments, an oligonucleotide may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to consecutive nucleotides of a target nucleic acid. In some embodiments, a complementary nucleotide sequence need not be 100% complementary to the sequence of the target to be specifically hybridizable to or specific for a target nucleic acid.

[0216] 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, 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.

[0217] b. Oligonucleotide Modifications: The oligonucleotides described herein may be modified, including, for example, modified sugar moieties, modified internucleoside linkages, modified nucleotides, and / or combinations thereof.In addition, in some embodiments, the oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immunostimulatory; are nuclease-resistant; have improved cellular uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; have improved endosomal exit 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.

[0218] In some embodiments, specific nucleotide modifications can be used to make the oligonucleotides that incorporate the modifications more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides remain intact for longer than unmodified oligonucleotides.Specific examples of modified oligonucleotides include those that include 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.As a result, the oligonucleotides of the present disclosure can be stabilized against nucleolytic degradation by modifications, such as incorporating nucleotide modifications.

[0219] In some embodiments, the oligonucleotide may be up to 50 nucleotides or up to 100 nucleotides in length, with 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30, 2-40, 2-45, or more nucleotides of the oligonucleotide being modified nucleotides. The oligonucleotide may be 8-30 nucleotides in length, with 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, or 2-30 nucleotides of the oligonucleotide being modified nucleotides. The oligonucleotide may be 8-15 nucleotides in length, with 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, or 2-14 nucleotides of the oligonucleotide being modified nucleotides. Optionally, the oligonucleotide may be modified at every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Oligonucleotide modifications are further described herein.

[0220] c modified nucleotides In some embodiments, the oligonucleotide includes 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamide (2'-O-NMA).

[0221] In some embodiments, the oligonucleotide may comprise at least one 2'-O-methyl-modified nucleotide, and in some embodiments, all of the nucleotides comprise 2'-O-methyl modification. In some embodiments, the oligonucleotide comprises a modified nucleotide in which the ribose ring comprises a bridging moiety connecting two atoms in the ring (for example, connecting the 2'-O atom to the 4'-C atom). In some embodiments, the oligonucleotide is "locked", for example, the ribose ring comprises a modified nucleotide in which the ribose ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4'-C atom. Examples of LNAs are described in International Patent Application Publication WO / 2008 / 043753, published on April 17, 2008, entitled "RNA Antagonist Compounds For The Modulation Of PCSK9," the contents of which are incorporated herein by reference in their entirety.

[0222] Other modifications that may be used in the oligonucleotides disclosed herein include ethylene-bridged nucleic acids (ENAs), which include, but are not limited to, 2'-O,4'-C-ethylene-bridged nucleic acids. Examples of ENAs are provided in International Patent Publication No. WO 2005 / 042777, published May 12, 2005, entitled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006; and Horie et al., Nucleic Acids Symp. Ser(Oxf), 49:171-172, 2005, the disclosures of which are incorporated herein by reference in their entireties.

[0223] In some embodiments, the oligonucleotide may comprise bridged nucleotides, such as locked nucleic acid (LNA) nucleotides, constrained ethyl (cEt) nucleotides, or ethylene-bridged nucleic acid (ENA) nucleotides. In some embodiments, the oligonucleotide comprises a modified nucleotide disclosed in one of the following U.S. patents or published patent applications: U.S. Patent 7,399,845, issued July 15, 2008, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,741,457, issued June 22, 2010, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 8,022,193, issued September 20, 2011, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,569,686, issued August 4, 2009, entitled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,335,765, issued February 26, 2008, entitled "Novel Nucleoside And Oligonucleotide U.S. Patent No. 7,314,923, issued January 1, 2008, entitled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent No. 7,816,333, issued October 19, 2010, entitled "Oligonucleotide Analogues And Methods Utilizing The Same" and U.S. Publication No. 2011 / 0009471, now U.S. Patent No. 8,957,201, issued 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.

[0224] In some embodiments, the oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, preferably a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro-modified nucleotide. In other preferred embodiments, the RNA modifications include 2'-fluoro, 2'-amino, and 2'-O-methyl modifications on the ribose of a pyrimidine, an abasic residue, or an inverted base at the 3' end of the RNA.

[0225] In some embodiments, an oligonucleotide may have at least one modified nucleotide that results in an increase in the Tm of the oligonucleotide in the range of 1°C, 2°C, 3°C, 4°C, or 5°C compared to an oligonucleotide that does not have at least one modified nucleotide. An oligonucleotide may have multiple modified nucleotides that, in total, result in an increase in the Tm of the oligonucleotide in the range of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or more, compared to an oligonucleotide that does not have the modified nucleotide.

[0226] The oligonucleotide may contain alternating nucleotides of different types. For example, the oligonucleotide may contain alternating deoxyribonucleotides or ribonucleotides and 2'-fluoro-deoxyribonucleotides. The oligonucleotide may contain alternating deoxyribonucleotides or ribonucleotides and 2'-O-methyl nucleotides. The oligonucleotide may contain alternating 2'-fluoro nucleotides and 2'-O-methyl nucleotides. The oligonucleotide may contain alternating bridged nucleotides and 2'-fluoro or 2'-O-methyl nucleotides.

[0227] d. Internucleotide linkage / backbone In some embodiments, the oligonucleotide may contain phosphorothioate or other modified internucleotide linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the oligonucleotide comprises modified internucleotide linkages at the first, second, and / or third internucleoside linkages at the 5' or 3' end of the nucleotide sequence.

[0228] Phosphorus-containing linkages that may be used include, but are not limited to, normal 3'-5' linkages, 2'-5' linked analogs thereof, 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'); 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; 5,39 See Nos. 9,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0229] 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).

[0230] 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 Dec;40(12):5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided that contain nucleoside units linked together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages. In some embodiments, such phosphorothioate oligonucleotides with substantially chiral pure intersugar linkages are prepared by enzymatic synthesis or chemical synthesis, for example, as described in U.S. Patent 5,587,261, issued December 12, 1996, the contents of which are incorporated herein by reference in their entirety. In some embodiments, chiral-controlled oligonucleotides provide selective cleavage patterns of target nucleic acids.For example, in some embodiments, chiral-controlled oligonucleotides provide a single cleavage site within the complementary sequence of nucleic acid, as described, for example, in U.S. Patent Application Publication No. 20170037399 A1, entitled "CHIRAL DESIGN," published on February 2, 2017 (the contents of which are incorporated herein by reference in their entirety).

[0231] 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).

[0232] 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.

[0233] h.Gapmer In some embodiments, the oligonucleotide is a gapmer. Gapmer oligonucleotides generally have the formula 5'-XYZ-3', with X and Z flanking regions surrounding gap region Y. In some embodiments, the Y region is a contiguous stretch of nucleotides, e.g., a region of at least 6 DNA nucleotides, capable of recruiting an RNAse, such as RNAse H. In some embodiments, the gapmer binds to the target nucleic acid at a point where the RNAse can be recruited and subsequently cleave the target nucleic acid. In some embodiments, the Y region is flanked on both the 5' and 3' sides by regions X and Z containing high-affinity modified nucleotides, e.g., 1 to 6 modified nucleotides. Examples of modified nucleotides include, but are not limited to, 2'MOE or 2'OMe, or locked nucleobases (LNAs). The flanking sequences X and Z may be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides in length in some embodiments. The flanking sequences X and Z may or may not be of similar length. The gap segment Y may in some embodiments be a nucleotide sequence that is 5 to 20 nucleotides, 6 to 12 nucleotides, or 6 to 10 nucleotides in length.

[0234] In some embodiments, the gap region of a gapmer oligonucleotide may contain modified nucleotides known to be acceptable for efficient RNase H activity, in addition to DNA nucleotides such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-configured nucleotides. In some embodiments, the gap region comprises one or more unmodified internucleosides. In some embodiments, one or both flanking regions each independently comprise one or more phosphorothioate 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 nucleotides. In some embodiments, the gap region and two flanking regions each independently contain 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 nucleotides.

[0235] Gapmers may be produced using suitable methods. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include, but are not limited to, U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; Nos. 6; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. Patent Publication Nos. US20090286969, US20100197762, and US20110112170; and PCT Publication Nos. WO2008049085 and WO2009090182, each of which is incorporated by reference herein in its entirety.

[0236] i.Mixmer In some embodiments, the oligonucleotides described herein may be mixmers or may include mixmer sequence patterns. Generally, mixmers are oligonucleotides that contain both naturally occurring and non-naturally occurring nucleotides, or oligonucleotides that contain two different types of non-naturally occurring nucleotides, typically in a staggered pattern. Mixmers generally have higher binding affinity than unmodified oligonucleotides and specifically bind to target molecules, for example, they may 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, WO2007 / 112754 or WO2007 / 112753.

[0237] In some embodiments, a mixmer comprises or consists of a repeating pattern of nucleotide analogs and naturally occurring nucleotides, or a repeating pattern of one type of nucleotide analog and another type of nucleotide analog. However, a mixmer need not comprise a repeating pattern, but instead can comprise any arrangement of modified nucleotides and naturally occurring nucleotides, or any arrangement of one type of modified nucleotide and another type of modified nucleotide. The repeating pattern can illustratively be a modified nucleotide such as LNA at every second or third nucleotide, with the remaining nucleotides being naturally occurring nucleotides such as DNA, or 2'-substituted nucleotide analogs such as 2'MOE or 2'fluoro analogs, or any other modified nucleotide described herein. It is recognized that a repeating pattern of modified nucleotides, such as LNA units, can be combined with modified nucleotides at fixed positions, e.g., at the 5' or 3' termini.

[0238] In some embodiments, the mixmer does not contain a region of more than five, more than four, more than three, or more than two consecutive naturally occurring nucleotides, such as DNA nucleotides. In some embodiments, the mixmer contains at least a region of at least two consecutive modified nucleotides, such as at least two consecutive LNAs. In some embodiments, the mixmer contains at least a region of at least three consecutive modified nucleotides, such as at least three consecutive LNAs.

[0239] In some embodiments, the mixmer does not contain a region of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive nucleotide analogs, such as LNA. In some embodiments, the LNA units may be replaced with other nucleotide analogs, such as those mentioned herein.

[0240] Mixmers may be designed to contain a mixture of affinity-enhancing modified nucleotides, such as, in non-limiting examples, LNA nucleotides and 2'-O-methyl nucleotides. 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 nucleotides.

[0241] 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.

[0242] In some embodiments, a mixmer comprises one or more morpholino nucleotides. For example, in some embodiments, a mixmer may comprise morpholino nucleotides mixed (e.g., in a staggered manner) with one or more other nucleotides (e.g., DNA, RNA nucleotides) or modified nucleotides (e.g., LNA, 2'-O-methyl nucleotides).

[0243] In some embodiments, mixmers are useful for splice correcting 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).

[0244] j.RNA interference (RNAi) In some embodiments, the oligonucleotides provided herein may be in the form of small interfering RNA (siRNA), also known as small interfering RNA or silencing RNA. siRNAs are a class of double-stranded RNA molecules, typically about 20-25 base pairs in length, that target nucleic acids (e.g., mRNA) for degradation via the RNA interference (RNAi) pathway in cells. The specificity of an siRNA molecule may be determined by the binding of the antisense strand to its target RNA. Effective siRNA molecules are generally less than 30-35 base pairs in length to avoid triggering nonspecific RNA interference pathways in cells via the interferon response, although longer siRNAs may also be effective.

[0245] Following selection of an appropriate target RNA sequence, siRNA molecules containing nucleotide sequences complementary to all or a portion of the target sequence, i.e., antisense sequences, can be designed and prepared using suitable methods (see, for example, PCT Publication No. WO2004 / 016735; and U.S. Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791).

