Fibrinogenated complexes and their use

Abstract

To provide complexes comprising a muscle-targeting agent covalently linked to a molecular payload.SOLUTION: In some embodiments, a muscle-targeting agent specifically binds to an internalizing cell surface receptor on muscle cells. In some embodiments, a molecular payload inhibits activity of a disease allele associated with muscle disease. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or RNAi oligonucleotide.SELECTED DRAWING: Figure 3

Description

Technical Field

[0001] Related Applications This application claims the benefit of the filing dates of U.S. Provisional Application No. 62 / 714,010, filed on August 2, 2018, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF"; U.S. Provisional Application No. 62 / 779,173, filed on December 13, 2018, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF"; U.S. Provisional Application No. 62 / 855,781, filed on May 31, 2019, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF"; U.S. Provisional Application No. 62 / 858,925, filed on June 7, 2019, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF"; and U.S. Provisional Application No. 62 / 859,694, filed on June 10, 2019, entitled "MUSCLE TARGETING COMPLEXES AND USES 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 (e.g., oligonucleotides) to cells, and their use, particularly for the treatment of diseases.

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

Background Art

[0004] Background of the Invention Muscle diseases are often associated with muscle weakness and / or muscle dysfunction that can lead to life-threatening complications. Many examples of such diseases have been characterized, including muscular dystrophies (such as Duchenne, facioscapulohumeral, myotonic, and oculopharyngeal), Pompe disease, central core myopathy, familial hypertrophic cardiomyopathy, Laing distal myopathy, progressive ossifying fibrodysplasia, Friedreich's ataxia, myofibrillar myopathy, and other various forms. These diseases are generally genetic but can occur spontaneously. They are often congenital but can also occur 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, the options for effective treatment 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 gene expression or activity in a subject having or suspected of having a muscle disease associated with that gene (e.g., the genes / diseases of 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 a muscle cell for the purpose of delivering a molecular payload to the muscle cell. In some embodiments, the complex is taken into the cell via receptor-mediated endocytosis (e.g., the transferrin receptor), and in response, the molecular payload may be released into the interior of the cell to perform its function. For example, a complex modified to deliver an oligonucleotide may release the oligonucleotide such 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 that connects the oligonucleotide of the complex to the muscle targeting agent.

[0006] In some embodiments, the method is provided for treating a subject diagnosed as having a muscular disease associated with a disease allele (e.g., a gain-of-function disease allele). In some embodiments, the method involves administering to the subject a complex comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit the expression or activity of the disease allele. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on the subject's muscle cells. In some embodiments, the muscular disease is genetic and may exhibit increased severity in a series of family generations of the subject. In some embodiments, the subject has been diagnosed as having a muscular 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, a method for treating a subject diagnosed as having a muscular disease (e.g., associated with a gain-of-function disease allele) is provided. In some embodiments, the method comprises administering to the subject a complex comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit the expression or activity of the disease allele. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on the subject's muscle cells.

[0008] In some embodiments, the muscular disease is genetic. In some embodiments, the muscular disease exhibits increased severity in a series of family generations of the subject. In some embodiments, the subject has been diagnosed as having a muscular 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, e.g., measurable by electromyogram, 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 tip domain of the transferrin receptor. In some embodiments, the muscle targeting antibody specifically binds to an epitope of a sequence in the range of C89 to F760 of SEQ ID NOs: 1-3. In some embodiments, the equilibrium dissociation constant (Kd) of the binding of the muscle targeting antibody to the transferrin receptor is 10 -11 M to 10 -6 M. In some embodiments, the muscle targeting antibody competes with the antibodies listed in Table 2 for specific binding to an epitope of the transferrin receptor.

[0010] In some embodiments, the muscle targeting antibody competes with a Kd of less than or equal to 10 -6 M for specific binding to an epitope of the transferrin receptor. In some embodiments, the Kd is in the range of 10 -11 M to 10 -6 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 extracellular epitopes of two or more of the transferrin receptors of humans, non-human primates, and rodents. In some embodiments, the method is configured to promote the internalization of a molecular payload mediated by the transferrin receptor 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 a ScFv, Fab fragment, Fab' fragment, F(ab')2 fragment, or 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 the 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 complex is administered to a subject by parenteral administration outside of muscle. In some embodiments, the complex is administered to a subject by intravenous administration. In some embodiments, the complex is administered to a subject by subcutaneous administration of the complex.

[0015] In some aspects, the complex is provided to comprise 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 muscle cells, and wherein the oligonucleotide comprises a region complementary to a muscle disease gene.

[0016] In some embodiments, the composition is provided to comprise a plurality of complexes, each complex comprising a muscle targeting agent covalently linked to two, at least three or more (by way of example, 2-6) oligonucleotides. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle cells of the subject, and each oligonucleotide comprises a region complementary to a muscle disease gene.

[0017] In some aspects, the complex is provided to comprise 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-secretory that functions within 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 includes an epitope of the tip domain of the transferrin receptor. In some embodiments, the muscle targeting antibody specifically binds to an epitope of a sequence within amino acids C89 - F760 of SEQ ID NOs: 1 - 3. In some embodiments, the equilibrium dissociation constant (Kd) of the binding of the muscle targeting antibody to the transferrin receptor is from 10 -11 M to 10 -6 M. In some embodiments, the muscle targeting antibody competes with the antibodies listed in Table 2 for specific binding to an epitope of the transferrin receptor. In some embodiments, the muscle targeting antibody competes with a Kd of less than or equal to 10 -6 M for specific binding to an epitope of the transferrin receptor. In some embodiments, the Kd is in the range of 10 -11 M to 10 -6 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 extracellular epitopes of two or more of the transferrin receptors of humans, non - human primates, and rodents.

[0020] In some embodiments, the complex is configured to facilitate the internalization of a molecular payload mediated by the transferrin receptor 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 a ScFv, Fab fragment, Fab' fragment, F(ab')2 fragment, or Fv fragment.

[0022] In some embodiments, the molecular payload is an oligonucleotide. In some embodiments, the oligonucleotide comprises a region complementary 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, the 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 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 targets RNAse H-mediated cleavage of the mRNA transcript encoded by the muscle disease gene in cells. In some embodiments, the gapmer oligonucleotide comprises a central portion of 5 to 15 deoxyribonucleotides flanked on either side by 2 to 8 modified nucleotides each.

[0027] In some embodiments, the modified nucleotides on both sides thereof are 2'-modified nucleotides. In some embodiments, the oligonucleotide is a mixmer oligonucleotide.

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

[0029] In some embodiments, the oligonucleotide is a double-stranded oligonucleotide 19 to 25 nucleotides in length. In some embodiments, the RNAi oligonucleotide contains 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 contains a guide sequence for a genome editing nuclease.

[0032] In some embodiments, the oligonucleotide is a phosphorodiamidate 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 an array cleavable by lysosomal protease and / or endosomal protease. In some embodiments, the protease-sensitive linker comprises a valine-citrulline peptide sequence. In some embodiments, the linker is a pH-sensitive linker 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 a non-natural 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 residue or a cysteine residue of the antibody. In some embodiments, the muscle targeting antibody is conjugated to cysteine via a maleimide-containing linker, optionally where 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 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-targeted antibody is an antibody that is glycosylated globally. In some embodiments, the muscle-targeted antibody is an antibody that is glycosylated partially. In some embodiments, the partially glycosylated antibody is produced via chemical or enzymatic means. In some embodiments, the partially glycosylated antibody is produced in a cell that is a cell lacking an enzyme in the N- or O-glycosylation pathway.

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

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

[0040] According to some aspects, a method for treating a subject having a muscle disease is provided. In some embodiments, the method comprises administering to the subject an effective amount of a complex 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 disease), myotonic dystrophy type I, myotonic dystrophy type II, myotubular myopathy, oculopharyngeal muscular dystrophy, and paramyotonia congenita. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Brief Description of the Drawings

Figure 1

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Figure 2A - B

[0043] Figure 2B depicts a non-limiting image of the SDS-PAGE analysis of the muscle-targeted complex.

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Figure 4D - E

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Figure 5A - B

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Figure 6A - B

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Figure 10

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Figure 11A - B

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Figure 12

Mode for Carrying Out the Invention

[0054] Detailed Description of the Invention While certain molecular payloads (e.g., oligonucleotides, peptides, small molecules) can have beneficial effects on muscle cells, effectively targeting such cells has proven extremely challenging. As described herein, the present disclosure provides a complex comprising a muscle targeting agent covalently linked to a molecular payload to overcome such challenges. In some embodiments, the complex is particularly useful for delivering a molecular payload that modulates the expression or activity of a target gene in muscle cells, e.g., in a subject having or suspected of having a muscle disease. For example, in some embodiments, the complex is useful for treating a subject having a rare muscle disease 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 complexes depending on the disease being treated. For example, if the causative 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 causative mutation results in a gain-of-function allele, an oligonucleotide (e.g., RNAi, PMO, ASO-gapmer) may be used to inhibit the expression or activity of the allele. In some embodiments, e.g., if the mutation results in a loss-of-function allele, the payload may include, e.g., an expression construct to express the wild-type version of the allele. In some embodiments, the payload may include, e.g., machinery (e.g., a guide nucleic acid, an expression construct encoding a gene editing enzyme) for correcting the causative defect by gene editing.

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

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

[0057] Approximately: As used herein, the term "approximately" or "about", when applied to one or more values of interest, refers to a value similar to the specified reference value. In certain embodiments, the term "approximately" or "about" refers to a broad range of values that fall within plus or minus (more or less) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the specified reference value, unless otherwise stated or apparent from the context (except when such numbers exceed 100% of the practicable 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, for example, 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, an F(ab')2 fragment, an Fv fragment, or a scFv fragment. In some embodiments, the antibody is a nanobody derived from an antibody of a camelid animal or a nanobody derived from a shark antibody. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody includes a framework having a human germline sequence. In another embodiment, the antibody includes a heavy chain constant region selected from the group consisting of the constant regions of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. In some embodiments, the antibody includes 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, the antibody includes a constant region, for example, an Fc region. An immunoglobulin constant region refers to a heavy chain or light chain constant region. The amino acid sequences of human IgG heavy and light chain constant regions and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of the antibodies described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibodies described herein can include a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In a specific embodiment, the antibodies described herein include human gamma 1 CH1, CH2, and / or CH3 domains. In some embodiments, the amino acid sequence of the VH domain includes the amino acid sequence of a human gamma (γ) heavy chain constant region, for example, 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 E A et al. (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, e.g., modified via glycosylation, phosphorylation, SUMOylation, and / or 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 mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the disclosure linked to a linker polypeptide or an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues joined by peptide bonds and is used to link to one or more antigen-binding portions. Examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R.J., et al. (1994) Structure 2:1121-1123).Furthermore, the antibody may be part of a larger immune adhesion molecule formed by covalent or non-covalent binding of the antibody or antibody portion to one or more other proteins or peptides. Examples of such immune adhesion molecules include the use of streptavidin core regions to create tetrameric scFv molecules (Kipriyanov, S.M., et al. (1995) Human Antibodies and Hybridomas 6:93-101), as well as the use of cysteine residues, marker peptides, and C-terminal polyhistidine tags to create divalent and biotinylated scFv molecules (Kipriyanov, S.M., et al. (1994) Mol. Immunol. 31:1047-1058).

[0059] CDR: As used herein, the term "CDR" refers to the complementarity determining regions within an antibody variable sequence. There are three CDRs in each of the variable regions of the heavy and light chains, and these are designated CDR1, CDR2, and CDR3 for each of the variable regions. The term "CDR set" as used herein refers to a group of the three CDRs that occur in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently according to various systems. The system described by Kabat (Kabat et al., Sequence of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides the exact residue boundaries that define the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Sub-portions of the CDRs may be designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with the Kabat CDRs are described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Still other CDR boundary definitions may not strictly follow one of the above systems, but still may overlap with the Kabat CDRs, and based on the prediction or experimental finding that specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding, they may be shortened or extended. The methods used herein may utilize CDRs defined according to any of these systems, but the preferred embodiments use CDRs defined by Kabat or Chothia.

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

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

[0062] Complementary: As used herein, the term "complementary" refers to the capacity for exact pairing between two nucleotides or two sets of nucleotides. In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in a bond between two nucleotides or two sets of nucleotides. For example, when the base of an oligonucleotide at a particular position is capable of hydrogen bonding with the base of a target nucleic acid (e.g., mRNA) at the corresponding position, then the bases are considered to be complementary to each other at that position. Base pairing may include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, as complementary base pairing, 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 universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any of A, C, U, or T and are considered to be complementary to them. Inosine (I) is also considered to be a universal base in the art and is considered to be complementary to any of A, C, U, or T.

[0063] Conservative amino acid substitution: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for changing polypeptide sequences known to those of skill in the art, for example, as found in references that compile such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions made with 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 that are linked together through at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond (such as a disulfide bond or disulfide bridge) that serves as an intermolecular linker. However, in some embodiments, two or more molecules can be covalently linked together through a molecule that serves as a linker that joins 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-react: As used herein, and in the context of a targeting agent (e.g., an antibody), the term "cross-reacting" refers to the property of an agent capable of specifically binding with similar affinity or binding activity to more than one antigen of the same type or class (e.g., antigens of multiple homologs, paralogs, or orthologs). For example, in some embodiments, an antibody that cross-reacts with antigens of the same type or class from humans and non-human primates (e.g., the human transferrin receptor and the transferrin receptor of non-human primates) is capable of binding to the human antigen and the non-human primate antigen with similar affinity or binding activity. In some embodiments, the antibody cross-reacts with human antigens and rodent antigens of the same type or class. In some embodiments, the antibody cross-reacts with rodent antigens and non-human primate antigens of the same type or class. In some embodiments, the antibody cross-reacts with human antigens, non-human primate antigens, and rodent antigens of the same type or class.