[0246] siRNA molecules can be double-stranded (i.e., dsRNA molecules containing an antisense strand and a complementary sense strand) or single-stranded (i.e., ssRNA molecules containing only the antisense strand). siRNA molecules can comprise duplexes, asymmetric duplexes, hairpins, or asymmetric hairpin secondary structures with self-complementary sense and antisense strands.

[0247] Double-stranded siRNA may comprise RNA strands of the same length or different lengths.Double-stranded siRNA molecules can also be assembled from a single oligonucleotide of stem-loop structure (where the self-complementary sense and antisense regions of siRNA molecules are linked using nucleic acid-based or non-nucleic acid-based linker(s)), and from circular single-stranded RNA (where circular RNA can be processed either in vivo or in vitro to produce active siRNA molecules that can mediate RNAi) with a stem that comprises two or more loop structures and self-complementary sense and antisense strands.Therefore, small hairpin RNA (shRNA) molecules are also contemplated herein.These molecules comprise a specific antisense sequence in addition to a reverse complementary (sense) sequence, and are typically separated by a spacer or loop sequence. Cleavage of the spacer or loop (optionally with additional processing steps that may result in the addition or removal of one, two, three, or more nucleotides from the 3' and / or 5' ends of one or both strands) provides the single-stranded RNA molecule and its reverse complement so that they can anneal to form a dsRNA molecule. The spacer can be long enough to allow the antisense and sense sequences to anneal to form a double-stranded structure (or stem) prior to cleavage of the spacer (and optionally with subsequent processing steps that may result in the addition or removal of one, two, three, four, or more nucleotides from the 3' and / or 5' ends of one or both strands). The spacer sequence can be an unrelated nucleotide sequence located between two complementary nucleotide sequence regions that, once annealed to form the double-stranded nucleic acid, comprise the shRNA.

[0248] The overall length of siRNA molecule can vary from about 14 nucleotides to about 100 nucleotides according to the type of siRNA molecule designed.Generally, between about 14 and about 50 of these nucleotides are complementary to RNA target sequence, that is, constitute the specific antisense sequence of siRNA molecule.For example, when siRNA is double-stranded siRNA or single-stranded siRNA, its length can vary from about 14 nucleotides to about 50 nucleotides, while when siRNA is shRNA or circular molecule, its length can vary from about 40 nucleotides to about 100 nucleotides.

[0249] The siRNA molecule may contain a 3' overhang at one end of the molecule, and the other end may be blunt or may also have an overhang (5' or 3'). When the siRNA molecule contains overhangs at both ends of the molecule, the lengths of the overhangs may be the same or different. In one embodiment, the siRNA molecule of the present disclosure contains a 3' overhang of about 1 to about 3 nucleotides on both ends of the molecule.

[0250] k. microRNA (miRNA) In some embodiments, the oligonucleotide may be a microRNA (miRNA). MicroRNAs (also referred to as "miRNAs") are small, non-coding RNAs that belong to a class of regulatory molecules that control gene expression by binding to complementary sites on target RNA transcripts. Typically, miRNAs are generated from large RNA precursors (called pri-miRNAs) that are processed in the nucleus to produce approximately 70-nucleotide pre-miRNAs, which fold into imperfect stem-loop structures. These pre-miRNAs typically undergo additional processing steps in the cytoplasm, where the mature miRNAs, 18-25 nucleotides in length, are excised from one side of the pre-miRNA hairpin by the RNase III enzyme Dicer.

[0251] As used herein, miRNA includes pri-miRNA, pre-miRNA, mature miRNA, and variant fragments thereof, or variant fragments thereof that retain the biological activity of mature miRNA. In one embodiment, the size range of miRNA can be from 21 nucleotides to 170 nucleotides. In one embodiment, the size range of miRNA is from 70 nucleotides to 170 nucleotides in length. In another embodiment, mature miRNAs of 21 nucleotides to 25 nucleotides in length can be used.

[0252] l. Aptamer In some embodiments, the oligonucleotides provided herein may be in the form of an aptamer. Generally, in the context of molecular payloads, an aptamer is any nucleic acid that specifically binds to a target, such as a small molecule, protein, or nucleic acid, in a cell. In some embodiments, the aptamer is a DNA aptamer or an RNA aptamer. In some embodiments, the nucleic acid aptamer is single-stranded DNA or RNA (ssDNA or ssRNA). It should be understood that single-stranded nucleic acid aptamers may form helix and / or loop structures. The nucleic acids forming the nucleic acid aptamers may include naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with hydrocarbon linkers (e.g., alkylene) or polyether linkers (e.g., PEG linkers) inserted between one or more nucleotides, modified nucleotides with hydrocarbon or PEG linkers inserted between one or more nucleotides, or combinations thereof. Exemplary publications and patents describing aptamers and methods of producing aptamers include, by way of example, Lorsch and Szostak, 1996; Jayasena, 1999; U.S. Patent Nos. 5,270,163; 5,567,588; 5,650,275; 5,670,637; 5,683,867; 5,696,249; 5,789,157; ​​5,843,653; 5,864,026; 5,989,823; 6,569,630; 8,318,438 and PCT application WO 99 / 31275, each of which is incorporated herein by reference.

[0253] m. ribozyme In some embodiments, the oligonucleotide provided herein can be in the form of ribozyme.Ribozyme (ribonucleic acid enzyme) is a molecule, typically an RNA molecule, that can carry out specific biochemical reactions similar to the action of protein enzymes.Ribozyme is a molecule with enzymatic activity that includes the ability to cleave specific phosphodiester bonds in the RNA molecules (such as mRNA, RNA-containing substrates, lncRNA, and ribozyme itself) that they hybridize with.

[0254] Ribozymes can take on one of several physical structures, one of which is called a "hammerhead." Hammerhead ribozymes consist of a conserved nine-base catalytic core, a double-stranded stem and loop structure (stem-loop II), and two flanking regions complementary to the target RNA region surrounding the catalytic core. The flanking regions enable the ribozyme to specifically bind to the target RNA by forming double-stranded stems I and III. Cleavage occurs in cis (i.e., cleavage of the same RNA molecule containing the hammerhead motif) or trans (cleavage of an RNA substrate other than the one containing the ribozyme) following a specific ribonucleotide triplet by transesterification of a 3',5'-phosphodiester to a 2',3'-cyclic phosphodiester. Without wishing to be bound by theory, this catalytic activity is believed to require the presence of a specific, highly conserved sequence in the catalytic region of the ribozyme.

[0255] Modifications in ribozyme structures also encompass the substitution or replacement of various non-core portions of molecules with non-nucleotide molecules.For example, Benseler et al. (J. Am. Chem. Soc. (1993) 115:8483-8484) disclosed a hammerhead-like molecule, in which two base pairs of stem II and all four nucleotides of loop II were replaced with non-nucleoside linkers based on hexaethylene glycol, propanediol, bis(triethylene glycol) phosphate, tris(propanediol) bisphosphate, or bis(propanediol) phosphate.Ma et al. (Biochem. (1993) 32:1751-1758; Nucleic Acids Res. (1993) 21:2585-2589) replaced the 6-nucleotide loop of the TAR ribozyme hairpin with a non-nucleotide linker based on ethylene glycol. Thomson et al. (Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear non-nucleotide linkers of 13, 17, and 19 atoms in length.

[0256] Ribozyme oligonucleotides can be prepared using well-known methods (see, for example, PCT publications WO9118624; WO9413688; WO9201806; and WO92 / 07065; and U.S. Patents 5,436,143 and 5,650,502) or purchased from commercial sources (e.g., US Biochemicals), and, if desired, can incorporate nucleotide analogs to increase the resistance of the oligonucleotide to degradation by cellular nucleases. Ribozymes can be synthesized in any known manner, for example, by using a commercially available synthesizer (e.g., manufactured by Applied Biosystems, Inc. or Milligen). Ribozymes can also be produced in recombinant vectors by conventional means. See Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current Edition). Ribozyme RNA sequences can be synthesized in conventional ways, for example, by using RNA polymerases such as T7 or SP6.

[0257] n. Guide nucleic acid In some embodiments, the oligonucleotide is a guide nucleic acid, for example, a guide RNA (gRNA) molecule. Generally, a guide RNA is a small synthetic RNA molecule composed of (1) a scaffold sequence that binds to a nucleic acid programmable DNA-binding protein (napDNAbp), such as Cas9, and (2) a nucleotide spacer portion that defines a DNA target sequence (e.g., a genomic DNA target) to which the gRNA binds to bring the nucleic acid programmable DNA-binding protein close to the DNA target sequence. In some embodiments, the napDNAbp is a nucleic acid programmable protein that forms a complex with (e.g., binds to or associates with) one or more RNA(s) that target the nucleic acid programmable protein to a target DNA sequence (e.g., a target genomic DNA sequence). In some embodiments, a nucleic acid programmable nuclease, when in a complex with an RNA, is sometimes referred to as a nuclease:RNA complex. A guide RNA can exist as a complex of two or more RNAs or as a single RNA molecule.

[0258] A guide RNA (gRNA) that exists as a single RNA molecule is sometimes referred to as a single guide RNA (sgRNA), although gRNA is also used to refer to a guide RNA that exists either as a single molecule or as a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that shares homology with the target nucleic acid (i.e., directs the binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. In some embodiments, domain (2) is identical to or homologous to tracrRNA as provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference.

[0259] In some embodiments, the gRNA comprises two or more of domains (1) and (2) and is sometimes referred to as an extended gRNA. For example, the extended gRNA will bind to two or more Cas9 proteins and bind to the target nucleic acid in two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence complementary to the target site that mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity for the nuclease:RNA complex.In some embodiments, the nuclease capable of programming RNA is a (CRISPR-related system) Cas9 endonuclease, e.g., Cas9 (Csn1) from Streptococcus pyogenes (e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE,Proc.Natl.Acad.Sci.USA98:4658-4663(2001);“CRISPR RNA maturation by See "trans-encoded small RNA and host factor RNase III." Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna JA, Charpentier E. Science 337:816-821 (2012) (the entire contents of each of which are incorporated herein by reference).

[0260] o. Splice-altering oligonucleotides In some embodiments, the oligonucleotides of the present disclosure (e.g., antisense oligonucleotides, including morpholinos) target splicing. In some embodiments, the oligonucleotides target splicing by inducing exon skipping and restoring the reading frame within the gene. As a non-limiting example, the oligonucleotides may induce skipping of exons encoding frameshift mutations and / or exons encoding premature stop codons. In some embodiments, the oligonucleotides may induce exon skipping by blocking spliceosome recognition of splice sites. In some embodiments, exon skipping results in a truncated but functional protein compared to a reference protein (e.g., the truncated but functional DMD protein described below). In some embodiments, the oligonucleotides promote the inclusion of a specific exon (e.g., exon 7 of the SMN2 gene described below). In some embodiments, the oligonucleotides may induce exon inclusion by targeting splice site inhibitory sequences. RNA splicing has been implicated in muscle diseases, including Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA).

[0261] Alterations (e.g., deletions, point mutations, and duplications) in the gene encoding dystrophin (DMD) cause DMD. These alterations can lead to frameshift mutations and / or nonsense mutations. In some embodiments, the oligonucleotides of the present disclosure promote skipping of one or more DMD exons (e.g., exon 8, exon 43, exon 44, exon 45, exon 50, exon 51, exon 52, exon 53, and / or exon 55), resulting in a functional truncated protein. For examples, see U.S. Patent No. 8,486,907, published July 16, 2013, and U.S. Patent No. 20140275212, published September 18, 2014.

[0262] In SMA, there is a loss of functional SMN1. Although the SMN2 gene is a paralog to SMN1, alternative splicing of the SMN2 gene primarily leads to skipping of exon 7 and the subsequent production of a truncated SMN protein that cannot compensate for the loss of SMN1. In some embodiments, the oligonucleotides of the present disclosure promote the inclusion of SMN2 exon 7. In some embodiments, the oligonucleotides are antisense oligonucleotides that target SMN2 splice site inhibitory sequences (see, e.g., U.S. Patent No. 7,838,657, published November 23, 2010).

[0263] p. multimer In some embodiments, the molecular payload may comprise a multimer (e.g., a concatemer) of two or more oligonucleotides connected by a linker. Thus, in some embodiments, the oligonucleotide loading of the complex / conjugate can be increased beyond the available linking sites on the targeting agent (e.g., available thiol sites on an antibody), or can be 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).

[0264] In some embodiments, a multimer comprises two or more oligonucleotides linked together by a cleavable linker. However, in some embodiments, a multimer comprises two or more oligonucleotides linked together by a non-cleavable linker. In some embodiments, a multimer comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, or more oligonucleotides linked together. In some embodiments, a multimer comprises 2-5, 2-10, or 4-20 oligonucleotides linked together.

[0265] 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.

[0266] Further examples of multimers that may be used in the conjugates provided herein are described, for example, in U.S. Patent Application No. 2015 / 0315588 A1, published November 5, 2015, entitled "Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers"; U.S. Patent Application No. 2015 / 0247141 A1, published September 3, 2015, entitled "Multimeric Oligonucleotide Compounds"; U.S. Patent Application No. US 2011 / 0158937 A1, published June 30, 2011, entitled "Immunostimulatory Oligonucleotide Multimers"; and U.S. Patent Application No. US 2011 / 0158937 A1, published December 2, 1997, entitled "Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers, Targeting Nucleic Acids Comprising Mixed Sequences of Purines and No. 5,693,773, entitled "Pyrimidines," the contents of each of which are incorporated herein by reference in their entirety.

[0267] ii.Small molecules: Any suitable small molecule may be used as a molecular payload, as described herein. Non-limiting examples are provided below for selected genes in Table 1.

[0268] DMPK / DM1 In some embodiments, for example, for the treatment of DM, the small molecule is as described in U.S. Patent Application Publication No. 2016052914A1, published February 25, 2016, entitled "Compounds And Methods For Myotonic Dystrophy Therapy." Further examples of small molecule payloads are provided in Lopez-Morato M, et al., "Small Molecules Which Improve Pathogenesis of Myotonic Dystrophy Type 1," (Review) Front. Neurol., 18 May 2018. For example, in some embodiments, the small molecule is an MBNL1 upregulator such as phenylbutazone, ketoprofen, ISOX, or vorinostat. In some embodiments, the small molecule is an H-Ras pathway inhibitor such as manumicin A. In some embodiments, the small molecule is a protein kinase modulator such as Ro-318220, C16, C51, metformin, AICAR, lithium chloride, TDZD-8, or Bio. In some embodiments, the small molecule is a plant alkaloid such as harmine. In some embodiments, the small molecule is a transcription inhibitor such as pentamidine, propamidine, heptamidiine, or actinomycin D.In some embodiments, the small molecule is an inhibitor of glycogen synthase kinase 3 beta (GSK3B), as disclosed, e.g., in Jones K, et al., GSK3β mediates muscle pathology in myotonic dystrophy. J Clin Invest. 2012 Dec;122(12):4461-72; and Wei C, et al., GSK3β is a new therapeutic target for myotonic dystrophy type 1. Rare Dis. 2013;1:e26555; and Palomo V, et al., Subtly Modulating Glycogen Synthase Kinase 3 β: Allosteric Inhibitor Development and Their Potential for the Treatment of Chronic Diseases. J Med Chem. 2017 Jun 22;60(12):4983-5001 (the entire contents of each of which are incorporated herein by reference). In some embodiments, the small molecule is a substituted pyrido[2,3-d]pyrimidine and pentamidine-like compound as disclosed in Gonzalez AL, et al., In silico discovery of substituted pyrido[2,3-d]pyrimidines and pentamidine-like compounds with biological activity in myotonic dystrophy models. PLoS One. 2017 Jun 5;12(6):e0178931, the entire contents of which are incorporated herein by reference.In some embodiments, the small molecule is an MBNL1 modulator, e.g., as disclosed in Zhange F, et al., A flow cytometry-based screen identifies MBNL1 modulators that rescue splicing defects in myotonic dystrophy type I. Hum Mol Genet. 2017 Aug 15;26(16):3056-3068, the entire contents of which are incorporated herein by reference.

[0269] DUX4 / FSHD In some embodiments, by way of example, for the treatment of FSHD, the small molecule payload is as described in U.S. Patent Application Publication No. 20170340606, published November 30, 2017, entitled "METHODS OF TREATING MUSCULAR DYSTROPHY," or as described in U.S. Patent Application Publication No. 20180050043, published February 22, 2018, entitled "INHIBITION OF DUX4 EXPRESSION USING BROMODOMAIN AND EXTRA-TERMINAL DOMAIN PROTEIN INHIBITORS (BETi)." Further examples of small molecule payloads are provided in Bosnakovski, D., et al., High-throughput screening identifies inhibitors of DUX4-induced myoblast toxicity, Skelet Muscle, February 2014, and Choi, S., et al., "Transcriptional Inhibitors Identified in a 160,000-Compound Small-Molecule DUX4 Viability Screen," Journal of Biomolecular Screening, 2016. For example, in some embodiments, the small molecule is a transcription inhibitor such as SHC351, SHC540, or SHC572. In some embodiments, the small molecule is STR00316, which increases the production or activity of another protein, such as an integrin. In some embodiments, the small molecule is a bromodomain inhibitor (BETi), such as JQ1, PF1-1, I-BET-762, I-BET-151, RVX-208, or CPI-0610.

[0270] DNM / CNM In some embodiments, e.g., for the treatment of CNM, the small molecule is as described in U.S. Patent Application Publication No. 20160264976, published on September 15, 2016, entitled "DYNAMIN 2 INHIBITOR FOR TREATMENT OF CENTRONUCLEAR MYOPATHIES." For example, in some embodiments, the small molecule is selected from the group consisting of 3-hydroxynaphthalene-2-carboxylic acid (3,4-dihydroxybenzylidene) hydrazide, 3-hydroxy-N'-[(2,4,5-trihydroxyphenyl)methylidene]naphthalene-2-carbohydro-azide. In some embodiments, the small molecule is as described in U.S. Patent Application Publication No. 20180000762, published on January 4, 2018, entitled "COMPOSITION AND METHOD FOR MUSCLE REPAIR AND REGENERATION." In some embodiments, the small molecule is a retinoid receptor agonist such as 4-[(E)-2-[5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-3-(1H-pyrazol-1-ylmethyl-)-2-naphthalenyl]-ethenyl]-benzoic acid. In some embodiments, the small molecule is as described in U.S. Patent Application Publication No. 20170119748, published May 4, 2017, entitled "METHODS, COMPOUNDS, AND COMPOSITIONS FOR THE TREATMENT OF MUSCULOSKELETAL DISEASES." The entire contents of each of these publications are incorporated herein.

[0271] Pompe disease In some embodiments, by way of example, for the treatment of Pompe disease, the small molecule is a 1-deoxynojirimycin (DNJ) derivative, such as N-butyl-DNJ, N-methyl-DNJ, or N-cyclopropylmethyl-DNJ, as described in U.S. Patent Application Publication No. 20160051528, published February 25, 2016, entitled "METHOD FOR TREATMENT OF POMPE DISEASE USING 1-DEOXYNOJIRIMYCIN DERIVATIVES." In some embodiments, the small molecule DNJ derivative is used as a molecular chaperone to increase the activity of GAA. In some embodiments, the non-inhibitory acid alpha glucosidase chaperone ML247 small molecule is utilized as described in Marugan, et al., "Discovery, SAR, and Biological Evaluation of a Non-Inhibitory Chaperone for Acid Alpha Glucosidase," published in Probe Reports from NIH Molecular Libraries in December 2011. For example, the small molecule chaperone ML247 is utilized to enhance the activity of PD-associated GAA alleles or wild-type GAA alleles. The entire contents of each of these publications are incorporated herein by reference.

[0272] FXN / Friedreich's ataxia In some embodiments, for example, for the treatment of Friedreich's ataxia, the small molecule is as described in Herman D. et al. "Histone deacetylase inhibitors reverse gene silencing in Friedreich's ataxia." Nat Chem Biol. 2006;2:551-558. In some embodiments, the small molecule is as described in Rai, M. et al. "HDAC inhibitors correct frataxin deficiency in a Friedreich ataxia mouse model." PLoS One. 2008 Apr 9;3(4):e1958. Further examples of small molecule payloads are provided in Richardson, TE et al., "Therapeutic strategies in Friedreich's ataxia," Brain Res. 2013 Jun 13; 1514: 91-97; Zeier Z et al. "Bromodomain inhibitors regulate the C9ORF72 locus in ALS," Exp Neurol. 2015 Sep; 271: 241-50; and Gottesfeld JM "Small molecules affecting transcription in Friedreich ataxia." Pharmacol Ther. 2007 Nov; 116(2): 236-48. For example, in some embodiments, the small molecule is an inhibitor of histone deacetylase, e.g., BML-210 and Compound 106. In some embodiments, the small molecule is 17β-estradiol or methylene blue. In some embodiments, the small molecule targets (e.g., binds to) disease-associated repeats and / or R-loops. In some embodiments, the small molecule is as described in WO 2004 / 003565, published 1 / 8 / 2004, "A screening method and compounds for treating friedreich ataxia."In some embodiments, the small molecule is a glutathione peroxidase mimetic.

[0273] DMD / Dystrophinopathy In some embodiments, the small molecule enhances exon skipping of mRNA expression from a mutant DMD allele. In some embodiments, the small molecule is as described in U.S. Patent Application Publication US20140080896A1, published March 20, 2014, entitled "IDENTIFICATION OF SMALL MOLECULES THAT FACILITATE THERAPEUTIC EXON SKIPPING." Further examples of small molecule payloads are provided in U.S. Patent No. 9,982,260, issued May 29, 2018, entitled "Identification of structurally similar small molecules that enhance therapeutic exon skipping." For example, in some embodiments, the small molecule is an exon skipping enhancer such as perphenazine, flupenthixol, zuclopenthixol, or corynanthine. In some embodiments, the exon skipping small molecule enhancer inhibits ryanodine receptors or calmodulin. In some embodiments, the small molecule is an H-Ras pathway inhibitor, such as manumicin A. In some embodiments, the small molecule is a stop codon suppressor and desensitizes the ribosome to premature stop codons. In some embodiments, the small molecule is ataluren, as described in McElroy SP et al. "A Lack of Premature Termination Codon Read Through Efficacy of PTC124 (Ataluren) in a Diverse Array of Reporter Assays," PLOS Biology, published June 25, 2013. In some embodiments, the small molecule is a corticosteroid, as described, for example, in Manzur, AY et al. "Glucocorticoid corticosteroids for Duchenne muscular dystrophy."In some embodiments, the small molecule upregulates the expression and / or activity of a gene that can replace the function of dystrophin, such as utrophin. In some embodiments, the utrophin modulator is as described in International Publication No. WO2007091106, entitled "Treatment of Duchenne Muscular Dystrophy," published on August 16, 2007, and / or International Publication No. WO / 2017 / 168151, entitled "Composition for the Treatment of Duchenne Muscular Dystrophy," published on October 5, 2017.