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

[0067] Disease-related repeat: As used herein, the term "disease-related repeat" refers to a repetitive nucleotide sequence at a genomic location where the number of units of the repetitive nucleotide sequence correlates with and / or directly or indirectly contributes to or causes a genetic disease. Each repeat unit of a disease-related repeat may be 2, 3, 4, 5 or more nucleotides in length. For example, in some embodiments, the disease-related repeat is a dinucleotide repeat. In some embodiments, the disease-related repeat is a trinucleotide repeat. In some embodiments, the disease-related repeat is a tetranucleotide repeat. In some embodiments, the disease-related repeat is a pentanucleotide repeat. In some embodiments, the disease-related repeat includes a CAG repeat, a CTG repeat, a CUG repeat, a CGG repeat, a CCTG repeat, or a nucleotide complement thereof. In some embodiments, the disease-related repeat is in the non-coding portion of a gene. However, in some embodiments, the disease-related repeat is in the coding region of a gene. In some embodiments, the disease-related 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-related repeat is in RNA (e.g., an RNA transcript). In some embodiments, the disease-related repeat is in DNA (e.g., a chromosome, a plasmid). In some embodiments, the disease-related repeat is expanded in the chromosomes of germline cells. In some embodiments, the disease-related repeat is expanded in the chromosomes of somatic cells. In some embodiments, the disease-related repeat is expanded to a number of repeat units associated with congenital onset. In some embodiments, the disease-related repeat is expanded to a number of repeat units associated with pediatric onset of a disease. In some embodiments, the disease-related repeat is expanded to a number of repeat units associated with adult onset of a disease.

[0068] Framework: As used herein, the terms "framework" or "framework array" refer to the remaining array of variable regions minus the CDRs. Since the precise definition of the CDR array can be determined by various systems, the meaning of the framework array depends on 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 positioned between FR1 and FR2, CDR2 is between FR2 and FR3, and CDR3 is between FR3 and FR4. A framework region that does not specify a particular subregion as FR1, FR2, FR3, or FR4 represents the combined FR(s) within the variable region of a naturally occurring single immunoglobulin chain when referred to otherwise. As used herein, FR represents one of the four subregions, and FR(s) represents two or more of the four subregions that contain the framework region. Human heavy and light chain acceptor sequences are known in the art. In one aspect, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0069] Human antibody: As used herein, the term "human antibody" is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences in the CDRs, particularly CDR3 (e.g., mutations introduced by random mutagenesis or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto a human framework sequence.

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

[0071] Internalizing cell surface receptor: As used herein, the term "internalizing cell surface receptor" refers to a cell surface receptor that is internalized by a cell upon, for example, an external stimulus (e.g., a ligand that binds to the receptor). In some aspects, the internalizing cell surface receptor is internalized by endocytosis. In some aspects, the internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some aspects, the internalizing cell surface receptor is internalized by a clathrin-independent pathway, such as phagocytosis, macropinocytosis, caveola- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some aspects, the internalizing cell surface receptor includes an intracellular domain, a transmembrane domain, and / or an extracellular domain, which may optionally further include a ligand-binding domain. In some aspects, the cell surface receptor becomes internalized by the cell after ligand binding. In some aspects, the ligand may be a muscle targeting agent or a muscle targeting antibody. In some aspects, the internalizing cell surface receptor is a transferrin receptor.

[0072] Isolated antibody: As used herein, an "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to the transferrin receptor is substantially free of antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to a transferrin receptor complex may have cross-reactivity to other antigens such as transferrin receptor molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular materials and / or chemicals.

[0073] Kabat numbering: The terms "Kabat numbering," "Kabat definition," and "Kabat labeling" are used interchangeably herein. These terms refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof, as recognized in the art (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable regions extend from amino acid positions 31-35 for CDR1, 50-65 for CDR2, and 95-102 for CDR3. In the light chain variable region, the hypervariable regions extend from amino acid positions 24-34 for CDR1, 50-56 for CDR2, and 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 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, the expression of a protein, or the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide that includes a strand having a region complementary to a target gene.

[0075] Muscle disease gene: 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, such 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 individuals. In some embodiments, the muscle disease is a single gene disorder. In some embodiments, the muscle disease gene is a gene listed in Table 1.

[0076] Muscle targeting agent: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on muscle cells. The antigen in or on the muscle cell may be a membrane protein, such as an integral membrane protein or a surface membrane protein. Typically, the muscle targeting agent specifically binds to an antigen on the muscle cell that facilitates internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent can specifically bind to an internalizing cell surface receptor on the muscle and be internalized into the muscle cell through receptor-mediated endocytosis. 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 antibody: As used herein, the term "muscle-targeting antibody" refers to a muscle targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, the muscle-targeting antibody specifically binds to an antigen on the muscle cell that facilitates internalization of the muscle-targeting antibody (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an internalizing cell surface receptor present on the muscle cell. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0078] Oligonucleotide: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound of up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, gapmer, mixmer, phosphorodiamidate morpholino, peptide nucleic acid, aptamer, guide nucleic acid (e.g., Cas9 guide RNA), and the like. The oligonucleotide may be single-stranded or double-stranded. In some embodiments, the oligonucleotide may contain one or more modified nucleotides (e.g., 2'-O-methyl sugar modification, purine or pyrimidine modification). In some embodiments, the oligonucleotide may contain one or more modified internucleotide linkages. In some embodiments, the oligonucleotide may contain one or more phosphorothioate linkages that may be in the Rp or Sp stereochemical configuration.

[0079] Recombinant antibody: As used herein, the term "recombinant human antibody" encompasses all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in more detail by the present disclosure), antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom H.R., (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith W.E., (2002) Clin. Biochem. 35:425-445; Gavilondo J.V., and Larrick J.W. (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from a human immunoglobulin transgenic animal (e.g., mouse) (see, for example, Taylor, L.D., et al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann S-A., and Green L.L. (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or antibodies prepared, expressed, created, or isolated by any other means involving splicing with other DNA sequences of the human immunoglobulin gene sequence. 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 mutagenesis in vitro (or, when a human Ig sequence transgenic animal is used, somatic mutagenesis in vivo), and thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that are derived from and related to the VH and VL sequences of the human germline but may not naturally occur within the germline repertoire of human antibodies in vivo.One aspect of the present disclosure provides a fully human antibody capable of binding to a human transferrin receptor that can be generated using techniques well known in the art, for example, but not limited to, techniques using a human Ig phage library (e.g., as disclosed in PCT Publication No. WO 2005 / 007699 A2 by Jermutus et al.).

[0080] Complementary region: As used herein, the term "complementary region" refers to a nucleotide sequence (e.g., the nucleotide sequence of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., the nucleotide sequence of a target nucleic acid) such that two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is completely complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary (e.g., at least 80%, 90%, 95%, or 99% complementary) to the cognate nucleotide sequence of the target nucleic acid. In some embodiments, the complementary region contains 1, 2, 3, or 4 mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0081] Specifically bind: As used herein, the term "specifically bind" refers to the ability of a molecule to bind to a binding partner with an affinity or binding activity such that, in a binding assay or other binding context, the molecule can distinguish the binding partner from appropriate controls. With respect to an antibody, the term "specifically bind" refers to the ability of the antibody to bind to a particular antigen with an affinity or binding activity (e.g., to the extent that it allows for preferential targeting to a particular cell (e.g., a muscle cell) through binding to the antigen as described herein) such that, compared to an appropriate reference antigen, or an antigen against which the antibody can be used to distinguish a particular antigen from other antigens, the antibody binds to the particular antigen. In some embodiments, when the antibody binds to the target, at least about 10-4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or K less than this D When having, the antibody specifically binds to the target. In some embodiments, the antibody specifically binds to an epitope of the tip domain of the transferrin receptor, for example, 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 or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient / animal suspected of having or having a disease, for example, a human patient. In some embodiments, the subject is a human patient suspected of having a muscle disease (for example, any of the diseases provided in Table 1) or FOP.

[0083] Transferrin receptor: As used herein, the term "transferrin receptor" (also known as TFRC, CD71, p90, or TFR1) refers to an internalizing cell surface receptor that binds to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may be of human origin (NCBI Gene ID 7037), non-human primate origin (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodent origin (e.g., NCBI Gene ID 22042). Additionally, multiple human transcript variants encoding various 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 complexes comprising a targeting agent, e.g., an antibody covalently linked to a molecular payload. In some embodiments, the complex comprises a muscle-targeting antibody covalently linked to an oligonucleotide. The complex may comprise an antibody that specifically binds to a single antigenic site, or an antibody that binds to at least two antigenic sites that may be present on the same antigen or different antigens. The complex 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 complex is responsible for the modulation of a 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 complex comprises a muscle targeting agent (such as an anti-transferrin receptor antibody) covalently linked to a molecular payload (such as an antisense oligonucleotide that targets a muscular disorder allele).

[0086] In some embodiments, the complex is useful for treating a muscular disorder in which the molecular payload affects the activity of the corresponding gene provided in Table 1. For example, depending on the disease, the molecular payload may modulate (such as decrease, increase) gene transcription or expression, modulate the expression of the 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 complementary to the target gene provided in Table 1.

[0087] Table 1 - List of Muscular Disorders and Corresponding Genes

Table 1-1

Table 1-2

Table 1-3

[0088] A. Muscle Targeting Agent Some aspects of the present disclosure provide muscle targeting agents, such as muscle targeting agents for delivering a molecular payload to muscle cells. In some embodiments, such muscle targeting agents are capable of binding to muscle cells, and delivering an associated molecular payload to muscle cells, for example, via specific binding to an antigen on the muscle cell. In some embodiments, the molecular payload is bound to the muscle targeting agent (e.g., covalently bound), and when the muscle targeting agent binds to an antigen on the muscle cell, it is internalized into the muscle cell, for example, 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 more 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 an antigen on muscle, such as skeletal muscle, smooth muscle, or cardiac muscle. In some embodiments, any of the muscle targeting agents provided herein bind to (e.g., specifically bind to) an antigen on skeletal muscle cells, smooth muscle cells, and / or cardiac muscle cells.

[0090] By interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, a molecule that is a substrate for a muscle uptake transporter is useful for delivering a molecular payload into muscle tissue. Binding to a muscle surface recognition element, followed by endocytosis, can allow even macromolecules such as antibodies to enter muscle cells. As another example, a molecular payload conjugated to transferrin or an anti-transferrin receptor antibody can be taken up by muscle cells via binding to the transferrin receptor and then endocytosed, for example, via clathrin-mediated endocytosis.

[0091] The use of a muscle targeting agent can also be useful for concentrating a molecular payload (e.g., an oligonucleotide) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, a muscle targeting agent concentrates the bound molecular payload in muscle cells as compared to another cell type in a subject. In some embodiments, a muscle targeting agent concentrates 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 more than in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or fat cells). In some embodiments, the toxicity of a molecular payload in a subject 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 it is delivered to the subject.

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

[0093] Muscle cells encompassed by the present 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 provides the potential to selectively target muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and / or cardiomyocytes). This specificity may also limit off-target toxicity. Examples of antibodies capable of targeting surface antigens of muscle cells have been reported and are within the scope of the present disclosure. For example, antibodies targeting the surface of muscle cells are described in Arahata K., et al. “Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide” Nature 1988; 333:861-3; Song K.S., et al. “Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins” J Biol Chem 1996; 271:15160-5; and Weisbart R.H. et al., “Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb” Mol Immunol. 2003 Mar, 39(13):78309; the entire contents of each of which are incorporated herein by reference.

[0095] a. Anti-transferrin receptor antibody Some aspects of the present disclosure are based on the recognition that agents that bind to the transferrin receptor, such as anti-transferrin receptor antibodies, can target muscle cells. The transferrin receptor is a cell surface receptor that transports transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels by internalization. Some aspects of the present disclosure provide transferrin receptor-binding proteins that are capable of binding to the transferrin receptor. As a result, aspects of the present disclosure provide binding proteins (such as antibodies) that bind to the transferrin receptor. In some embodiments, the binding protein that binds to the transferrin receptor is internalized into muscle cells along with any molecular payload to which it is bound. As used herein, an antibody that binds to the transferrin receptor may also be referred to as an anti-transferrin receptor antibody. An antibody that binds to, such as specifically binds to, the transferrin receptor may be internalized into cells, such as through receptor-mediated endocytosis, when bound to the transferrin receptor.

[0096] It should be understood that anti-transferrin receptor antibodies may be produced, synthesized, and / or derivatized using several known methodologies, such as library design using phage display. Exemplary methodologies are characterized in the art and incorporated by reference (Diez, P. et al. “High-throughput phage-display screening in array format”, Enzyme and microbial technology, 2015, 79, 34-41.; Christoph M. H. and Stanley, J. R. “Antibody Phage Display: Technique and Applications” J Invest Dermatol. 2014, 134:2.; Engleman, Edgar (Ed.) “Human Hybridomas and Monoclonal Antibodies.” 1985, Springer). In other embodiments, anti-transferrin antibodies have been previously characterized or disclosed.Antibodies that specifically bind to the transferrin receptor are known in the art (see, for example, U.S. Patent No. 4,364,934, filed December 4, 1979, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Patent No. 8,409,573, filed June 14, 2006, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Patent No. 9,708,406, filed May 20, 2014, "Anti-transferrin receptor antibodies and methods of use"; U.S. Patent 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 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 complexes disclosed herein. Examples of anti-transferrin receptor antibodies are listed in Table 2, including related reference epitopes and binding epitopes. 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, such as the anti-transferrin receptor antibodies listed in Table 2.

[0098] Table 2 - List of anti-transferrin receptor antibody clones, including related 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 an epitope (including the apical domain, transferrin binding domain, and protease-like domain) that becomes exposed to the antibody. In some embodiments, the anti-transferrin receptor antibody binds to an amino acid segment of the transferrin receptor of a human or non-human primate animal as provided in SEQ ID NOs: 1-3 in the range of amino acids C89 - F760. In some embodiments, the anti-transferrin receptor antibody specifically binds with a binding affinity of 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. The anti-transferrin receptor antibody used herein may be capable of competing for binding with other anti-transferrin receptor antibodies (such as OKT9, 8D3) that bind to the transferrin receptor with a binding affinity of 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, or less.

[0100] An example of the amino acid sequence of the human transferrin receptor corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAVDEEENADNNTKANVTKPKRCSGSICYGTIAVIVFFLIGFMIGYLGYCKGVEPKTECERLAGTESPVREEPGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 1).