[0274] MYH7 / hypertrophic cardiomyopathy In some embodiments, the small molecule is a hypomethylating agent such as 5-azacytidine or 5-aza-2'-deoxycytidine that modulates expression of the MYH7 gene, such as U.S. Patent Application Publication No. 20160106771, published April 21, 2016, entitled Therapies for Cardiomyopathy; in some embodiments, the small molecule is a JAK-STAT inhibitor such as nifuroxazide, ketoprofen, sulfasalazine, 5,15-diphenylporphyrin, or AG490, such as U.S. Patent Application Publication No. 20180185478, published July 5, 2018, entitled Treatment for Myopathy; in some embodiments, the small molecule is a JAK-STAT inhibitor such as Tang, W., et al. "Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels," Front. Physiol. 2016 (7): 659, the entire contents of both of which are incorporated herein by reference, para-Nitroblebbistatin, which reduces the force of myosin contraction while not altering ADP dissociation.

[0275] iii. Peptides / proteins Any suitable peptide or protein may be used as a molecular payload as described herein. In some embodiments, the protein is an enzyme (e.g., acid alpha-glucosidase, e.g., encoded by the GAA gene). These peptides or proteins may be produced, synthesized, and / or derivatized using several methodologies, such as phage-displayed peptide libraries, one-bead-one-compound peptide libraries, or position-scanning synthetic peptide combinatorial libraries. Exemplary methodologies have been characterized in the art and are incorporated by reference (Gray, BP and Brown, KC "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides" Chem Rev. 2014, 114:2, 1020-1081; Samoylova, TI and Smith, BF "Elucidation of muscle-binding peptides by phage display screening." Muscle Nerve, 1999, 22:4, 460-6).

[0276] Non-limiting examples are provided below for selected genes in Table 1.

[0277] DMPK / DM1 The peptide or protein payload may correspond to a sequence of a protein that preferentially binds to a nucleic acid (e.g., a disease-associated repeat) or a protein (e.g., MBNL1 found in muscle cells), e.g., for the treatment of DM1. In some embodiments, the peptide is as described in U.S. Patent Application 2018 / 0021449, published 1 / 25 / 2018, "Antisense conjugates for decreasing expression of DMPK." In some embodiments, the peptide is as described in Garcia-Lopez et al., "In vivo discovery of a peptide that prevents CUG-RNA hairpin formation and reverses RNA toxicity in myotonic dystrophy models," PNAS July 19, 2011. 108 (29) 11866-11871. In some embodiments, the peptide or protein may target (e.g., bind to) a disease-associated repeat, e.g., an RNA CUG repeat expansion.

[0278] In some embodiments, for example, for the treatment of DM1, the peptide or protein comprises an MBNL protein, e.g., a fragment of MBNL1. In some embodiments, the peptide or protein comprises at least one zinc finger. In some embodiments, the peptide or protein may comprise about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. The peptide or protein may comprise naturally occurring amino acids, e.g., 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 peptide may be linear; in other embodiments, the peptide may be cyclic, e.g., bicyclic.

[0279] DUX4 / FSHD In some embodiments, for example, for the treatment of FSHD, a peptide or protein may bind to a DME1 or DME2 enhancer to inhibit DUX4 expression, for example, by blocking binding of an activator.

[0280] DNM2 / CNM In some embodiments, by way of example, for the treatment of CNM, the peptide is a dynamin inhibitor peptide with the amino acid sequence QVPSRPNRAP, as described in U.S. Patent Application Publication No. 20160264976, published September 15, 2016, entitled "DYNAMIN 2 INHIBITOR FOR TREATMENT OF CENTRONUCLEAR MYOPATHIES."

[0281] Pompe disease In some embodiments, by way of example, for the treatment of Pompe disease, the molecular payload is a protein or enzyme such as acid alpha-glucosidase or wild-type GAA protein or an active fragment thereof, as in U.S. Patent Application Publication No. 20160346363, published December 1, 2016, entitled "METHODS AND ORAL FORMULATIONS FOR ENZYME REPLACEMENT THERAPY OF HUMAN LYSOSOMAL AND METABOLIC DISEASES," U.S. Patent Application Publication No. 20160279254, published September 29, 2016, entitled "METHODS AND MATERIALS FOR TREATMENT OF POMPE'S DISEASE," or U.S. Patent Application Publication No. 20130243746, published September 19, 2013, entitled "METHODS AND MATERIALS FOR TREATMENT OF POMPE'S DISEASE." In some embodiments, the acid alpha-glucosidase or wild-type GAA protein increases GAA activity in a subject. In some embodiments, the acid alpha-glucosidase or wild-type GAA protein is encoded by a GAA gene.

[0282] ACVR1 / FOP In some embodiments, for example, for the treatment of FOP, the peptide or protein is a BMP inhibitor, such as regulatory SMAD6 and 7 or a fragment thereof. Additional examples of peptides or proteins are included in Cappato, S. et al. "The Horizon of a Therapy for Rare Genetic Diseases: A "Druggable" Future for Fibrodysplasia Ossificans Progressiva" Int. J. Mol. Sci. 2018, 19(4), 989. The entire contents of each of the above are incorporated herein by reference.

[0283] FXN / Friedreich's ataxia In some embodiments, for example, for the treatment of Friedreich's ataxia, the peptide is as described in U.S. Patent No. 8,815,230, filed 8 / 30 / 2010, entitled "Methods for treating Friedreich's ataxia with interferon gamma." In some embodiments, the peptide is as described in Britti, E. et al., "Frataxin-deficient neurons and mice models of Friedreich ataxia are improved by TAT-MTScs-FXN treatment." J Cell Mol Med. 2018 Feb;22(2):834-848. In some embodiments, the peptide is as described in Zhao, H. et al., "Peptide SS-31 upregulates frataxin expression and improves the quality of mitochondria: implications in the treatment of Friedreich ataxia," Sci Rep. 2017 Aug 29;7(1):9840. In some embodiments, the peptide is as described in Vyas, PM et al. "A TAT-frataxin fusion protein increases lifespan and cardiac function in a conditional Friedreich's ataxia mouse model," Hum Mol Genet. 2012 Mar 15;21(6):1230-47. In some embodiments, the peptide or protein may target (e.g., bind to) a disease-associated repeat, e.g., a GAA repeat expansion.

[0284] DMD / Dystrophinopathy In some embodiments, for example, for the treatment of dystrophinopathy, such as Duchenne muscular dystrophy, the peptide may facilitate exon skipping in the mRNA expressed from mutant DMD alleles.In some embodiments, the peptide may promote the expression of functional dystrophin and / or the expression of a protein that can function in place of dystrophin.In some embodiments, the payload is a protein that is a functional fragment of dystrophin, for example, the amino acid segment of functional dystrophin protein.

[0285] iv. Nucleic acid constructs Any suitable gene expression construct may be used as a molecular payload as described herein. In some embodiments, the gene expression construct may be a vector or a cDNA fragment. In some embodiments, the gene expression construct may be messenger RNA (mRNA). In some embodiments, the mRNA used herein may be modified mRNA, e.g., as described in U.S. Patent 8,710,200, issued April 24, 2014, entitled "Engineered nucleic acids encoding a modified erythropoietin and their expression." In some embodiments, the mRNA may include a 5' methyl cap. In some embodiments, the mRNA may include a polyA tail, optionally up to 160 nucleotides in length. The gene expression construct may encode a protein sequence deficient in a muscle disease. In some embodiments, the gene expression construct may be expressed in the nucleus of a muscle cell, e.g., overexpressed. In some embodiments, the gene expression construct may encode a gene deficient in a muscle disease. In some embodiments, the gene expression construct encodes a protein comprising at least one zinc finger. In some embodiments, the gene expression construct encodes a protein that binds to a gene in Table 1. In some embodiments, the gene expression construct encodes a protein that leads to reduced expression of a protein (e.g., a mutant protein) encoded by a gene in Table 1. In some embodiments, the gene expression construct encodes a gene-editing enzyme.Additional examples of nucleic acid constructs that may be used as molecular payloads are provided in International Patent Application Publication WO2017152149A1, published September 19, 2017, entitled "CLOSED-ENDED LINEAR DUPLEX DNA FOR NON-VIRAL GENE TRANSFER"; U.S. Patent No. 8,853,377B2, issued October 7, 2014, entitled "MRNA FOR USE IN TREATMENT OF HUMAN GENETIC DISEASES"; and U.S. Patent No. US8822663B2, issued September 2, 2014, entitled "ENGINEERED NUCLEIC ACIDS AND METHODS OF USE THEREOF," the contents of each of which are incorporated herein by reference in their entirety.

[0286] Further non-limiting examples are provided below for selected genes / diseases in Table 1.

[0287] DMPK / DM1 In some embodiments, eg, for the treatment of DM, the gene expression construct encodes an MBNL protein, eg, MBNL1. DUX4 / FSHD In some embodiments, e.g., for the treatment of FSHD, the gene expression construct encodes an oligonucleotide (e.g., an shRNA targeting DUX4) or a protein that downregulates the expression of DUX4 (e.g., a peptide or protein that binds to the DME1 or DME2 enhancer to inhibit DUX4 expression, e.g., by blocking the binding of an activator). DNM2 / CNM In some embodiments, for example, for the treatment of CNM1, the gene expression construct may encode a protein sequence that downregulates the expression of mutant DNM2 protein or expresses wild-type DNM2. In some embodiments, the gene expression construct encodes an oligonucleotide (e.g., shRNA) that inhibits the expression of DNM2. However, in some embodiments, the expression construct encodes a spliceosome-mediated RNA trans-splicing component that can be used to initialize mutant DNM2-mRNA, as described in Trochet D., et al., Reprogramming the Dynamin 2 mRNA by Spliceosome-mediated RNA Trans-splicing Mol Ther Nucleic Acids. 2016 Sep; 5(9): e36 (the contents of which are incorporated herein by reference).

[0288] Pompe disease In some embodiments, for example, for the treatment of Pompe disease, the gene expression construct encodes a wild-type GAA protein. The gene expression construct may encode a protein sequence that leads to reduced expression of the ACVR1 gene or reduced activity of the GYS1 protein. In some embodiments, for example, for the treatment of Pompe disease, the gene expression construct encodes an oligonucleotide (e.g., shRNA) that inhibits the expression of GYS1.

[0289] ACVR1 / FOP The gene expression construct may encode a protein sequence that leads to decreased expression of the ACVR1 gene or decreased activity of the ACVR1 protein. In some embodiments, the gene expression construct encodes a protein, such as a histone deacetylase, that leads to a reduction in the expression of an epigenetic regulator that negatively regulates ACVR1 expression. In some embodiments, the gene expression construct encodes an oligonucleotide (e.g., an shRNA) that inhibits ACVR1 expression.

[0290] FXN / Friedreich's ataxia The gene expression construct may encode a protein sequence that leads to increased expression of frataxin. In some embodiments, the gene expression construct may be expressed, e.g., overexpressed, in the nucleus of a muscle cell. In some embodiments, the gene expression construct encodes frataxin. In some embodiments, the gene expression construct encodes a protein, e.g., a histone deacetylase, that inhibits the function of an epigenetic regulator that negatively regulates the expression of FXN. In some embodiments, the gene expression construct encodes a protein that binds to a disease-associated repeat expansion of GAA trinucleotide. In some embodiments, the gene expression construct encodes a protein, e.g., a histone deacetylase, that leads to a reduction in the expression of an epigenetic regulator that negatively regulates the expression of FXN. In some embodiments, the gene expression construct encodes a gene editing enzyme. In some embodiments, the gene expression construct encodes erythropoietin (see, e.g., Miller, JL et al, "Erythropoietin and small molecule agonists of the tissue-protective erythropoietin receptor increase FXN expression in neuronal cells in vitro and in FXN-deficient KIKO mice in vivo," Neuropharmacology. 2017 Sep 1;123:34-45). In some embodiments, the gene expression construct encodes interferon gamma (see, e.g., U.S. Patent No. 8,815,230, 8 / 30 / 2010 application, "Methods for treating Friedreich's ataxia with interferon gamma").