[0101] An example of the amino acid sequence of the non-human primate transferrin receptor corresponding to NCBI sequence NP_001244232.1 (transferrin receptor protein 1, Macaca mulatta) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 2).

[0102] An example of a non-human primate transferrin receptor amino acid sequence corresponding to NCBI accession XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKANGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKIQVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNVLKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 3).

[0103] An example of the mouse transferrin receptor amino acid sequence corresponding to NCBI accession NP_001344227.1 (transferrin receptor protein 1, mus musculus) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYVKIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKNVLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTRLDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF (SEQ ID NO: 4). In some embodiments, the anti-transferrin receptor antibody is directed against the following receptor amino acid segments: Binds to FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 5) and does not inhibit the binding interaction between the transferrin receptor and transferrin and / or human hemochromatosis protein (also known as HFE).

[0104] Suitable methodologies may be used, for example, to obtain and / or produce antibodies, antibody fragments, or antigen-binding agents 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). Antigens of interest may be used as immunogens of any type or entity, for example, recombinant or naturally occurring types or entities. Hybridomas are screened using standard methods, such as ELISA screening, to find at least one hybridoma that produces an antibody that targets a specific antigen. Antibodies may also be produced through the screening of protein expression libraries that express antibodies (for example, phage display libraries). Phage display library design may also be used in some embodiments (see, for example, U.S. Patent No. 5,223,409, filed 3 / 1 / 1991, “Directed evolution of novel binding proteins”; WO 1992 / 18619, filed 4 / 10 / 1992, “Heterodimeric receptor libraries using phagemids”; WO 1991 / 17271, filed 5 / 1 / 1991, “Recombinant library screening methods”; WO 1992 / 20791, filed 5 / 15 / 1992, “Methods for producing members of specific binding pairs”; and WO 1992 / 15679, filed 2 / 28 / 1992, “Improved epitope displaying phage”). In some embodiments, antigens of interest may be used to immunize non-human animals, such as rodents or goats.In some embodiments, once the antibody is obtained from a non-human animal, it may optionally be modified using any number of methodologies, such as, by way of example, 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 mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, there are about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 3, or about 2 sugar molecules present. In some embodiments, the glycosylated antibody is glycosylated either wholly or in part. In some embodiments, the antibody is glycosylated by a chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or inside a cell (optionally lacking enzymes in the N- or O-glycosylation pathway, e.g., glycosyltransferases). 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 the transferrin receptor (e.g., the extracellular portion of the transferrin receptor). In some embodiments, the anti-transferrin receptor antibodies provided herein specifically bind to a transferrin receptor (e.g., a human transferrin receptor). The transferrin receptor is an internalizing cell surface receptor that transports transferrin across the cell membrane and contributes to 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, and the like. 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 human transferrin receptor. In some embodiments, the anti-transferrin receptor antibodies provided herein 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, the anti-transferrin receptor antibodies of the present disclosure include one or more of the CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the 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, the 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, the 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 includes any nucleic acid sequence encoding a molecule comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3 as provided in any one of the anti-transferrin receptor antibodies selected from Table 2. In some embodiments, the antibody heavy and light chain CDR3 domains may specifically play an important role in the binding specificity / affinity of the antibody to the antigen. As a result, the anti-transferrin receptor antibodies of the present disclosure may include at least the heavy chain 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 has one or more CDR (e.g., CDR-H or CDR-L) sequences that are substantially similar to any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 sequences of an anti-transferrin receptor antibody 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) region, and / or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) region of the antibodies described herein can vary by one, two, three, four, five, or six amino acid positions, so long as immunospecific binding to the 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% of the binding of the original antibody from which it is derived). For example, in some embodiments, the position defining the CDR of any of the antibodies described herein can vary by one, two, three, four, five, or six amino acid positions compared to the CDR position of any one of the antibodies described herein, by shifting the N-terminus and / or C-terminus boundaries of the CDR, so long as immunospecific binding to the 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% of the binding of the original antibody from which it is derived).In another aspect, the length of one or more CDRs along the VH (e.g., CDR-H1, CDR-H2, or CDR-H3) region and / or VL (e.g., CDR-L1, CDR-L2, or CDR-L3) region of the antibodies described herein can vary by 1, 2, 3, 4, 5 amino acids, or more amino acids (e.g., become shorter or longer), as long as immunospecific binding to the 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% of the binding of the original antibody from which it is derived).

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

[0110] In some examples, any of the anti-transferrin receptor antibodies of the present disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences that are substantially the same as any one of the anti-transferrin receptor antibodies selected from Table 2. For example, the antibody maintains (e.g., substantially maintains, 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) immunospecific binding to the transferrin receptor (e.g., human transferrin receptor). As long as it is maintained), 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), it may contain up to 5, 4, 3, 2, or 1 amino acid residue variations. One or more CDR sequences (singular or plural) from any of the anti-transferrin receptor antibodies selected from Table 2 may be included. In some embodiments, any of the amino acid variations in any of the CDRs provided herein may be conservative variations. Conservative variations can be introduced into the CDR at positions where the residue does not appear to be involved in the interaction with the transferrin receptor protein (e.g., human transferrin receptor protein), as determined, for example, based on the crystal structure. Some aspects of the present disclosure provide anti-transferrin receptor antibodies that include 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 includes one or more of the CDR-H sequences provided herein (e.g., CDR-H1, CDR-H2, and CDR-H3) (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 includes one or more of the CDR-L sequences provided herein (e.g., CDR-L1, CDR-L2, and CDR-L3) (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, the anti-transferrin receptor antibodies of the present disclosure include any antibody that includes the heavy chain variable domain and / or the 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, the anti-transferrin receptor antibodies of the present disclosure include any antibody that includes any pair of heavy chain variable and light chain variable 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 that are 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 and / or a light chain variable sequence that is at least 75% (by way of example, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy chain variable sequence or 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 sequences do not vary within the scope of any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (by way of example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur within the scope of the heavy chain variable and / or light chain variable sequences that exclude any of the CDR sequences provided herein. In some embodiments, the heavy chain variable sequence and light chain variable sequence include a framework sequence that is at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 2, of any of the anti-transferrin receptor antibodies provided herein.

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

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

[0115] In some embodiments, any of the anti-transferrin receptor antibodies provided herein includes a variable light chain domain that further includes 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, by way of example, from a human, monkey, rat, or 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 includes an amino acid sequence that is 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 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 a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some embodiments, the anti-transferrin receptor antibody comprises either a VL domain, or a variant of the VL domain, and either a VH domain, or a 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 an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, the constant region amino acid sequence of an immunoglobulin molecule of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b). Non-limiting examples of human constant regions are described in the art, see, e.g., Kabat E A et al. (1991) supra.

[0118] In some embodiments, the antibodies of the disclosure have a 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 DIt can bind to a target antigen (e.g., transferrin receptor). For example, an anti-transferrin receptor antibody can bind to a transferrin receptor protein (e.g., human transferrin receptor) with an affinity between 5 pM and 500 nM, such as between 50 pM and 100 nM, such as 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 kinetics of the anti-transferrin receptor antibody can be tested using any suitable method including, but not limited to, biosensor technologies (e.g., OCTET or BIACORE).

[0119] In some embodiments, the antibodies of the present disclosure can bind to a target antigen, e.g., at a relatively high affinity of, for example, -6 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, or lower K D It can bind to a target antigen (e.g., transferrin receptor). For example, an anti-transferrin receptor antibody can bind to a transferrin receptor protein (e.g., human transferrin receptor) with an affinity between 5 pM and 500 nM, such as between 50 pM and 100 nM, such as 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 kinetics of the anti-transferrin receptor antibody can be tested using any suitable method including, but not limited to, biosensor technologies (e.g., OCTET or BIACORE).

[0120] In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody (for example, an antibody and its variants as described in International Application Publication No. 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. For example, (for example, Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242, Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). Also see hgmp.mrc.ac.uk and bioinf.org.uk / abs). Table 1.1 Heavy and light chain CDRs of mouse anti-transferrin receptor antibody [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, the anti-transferrin receptor antibodies of the disclosure comprise CDR-H1, CDR-H2, and CDR-H3 that are the same as CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1. Alternatively or in addition, the anti-transferrin receptor antibodies of the disclosure comprise CDR-L1, CDR-L2, and CDR-L3 that are the same as CDR-L1, CDR-L2, and CDR-L3 shown in Table 1.1.

[0126] In some embodiments, the anti-transferrin receptor antibodies of the disclosure comprise CDR-H1, CDR-H2, and CDR-H3 that contain a total of only 5 amino acid variations (e.g., only 5, 4, 3, 2, or 1 amino acid variations) in combination when compared to CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1. "In combination" means that the total number of amino acid variations in all three heavy chain CDRs is within the defined range. Alternatively or in addition, the anti-transferrin receptor antibodies of the disclosure may comprise CDR-L1, CDR-L2, and CDR-L3 that contain a total of only 5 amino acid variations (e.g., only 5, 4, 3, 2, or 1 amino acid variations) in combination when compared to CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1.

[0127] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include CDR-H1, CDR-H2, and CDR-H3, and at least one of these contains only 3 amino acid variations (e.g., only 3, 2, or 1 amino acid variations) when compared to the corresponding heavy chain CDRs as shown in Table 1.1. Alternatively or in addition, the anti-transferrin receptor antibodies of the present disclosure may include CDR-L1, CDR-L2, and CDR-L3, and at least one of these contains only 3 amino acid variations (e.g., only 3, 2, or 1 amino acid variations) when compared to the corresponding light chain CDRs as shown in Table 1.1.

[0128] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include CDR-L3, which contains only 3 amino acid variations (e.g., only 3, 2, or 1 amino acid variations) when compared to CDR-L3 as shown in Table 1.1. In some embodiments, the transferrin receptor antibodies of the present disclosure include CDR-L3 that contains 1 amino acid variation when compared to CDR-L3 as shown in Table 1.1. In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include CDR-L3 of QHFAGTPLT (SEQ ID NO: 31, according to the Kabat and Chothia definition systems) or QHFAGTPL (SEQ ID NO: 32, according to the Contact definition system). In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 as CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1, and include CDR-L3 of QHFAGTPLT (SEQ ID NO: 31, according to the Kabat and Chothia definition systems) or QHFAGTPL (SEQ ID NO: 32, according to the Contact definition system).

[0129] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure comprise heavy chain CDRs that 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, the anti-transferrin receptor antibodies of the present disclosure comprise light chain CDRs that 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, the anti-transferrin receptor antibodies of the present disclosure comprise a VH comprising the amino acid sequence of SEQ ID NO: 33. Alternatively or in addition, the anti-transferrin receptor antibodies of the present disclosure comprise a VL comprising the amino acid sequence of SEQ ID NO: 34.

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

[0132] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure comprise a VH having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VH as represented by SEQ ID NO: 33. Alternatively or in addition, the anti-transferrin receptor antibodies of the present disclosure comprise a VL having an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the VL as represented by 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 the antibodies). In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1, the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, and include a humanized heavy chain variable region and / or a humanized light chain variable region.

[0134] A humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity determining regions (CDRs) are replaced by residues from the CDRs of a non-human species (donor antibody), such as a mouse, rat, or rabbit, that has the desired specificity, affinity, and capacity. In some embodiments, residues in the Fv framework region (FR) of the human immunoglobulin are replaced by the corresponding non-human residues. Additionally, a humanized antibody may also contain residues not found in the recipient antibody or residues not found in the imported CDR or framework sequences, but residues included to further refine and optimize the performance of the antibody. Generally, a humanized antibody will substantially comprise all of at least one, typically two variable domains, wherein in said variable domains all or substantially all of the CDR regions correspond to the CDR regions of a non-human immunoglobulin and all or substantially all of the FR regions are the FR regions of a human immunoglobulin consensus sequence. A humanized antibody will also optimally include at least a portion of the constant region or domain (Fc) of an immunoglobulin (typically a human immunoglobulin). 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 have been altered with respect to the original antibody, and these are also referred to as one or more CDRs that are derived from one or more CDRs from the original antibody. Humanized antibodies may also be accompanied by affinity maturation.

[0135] In some embodiments, humanization is achieved by joining the CDRs (such as those shown in Table 1.1) into the IGKV1-NL1*01 and IGHV1-3*01 human variable domains. In some embodiments, the anti-transferrin receptor antibodies of the present disclosure are humanized variants that include one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 (when compared to VL as represented by 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 VH as represented by SEQ ID NO: 33). In some embodiments, the anti-transferrin receptor antibodies of the present disclosure are humanized variants that include amino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and 70 (when compared to VL as represented by 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 VH as represented by SEQ ID NO: 33).

[0136] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure are humanized antibodies that contain the residues at positions 43 and 48 of VL as represented by SEQ ID NO: 34. Alternatively or in addition, the anti-transferrin receptor antibodies of the present disclosure are humanized antibodies that contain the residues at positions 48, 67, 69, 71, and 73 of VH as represented by SEQ ID NO: 33.

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

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

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

[0140] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 35. Alternatively or additionally, the 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, the anti-transferrin receptor antibody of the present disclosure comprises a VH that contains only 20 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) when compared to the VH as represented by SEQ ID NO: 35. Alternatively or additionally, the anti-transferrin receptor antibody of the present disclosure comprises a VL that contains only 15 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) when compared to the VL as represented by SEQ ID NO: 36.

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

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

[0144] In some embodiments, the anti-transferrin receptor antibodies of the present 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 VL as represented by 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 VH as represented by SEQ ID NO: 33).

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

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

[0147] In some embodiments, any heavy chain of the anti-transferrin receptor antibodies as described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can belong to any suitable origin, e.g., human, mouse, rat, or rabbit. In a 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 given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 37)

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

[0149] The 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 anti-transferrin receptor antibodies described are provided below:

[0151] Heavy chain (VH + human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(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) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(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 include a heavy chain comprising an amino acid sequence that is at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to SEQ ID NO: 39. Alternatively or additionally, the anti-transferrin receptor antibodies described herein include a light chain comprising an amino acid sequence that is 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 include a heavy chain comprising the amino acid sequence of SEQ ID NO: 39. Alternatively or additionally, the anti-transferrin receptor antibodies described herein include a light chain comprising the amino acid sequence of SEQ ID NO: 40.