[0291] DMD / Dystrophinopathy The gene expression construct may encode the sequence of a dystrophin protein, a dystrophin fragment, a mini-dystrophin, a utrophin protein, or any protein that shares a common function with dystrophin. In some embodiments, the gene expression construct may be expressed, e.g., overexpressed, in the nucleus of a muscle cell. In some embodiments, the gene expression construct encodes a protein that includes at least one zinc finger. In some embodiments, the gene expression construct encodes a protein that promotes the expression of dystrophin or a protein that shares a function with dystrophin, e.g., utrophin. In some embodiments, the gene expression construct encodes a gene editing enzyme. In some embodiments, the gene expression construct is as described in the expression cassettes disclosed in U.S. Patent Application Publication US20170368198A1, published December 28, 2017, entitled "Optimized mini-dystrophin genes and expression cassettes and their use"; Duan D. "Myodys, a full-length dystrophin plasmid vector for Duchenne and Becker muscular dystrophy gene therapy." Curr Opin Mol Ther 2008;10:86-94; and Tang, Y. et al., "AAV-directed muscular dystrophy gene therapy" Expert Opin Biol Ther. 2010 Mar;10(3):395-408, the entire contents of each of which are incorporated herein by reference.

[0292] C. Linker The conjugates described herein generally include a linker connecting the muscle-targeting agent to the molecular payload. The linker includes at least one covalent bond. In some embodiments, the linker may be a single bond, such as a disulfide bond or disulfide bridge, connecting the muscle-targeting agent to the molecular payload. However, in some embodiments, the linker may connect the muscle-targeting agent to the molecular payload through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker. Linkers are generally stable in vitro and in vivo and may be stable in certain cellular environments. In addition, linkers generally do not negatively affect the functional properties of either the muscle-targeting agent or the molecular payload. Examples and methods for 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).

[0293] The linker precursor will typically contain two different reactive species that can attach to both the muscle-targeting agent and the molecular payload. In some embodiments, the two different reactive species may be nucleophiles and / or electrophiles. In some embodiments, the linker is connected to the muscle-targeting agent via conjugation to a lysine or cysteine ​​residue of the muscle-targeting agent. In some embodiments, the linker is connected to the cysteine ​​residue of the muscle-targeting agent via a maleimide-containing linker, where the maleimide-containing linker optionally contains a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some embodiments, the linker is connected to the cysteine ​​residue of the muscle-targeting agent or to a thiol-functionalized molecular payload via a 3-arylpropionitrile functional group. In some embodiments, the linker is connected to the muscle-targeting agent and / or molecular payload via an amide bond, hydrazide, triazole, thioether, or disulfide bond.

[0294] i. Cleavable linker The cleavable linker may be a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker, which are generally only cleavable intracellularly and are preferably stable in an extracellular environment, e.g., outside of a muscle cell.

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

[0296] A pH-sensitive linker is a covalent linkage that is 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.

[0297] 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.

[0298] In some embodiments, the linker is a Val-cit linker (e.g., as described in US Patent 6,214,345, incorporated herein by reference). In some embodiments, the val-cit linker prior to conjugation has the following structure: [ka]

[0299] In some embodiments, the val-cit linker after conjugation has the following structure: [ka]

[0300] 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 (SEQ ID NO: 15), a thioether, biotin, 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 a muscle-targeting agent comprising an LPXT sequence to (G). n(See, e.g., Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1):1-10.) In some embodiments, the linker comprises the sequence LPXTG (SEQ ID NO: 16), where X is any amino acid.

[0301] 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.

[0302] iii. Linker conjugation In some embodiments, the linker is connected to the muscle-targeting agent and / or molecular payload via a phosphate, thioether, ether, carbon-carbon, or amide bond. In some embodiments, the linker is connected to the oligonucleotide through a phosphate or phosphorothioate group, e.g., a phosphate at the end of the oligonucleotide backbone. In some embodiments, the linker is connected to the muscle-targeting agent, e.g., an antibody, through a lysine or cysteine ​​residue present on the muscle-targeting agent.

[0303] In some embodiments, the linker is connected to the muscle-targeting agent and / or molecular payload via a cycloaddition reaction between an azide and an alkyne to form a triazole, where the azide and alkyne may be located on the muscle-targeting agent, molecular payload, or linker. In some embodiments, the alkyne may be a cyclic alkyne, such as a cyclooctyne. In some embodiments, the alkyne may be a bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctane is as described in International Patent Application Publication WO2011136645, entitled "Fused Cyclooctyne Compounds and Their Use in Metal-free Click Reactions," published November 3, 2011. In some embodiments, the azide may be an azide-containing sugar or carbohydrate molecule. In some embodiments, the azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the azide-containing sugar or carbohydrate molecule is as described in International Patent Application Publication WO2016170186, published October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase."In some embodiments, the cycloaddition reaction between an azide and an alkyne to form a triazole (wherein the azide and alkyne can be located on the muscle-targeting agent, molecular payload, or linker) is as described in International Patent Application Publication No. WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or International Patent Application Publication No. WO2016170186, published October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase."

[0304] In some embodiments, the linker further comprises a spacer, e.g., a polyethylene glycol spacer or an acyl / carbamoyl sulfamide spacer, e.g., a HydraSpace™ spacer. In some embodiments, the spacer is as described in Verkade, JMM et al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturing Ability, Stabilization Ability, and Therapeutic Index of Antibody-Drug Conjugates," Antibodies, 2018, 7, 12.

[0305] In some embodiments, the linker is connected to the muscle-targeting agent and / or molecular payload via a Diels-Alder reaction between a dienophile and a diene / hetero-diene, where the dienophile and diene / hetero-diene may be located on the muscle-targeting agent, molecular payload, or linker. In some embodiments, the linker is connected to the muscle-targeting agent and / or molecular payload via other pericyclic reactions, such as an ene reaction. In some embodiments, the linker is connected to the muscle-targeting agent and / or molecular payload via an amide, thioamide, or sulfonamide coupling reaction. In some embodiments, the linker is connected to the muscle-targeting agent and / or molecular payload via a condensation reaction to form an oxime, hydrazone, or semicarbazide group present between the linker and the muscle-targeting agent and / or molecular payload.

[0306] In some embodiments, the linker is connected to the muscle-targeting agent and / or molecular payload via a conjugate addition reaction between a nucleophile (e.g., an amine group or a hydroxyl group) and an electrophile (e.g., a carboxylic acid or an aldehyde). In some embodiments, the nucleophile may be present on the linker, and the electrophile may be present on the muscle-targeting agent or molecular payload, prior to the reaction between the linker and the muscle-targeting agent or molecular payload. In some embodiments, the electrophile may be present on the linker, and the nucleophile may be present on the muscle-targeting agent or molecular payload, prior to the reaction between the linker and the muscle-targeting agent or molecular payload. In some embodiments, the electrophile may be an azide, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimidyl ester, sulfosuccinimidyl ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center, and / or activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilido group, or a thiol group.

[0307] D. Examples of antibody-molecular payload conjugates Another aspect of the present disclosure provides a conjugate comprising any one of the muscle-targeting agents described herein (e.g., an anti-transferrin receptor antibody) covalently linked to any of the molecular payloads described herein (e.g., an oligonucleotide). In some embodiments, the muscle-targeting agent (e.g., an anti-transferrin receptor antibody) is covalently linked to the molecular payload (e.g., an oligonucleotide) via a linker. Any of the linkers described herein may be used. In some embodiments, the linker is linked to the 5' end, 3' end, or internally of the oligonucleotide. In some embodiments, the linker is linked to the antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody).

[0308] An exemplary structure of a conjugate comprising an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker is provided below: [ka] wherein the linker is attached to the 5' end, 3' end, or internally of the oligonucleotide, and wherein the linker is attached to the antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody).

[0309] It should be understood that antibodies can be linked to oligonucleotides with various stoichiometries; this characteristic is sometimes referred to as the drug-antibody ratio (DAR), where "drug" is the oligonucleotide. In some embodiments, one oligonucleotide is linked to the antibody (DAR=1). In some embodiments, two oligonucleotides are linked to the antibody (DAR=2). In some embodiments, three oligonucleotides are linked to the antibody (DAR=3). In some embodiments, four oligonucleotides are linked to the antibody (DAR=4). In some embodiments, a mixture of different conjugates, each having a different DAR, is provided. In some embodiments, the average DAR of the conjugates in such a mixture may range from 1 to 3, 1 to 4, 1 to 5, or more. The DAR may be increased by conjugating oligonucleotides to various sites on the antibody and / or by conjugating multimers to more than one site on the antibody. For example, a DAR of 2 may be achieved by conjugating a single oligonucleotide to two different sites on the antibody or by conjugating a dimeric oligonucleotide to a single site on the antibody.

[0310] In some embodiments, the conjugates described herein comprise an anti-transferrin receptor antibody (e.g., an antibody as described herein, or any variant thereof) covalently linked to an oligonucleotide. In some embodiments, the conjugates described herein comprise an anti-transferrin receptor antibody (e.g., an antibody as described herein, or any variant thereof) covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the 5' end, 3' end, or internally of the oligonucleotide. In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an antibody as described herein, or any variant thereof) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody).

[0311] In some embodiments, the conjugates described herein comprise an anti-transferrin receptor antibody covalently linked to an oligonucleotide, wherein the anti-transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to those shown in Table 1.1; and CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 1.1.

[0312] In some embodiments, the conjugates described herein comprise an anti-transferrin receptor antibody covalently linked to an oligonucleotide, wherein the anti-transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 33 and a VL having the amino acid sequence of SEQ ID NO: 34. In some embodiments, the conjugates described herein comprise an anti-transferrin receptor antibody covalently linked to an oligonucleotide, wherein the anti-transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 35 and a VL having the amino acid sequence of SEQ ID NO: 36.

[0313] In some embodiments, the conjugates described herein comprise an anti-transferrin receptor antibody covalently linked to an oligonucleotide, wherein the anti-transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 39 and a light chain having the amino acid sequence of SEQ ID NO: 40. In some embodiments, the conjugates described herein comprise an anti-transferrin receptor antibody covalently linked to an oligonucleotide, wherein the anti-transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 41 and a light chain having the amino acid sequence of SEQ ID NO: 42.

[0314] In some embodiments, the conjugates described herein comprise an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the anti-transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to those shown in Table 1.1; and CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 1.1.

[0315] In some embodiments, a conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the anti-transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 33 and a VL having the amino acid sequence of SEQ ID NO: 34. In some embodiments, a conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the anti-transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 35 and a VL having the amino acid sequence of SEQ ID NO: 36.

[0316] In some embodiments, a conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the anti-transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 39 and a light chain having the amino acid sequence of SEQ ID NO: 40. In some embodiments, a conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker), wherein the anti-transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 41 and a light chain having the amino acid sequence of SEQ ID NO: 42.

[0317] In some embodiments, a conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the anti-transferrin receptor antibody comprises CDR-H1, CDR-H2, and CDR-H3 identical to CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1; and CDR-L1, CDR-L2, and CDR-L3 identical to CDR-L1, CDR-L2, and CDR-L3 shown in Table 1.1, and wherein the conjugate comprises the following structure: [ka] wherein the linker Val-cit linker is linked to the 5' end, 3' end, or internally of the oligonucleotide, and wherein the Val-cit linker is linked to an antibody (e.g., an antibody as described herein or any variant thereof) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody).