[0156] In some embodiments, the anti-transferrin receptor antibodies of the disclosure include a heavy chain that contains only 20 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) when compared to the heavy chain as represented by SEQ ID NO: 39. Alternatively or additionally, the anti-transferrin receptor antibodies of the disclosure include a light chain that contains only 15 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) when compared to the light chain as represented by SEQ ID NO: 40.

[0157] In some embodiments, the anti-transferrin receptor antibodies described herein include a heavy chain comprising an amino acid sequence that is 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 include a light chain comprising an amino acid sequence that is 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 include a heavy chain comprising the amino acid sequence of SEQ ID NO: 41. Alternatively or in addition, the anti-transferrin receptor antibodies described herein include a light chain comprising the amino acid sequence of SEQ ID NO: 42.

[0158] In some embodiments, the anti-transferrin receptor antibodies of the disclosure include a heavy chain that contains only 20 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) when compared to the heavy chain of the humanized antibody as represented by SEQ ID NO: 39. Alternatively or in addition, the anti-transferrin receptor antibodies of the disclosure include a light chain that contains only 15 amino acid variations (e.g., only 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) when compared to the light chain of the humanized antibody as represented by 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). The antigen-binding fragment of an intact antibody (full-length antibody) can be prepared via conventional methods. For example, an F(ab')2 fragment can be produced by pepsin digestion of the antibody molecule, and a Fab fragment 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 + a part of human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (SEQ ID NO: 43)

[0161] Heavy chain FAB (humanized VH + a 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, their antigen-binding fragments (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 scFv. In some embodiments, the anti-transferrin receptor antibodies described herein are scFv-Fab (for example, scFv conjugated with a part of the constant region). In some embodiments, the anti-transferrin receptor antibodies described herein are scFv conjugated with a constant region (for example, the human IgG1 constant region as represented by SEQ ID NO: 39).

[0163] b. Other muscle-targeting antibodies In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to hemojuvelin, caveolin-3, dystrophin, myosin Iib, or CD63. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a myogenic precursor protein. Exemplary myogenic precursor proteins include, without limitation, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha7, integrin alpha7beta1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a skeletal muscle protein. Exemplary skeletal muscle proteins include, without limitation, alpha-sarcoglycan, beta-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin 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, without limitation, alpha-smooth muscle actin, VE-cadherin, caldesmon / CALD1, calponin 1, desmin, histamine H2 R, motilin R / GPR38, transgelin / TAGLN, and vimentin. However, it should be understood that antibodies to additional targets are within the scope of the present disclosure and that the exemplary lists of targets provided herein are not intended to be limiting.

[0164] c. Antibody characteristics / modifications In some embodiments, conservative mutations can be introduced into the antibody sequence (e.g., CDR or framework region) at positions where the residue is unlikely to be involved in the interaction with the target antigen (e.g., transferrin receptor), when determined based on, for example, the crystal structure. In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeting antibodies described herein (e.g., according to the numbering following the Kabat numbering system (e.g., the EU index of Kabat), 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) to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity to cells.

[0165] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine residues in the hinge region can be varied (e.g., increased or decreased) as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine residues in the hinge region of the CH1 domain can be altered, for example, to facilitate the 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 the muscle-targeting antibodies described herein (e.g., numbered according to the Kabat numbering system (e.g., Kabat's EU index), in the CH2 domain (residues 231-340 of human IgG1), and / or in the CH3 domain (residues 341-447 of human IgG1), and / or in the hinge region) to increase or decrease the affinity of the antibody for Fc receptors on effector cells (e.g., activated Fc receptors). Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof, are known to those of skill in the art. Examples of mutations in Fc receptors of an antibody that can be made to alter the affinity of the antibody for Fc receptors are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publications No. WO 02 / 060919; No. WO 98 / 23289; and No. 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 the IgG constant region or its FcRn-binding fragment (preferably, the Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the in vivo half-life of the antibody. For example, for mutations that would alter (e.g., increase or decrease) the in vivo half-life of an antibody, see, for example, International Publications No. WO 02 / 060919; No. WO 98 / 23289; and No. WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745.

[0168] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably, the Fc or hinge-Fc domain fragment) to decrease the in vivo half-life of an anti-transferrin receptor antibody. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably, the Fc or hinge-Fc domain fragment) to increase the in vivo half-life of an antibody. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1) according to the Kabat EU numbering (Kabat E A et al. (1991) supra). In some embodiments, the IgG1 constant region of the antibodies described herein comprises a substitution of methionine (M) to tyrosine (Y) at position 252, a substitution of serine (S) to threonine (T) at position 254, and a substitution of threonine (T) to glutamic acid (E) at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, designated the "YTE mutant," has been shown to exhibit a four-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua W F et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant 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 Kabat EU index.

[0169] In some embodiments, one or more amino acid substitutions are introduced into the Fc region of the IgG constant region to alter one or more effector functions of the anti-transferrin receptor antibody. Effector ligands with altered affinity for themselves can be, for example, Fc receptors or the C1 component of complement. This approach is described in more detail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation (through point mutations or other means) of the constant region domain can reduce binding of the circulating antibody to Fc receptors, thereby increasing tumor localization. For a description of mutations that delete or inactivate the constant region and thereby increase tumor localization, see, for example, U.S. Pat. Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of the antibodies described herein to remove potential glycosylation sites on the Fc region (which may also reduce binding to Fc receptors) (see, for example, Shields R L et al., (2001) J Biol Chem 276:6591-604).

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

[0171] In some embodiments, the heavy chain and / or light chain variable domain(s) sequence(s) of the antibodies provided herein may be used, as described elsewhere herein, to generate, for example, CDR-grafted antibodies, chimeric antibodies, humanized antibodies, or bispecific human antibodies, or antigen-binding fragments. As will be understood by those skilled in the art, any variant, CDR-grafted antibody, chimeric antibody, humanized antibody, or bispecific antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, provided that the variant, CDR-grafted antibody, chimeric antibody, humanized antibody, or bispecific antibody maintains specific binding ability to the transferrin receptor such that it has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it is derived.

[0172] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibody. For example, to avoid potential complications arising from Fab-arm exchange, which is known to occur with 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), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include the stabilizing “Adair” mutation.

[0173] As provided herein, the antibodies of the disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be attached at its C-terminus to a light chain constant region-like Cκ or Cλ. Similarly, the VH domain or a portion thereof may be attached to all or some of the heavy chain-like IgA, IgD, IgE, IgG, and IgM, and to any isotype subclass. The antibody may include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of the disclosure may be combined with any suitable constant region and include the VH and VL domains, or the antigen-binding portion thereof.

[0174] ii. Muscle targeting peptide Some aspects of the present disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences that bind to specific cell types (e.g., peptide sequences that are 5 to 20 amino acids in length) are described. For example, cell-targeting peptides are described in Vines e.,et al.,A.“Cell-penetrating and cell-targeting peptides in drug delivery”Biochim Biophys Acta 2008,1786:126-38;Jarver P.,et al.,“In vivo biodistribution and efficacy of peptide mediated delivery”Trends Pharmacol Sci 2010;31:528-35;Samoylova T.I.,et al.,“Elucidation of muscle-binding peptides by phage display screening”Muscle Nerve 1999;22:460-6;U.S. Patent No. 6,329,501, issued December 11, 2001, titled “METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE”; and Samoylov A.M.,et al.,“Recognition of cell-specific binding of phage display derived peptides using an acoustic wave sensor.”Biomol Eng 2002;18:269-72; the entire contents of each of which are incorporated herein by reference. Selectivity for a desired tissue, e.g., muscle, can be obtained by designing the peptide to interact with a specific cell surface antigen (e.g., a receptor). Skeletal muscle targeting has been investigated and a wide range of molecular payloads can be delivered. These approaches, which do not have many of the practical disadvantages associated with 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 that is 4 to 50 amino acids in length.In some embodiments, the muscle-targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. The muscle-targeting peptide can be generated using any of several methods such as phage display.

[0175] In some embodiments, the muscle-targeting peptide may bind to an internalizing cell surface receptor (e.g., the transferrin receptor) that is overexpressed or relatively highly expressed in muscle cells compared to other cells. In some embodiments, the muscle-targeting peptide may target the transferrin receptor (e.g., may bind to the transferrin receptor). In some embodiments, the peptide that targets the transferrin receptor may include a segment of a naturally occurring ligand, e.g., transferrin. In some embodiments, the peptide that targets the transferrin receptor is as described in U.S. Patent No. 6,743,893, filed November 30, 2000, "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR". In some embodiments, the peptide that targets the transferrin receptor is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells." BMC Cancer. 2011 Aug 18;11:359. In some embodiments, the peptide that targets the transferrin receptor is as described in U.S. Patent No. 8,399,653, filed May 20, 2011, "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY".

[0176] As described above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides were identified using a phage display library that presents peptapeptides on the surface. 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. As a result, in some embodiments, the muscle-targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 6). This peptide exhibited improved specificity for binding to myocardial and skeletal muscle tissues after intravenous injection into mice, with reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have been identified using phage display. For example, in the context of treatment of DMD, a 12-amino acid peptide was identified by a phage display library for muscle targeting. See Yoshida D., et al., “Targeting of salicylate to skin and muscle following topical injections in rats.” Int J Pharm 2002;231:177-84; the entire content of which is hereby incorporated by reference. Here, 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] Any additional methods for identifying peptides selective for muscle (e.g., skeletal muscle) over other cell types involve in vitro selection, which is described in Ghosh D., et al., “Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting” J Virol 2005;79:13667-72; the entire content of which is incorporated herein by reference. Nonspecific cell binders were selected by pre-incubating 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 a peptide. The muscle targeting peptide may correspond to the sequence of a protein that preferentially binds to a protein receptor found in muscle cells. In some embodiments, the muscle targeting peptide highly contains the propensity of hydrophobic amino acids (e.g., valine) so that the peptide can preferentially target muscle cells. In some embodiments, the muscle targeting peptides have not been characterized or disclosed heretofore. These peptides may be evoked, produced, synthesized, and / or derivatized using any of several methodologies, e.g., phage display peptide libraries, one-bead one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. Exemplary methodologies are characterized in the art and incorporated by reference (Gray, B.P. and Brown, K.C. “Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides” Chem Rev. 2014, 114:2, 1020-1081.; Samoylova, T.I. and Smith, B.F. “Elucidation of muscle-binding peptides by phage display screening.” Muscle Nerve, 1999, 22:4. 460-6).In some embodiments, muscle-targeting peptides have been previously disclosed (see, for example, Writer M.J. et al. “Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting 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, M.J. et al. “In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo.” J Mol Biol. 2004,342:1,171-82). Exemplary muscle-targeting peptides include the amino acid sequences of the following groups: CQAQGQLVC (SEQ ID NO: 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, the muscle-targeting peptide may comprise about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. The muscle-targeting peptide may comprise naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring amino acids, or modified amino acids.Non-naturally occurring amino acids include β - amino acids, homo - amino acids, proline derivatives, 3 - substituted alanine derivatives, linear core amino acids, N - methyl amino acids, and other amino acids known in the art. In some embodiments, the muscle - targeting peptide may be linear; in other embodiments, the muscle - targeting peptide may be cyclic (e.g., bicyclic) (see, e.g., Silvana, M.G. et al. Mol. Therapy, 2018, 26:1, 132 - 147).

[0179] iii. Muscle - targeting receptor ligand The muscle - targeting agent may be a ligand, e.g., a ligand that binds to a receptor protein. The muscle - targeting ligand may be a protein that binds to an internalizing cell - surface receptor expressed by muscle cells, e.g., transferrin. Consequently, in some embodiments, the muscle - targeting agent is transferrin or a derivative thereof that binds to the transferrin receptor. The muscle - targeting ligand may alternatively be a small molecule, e.g., a lipophilic small molecule that preferentially targets muscle cells compared to other cell types. Exemplary lipophilic small molecules that may target muscle cells include cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolenic acid, linoleic acid, myristic acid, sterol, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and compounds containing alkoxy acids.

[0180] iv. Muscle - targeting aptamer The muscle targeting agent may be an aptamer, such as an RNA aptamer, that preferentially targets muscle cells compared to other cell types. In some embodiments, the muscle targeting aptamers have not been previously characterized or disclosed. These aptamers may be recalled, generated, synthesized, and / or derivatized using any of several methodologies, such as Systematic Evolution of Ligands by Exponential Enrichment. Exemplary methodologies are characterized in the art and incorporated by reference (Yan, A.C. and Levy, M. “Aptamers and aptamer targeted delivery” RNA biology, 2009, 6:3, 316 - 20.; Germer, K. et al. “RNA aptamers and their therapeutic and diagnostic applications.” Int. J. Biochem. Mol. Biol. 2013;4:27 - 40). In some embodiments, the muscle targeting aptamers have been previously disclosed (see, for example, 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 substrates of muscle transporter proteins such as transporter proteins expressed on the muscle fiber sheath. 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 muscle fiber sheath of skeletal muscle are: (1) the ATP-binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue, and (2) the solute carrier (SLC) superfamily, which may facilitate influx of substrates into skeletal muscle. In some embodiments, the muscle targeting agent is a substrate that binds to the ABC or SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a naturally occurring substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-naturally occurring substrate, e.g., a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0182] In some embodiments, the muscle targeting agent is a substrate of the SLC superfamily of transporters. The SLC transporters are either equilibrium type or use a proton or sodium ion gradient created across the membrane to drive substrate transport. Exemplary SLC transporters having high expression in skeletal muscle include, without limitation, the SATT transporter (ASCT1; SLC1A4), 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 can provide an opportunity for muscle targeting by facilitating the influx of the substrate into skeletal muscle.

[0183] In some embodiments, the muscle targeting agent is a substrate of the equilibrative nucleoside transporter 2 (ENT2) transporter. Compared to other transporters, ENT2 has one of the highest expressed mRNAs in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs such as the brain, heart, placenta, thymus, pancreas, prostate, and kidney, but is particularly abundant in skeletal muscle. Human ENT2 facilitates the uptake of its substrates according to their concentration gradients. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. The hENT2 transporter has low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except inosine. As a result, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, without limitation, inosine, 2',3'-dideoxyinosine, and clofarabine. In some embodiments, any of the muscle targeting agents provided herein are related to a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular payload.