[0318] In some embodiments, a conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the anti-transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 33 and a VL having the amino acid sequence of SEQ ID NO: 34, and wherein the conjugate comprises the following structure: [ka] wherein the linker Val-cit linker is linked to the 5' end, 3' end, or internally of the oligonucleotide, and wherein the Val-cit linker is linked to an antibody (e.g., an antibody as described herein or any variant thereof) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody).

[0319] In some embodiments, a conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the anti-transferrin receptor antibody comprises a VH having the amino acid sequence of SEQ ID NO: 35 and a VL having the amino acid sequence of SEQ ID NO: 36, and wherein the conjugate comprises the following structure: [ka] wherein the linker Val-cit linker is linked to the 5' end, 3' end, or internally of the oligonucleotide, and wherein the Val-cit linker is linked to an antibody (e.g., an antibody as described herein or any variant thereof) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody).

[0320] In some embodiments, a conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the anti-transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:39 and a light chain having the amino acid sequence of SEQ ID NO:40, and wherein the conjugate comprises the following structure: [ka] wherein the linker Val-cit linker is linked to the 5' end, 3' end, or internally of the oligonucleotide, and wherein the Val-cit linker is linked to an antibody (e.g., an antibody as described herein or any variant thereof) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody).

[0321] In some embodiments, a conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the anti-transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO:41 and a light chain having the amino acid sequence of SEQ ID NO:42, and wherein the conjugate comprises the following structure: [ka] wherein the linker Val-cit linker is linked to the 5' end, 3' end, or internally of the oligonucleotide, and wherein the Val-cit linker is linked to an antibody (e.g., an antibody as described herein or any variant thereof) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody).

[0322] 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 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.

[0323] It should be understood that in some embodiments, a composition may individually include one or more components of the conjugates provided herein (e.g., a muscle-targeting agent, a linker, a molecular payload, or a precursor molecule of any one of these).

[0324] 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 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).

[0325] In some embodiments, the complex or a component thereof (e.g., an oligonucleotide or an antibody) is lyophilized to extend its shelf life and then brought into solution prior to use (e.g., administration to a subject). Consequently, the excipient in a composition comprising the complex or a component thereof described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0326] 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. In some embodiments, the route of administration is intramuscular parenteral administration.

[0327] 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 injectable 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 injectable 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.

[0328] In some embodiments, the composition may contain at least about 0.1% of the complex or its components, although the percentage of active ingredient(s) may be between about 1% and about 80% or more by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will be taken into account by those skilled in the art when preparing such pharmaceutical formulations. Therefore, different dosages and treatment regimens may be desired.

[0329] IV. Method of Use / Treatment Conjugates comprising a muscle-targeting agent covalently linked to a molecular payload as described herein are effective for targeting FOP. In some embodiments, the conjugates are effective for treating typical FOP or atypical FOP. In some embodiments, FOP is associated with mutations in the ACVR1 gene that lead to mutations in the ACVR1 protein, such as R206H, Q207E, G328R, G328W, G328E, G356D, R375P, and ΔP197-F198.

[0330] 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 a muscle disorder provided in Table 1.

[0331] Aspects of the present disclosure include methods involving administering to a subject an effective amount of a conjugate as described herein. In some embodiments, an effective amount of a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent covalently attached 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, intravenous administration can be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, 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.

[0332] Compositions for intravenous administration may contain a variety of carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). Water-soluble antibodies for intravenous injection may be administered by infusion, whereby a pharmaceutical formulation containing the antibody and a pharmaceutically acceptable excipient is infused. Pharmaceutically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. 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.

[0333] In some embodiments, pharmaceutical compositions comprising a conjugate comprising a muscle-targeting agent covalently attached to a molecular payload are administered via site-specific or localized delivery techniques, examples of which include an implanted depot source of the conjugate, a localized delivery catheter, a site-specific carrier, direct injection, or direct application.

[0334] 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 minimal 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.

[0335] Empirical considerations, such as the half-life of the conjugate in the subject, will generally contribute to determining the concentration of pharmaceutical composition used for treatment. Dosage frequency may be empirically determined and adjusted to maximize the efficacy of treatment.

[0336] Generally, for administration of any of the conjugates described herein, the initial candidate dosage may be about 1-100 mg / kg, or may be higher, depending on the factors described above, e.g., safety or efficacy. In some embodiments, treatment will be administered once. In some embodiments, treatment will be administered daily, biweekly, weekly, bimonthly, monthly, or at any time interval that provides maximum efficacy to the subject while minimizing safety risks. Generally, efficacy and treatment and safety risks may be monitored throughout the course of treatment.

[0337] The efficacy of treatment may be assessed using any suitable method. In some embodiments, the efficacy of treatment may be assessed by evaluation or observation of symptoms associated with FOP. In some embodiments, symptoms associated with FOP may include the gradual replacement of muscle tissue with bone, limited movement, loss of mobility, and / or difficulty breathing and eating.

[0338] In some embodiments, a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent covalently attached to a molecular payload described herein is administered to a subject at an effective concentration sufficient to inhibit target gene activity or expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% as compared to a control (e.g., a baseline level of gene expression prior to treatment).

[0339] In some embodiments, a single dose or administration to a subject of a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent covalently attached to a molecular payload described herein is sufficient to inhibit target gene activity or expression for at least 1-5 days, 1-10 days, 5-15 days, 10-20 days, 15-30 days, 20-40 days, 25-50 days, or longer. In some embodiments, a single dose or administration to a subject of a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent covalently attached to a molecular payload described herein is sufficient to inhibit target gene activity or expression for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks. In some embodiments, a single dose or administration to a subject of a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent covalently attached to a molecular payload described herein is sufficient to inhibit target gene activity or expression for at least 1, 2, 3, 4, 5, or 6 months.

[0340] In some embodiments, the pharmaceutical composition may include more than one conjugate comprising a muscle-targeting agent covalently attached to a molecular payload. In some embodiments, the pharmaceutical composition may further include any other suitable therapeutic agent for treating a subject, e.g., a human subject, having a muscle disease (e.g., a muscle disease provided in Table 1). In some embodiments, the other therapeutic agent may enhance or complement the effectiveness of the conjugates described herein. In some embodiments, the other therapeutic agent may function to treat a different condition or disease than the conjugates described herein.

[0341] example Example 1: Targeting DMPK with transfected antisense oligonucleotides Gapmer antisense oligonucleotides targeting both wild-type and mutant alleles of DMPK (DTX-P-060) were tested in vitro for their ability to reduce DMPK expression levels in immortalized cell lines. Briefly, Hepa1-6 cells were transfected with DTX-P-060 (100 nM) formulated in Lipofectamine 2000. DMPK expression levels were assessed 72 hours after transfection. A control experiment was also performed in which vehicle (phosphate-buffered saline) was delivered to Hepa1-6 cells in culture and the cells were maintained for 72 hours. As shown in Figure 1, DTX-P-060 was found to reduce DMPK expression levels by approximately 90% compared to the control.

[0342] Example 2: Targeting DMPK with a muscle-targeting complex A muscle-targeting conjugate was generated containing the DMPK ASO used in Example 1 (DTX-P-060) covalently linked to the anti-transferrin receptor antibody DTX-A-002 (RI7 217 (Fab)) via a cathepsin-cleavable linker.

[0343] Briefly, a maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate (MC-Val-Cit-PABC-PNP) linker molecule was coupled to NH-C-DTX-P-060 using an amide coupling reaction. Excess linker and organic solvent were removed by gel permeation chromatography. The purified Val-Cit-linker-DTX-P-060 was then coupled to a thiol-reactive anti-transferrin receptor antibody (DTX-A-002).

[0344] The product of the antibody coupling reaction was subjected to hydrophobic interaction chromatography (HIC-HPLC). Figure 2A shows the resulting HIC-HPLC trace, in which fraction B7-C2 of the trace (indicated by the vertical line) contained an ASO-to-antibody ratio of 1 or 2, as determined by SDS-PAGE. These fractions were pooled to arrive at the final muscle-targeting complex, designated DTX-C-008. Concentration measurements confirmed that DTX-C-008 had an average ASO-to-antibody ratio of 1.48, and SDS-PAGE revealed a purity of 86.4% (Figure 2B).

[0345] The same approach was used to generate a control conjugate containing the DMPK ASO used in Example 1 (DTX-P-060) covalently linked to an IgG2a (Fab) antibody (DTX-C-007) via a Val-Cit linker.

[0346] Purified DTX-C-008 was then tested for cellular internalization and DMPK inhibition. Hepa1-6 cells, which have relatively high expression levels of the transferrin receptor, were incubated for 72 hours in the presence of vehicle control, DTX-C-008 (100 nM), or DTX-C-007 (100 nM). After 72 hours of incubation, cells were isolated and assayed for DMPK expression levels (Figure 3). Cells treated with DTX-C-008 demonstrated a ~65% reduction in DMPK expression compared to cells treated with the vehicle control. Meanwhile, cells treated with DTX-C-007 had DMPK expression levels comparable to the vehicle control (no reduction in DMPK expression). These data suggest that the anti-transferrin receptor antibody in DTX-C-008 allows cellular internalization of the complex, thereby enabling the DMPK ASO to inhibit DMPK expression.

[0347] Example 3: Targeting DMPK in mouse muscle tissue with a muscle-targeting complex DTX-C-008, the muscle-targeting conjugate described in Example 2, was tested for inhibition of DMPK in mouse tissues. C57BL / 6 wild-type mice were intravenously injected with a single dose of vehicle control, DMPK-1 (3 mg / kg RNA), DTX-C-008 (3 mg / kg RNA, equivalent to 20 mg / kg antibody conjugate), or DTX-C-007 (3 mg / kg RNA, equivalent to 20 mg / kg antibody conjugate). DTX-P-060, a DMPK ASO described in Example 1, was used as a control. Each experimental condition was replicated in three individual C57BL / 6 wild-type mice. Seven days after injection, mice were euthanized and dissected into isolated tissue types. Individual tissue samples were then assayed for DMPK expression levels (Figures 4A-4E and 5A-5B).

[0348] Mice treated with the DTX-C-008 conjugate demonstrated reduced DMPK expression in various skeletal, cardiac, and smooth muscle tissues. For example, as shown in Figures 4A-4E, DMPK expression levels were significantly reduced in gastrocnemius (50% reduction), cardiac (30% reduction), esophageal (45% reduction), tibialis anterior (47% reduction), and soleus (31% reduction) tissues compared to mice treated with vehicle control. Meanwhile, mice treated with the DTX-C-007 conjugate had DMPK expression levels comparable to vehicle control (no reduction in DMPK expression) for all muscle tissue types assayed.

[0349] Mice treated with the DTX-C-008 conjugate demonstrated no change in DMPK expression in non-muscle tissues such as spleen and brain tissue (FIGS. 5A and 5B).

[0350] These data suggest that the anti-transferrin receptor antibody in DTX-C-008 enables cellular internalization of the complex into muscle-specific tissues in an in vivo mouse model, thereby inhibiting DMPK expression, which is inhibited by the DMPK ASO. These data further demonstrate that the DTX-C-008 complex can specifically target muscle tissue.

[0351] Example 4: Targeting DMPK in mouse muscle tissue with a muscle-targeting complex DTX-C-008, the muscle-targeting conjugate described in Example 2, was tested for dose-dependent inhibition of DMPK in mouse tissues. C57BL / 6 wild-type mice were intravenously injected with a single dose of vehicle control (phosphate-buffered saline, PBS), DTX-P-060 (10 mg / kg RNA), DTX-C-008 (3 mg / kg or 10 mg / kg RNA, where 3 mg / kg corresponds to 20 mg / kg antibody conjugate), or DTX-C-007 (3 mg / kg or 10 mg / kg RNA, where 3 mg / kg corresponds to 20 mg / kg antibody conjugate). DTX-P-060, the DMPK ASO described in Example 1, was used as a control. Each experimental condition was replicated in five individual C57BL / 6 wild-type mice. After a 7-day period following injection, the mice were euthanized and dissected into isolated tissue types. Individual tissue samples were then assayed for DMPK expression levels (FIGS. 6A-6F).