[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 any derivative thereof that binds to OCTN2. In some embodiments, carnitine, mildronate, acetylcarnitine, or their derivatives are covalently linked to the molecular payload (e.g., an oligonucleotide payload).

[0185] The muscle targeting agent may be a protein present in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be the protein hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein) that is involved in iron overload and homeostasis. In some embodiments, hemojuvelin may be full-length or a fragment, or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to the functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack N-terminal signaling, and / or may lack a C-terminal anchoring domain. In some embodiments, hemojuvelin may be annotated with GenBank RefSeq accession numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It should be understood that hemojuvelin may be of human, non-human primate, or rodent origin.

[0186] B. Molecular Payload Some aspects of the present disclosure provide a molecular payload, e.g., a molecular payload for modulating a biological outcome (e.g., transcription of a DNA sequence, protein expression, or protein activity). In some embodiments, the molecular payload is linked or otherwise associated with a muscle targeting agent. In some embodiments, such a molecular payload can target muscle cells via specific binding to a nucleic acid or protein in a muscle cell that has received delivery to the muscle cell by the associated muscle targeting agent. It will be appreciated that various types of muscle targeting agents may be used in accordance with the present disclosure. For example, the molecular payload may include, 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 the gene provided in Table 1. Exemplary molecular payloads are described in more 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 1 (by way of example, at least 2, at least 3, at least 4, at least 5, at least 10) molecular payloads (by way of example, oligonucleotides) are linked to the muscle targeting agent. In some embodiments, all of the molecular payloads linked to the muscle targeting agent are the same and, by way of example, target the same gene. In some embodiments, all of the molecular payloads linked to the muscle targeting agent are different; for example, the molecular payloads may target different portions of the same target gene, or the molecular payloads may target at least two different target genes. In some embodiments, the muscle targeting agent may be linked to some molecular payloads that are the same and some molecular payloads that are different.

[0188] The present disclosure also provides a composition comprising a plurality of complexes, wherein at least 80% (by way of example, 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 complexes comprise a muscle targeting agent linked to the same number of molecular payloads (by way of example, oligonucleotides).

[0189] i. Oligonucleotide 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 and block its translation. In some embodiments, the oligonucleotide may be a guide nucleic acid (e.g., guide RNA) for directing the activity of an enzyme (e.g., gene editing enzyme). Other examples of oligonucleotides are provided herein. In some embodiments, it will be understood that an oligonucleotide of one format (e.g., antisense oligonucleotide) may be suitably adapted to another format (e.g., siRNA oligonucleotide) by incorporating a functional sequence (e.g., antisense strand sequence) from one format into the other format.

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

[0191] DMPK / DM1 In some embodiments, for targeting DMPK, for example for the treatment of DM1, examples of useful oligonucleotides are 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"; U.S. Patent Application Publication No. 20150238627A1, published August 27, 2015, entitled "Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy"; Pandey, S.K. 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, S.A. et al. "Triplet-repeat oligonucleotide-mediated reversal of RNA toxicity in myotonic dystrophy" PNAS, 2009, 106:33, 13915-13920.; Wheeler, T.M. et al., "Targeting nuclear RNA for in vivo correction of myotonic dystrophy" Nature, 2012, 488(7409):111-115.; and U.S. Patent Application Publication No. 20160304877A1, published on October 20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression", the entire contents of each of which are incorporated herein by reference.

[0192] Examples of oligonucleotides for promoting DMPK gene editing include U.S. Patent Application Publication No. 20170088819A1, published on March 3, 2017, entitled "Genetic Correction Of Myotonic Dystrophy Type 1"; and International Patent Application Publication No. WO18002812A1, published on 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 regions complementary to mutant forms of DMPK, such as those 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 that target DMPK. In some embodiments, oligonucleotide targeting is any one of the antisense oligonucleotides (e.g., Gapmer) that target DMPK as described in U.S. Patent Application Publication No. US20160304877A1, published October 20, 2016, entitled “Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression,” 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 number NM_001081560.2 or represented by Genbank accession number NG_009784.1.

[0195] In some embodiments, the DMPK-targeting oligonucleotide comprises a nucleotide sequence comprising a region complementary to a target region that is at least 10 consecutive nucleotides (e.g., at least 10, at least 12, at least 14, at least 16, or more consecutive nucleotides) in Genbank accession number NM_001081560.2.

[0196] In some embodiments, the DMPK-targeting oligonucleotide comprises a gapmer motif. A "gapmer" refers to a chimeric antisense compound in which an internal region having a plurality of nucleotides that support RNase H cleavage is positioned between outer regions having one or more nucleotides, wherein the nucleotides comprising the internal region are chemically distinct from the nucleotides of the outer region. The internal region may be referred to as the "gap segment" and the outer region may be referred to as the "wing segment". In some embodiments, the DMPK-targeting oligonucleotide comprises one or more modified nucleotides and / or one or more internucleotide linkages. In some embodiments, the internucleotide linkage is a phosphorothioate linkage. In some embodiments, the oligonucleotide comprises 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 comprises 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, for example, for the treatment of FSHD, include U.S. Patent No. 9,988,628, issued February 2, 2017, titled “AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; U.S. Patent No. 9,469,851, issued October 30, 2014, titled “RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4”; U.S. Patent Application Publication No. 20120225034, published September 6, 2012, titled “AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; PCT Patent Application Publication No. WO 2013 / 120038, published August 15, 2013, titled “MORPHOLINO TARGETING DUX4 FOR TREATING FSHD”; Chen et al., “Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics,” Molecular Therapy, 2016, 24:8, 1405-1411.; and Ansseau et al., “Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy (FSHD),” Genes, 2017, 8, 93. The entire contents of each of these are incorporated herein by reference. 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, by way of example, for the treatment of FSHD, the oligonucleotide may have a region complementary to hypomethylated and reduced D4Z4 repeats, as described in Daxinger, et al., “Genetic and Epigenetic Contributors to FSHD,” Lim J-W, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1; 24(17): 4817-4828, which is incorporated herein by reference in its entirety.

[0199] DNM2 / CNM In some embodiments, for targeting DNM2, for example, for the treatment of CNM, examples of useful oligonucleotides are provided in US Patent Application Publication No. 20180142008, published on May 24, 2018, entitled "DYNAMIN 2 INHIBITOR FOR THE TREATMENT OF DUCHENNE'S MUSCULAR DYSTROPHY", and PCT Application Publication No. WO 2018 / 100010A1, published on 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 a 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 by reference.

[0200] In some embodiments, by way of example, for the treatment of CNM, the oligonucleotide may have a region complementary to a mutant in DNM2 associated with CNM, as described 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 by reference).

[0201] Pompe disease In some embodiments, by way of example, for the treatment of Pompe disease, the oligonucleotide mediates exon 2 inclusion in the GAA disease allele as described 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 content of which is incorporated herein by reference). Consequently, in some embodiments, the oligonucleotide may have a region complementary to the GAA disease allele.

[0202] In some embodiments, by way of example, for the treatment of Pompe disease, oligonucleotides such as RNAi or antisense oligonucleotides are utilized to suppress the 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 U.S. Patent Application Publication No. 2017 / 182189, published Jun. 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 with a region complementary 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, for the treatment of FOP, examples of oligonucleotides useful for targeting ACVR1 are U.S. Patent Application 2009 / 0253132, published 10 / 8 / 2009, “Mutated ACVR1 for diagnosis and treatment of fibrodyplasia ossificans progressiva (FOP)”; WO 2015 / 152183, published 10 / 8 / 2015, “Prophylactic agent and therapeutic agent for fibrodysplasia ossificans progressive”; Lowery, J.W. 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 (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.; provided in US Patent Application 2017 / 0159056, published 6 / 8 / 2017, "Antisense oligonucleotides and methods of use thereof"; US Patent No. 8,859,752, issued 10 / 4 / 2014, "SIRNA-based therapy of Fibrodyplasia Ossificans Progressiva (FOP)"; WO2004 / 094636, published 11 / 4 / 2004, "Effective sirna knock-down constructs", the contents of each of which are hereby incorporated by reference in their entirety.

[0204] FXN / Friedreich's ataxia In some embodiments, examples of useful oligonucleotides for targeting FXN and / or otherwise compensating for frataxin deficiency, e.g., for the treatment of Friedreich's ataxia, include 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 2008 / 018795, published 2 / 14 / 2008, “Methods and means for treating dna repeat instability associated genetic disorders”; US Patent Application 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”; US Patent Application 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 on 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. are provided, and the entire contents of each of these are incorporated herein by reference.

[0205] In some embodiments, the oligonucleotide payload is configured (e.g., as a gapmer or RNAi oligonucleotide) to inhibit the expression of a natural antisense transcript that inhibits FXN expression, as disclosed, for example, in U.S. Patent No. 9,593,330, filed June 9, 2011, “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 promoting FXN gene editing include WO 2016 / 094845, published 6 / 16 / 2016, "Compositions and methods for editing nucleic acids in cells utilizing oligonucleotides"; WO 2015 / 089354, published 6 / 18 / 2015, "Compositions and methods of use of CRISPR-Cas systems in nucleotide repeat disorders"; WO 2015 / 139139, published 9 / 24 / 2015, "CRISPR-based methods and products for increasing frataxin levels and uses thereof"; and WO 2018 / 002783, published 1 / 4 / 2018, "Materials and methods for treatment of Friedreich ataxia and other related disorders", the entire contents of each of which are incorporated herein by reference.

[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, "Epigenetic regulators of frataxin", the entire contents of which are incorporated herein by reference.

[0208] In some embodiments, the oligonucleotide may have a region complementary to a sequence defined as the human-derived FXN gene (Gene ID 2395; NC_000009.12) and / or the mouse-derived FXN gene (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 content of each of which is incorporated herein by reference).

[0209] DMD / dystrophinopathy Examples of useful oligonucleotides for targeting DMD include U.S. Patent Application Publication No. US20100130591A1, published May 27, 2010, entitled "MULTIPLE EXON SKIPPING COMPOSITIONS FOR DMD"; U.S. Patent No. 8,361,979, registered 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"; U.S. Patent Application Publication No. 20140329881, published November 6, 2014, entitled "EXON SKIPPING COMPOSITIONS FOR TREATING MUSCULAR DYSTROPHY"; U.S. Patent No. 8,232,384, registered July 31, 2012, entitled "ANTISENSE OLIGONUCLEOTIDES FOR INDUCING EXON SKIPPING AND METHODS OF USE THEREOF"; U.S. Patent Application Publication No. 20120022134A1, published January 26, 2012, entitled "METHODS AND MEANS FOR EFFICIENT SKIPPING OF EXON 45 IN DUCHENNE MUSCULAR DYSTROPHY PRE-MRNA"; U.S. Patent Application Publication No. 20120077860, published March 29, 2012, entitled "ADENO-ASSOCIATED VIRAL VECTOR FOR EXON SKIPPING IN A GENE ENCODING A DISPENSABLE DOMAN PROTEIN"; U.S. Patent No. 8,324,371, registered December 4, 2012, entitled "OLIGOMERS"; U.S. Patent No. 9,078,911, registered July 14, 2015, entitled "ANTISENSE OLIGONUCLEOTIDES";U.S. Patent No. 9,079,934, registered on July 14, 2015, with the title "Antisense Nucleic Acids"; U.S. Patent No. 9,034,838, registered on May 19, 2015, with the title "Mir-31 in Duchenne Muscular Dystrophy Therapy"; and International Patent Publication WO2017062862A3, published on April 13, 2017, with the title "Oligonucleotide Compositions and Methods Thereof", the entire contents of each of which are incorporated herein by reference.;

[0210] Examples of oligonucleotides for promoting DMD gene editing include International Patent Publication WO2018053632A1 published on March 29, 2018, with the title "METHODS OF MODIFYING THE DYSTROPHIN GENE AND RESTORING DYSTROPHIN EXPRESSION AND USES THEREOF"; International Patent Publication WO2017049407A1 published on March 30, 2017, with the title "MODIFICATION OF THE DYSTROPHIN GENE AND USES THEREOF"; International Patent Publication WO2016161380A1 published on October 6, 2016, with the title "CRISPR / CAS-RELATED METHODS AND COMPOSITIONS FOR TREATING DUCHENNE MUSCULAR DYSTROPHY AND BECKER MUSCULAR DYSTROPHY"; International Patent Publication WO2017095967 published on June 8, 2017, with the title "THERAPEUTIC TARGETS FOR THE CORRECTION OF THE HUMAN DYSTROPHIN GENE BY GENE EDITING AND METHODS OF USE"; International Patent Publication WO2017072590A1 published on May 4, 2017, with the title "MATERIALS AND METHODS FOR TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY"; International Patent Publication WO2018098480A1 published on May 31, 2018, with the title "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CPF1-MEDIATED GENE EDITING"; and U.S. Patent Application Publication US20170266320A1 published on September 21, 2017, with the title "RNA-Guided Systems for In Vivo Gene Editing".International Patent Publication WO2016025469A1 published on February 18, 2016 under the title "PREVENTION OF MUSCULAR DYSTROPHY BY CRISPR / CAS9-MEDIATED GENE EDITING"; US Patent Application Publication 2016 / 0201089 published on July 14, 2016 under the title "RNA-GUIDED GENE EDITING AND GENE REGULATION"; and US Patent Application Publication 2013 / 0145487 published on June 6, 2013 under the title "MEGANUCLEASE VARIANTS CLEAVING A DNA TARGET SEQUENCE FROM THE DYSTROPHN GENE AND USES THEREOF", the entire contents of each of which are incorporated herein by reference. In some embodiments, the oligonucleotide may have regions complementary to DMD gene sequences of multiple species, selected, by way of example, from human, mouse, and non-human species.;

[0211] In some embodiments, the oligonucleotide may have regions complementary to mutant DMD alleles, such as DMD alleles with at least one mutation in any of exons 1-79 of DMD in humans that lead to frameshifts and inappropriate RNA splicing / processing.;

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

[0213] In some embodiments, the oligonucleotide may target an lncRNA or mRNA, for example, for degradation. In some embodiments, the oligonucleotide may target a nucleic acid encoding a protein involved in the mismatch repair pathway, for example, MSH2, MutL alpha, MutS beta, MutL alpha, for example, for degradation. Non-limiting examples of proteins involved in the mismatch repair pathway (the mRNA encoding such proteins may be targeted by the oligonucleotides described herein) are described in Iyer, R.R. et al., “DNA triplet repeat expansion and mismatch repair” Annu Rev Biochem. 2015;84:199-226; and Schmidt M.H. and Pearson C.E., “Disease-associated repeat instability and mismatch repair” DNA Repair (Amst). 2016 Feb;38:117-26.

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

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

[0216] In some embodiments, the oligonucleotide comprises a region complementary to a target nucleic acid having a length in the range of 8-15, 8-30, 8-40, or 10-50, or 5-50, or 5-40 nucleotides. In some embodiments, the region of the oligonucleotide that is complementary 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 complementary region is complementary to at least 8 contiguous nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain 1, 2 or 3 base mismatches compared to a contiguous portion of nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

[0217] b. Oligonucleotide modification: The oligonucleotides described herein may be modified and include, by way of example, modified sugar moieties, modified internucleoside linkages, modified nucleotides, and / or combinations thereof. In addition, in some embodiments, the oligonucleotide may exhibit one or more of the following properties: does not mediate alternative splicing; is not immunostimulatory; is nuclease resistant; has improved cellular uptake compared to unmodified oligonucleotides; is not toxic to cells or mammals; has improved endosomal exit into cells; minimizes TLR stimulation; or avoids pattern recognition receptors. Any of the modified chemical properties or formats of the oligonucleotides described herein may be combined with each other. For example, 1, 2, 3, 4, 5, or more different types of modifications may be incorporated within the same oligonucleotide.

[0218] In some embodiments, certain nucleotide modifications may be used that render the oligonucleotide into which the modification is incorporated more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides survive intact for longer periods of time than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those with modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short chain alkyl or cycloalkyl sugar linkages, or short chain heteroatomic or heterocyclic sugar linkages. As a result, the oligonucleotides of the present disclosure can be stabilized against nuclease degradation, for example, by incorporation of nucleotide modifications.

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

[0220] c. Modified nucleotide In some embodiments, the oligonucleotide comprises 2'-modified nucleotides, such as, by way of example, 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 a 2'-O-methyl modification. In some embodiments, the oligonucleotide comprises modified nucleotides that include a bridging moiety in which the ribose ring connects two atoms in the ring (e.g., the 2'-O atom is connected to the 4'-C atom). In some embodiments, the oligonucleotide is "locked," e.g., it comprises modified nucleotides that are "locked" by a methylene bridge that connects the 2'-O atom and the 4'-C atom of the ribose ring. Examples of LNA are described in International Patent Application Publication WO / 2008 / 043753, published Apr. 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 (ENA). ENA includes, but is not limited to, 2'-O,4'-C-ethylene-bridged nucleic acids. Examples of ENA 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 entirety.

[0223] In some embodiments, the oligonucleotide may comprise a bridged nucleotide such as a locked nucleic acid (LNA) nucleotide, a constrained ethyl (cEt) nucleotide, or an ethylene-bridged nucleic acid (ENA) nucleotide. In some embodiments, the oligonucleotide comprises a modified nucleotide disclosed in one of the following U.S. patents or patent application publications: U.S. Patent 7,399,845, issued July 15, 2008, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,741,457, issued June 22, 2010, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 8,022,193, issued September 20, 2011, titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,569,686, issued August 4, 2009, titled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,335,765, issued February 26, 2008, titled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent 7,314,923, issued January 1, 2008, titled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent 7,816,333, issued October 19, 2010, titled "Oligonucleotide Analogues And Methods Utilizing The Same" and U.S. Publication No. 2011 / 0009471, now U.S. Patent 8,957,201, issued February 17, 2015, titled "Oligonucleotide Analogues And Methods Utilizing The Same", in their entireties for all purposes, the entire contents of each of which are incorporated herein by reference.

[0224] In some embodiments, the oligonucleotide comprises at least 1 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 pyrimidines, abasic residues, or inverted bases at the 3'-end of the RNA.

[0225] In some embodiments, the oligonucleotide may have at least 1 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 having no modified nucleotides. The oligonucleotide may have a plurality of modified nucleotides that result in an increase in the Tm of the oligonucleotide in the range of a total of 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 15 °C, 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, or more compared to an oligonucleotide having no modified nucleotides.

[0226] The oligonucleotide may include alternating different types of nucleotides. For example, the oligonucleotide may include alternating deoxyribonucleotides or ribonucleotides with 2'-fluoro-deoxyribonucleotides. The oligonucleotide may include alternating deoxyribonucleotides or ribonucleotides with 2'-O-methyl nucleotides. The oligonucleotide may include alternating 2'-fluoro nucleotides with 2'-O-methyl nucleotides. The oligonucleotide may include alternating cross-linked nucleotides with 2'-fluoro or 2'-O-methyl nucleotides.

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

[0228] Linkages containing phosphorus that may be used include, but are not limited to, normal 3'-5' linkages, phosphorothioates having 2'-5' linkage analogs thereof, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates (including 3' alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, and those having reverse polarity thereof (wherein adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'); see U.S. Patent Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0229] In some embodiments, the oligonucleotide may have a heteroatom backbone such as a methylene(methylimino) or MMI backbone; an amide backbone (see De Mesmaeker et al. Ace.Chem.Res.1995,28:366-374); a morpholino backbone (see Summerton and Weller, U.S. Patent No. 5,034,506); or a peptide nucleic acid (PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone and the nucleotides are directly or indirectly attached to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991,254,1497).

[0230] e. Stereospecific oligonucleotides In some embodiments, the phosphorus atom between nucleotides of the oligonucleotide is chiral, and the properties of the oligonucleotide are modulated based on the configuration of the chiral phosphorus atom. In some embodiments, suitable methods may be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (such as, for example, as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms. Chem Soc Rev.2011 Dec;40(12):5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided that include nucleoside units joined together by either substantially all Sp phosphorothioate internucleoside linkages or substantially all Rp phosphorothioate internucleoside linkages. In some embodiments, such phosphorothioate oligonucleotides having substantially enantiopure internucleoside linkages are prepared by enzymatic synthesis or chemical synthesis, such as, 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, chirally controlled oligonucleotides provide a selective cleavage pattern of a target nucleic acid. For example, in some embodiments, a chirally controlled oligonucleotide provides a single cleavage site within a complementary sequence of a nucleic acid, such as, for example, as described in U.S. Patent Application Publication 20170037399 A1, entitled "CHIRAL DESIGN," published February 2, 2017, the contents of which are incorporated herein by reference in their entirety.

[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) (as described, for example, in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entirety).

[0232] g. Peptide nucleic acid (PNA) In some embodiments, both the sugar of the nucleotide units of the oligonucleotide and the internucleoside linkage (backbone) are replaced with novel groups. In some embodiments, the base units are maintained for hybridization to a suitable nucleic acid target compound. An oligonucleotide mimetic, one such oligomeric compound, which has been shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). The sugar-backbone of the oligonucleotide in the PNA compound is replaced with an amide-containing backbone, for example, an aminoethylglycine backbone. The nucleobases are retained and are attached directly or indirectly to the azanitrogen atoms of the amide portions 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 of 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'-X-Y-Z-3' and have X and Z as flanking regions on both sides surrounding the gap region Y. In some embodiments, the Y region is a continuous stretch of nucleotides, for example, a region of at least 6 DNA nucleotides that is 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 is recruited and then cleaves the target nucleic acid. In some embodiments, the Y region is flanked on both the 5' and 3' sides by the regions X and Z containing high affinity modified nucleotides, for example, 1 to 6 modified nucleotides. Examples of modified nucleotides include, but are not limited to, 2'MOE or 2'OMe, or locked nucleic acid bases (LNA). The flanking sequences X and Z may, in some embodiments, be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides in length. The flanking sequences X and Z may be of the same length or may not be of the same length. The gap segment Y may, in some embodiments, be a nucleotide sequence 5 to 20 nucleotides, 6 to 12 nucleotides, or 6 to 10 nucleotides in length.

[0234] In some embodiments, the gap region of the gapmer oligonucleotide may contain modified nucleotides that, in addition to DNA nucleotides such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-configured nucleotides, are known to meet the conditions for efficient RNase H action. In some embodiments, the gap region includes between one or more unmodified nucleosides. In some embodiments, the one or both flanking regions each independently include at least two, at least three, at least four, at least five, or more internucleotide phosphorothioate linkages (e.g., internucleotide phosphorothioate linkages or other linkages). In some embodiments, the gap region and the two flanking regions each independently include at least two, at least three, at least four, at least five, or more internucleotide modified nucleoside linkages (e.g., internucleotide phosphorothioate linkages or other linkages).

[0235] The gapmer 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; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. Patent Publications Nos. US20090286969, US20100197762, and US20110112170; and PCT Publications Nos. WO2008049085 and WO2009090182, each of which is hereby incorporated by reference in its entirety.

[0236] i.Mixmer In some embodiments, the oligonucleotides described herein may be mixmers or may contain mixmer array patterns. Generally, a mixmer is an oligonucleotide that contains both naturally occurring nucleotides and non-naturally occurring nucleotides, or an oligonucleotide that contains two different types of non-naturally occurring nucleotides, typically in an alternating pattern. Mixmers generally have higher binding affinity than unmodified oligonucleotides and specifically bind to target molecules and may be used, for example, to block binding sites on target molecules. Generally, mixmers do not recruit RNase to the target molecule and thus do not promote cleavage of the target molecule. 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 an alternating pattern of nucleotide analogs and naturally occurring nucleotides, or an alternating pattern of one type of nucleotide analog and another type of nucleotide analog. However, a mixmer need not contain an alternating pattern and instead can include 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 alternating pattern can, for example, be a modified nucleotide such as LNA every two or three nucleotides, 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 an alternating pattern of modified nucleotides such as LNA units may be combined with modified nucleotides at fixed positions, for example, at the 5' or 3' terminus.

[0238] In some embodiments, a mixmer does not include a region of naturally occurring nucleotides, such as DNA nucleotides, that is more than 5, more than 4, more than 3, or more than 2 consecutive. In some embodiments, a mixmer includes at least a region consisting of at least 2 consecutive modified nucleotides, such as at least 2 consecutive LNAs. In some embodiments, a mixmer includes at least a region consisting of at least 3 consecutive modified nucleotides, such as at least 3 consecutive LNAs.

[0239] In some embodiments, a mixmer does not include a region of nucleotide analogs, such as LNAs, that is more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 consecutive. In some embodiments, the LNA units may be replaced by other nucleotide analogs, such as the nucleotide analogs referred to herein.

[0240] A mixmer may be designed to include a mixture of affinity-enhancing modified nucleotides, such as LNA nucleotides and 2'-O-methyl nucleotides, in non-limiting examples. In some embodiments, a mixmer includes a modified internucleoside linkage (e.g., phosphorothioate internucleoside linkage or other linkages) between at least 2, at least 3, at least 4, at least 5, or more nucleotides.

[0241] A mixmer 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. 7687617.

[0242] In some embodiments, the mixmer comprises one or more morpholino nucleotides. For example, in some embodiments, the mixmer may comprise morpholino nucleotides mixed (e.g., in an alternating 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, the mixmer is 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), 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 (each of which is incorporated herein by reference).

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

[0245] Upon receiving a suitable target RNA sequence, siRNA molecules comprising nucleotide sequences complementary to all or part of the target sequence, i.e., antisense sequences, can be designed and prepared using suitable methods (see, e.g., PCT Publication No. WO2004 / 016735; and U.S. Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791).

[0246] The siRNA molecule can be double-stranded (i.e., a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single-stranded (i.e., an ssRNA molecule comprising only the antisense strand). The siRNA molecule can contain a duplex, asymmetric duplex, hairpin, or asymmetric hairpin secondary structure having self-complementary sense and antisense strands.

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

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

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

[0250] k. MicroRNA (miRNA) In some aspects, the oligonucleotide may be a microRNA (miRNA). MicroRNAs (referred to as "miRNAs") are small non-coding RNAs that belong to the 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), which are processed in the nucleus to become approximately 70-nucleotide pre-miRNAs, which fold into an imperfect stem-loop structure. These pre-miRNAs typically undergo additional processing steps in the cytoplasm, where the mature miRNA, which is 18 - 25 nucleotides in length, is 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, fragments of its variants, or fragments of its variants that retain the biological activity of mature miRNA. In one aspect, the size range of miRNA can be from 21 nucleotides to 170 nucleotides. In one aspect, the size range of miRNA is from 70 nucleotides to 170 nucleotides in length. In another aspect, mature miRNA with a length from 21 nucleotides to 25 nucleotides can be used.

[0252] l. Aptamer In some embodiments, the oligonucleotides provided herein may be in the form of aptamers. Generally, in the context of a molecular payload, an aptamer is any nucleic acid that specifically binds to a target such as a small molecule, protein, nucleic acid, etc. 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 helical and / or loop structures. The nucleic acids forming the nucleic acid aptamer may include naturally occurring nucleotides, modified nucleotides, hydrocarbon linkers (e.g., alkylene) or polyether linkers (e.g., PEG linker) inserted between one or more nucleotides, naturally occurring nucleotides with hydrocarbon or PEG linker inserted between one or more nucleotides, modified nucleotides with hydrocarbon or PEG linker inserted between one or more nucleotides, or combinations thereof. Exemplary publications and patents that describe aptamers and methods for producing aptamers include, for 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. Ribozymes In some embodiments, the oligonucleotides provided herein may be in the form of ribozymes. A ribozyme (ribonucleic acid enzyme) is a molecule, typically an RNA molecule, capable of performing specific biochemical reactions similar to the action of protein enzymes. Ribozymes are molecules having enzymatic activity including the ability to cleave specific phosphodiester linkages in RNA molecules (such as mRNA, RNA-containing substrates, lncRNA, and the ribozyme itself) to which they hybridize.