[0352] Mice treated with DTX-C-008 conjugates demonstrated reduced DMPK expression in various skeletal muscle tissues. As shown in Figures 6A-6F, DMPK expression levels were significantly higher in mice treated with vehicle control, tibialis anterior (58% and 75% reduction for 3 mg / kg and 10 mg / kg DTX-C-008, respectively), soleus (55% and 66% reduction for 3 mg / kg and 10 mg / kg DTX-C-008, respectively), and extensor digitorum longus (EDL) (55% and 66% reduction for 3 mg / kg and 10 mg / kg DTX-C-008, respectively). DTX-C-008 levels were significantly reduced in the following tissues: calf (52% and 72% reduction for 3 mg / kg and 10 mg / kg DTX-C-008, respectively), gastrocnemius (55% and 77% reduction for 3 mg / kg and 10 mg / kg DTX-C-008, respectively), heart (19% and 35% reduction for 3 mg / kg and 10 mg / kg DTX-C-008, respectively), and diaphragm (53% and 70% reduction for 3 mg / kg and 10 mg / kg DTX-C-008, respectively). Notably, all muscle tissue types assayed experienced dose-dependent inhibition of DMPK, with a greater reduction in DMPK levels with the 10 mg / kg antibody conjugate compared to the 3 mg / kg antibody conjugate.

[0353] In contrast, mice treated with the control DTX-C-007 conjugate had DMPK expression levels comparable to the vehicle control (no reduction in DMPK expression) for all muscle tissue types assayed. These data suggest that the anti-transferrin receptor antibody in DTX-C-008 enables cellular internalization of the conjugate into muscle-specific tissues in an in vivo mouse model, thereby enabling the DMPK ASO to inhibit DMPK expression. These data further demonstrate that the DTX-C-008 conjugate can specifically target muscle tissue for dose-dependent inhibition of DMPK.

[0354] Example 5: Targeting DMPK in cynomolgus monkey muscle tissue with a muscle-targeting conjugate A muscle-targeting conjugate containing DTX-P-060 (DTX-C-012) was produced and purified using the method described in Example 2. DTX-C-012 is a conjugate containing a human anti-transferrin antibody covalently linked via a cathepsin-cleavable Val-Cit linker to DTX-P-060, an antisense oligonucleotide targeting DMPK. Following HIC-HPLC purification, concentration measurements confirmed that DTX-C-012 had an average ASO-to-antibody ratio of 1.32, and SDS-PAGE revealed a purity of 92.3%.

[0355] DTX-C-012 was tested for dose-dependent inhibition of DMPK in male cynomolgus monkey tissues. Male cynomolgus monkeys (19-31 months old; 2-3 kg) were intravenously injected with a single dose of saline control, DTX-P-060 (naked DMPK ASO) (10 mg / kg RNA), or DTX-C-012 (10 mg / kg RNA) on day 0. Each experimental condition was replicated in three individual male cynomolgus monkeys. Tissue biopsies (containing muscle tissue) were collected on day 7 post-injection. DMPK mRNA expression levels, ASO detection assays, serum clinical chemistry, tissue histology, clinical findings, and body weight were analyzed. Monkeys were euthanized on day 14.

[0356] Compared with saline controls, significant knockdown (KD) of DMPK mRNA expression using DTX-C-012 was observed in the soleus, flexor digitorum profundus, and masseter muscles at 39%, 62%, and 41% KD, respectively (Figures 7A-7C). Robust knockdown of DMPK mRNA expression was further observed in the gastrocnemius (62% KD; Figure 7D), EDL (29% KD; Figure 7E), tibialis anterior (23% KD; Figure 7F), diaphragm (54% KD; Figure 7G), tongue (43% KD; Figure 7H), cardiac muscle (36% KD; Figure 7I), quadriceps (58% KD; Figure 7J), biceps brachii (51% KD; Figure 7K), and deltoid (47% KD; Figure 7L). Knockdown of DMPK mRNA expression in smooth muscle by DTX-C-012 was also observed in the intestine, with a 63% KD in the terminal jejunum-duodenum (Figure 8A) and a 70% KD in the ileum (Figure 8B). Notably, the naked DMPK ASO (i.e., not linked to a muscle-targeting agent), DTX-P-060, had minimal effect on DMPK expression levels compared to vehicle control (i.e., little or no reduction in DMPK expression) in all muscle tissue types assayed. Monkeys treated with the DTX-C-012 conjugate demonstrated no change in DMPK expression in nonmuscle tissues, such as liver, kidney, brain, and spleen tissues (Figures 9A-9D). Additional tissues were examined, as depicted in Figure 10, which shows normalized DMPK mRNA tissue expression levels across several tissue types in cynomolgus monkeys. (N = 3 male cynomolgus monkeys)

[0357] Prior to euthanasia, all monkeys were tested for reticulocyte levels, platelet levels, hemoglobin expression, alanine aminotransferase (ALT) expression, aspartate aminotransferase (AST) expression, and blood urea nitrogen (BUN) levels on days 2, 7, and 14 after dosing. As shown in Figure 12, monkeys administered the antibody-oligonucleotide conjugate had normal reticulocyte levels, platelet levels, hemoglobin expression, alanine aminotransferase (ALT) expression, aspartate aminotransferase (AST) expression, and blood urea nitrogen (BUN) levels throughout the experiment. These data indicate that a single dose of the conjugate containing DTX-P-060 is safe and tolerated in cynomolgus monkeys.

[0358] These data suggest that the anti-transferrin receptor antibody in the DTX-C-012 conjugate These data suggest that the DTX-C-012 conjugate can specifically target muscle tissue due to dose-dependent inhibition of DMPK, thereby enabling the DMPK ASO (DTX-P-060) to inhibit DMPK expression in an in vivo cynomolgus monkey model. These data further demonstrate that the DTX-C-012 conjugate is capable of specifically targeting muscle tissue without substantially affecting non-muscle tissue due to dose-dependent inhibition of DMPK. This is in direct contrast to the limited ability of the naked DMPK ASO (i.e., not linked to a muscle-targeting agent), DTX-P-060, to inhibit DMPK expression in muscle tissue in an in vivo cynomolgus monkey model.

[0359] Example 6: Targeting DMPK in mouse muscle tissue with a muscle-targeting complex The muscle-targeting conjugate, DTX-C-008, described in Example 2, was tested for time-dependent inhibition of DMPK in mouse tissues. C57BL / 6 wild-type mice were intravenously injected with a single dose of vehicle control (saline), DTX-P-060 (10 mg / kg RNA), or DTX-C-008 (10 mg / kg RNA), as described in Table 2, and euthanized after the indicated time periods. Following euthanasia, mice were dissected into isolated tissue types, and tissue samples were subsequently assayed for DMPK expression levels (Figures 11A-11B). Table 2 - Experimental conditions [Table 4] Mice treated with the DTX-C-008 conjugate demonstrated an approximately 50% reduction in DMPK expression in the gastrocnemius (Figure 11A) and tibialis anterior (Figure 11B) muscles compared to vehicle for all groups 9-12 (3-28 days between injection and euthanasia). Mice treated with the DTX-P-060 naked oligonucleotide did not demonstrate a significant reduction in DMPK expression.

[0360] Equivalents and Terminology The present disclosure, as preferably illustrated and described herein, can 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 with either of the other two terms. The terms and expressions employed are used as descriptive terms, not limiting terms, and there is no intention to use such terms and expressions to exclude any equivalents of the features shown and described or portions thereof, although it is recognized that various modifications are possible within the scope of the present disclosure. Thus, while the present disclosure has been specifically disclosed by preferred embodiments, it should be understood that modifications and variations of any features and concepts disclosed herein may be reclassified by those skilled in the art, and that such modifications and variations are deemed to be within the scope of the present disclosure.

[0361] Additionally, when features or aspects of the present disclosure are described in terms of a Markush group or other alternative group, those skilled in the art will recognize that the present disclosure is thereby also described in terms of any individual member or subgroup member of the Markush group or other group.

[0362] It should be understood that in some embodiments, the sequences presented in the sequence listing may be referred to in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have, compared to a particular sequence, one or more alternative nucleotides (e.g., RNA equivalents of DNA nucleotides or DNA equivalents of RNA nucleotides), and / or one or more modified nucleotides, and / or one or more modified internucleotide linkages, and / or one or more other modifications while retaining essentially the same or similar complementary properties as the particular sequence.

[0363] 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 claims below), 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 is merely intended to serve as a shorthand method of individually referring to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated herein even if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better describe the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0364] Aspects of the invention are described herein. Variations of these aspects may become apparent to those of skill in the art upon reading the above description.

[0365] The inventors expect those skilled in the art to adopt such variations as appropriate, and the inventors intend that the invention may 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 practicable variations thereof is covered 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

1. 1. A method for treating a subject diagnosed with a muscle disease associated with a gain-of-function disease allele, the method comprising administering to the subject a conjugate comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit expression or activity of the disease allele, wherein the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle cells of the subject.

2. 10. The method of claim 1, wherein the muscle disease is hereditary.

3. 3. The method of claim 1 or 2, wherein the muscle disease manifests with increasing severity in successive family generations of the subject.

4. 4. The method of any one of claims 1 to 3, wherein the subject has been diagnosed as having a muscle disease based on genetic analysis of disease alleles.

5. 5. The method of any one of claims 1 to 4, wherein the subject exhibits progressive muscle wasting and / or sarcopenia prior to administration.

6. 6. The method of any one of claims 1 to 5, wherein the subject exhibits myotonia, e.g., measurable by electromyography, prior to administration.

7. 7. The method of any one of claims 1 to 6, wherein the muscle-targeting agent is a muscle-targeting antibody.

8. 8. The method of claim 7, wherein the muscle-targeting antibody specifically binds to an extracellular epitope of the transferrin receptor.

9. The method of claim 8, wherein the extracellular epitope of the transferrin receptor comprises an epitope of the apical domain of the transferrin receptor.

10. 10. The method of claim 8 or 9, wherein the muscle-targeting antibody specifically binds to an epitope of a sequence ranging from C89 to F760 of SEQ ID NOs: 1 to 3.

11. The equilibrium dissociation constant (Kd) of the muscle-targeting antibody binding to the transferrin receptor is 10 -11 M to 10 -6 The method of any one of claims 8 to 10, wherein the range is from 1 to M.

12. 12. The method of any one of claims 8-11, wherein the muscle-targeting antibody competes with an antibody listed in Table 2 for specific binding to an epitope of the transferrin receptor.

13. Muscle-targeting antibodies have been shown to specifically bind to epitopes on the transferrin receptor. -6 The method of claim 12, which competes with a Kd less than or equal to M.

14. Kd is 10 -11 M to 10 -6 The method of claim 13, wherein the saturation temperature is in the range of M.

15. 15. The method of any one of claims 7-14, wherein the muscle-targeting antibody does not specifically bind to the transferrin binding site of the transferrin receptor and / or the muscle-targeting antibody does not inhibit the binding of transferrin to the transferrin receptor.

16. 16. The method of any one of claims 7 to 15, wherein the muscle-targeting antibody cross-reacts with extracellular epitopes of two or more of the transferrin receptors of humans, non-human primates, and rodents.