[0254] A ribozyme can adopt one of several physical structures, one of which is called the "hammerhead." The hammerhead ribozyme consists of a catalytic core containing a conserved 9-base sequence, a double-stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA regions on both sides surrounding the catalytic core. By means of the regions on both sides, the ribozyme can specifically bind to the target RNA by forming double-stranded stems I and III. Cleavage occurs next to a specific ribonucleotide triplet by an ester exchange reaction from a 3',5'-phosphodiester to a 2',3'-cyclic phosphodiester, either 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). Although not wishing to be bound by theory, this catalytic activity is thought to require the presence of specific sequences highly conserved in the catalytic region of the ribozyme.

[0255] Modifications in ribozyme structures also include substitution or replacement of non-core portions of various molecules with non-nucleotide molecules. For example, Benseler et al. (J. Am. Chem. Soc. (1993) 115:8483 - 8484) disclosed a hammerhead-like molecule where all 2 base pairs of stem II and 4 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 regarding ethylene glycol. Thomson et al. (Nucleic Acids Res. (1993) 21:5600 - 5603) replaced loop II with linear non-nucleotide linkers of lengths 13, 17, and 19 atoms.

[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 can be purchased from commercial sources (e.g., US Biochemicals), and, if desired, nucleotide analogs can be incorporated to increase the resistance of the oligonucleotide to degradation by nucleases in cells. Ribozymes can be synthesized in any known manner, for example, using a commercially available synthesizer (manufactured by, for example, 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). The ribozyme RNA sequence can be synthesized by conventional methods, for example, using an RNA polymerase such as T7 or SP6.

[0257] n. guide nucleic acid In some embodiments, the oligonucleotide is a guide nucleic acid, e.g., a guide RNA (gRNA) molecule. Generally, a guide RNA is a short synthetic RNA 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 to the DNA target sequence. In some embodiments, the napDNAbp is a nucleic acid-programmable protein that forms a complex with one or more RNAs (singular or plural) that target a target DNA sequence (e.g., a target genomic DNA sequence) to the nucleic acid-programmable protein (e.g., binds to or associates with this). In some embodiments, a nucleic acid-programmable nuclease, when in a complex with RNA, may also be referred to as a nuclease:RNA complex. The 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 may also be referred to as a single guide RNA (sgRNA), but 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 a target nucleic acid (i.e., directs 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 includes a stem-loop structure. In some embodiments, domain (2) is identical or homologous to tracrRNA as provided in Jinek et al., Science 337:816-821 (2012) (the entire content of which is incorporated herein by reference).

[0259] In some embodiments, the gRNA comprises two or more of domains (1) and (2) and may be referred to as an extended gRNA. For example, an extended gRNA will bind to two or more Cas9 proteins and bind to a target nucleic acid at two or more distinct regions, as described herein. The gRNA comprises a nucleotide sequence complementary to the target site that mediates binding of the nuclease / RNA complex to the target site and provides sequence specificity to the nuclease:RNA complex.In some embodiments, the nuclease capable of programming RNA is a (CRISPR-associated) Cas9 endonuclease, such as Cas9 (Csn1) from Streptococcus pyogenes (see, for example, “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti J.J., McShan W.M., Ajdic D.J., Savic D.J., Savic G., Lyon K., Primeaux C., Sezate S., Suvorov A.N., Kenton S., Lai H.S., Lin S.P., Qian Y., Jia H.G., Najar F.Z., Ren Q., Zhu H., Song L., White J., Yuan X., Clifton S.W., Roe B.A., McLaughlin R.E., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma C.M., Gonzales K., Chao Y., Pirzada Z.A., Eckert M.R., 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 J.A., Charpentier E. Science 337:816-821 (2012), each of which is incorporated herein by reference in its entirety).

[0260] o. Splice-modifying 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 a gene. By way of non-limiting example, the oligonucleotides may induce the skipping of an exon encoding a frameshift mutation and / or an exon encoding a premature stop codon. 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 (e.g., the truncated but functional DMD protein described below) compared to the reference protein. In some embodiments, the oligonucleotides promote the inclusion of a particular 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] Changes (e.g., deletions, point mutations, and duplications) in the gene encoding dystrophin (DMD) cause DMD. These changes can lead to frameshift and / or nonsense mutations. In some embodiments, the oligonucleotides of the present disclosure promote the 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. See, e.g., U.S. Patent No. 8,486,907, published July 16, 2013, and U.S. 20140275212, published September 18, 2014.

[0262] In SMA, there is a loss of functional SMN1. The SMN2 gene is a paralog of SMN1, but alternative splicing of the SMN2 gene leads primarily to skipping of exon 7 and subsequent production of a truncated SMN protein that cannot compensate for the loss of SMN1. In some embodiments, the oligonucleotides of the disclosure promote inclusion of SMN2 exon 7. In some embodiments, the oligonucleotide is an antisense oligonucleotide that targets an SMN2 splice site inhibitor sequence (see, for example, U.S. Patent No. 7,838,657, published November 23, 2010).

[0263] p. multimer In some embodiments, the molecular payload may include a multimer (e.g., a concatemer) of two or more oligonucleotides connected by a linker. Thus, in some embodiments, the loading of oligonucleotides in the complex / conjugate can be increased beyond the available ligation sites on the targeting agent (e.g., available thiol sites on an antibody) or otherwise arranged to reach a specific payload loading amount. 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, the multimer includes two or more oligonucleotides linked together by a cleavable linker. However, in some embodiments, the multimer includes two or more oligonucleotides linked together by a non-cleavable linker. In some embodiments, the multimer includes two, three, four, five, six, seven, eight, nine, ten, or more oligonucleotides linked together. In some embodiments, the multimer includes two to five, two to ten, or four to twenty oligonucleotides linked together.

[0265] In some embodiments, the multimer comprises two or more oligonucleotides linked end-to-end (in a linear arrangement). In some embodiments, the 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, the multimer comprises the 5' end of one oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, the multimer comprises the 3' end of one oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, the multimer comprises the 5' end of one oligonucleotide linked to the 5' end of another oligonucleotide. Additionally, in some embodiments, the multimer can comprise a branched structure that includes multiple oligonucleotides linked together by a branched linker.

[0266] Further examples of multimers that may be used in the complexes provided herein are disclosed, 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 No. 5,693,773, issued December 2, 1997, entitled Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers Targeting Nucleic Acids Comprising Mixed Sequences Of Purines And Pyrimidines, the contents of each of which are incorporated herein by reference in their entirety.

[0267] ii. Small molecule: 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, by way of example, for the treatment of DM, small molecules are as described in US Patent Application Publication No. 2016052914A1, published on 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 molecules are MBNL1 upregulators such as phenylbutazone, ketoprofen, ISOX, or vorinostat. In some embodiments, the small molecules are H-Ras pathway inhibitors such as manumycin A. In some embodiments, the small molecules are protein kinase modulators such as Ro-318220, C16, C51, metformin, AICAR, lithium chloride, TDZD-8 or Bio. In some embodiments, the small molecules are plant alkaloids such as harmine. In some embodiments, the small molecules are transcription inhibitors 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, for example, 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, as disclosed, for example, in Zhang 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 content of which is 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, Feb 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 transcriptional inhibitor such as SHC351, SHC540, SHC572. In some embodiments, the small molecule is STR00316, which increases the production or activity of another protein such as 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, by way of example, for the treatment of CNM, small molecules are 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, small molecules are 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-carbohydrazide azide. In some embodiments, small molecules are 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, small molecules are retinoid receptor agonists 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, small molecules are as described in U.S. Patent Application Publication No. 20170119748, published on May 4, 2017, entitled "METHODS, COMPOUNDS, AND COMPOSITIONS FOR THE TREATMENT OF MUSCULOSKELETAL DISEASES". The entire content of each of these publications is incorporated herein by reference.

[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 small molecule ML247 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 increase the activity of PD-related GAA alleles or wild-type GAA alleles. The entire content of each of these publications is incorporated herein by reference.

[0272] FXN / Friedreich ataxia In some embodiments, by way of example, for the treatment of Friedreich's ataxia, small molecules are 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, small molecules are 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, T.E. 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 J.M. “Small molecules affecting transcription in Friedreich ataxia.” Pharmacol Ther. 2007 Nov;116(2):236-48. For example, in some embodiments, small molecules are inhibitors of histone deacetylase and are, by way of example, BML-210 and compound 106. In some embodiments, small molecules are 17β-estradiol or methylene blue. In some embodiments, small molecules target (e.g., bind to) disease-related repeats and / or R-loops. In some embodiments, small molecules are 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 / dystrophin disorder In some embodiments, the small molecule enhances exon skipping of mRNA expression from the mutant DMD allele. In some embodiments, the small molecule is as described in U.S. Patent Application Publication No. 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 enhancer of exon skipping such as perphenazine, flupenthixol, zuclopenthixol, or corynanthine. In some embodiments, the small molecule enhancer of exon skipping inhibits the ryanodine receptor or calmodulin. In some embodiments, the small molecule is an H-Ras pathway inhibitor such as manumycin A. In some embodiments, the small molecule is a suppressor of the stop codon and desensitizes ribosomes to premature stop codons. In some embodiments, the small molecule is ataluren as described in McElroy S.P. 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, A.Y. 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, published August 16, 2007, entitled "TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY" and / or International Publication No. WO / 2017 / 168151, published October 5, 2017, entitled "COMPOSITION FOR THE TREATMENT OF DUCHENNE MUSCULAR DYSTROPHY".

[0274] MYH7 / hypertrophic cardiomyopathy In some embodiments, the small molecule is a hypomethylating agent such as 5-azacitidine or 5-aza-2'-deoxycytidine that modulates the 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 nifroxide, 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 para-Nitroblebbistatin, which reduces the force of myosin contraction without changing the dissociation of ADP, as described in Tang, W., et al. “Modulating Beta-Cardiac Myosin Function at the Molecular and Tissue Levels,” Front. Physiol. 2016 (7): 659 (the entire contents of which are incorporated herein by reference).

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

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

[0277] DMPK / DM1 The peptide or protein payload may, by way of example, correspond to the sequence of a protein that preferentially binds to a nucleic acid (such as a disease-related repeat) or a protein (such as MBNL1 found in muscle cells) 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 (such as bind to) a disease-related repeat, such as an RNA CUG repeat expansion.

[0278] In some embodiments, by way of example, for the treatment of DM1, the peptide or protein comprises a fragment of an MBNL protein, such as MBNL1. In some embodiments, the peptide or protein comprises at least one zinc finger. In some embodiments, the peptide or protein may comprise from about 2 to 25 amino acids, from about 2 to 20 amino acids, from about 2 to 15 amino acids, from about 2 to 10 amino acids, or from about 2 to 5 amino acids. The peptide or protein may comprise naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the peptide may be linear; in other embodiments, the peptide may be cyclic, such as 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 that inhibits DUX4 expression, for example, by blocking the binding of an activator.

[0280] DNM2 / CNM In some embodiments, for example, for the treatment of CNM, a peptide is a dynamin inhibitor peptide with the amino acid sequence QVPSRPNRAP as described in US Patent Application Publication No. 20160264976, published on 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 described 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 the GAA activity of the subject. In some embodiments, the acid alpha-glucosidase or wild-type GAA protein is encoded by the GAA gene.

[0282] ACVR1 / FOP In some embodiments, by way of example, for the treatment of FOP, the peptide or protein is a BMP inhibitor such as regulatory SMAD6 and 7 or fragments 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 ataxia In some embodiments, by way of example, for the treatment of Friedreich's ataxia, the peptide is as described in U.S. Patent No. 8,815,230, filed August 30, 2010, "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, P.M. 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) disease-related repeats, such as GAA repeat expansions.

[0284] DMD / dystrophinopathy In some embodiments, for example, for the treatment of dystrophinopathies such as Duchenne muscular dystrophy, the peptide may facilitate exon skipping in the mRNA expressed from the mutant DMD allele. 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, such as an amino acid segment of the functional dystrophin protein.

[0285] iv. Nucleic acid construct 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, such as those described in U.S. Patent 8,710,200, issued April 24, 2014, titled "Engineered nucleic acids encoding a modified erythropoietin and their expression". In some embodiments, the mRNA may contain a 5' methyl cap. In some embodiments, the mRNA may contain a polyA tail, optionally up to 160 nucleotides in length. The gene expression construct may encode the sequence of a protein that is deficient in a muscle disease. In some embodiments, the gene expression construct may be expressed in the nucleus of a muscle cell, and for example, may be overexpressed. In some embodiments, the gene expression construct may encode a gene that is deficient in a muscle disease. In some embodiments, the gene expression construct encodes a protein that contains at least one zinc finger. In some embodiments, the gene expression construct encodes a protein that binds to the gene of Table 1. In some embodiments, the gene expression construct encodes a protein that leads to a reduction in the expression of a protein (e.g., a mutant protein) encoded by the gene of 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, titled "CLOSED-ENDED LINEAR DUPLEX DNA FOR NON-VIRAL GENE TRANSFER," published on September 19, 2017; U.S. Patent 8,853,377B2, titled "MRNA FOR USE IN TREATMENT OF HUMAN GENETIC DISEASES," issued on October 7, 2014; and U.S. Patent US8822663B2, "ENGINEERED NUCLEIC ACIDS AND METHODS OF USE THEREOF," issued on September 2, 2014, the contents of each of which are incorporated herein by reference in their entirety.

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

[0287] DMPK / DM1 In some embodiments, by way of example, for the treatment of DM, the gene expression construct encodes an MBNL protein, such as MBNL1. DUX4 / FSHD In some embodiments, by way of example, 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, such as by blocking the binding of an activator). DNM2 / CNM In some embodiments, by way of example, for the treatment of CNM1, the gene expression construct may encode a protein that downregulates the expression of mutant DNM2 protein or that encodes the sequence of a protein that 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 spliceosome-mediated RNA trans-splicing components that may be used to prime 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, by way of example, for the treatment of Pompe disease, the gene expression construct encodes wild-type GAA protein. The gene expression construct may encode a protein sequence that leads to decreased expression of the ACVR1 gene or decreased activity of the GYS1 protein. In some embodiments, by way of 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 the expression of ACVR1. In some embodiments, the gene expression construct encodes an oligonucleotide (e.g., shRNA) that inhibits the expression of ACVR1.