17. 17. The method of any one of claims 7 to 16, wherein the method is configured to promote transferrin receptor-mediated internalization of a molecular payload into muscle cells.

18. 18. The method of any one of claims 7 to 17, wherein the muscle-targeting antibody is a chimeric antibody, and optionally the chimeric antibody is a humanized monoclonal antibody.

19. Muscle-targeting antibodies include ScFv, Fab fragments, Fab' fragments, and F(ab') 2 The method of any one of claims 7 to 18, wherein the antibody is in the form of a fragment or an Fv fragment.

20. 20. The method of any one of claims 1 to 19, wherein the molecular payload is an oligonucleotide.

21. 21. The method of claim 20, wherein the oligonucleotide comprises a region of complementarity to a gene listed in Table 1 or an mRNA encoded therefrom.

22. 22. The method of claim 20 or 21, wherein the oligonucleotide is a gapmer oligonucleotide, a mixmer oligonucleotide, an antisense oligonucleotide, an RNAi oligonucleotide, a messenger RNA (mRNA), or a guide sequence.

23. 23. The method of any one of claims 1 to 22, wherein the conjugate is administered to the subject by intramuscular parenteral administration.

24. 24. The method of claim 23, wherein the conjugate is administered to the subject by intravenous administration.

25. 24. The method of claim 23, wherein the conjugate is administered to the subject by subcutaneous administration of the conjugate.

26. A conjugate comprising a muscle targeting agent linked to a single-stranded oligonucleotide, wherein the muscle targeting agent specifically binds to an internalizing cell surface receptor on a muscle cell, and wherein the oligonucleotide comprises a region complementary to a muscle disease gene.

27. 1. A composition comprising a plurality of conjugates, each conjugate comprising a muscle targeting agent covalently linked to at least three oligonucleotides, where the muscle targeting agent specifically binds to an internalizing cell surface receptor on a muscle cell of a subject, and where each oligonucleotide comprises a region of complementarity to a muscle disease gene.

28. A conjugate comprising a muscle targeting agent covalently linked to a molecular payload configured to modulate the expression or activity of a muscle disease gene encoding a non-secreted substance that functions in muscle cells, wherein the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle cells.

29. 29. The conjugate of claim 28, wherein the muscle-targeting agent is a muscle-targeting antibody.

30. 30. The conjugate of claim 29, wherein the muscle-targeting antibody specifically binds to an extracellular epitope of the transferrin receptor.

31. 31. The complex of claim 30, wherein the extracellular epitope of the transferrin receptor comprises an epitope of the apical domain of the transferrin receptor.

32. 32. The conjugate of claim 30 or 31, wherein the muscle-targeting antibody specifically binds to an epitope of a sequence within amino acids C89 to F760 of SEQ ID NOs: 1 to 3.

33. The equilibrium dissociation constant (Kd) of the muscle-targeting antibody binding to the transferrin receptor is 10 -11 M to 10 -6 The conjugate of any one of claims 30 to 32, wherein M is in the range of 0 to M.

34. 34. The conjugate of any one of claims 30-33, wherein the muscle-targeting antibody competes with an antibody listed in Table 1 for specific binding to an epitope of the transferrin receptor.

35. Muscle-targeting antibodies have been shown to specifically bind to epitopes on the transferrin receptor. -6 35. The complex of claim 34, which competes with a Kd less than or equal to M.

36. Kd is 10 -11 M to 10 -6 36. The conjugate of claim 35, wherein the range is M.

37. 37. The conjugate of any one of claims 30-36, wherein the muscle-targeting antibody does not specifically bind to the transferrin binding site of the transferrin receptor and / or the muscle-targeting antibody does not inhibit the binding of transferrin to the transferrin receptor.

38. 38. The conjugate of any one of claims 30 to 37, wherein the muscle-targeting antibody cross-reacts with extracellular epitopes of two or more of the transferrin receptors of humans, non-human primates, and rodents.

39. 39. The conjugate of any one of claims 30 to 38, wherein the conjugate is configured to promote transferrin receptor-mediated internalization of the molecular payload into muscle cells.

40. 40. The conjugate of any one of claims 29 to 39, wherein the muscle-targeting antibody is a chimeric antibody.

41. 41. The conjugate of claim 40, wherein the chimeric antibody is a humanized monoclonal antibody.

42. Muscle-targeting antibodies include ScFv, Fab fragments, Fab' fragments, and F(ab') 2 42. The conjugate of any one of claims 29 to 41, in the form of a fragment or an Fv fragment.

43. The conjugate of any one of claims 28 to 42, wherein the molecular payload is an oligonucleotide.

44. 44. The conjugate of claim 43, wherein the oligonucleotide comprises a region of complementarity to a muscle disease gene having a gain-of-function disease allele.

45. The conjugate of any one of claims 28 to 42, wherein the molecular payload is a polypeptide.

46. 46. ​​The conjugate of claim 45, wherein the polypeptide is an E3 ubiquitin ligase inhibitor peptide.

47. 45. The conjugate of claim 43 or 44, wherein the oligonucleotide comprises at least one modified internucleotide linkage.

48. 48. The conjugate of claim 47, wherein at least one modified internucleotide linkage is a phosphorothioate linkage.

49. 49. The conjugate of claim 48, wherein the oligonucleotide comprises phosphorothioate linkages in the Rp stereochemical configuration and / or in the Sp stereochemical configuration.

50. 50. The conjugate of claim 49, wherein the oligonucleotide comprises phosphorothioate linkages that are all in the Rp stereochemical configuration or all in the Sp stereochemical configuration.

51. 51. The conjugate of any one of claims 43, 44 or 47-50, wherein the oligonucleotide comprises one or more modified nucleotides.

52. 52. The conjugate of claim 51, wherein the one or more modified nucleotides are 2'-modified nucleotides.

53. 53. The conjugate of any one of claims 43, 44 or 47-52, wherein the oligonucleotide is a gapmer oligonucleotide that directs RNAse H-mediated cleavage of an mRNA transcript encoded by the myopathy gene in cells.

54. 54. The conjugate of claim 53, wherein the gapmer oligonucleotide comprises a core of 5 to 15 deoxyribonucleotides flanked on each side by 2 to 8 modified nucleotides.

55. 55. The conjugate of claim 54, wherein the flanking modified nucleotides are 2'-modified nucleotides.

56. 53. The conjugate of any one of claims 43, 44 or 47 to 52, wherein the oligonucleotide is a mixmer oligonucleotide.

57. 57. The conjugate of claim 56, wherein the mixmer oligonucleotide comprises two or more different 2'-modified nucleotides.

58. 53. The conjugate of any one of claims 43, 44, or 47-52, wherein the oligonucleotide is an RNAi oligonucleotide that promotes RNAi-mediated cleavage of an mRNA transcript encoded by the muscle disease gene.

59. 59. The complex of claim 58, wherein the RNAi oligonucleotide is a double-stranded oligonucleotide 19 to 25 nucleotides in length.

60. 60. The complex of claim 58 or 59, wherein the RNAi oligonucleotide comprises at least one 2' modified nucleotide.

61. 61. The conjugate of any one of claims 52, 55, 57, or 60, wherein each 2'-modified nucleotide is selected from the group consisting of 2'-O-methyl, 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-MOE), and 2',4'-bridged nucleotides.

62. 52. The conjugate of claim 51, wherein one or more modified nucleotides is a bridged nucleotide.

63. 61. The conjugate of any one of claims 52, 55, 57 or 60, wherein at least one 2'-modified nucleotide is a 2',4'-bridged nucleotide selected from 2',4'-constrained 2'-O-ethyl (cEt) and locked nucleic acid (LNA) nucleotides.

64. 53. The complex of any one of claims 43, 44, or 47-52, wherein the oligonucleotide comprises a guide sequence for a genome-editing nuclease.

65. 53. The conjugate of any one of claims 43, 44 or 47-52, wherein the oligonucleotide is a phosphorodiamidite morpholino oligomer.

66. 66. The conjugate of any one of claims 28-65, wherein the muscle targeting agent is covalently linked to the molecular payload via a cleavable linker.

67. 67. The conjugate of claim 66, wherein the cleavable linker is selected from a protease-sensitive linker, a pH-sensitive linker, and a glutathione-sensitive linker.

68. 68. The conjugate of claim 67, wherein the cleavable linker is a protease-sensitive linker.

69. 69. The conjugate of claim 68, wherein the protease-sensitive linker comprises a sequence cleavable by a lysosomal and / or endosomal protease.

70. 69. The conjugate of claim 68, wherein the protease-sensitive linker comprises a valine-citrulline dipeptide sequence.

71. 68. The conjugate of claim 67, wherein the linker is a pH-sensitive linker that is cleaved at a pH in the range of 4 to 6.

72. 66. The conjugate of any one of claims 28-65, wherein the muscle targeting agent is covalently linked to the molecular payload via a non-cleavable linker.

73. 73. The conjugate of claim 72, wherein the non-cleavable linker is an alkane linker.

74. 74. The conjugate of any one of claims 29-73, wherein the muscle-targeting antibody comprises an unnatural amino acid to which the oligonucleotide is covalently linked.

75. 75. The conjugate of any one of claims 29 to 74, wherein the muscle-targeting antibody is covalently linked to the oligonucleotide via conjugation to a lysine or cysteine ​​residue of the antibody.

76. 76. The conjugate of claim 75, wherein the muscle-targeting antibody is conjugated to the cysteine ​​via a maleimide-containing linker, optionally the maleimide-containing linker comprising a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group.

77. 77. The conjugate of any one of claims 29 to 76, wherein the muscle-targeting antibody is a glycosylated antibody comprising at least one sugar moiety to which the oligonucleotide is covalently linked.

78. 78. The conjugate of claim 77, wherein the sugar moiety is a branched mannose.

79. 79. The conjugate of Claim 77 or 78, wherein the muscle-targeting antibody is a glycosylated antibody comprising 1 to 4 sugar moieties, each of which is covalently linked to a separate oligonucleotide.

80. 78. The conjugate of claim 77, wherein the muscle-targeting antibody is a globally glycosylated antibody.

81. 78. The conjugate of claim 77, wherein the muscle-targeting antibody is a partially glycosylated antibody.

82. 82. The conjugate of claim 81, wherein the partially glycosylated antibody is produced via chemical or enzymatic means.

83. 82. The conjugate of claim 81, wherein the partially glycosylated antibody is produced in a cell that is deficient in an enzyme in the N- or O-glycosylation pathway.

84. 84. A method of delivering a molecular payload to a transferrin receptor expressing cell, the method comprising contacting the cell with a complex of any one of claims 29 to 83.

85. 84. A method of inhibiting the expression or activity of a myopathy gene in a cell, the method comprising contacting the cell with a complex of any one of claims 29-83 in an amount effective to promote internalization of the molecular payload into the cell.

86. 86. The method of claim 85, wherein the cell is in vitro.

87. 86. The method of claim 85, wherein the cell is in a subject.

88. 88. The method of claim 87, wherein the subject is a human.

89. 84. A method of treating a subject having a muscular disease, the method comprising administering to the subject an effective amount of a complex of any one of claims 29 to 83.

90. 90. The method of claim 89, wherein the muscle disease is a disease listed in Table 1.

91. 90. The method of claim 89, wherein the muscle disease is selected from the group consisting of adult Pompe disease, centronuclear myopathy (CNM), Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy (FSHD), familial hypertrophic cardiomyopathy, fibrodysplasia ossificans progressiva (FOP), Friedreich's ataxia (FRDA), inclusion body myopathy 2, Laing distal myopathy, myofibrillar myopathy, myotonia congenita (autosomal dominant, Thomsen's disease), myotonia dystrophy type I, myotonia dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and congenital myotonia.

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