[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, for example overexpressed, in the nucleus of muscle cells. In some embodiments, the gene expression construct encodes frataxin. In some embodiments, the gene expression construct encodes a protein that inhibits the function of an epigenetic regulator that negatively regulates the expression of FXN, for example, a histone deacetylase. In some embodiments, the gene expression construct encodes a protein that binds to the disease-associated repeat expansion of GAA trinucleotides. In some embodiments, the gene expression construct encodes a protein that leads to a reduction in the expression of an epigenetic regulator that negatively regulates the expression of FXN, for example, a histone deacetylase. In some embodiments, the gene expression construct encodes a gene editing enzyme. In some embodiments, the gene expression construct encodes erythropoietin (see, for example, Miller, J.L. 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, for example, U.S. Patent No. 8,815,230, filed 8 / 30 / 2010, “Methods for treating Friedreich's ataxia with interferon gamma”).

[0291] DMD / dystrophinopathy The gene expression construct may encode the sequence of any protein that shares a common function with dystrophin protein, dystrophin fragments, mini-dystrophin, utrophin protein, or dystrophin. In some embodiments, the gene expression construct may be expressed, for example, overexpressed, in the nucleus of muscle cells. 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, such as 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 No. US20170368198A1, published on 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 that connects a muscle targeting agent to a molecular payload. The linker includes at least one covalent bond. In some embodiments, the linker may be a single bond, such as a disulfide bond or disulfide bridge, that connects 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. The linker is generally stable in vitro and in vivo and may be stable in a particular cellular environment. In addition, generally the linker does not negatively affect the functional properties of either the muscle targeting agent or the molecular payload. Examples and methods of linker synthesis are known in the art (see, for example, Kline, T. et al. “Methods to Make Homogenous Antibody Drug Conjugates.” Pharmaceutical Research, 2015, 32:11, 3480-3493.; Jain, N. et al. “Current ADC Linker Chemistry” Pharm Res. 2015, 32:11, 3526-3540.; McCombs, J.R. and Owen, S.C. “Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry” AAPS J. 2015, 17:2, 339-351.).

[0293] The linker precursor will typically contain two different highly reactive species that can attach to both the muscle targeting agent and the molecular payload. In some embodiments, the two different highly 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 residue or cysteine residue of the muscle targeting agent. In some embodiments, the linker is connected to a cysteine residue of the muscle targeting agent via a maleimide-containing linker, where optionally, the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some embodiments, the linker is connected to a cysteine residue of the muscle targeting agent or 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 the 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. These linkers are generally cleavable only intracellularly and are preferably stable in the extracellular environment, for example, extracellularly in muscle cells.

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

[0296] pH-sensitive linkers are covalent linkages that are readily degraded in high or low pH environments. In some embodiments, the pH-sensitive linker may be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker contains a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved within endosomes or lysosomes.

[0297] In some embodiments, the glutathione-sensitive linker contains a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with glutathione species inside the cell. In some embodiments, the disulfide moiety further contains at least 1 amino acid, such as a cysteine residue.

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

Chemical formula

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

Chemical formula

[0300] ii. Non-cleavable linker In some embodiments, a non-cleavable linker may be used. Generally, a non-cleavable linker cannot be easily decomposed in a cellular or physiological environment. In some embodiments, a non-cleavable linker contains an optionally substituted alkyl group, where the substitution may include halogen, hydroxyl group, oxygen species, and other common substitutions. In some embodiments, the linker may include an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, heteroarylene, a peptide sequence containing at least one non-natural amino acid, a truncated glycan, a sugar (s) that cannot be enzymatically degraded, an azide, an alkyne-azide, a peptide sequence containing an LPXT sequence (SEQ ID NO: 15), a thioether, biotin, biphenyl, a polyethylene glycol or equivalent compound of repeating units, an acid ester, an acid amide, a sulfamide, and / or an alkoxy-amine linker. In some embodiments, sortase-mediated ligation uses a muscle targeting agent containing an LPXT sequence (G) nIt will be utilized for ligation to a molecular payload containing an array (see, for example, Profit 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 LPXTG sequence (SEQ ID NO: 16), where X is any amino acid.

[0301] In some embodiments, the linker is a substituted alkylene, optionally substituted alkenylene, optionally substituted alkynylene, optionally substituted cycloalkylene, optionally substituted cycloalkenylene, optionally substituted arylene, optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; imino, optionally substituted nitrogen species, optionally substituted oxygen species O, optionally substituted sulfur species, or may comprise poly(alkylene oxide), for example, polyethylene oxide or polypropylene oxide.

[0302] iii. Linker conjugation In some embodiments, the linker is connected to the muscle targeting agent and / or the 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, for example, the phosphate at the end of the oligonucleotide backbone. In some embodiments, the linker is connected to a muscle targeting agent, such as an antibody, through a lysine residue or a cysteine residue present on the muscle targeting agent.

[0303] In some embodiments, the linker is connected to the muscle targeting agent and / or the molecular payload by a cycloaddition reaction between an azide and an alkyne that forms a triazole, where the azide and the alkyne may be located on the muscle targeting agent, the molecular payload, or the linker. In some embodiments, the alkyne may be a cyclic alkyne, such as cyclooctyne by way of example. In some embodiments, the alkyne may be bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctane is as described in International Patent Application Publication WO2011136645, published November 3, 2011, entitled "Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions". In some embodiments, the azide may be a sugar or carbohydrate molecule containing an azide. In some embodiments, the azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the sugar or carbohydrate molecule containing an azide is as described in International Patent Application Publication WO2016170186, published October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase".In some embodiments, the cycloaddition reaction between an azide and an alkyne that forms a triazole (where the azide and alkyne may be located on a muscle targeting agent, molecular payload, or linker) is as described in International Patent Application Publication WO2014065661, published May 1, 2014, titled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or International Patent Application Publication WO2016170186, published October 27, 2016, titled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase".

[0304] In some embodiments, the linker further includes a spacer, such as a polyethylene glycol spacer or an acyl / carbamoyl sulfamide spacer, such as a HydraSpace™ spacer. In some embodiments, the spacer is as described in Verkade, J.M.M. et al., “A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates”, Antibodies, 2018, 7, 12.

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

[0306] In some embodiments, the linker is connected to the muscle targeting agent and / or the molecular payload by a conjugate addition reaction between a nucleophilic reagent (e.g., an amine group or a hydroxyl group) and an electrophilic reagent (e.g., a carboxylic acid or an aldehyde). In some embodiments, prior to the reaction between the linker and the muscle targeting agent or the molecular payload, the nucleophilic reagent may be present on the linker and the electrophilic reagent may be present on the muscle targeting agent or the molecular payload. In some embodiments, prior to the reaction between the linker and the muscle targeting agent or the molecular payload, the electrophilic reagent may be present on the linker and the nucleophilic reagent may be present on the muscle targeting agent or the molecular payload. In some embodiments, the electrophilic reagent may be an azide, a silicon center, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidyl ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl, an activated phosphorus center, and / or an activated sulfur center. In some embodiments, the nucleophilic reagent 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 complex comprising any one of the muscle targeting agents (such as an anti-transferrin receptor antibody) described herein covalently linked to any of the molecular payloads (such as oligonucleotides) described herein. In some embodiments, the muscle targeting agent (such as an anti-transferrin receptor antibody) is covalently linked to the molecular payload (such as 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 (such as via a cysteine in the antibody).

[0308] An exemplary structure of a complex comprising an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker is provided below:

Chemical formula

[0309] Antibodies are understood to be linkable to oligonucleotides in various stoichiometries, and said property may also be referred to as the drug-to-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 complexes each having a different DAR is provided. In some embodiments, the average DAR of the complexes in such a mixture may be in the range of 1-3, 1-4, 1-5 or more. The DAR may be increased by conjugating the oligonucleotide to various sites on the antibody and / or by conjugating a multimer to one or more sites 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 dimer oligonucleotide to a single site on the antibody.

[0310] In some embodiments, the complexes 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 complexes 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 internal portion 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 conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide, wherein the anti-transferrin receptor antibody comprises the same CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1; and the same CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1.

[0312] In some embodiments, the conjugate described herein comprises 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 conjugate described herein comprises 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 conjugate described herein comprises 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 conjugate described herein comprises 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 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 the same CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1; and the same CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1.

[0315] In some embodiments, the 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, the 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, the 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, the 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, the conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, where the anti-transferrin receptor antibody comprises the same CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1; and the same CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1, and where the conjugate comprises the following structure: [Chemical formula] Here, the Val-cit linker is linked to the 5'-end, 3'-end, or internally of the oligonucleotide, and here 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, the conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, where 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 where the conjugate comprises the following structure: [Chemical formula] Here, the Val-cit linker is linked to the 5'-end, 3'-end, or internally of the oligonucleotide, and here 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, the conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, where 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 where the conjugate comprises the following structure: [Chemical formula] where the linker Val-cit linker is linked to the 5'-end, 3'-end, or internally of the oligonucleotide, and where the 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).

[0320] In some embodiments, the conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, where 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 where the conjugate comprises the following structure: [Chemical formula] where the linker Val-cit linker is linked to the 5'-end, 3'-end, or internally of the oligonucleotide, and where the 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).

[0321] In some embodiments, the conjugate described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, where 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 where the conjugate comprises the following structure:

Chemical formula

[0322] III. Formulations 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 conjugate can be delivered to a subject using formulations that minimize degradation and facilitate delivery and / or uptake, or that provide other beneficial properties to the conjugate in the formulation. In some embodiments, what is provided herein is a composition comprising a conjugate and a pharmaceutically acceptable carrier. Such a composition can be suitably formulated such that a sufficient amount of the conjugate can penetrate into target muscle cells when administered either in the environment around the target cells of the subject or systemically in the subject. In some embodiments, the conjugate is formulated in a buffered solution such as phosphate buffered saline, in liposomes, in micellar structures, and in capsids.

[0323] It should be understood that in some embodiments, the composition may individually comprise one or more components of the conjugate 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, the formulations as disclosed herein include excipients. In some embodiments, the excipients impart 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 its components (e.g., an oligonucleotide or an antibody) are lyophilized to extend their shelf life and then reconstituted into a solution prior to use (e.g., administration to a subject). Consequently, the excipient in a composition containing the complex or its components 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, e.g., intravenous administration, intradermal administration, subcutaneous administration. Typically, the route of administration is intravenous administration or subcutaneous administration. In some embodiments, the route of administration is extra-muscular parenteral administration.

[0327] A pharmaceutical composition suitable for use in an injection comprises a sterile aqueous solution (soluble in water here) or a dispersion solution, and a sterile powder for the immediate preparation of a sterile injectable solution or dispersion solution. The carrier can be, for example, a solvent or a dispersion medium containing water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, etc.), and suitable mixtures thereof. In some embodiments, the formulation comprises an isotonic agent in the composition, such as polyalcohols like sugar, mannitol, sorbitol, etc., and sodium chloride. The sterile injectable solution can be prepared by incorporating the required amount of the conjugate with one or a combination of the components listed above into a selected solvent and, if required, subsequently filter-sterilizing it.

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

[0329] IV. Method of Use / Treatment A conjugate comprising a muscle targeting agent covalently to the molecular payload as described herein is effective in targeting FOP. In some embodiments, the conjugate is effective in treating typical FOP or atypical FOP. In some embodiments, FOP is associated with mutations in the ACVR1 protein, for example, mutations in the ACVR1 gene leading to R206H, Q207E, G328R, G328W, G328E, G356D, R375P, 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 animal subject. In some embodiments, the subject may have a muscle disorder provided in Table 1.

[0331] Aspects of the disclosure include methods involving administering to a subject an effective amount of a complex as described herein. In some embodiments, an effective amount of a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently bound to a molecular payload can be administered to a subject in need of treatment. In some embodiments, a pharmaceutical composition comprising a complex as described herein may be administered by a suitable route including, for example, intravenous administration, as a bolus, or by continuous infusion over a period of time. In some embodiments, intravenous administration may be effected by an intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, or intrathecal route. In some embodiments, the pharmaceutical composition may be in solid, aqueous, or liquid form. In some embodiments, the aqueous or liquid form may be sprayed or lyophilized. In some embodiments, the spray or lyophilized form may be reconstituted with an aqueous or liquid solution.

[0332] Compositions for intravenous administration may contain various carriers such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). Water-soluble antibodies for intravenous injection can be administered by the drip method, whereby a pharmaceutical preparation 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. Sterile preparations of suitable soluble salt forms of antibodies for intramuscular use can be administered by dissolving in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0333] In some embodiments, a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently attached to a molecular payload is administered via a site-specific or local delivery technique. Examples of these techniques include an implantable depot source of the complex, a local delivery catheter, a site-specific carrier, direct injection, or direct application.

[0334] In some embodiments, a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently attached to a molecular payload is administered at an effective concentration to impart a therapeutic effect to a subject. The effective amount will vary according to factors recognized by those of ordinary skill in the art, such as the severity of the disease, the particular characteristics of the subject being treated, e.g., age, physical condition, state of health, or weight, the duration of treatment, the nature of any concomitant treatment, the route of administration, and related factors. These related factors are known to those of ordinary skill in the art and can be addressed with a few routine experimental methods. In some embodiments, the effective concentration is the maximum dose considered safe for the patient. In some embodiments, the effective concentration will be the lowest practicable concentration that provides the maximum effect.

[0335] Empirical considerations, e.g., the half-life of the complex in the subject will generally contribute to the determination of the concentration of the pharmaceutical composition used for treatment. The dosing frequency may be determined and adjusted empirically to maximize the effectiveness of the treatment.

[0336] Generally, for any administration of a complex described herein, the initial candidate dosage may be about 1-100 mg / kg, or more, depending on factors such as safety or effectiveness as described above. In some embodiments, the treatment will be administered once. In some embodiments, the treatment will be administered daily, bi-weekly, weekly, bi-monthly, monthly, or at any time interval that provides the maximum effectiveness to the subject while minimizing the risk to safety. Generally, the effectiveness as well as the risk to treatment and safety may also be monitored throughout the course of the treatment.

[0337] The efficacy of the treatment may be assayed using any suitable method. In some embodiments, the efficacy of the treatment may be assayed by evaluation or findings of symptoms related to FOP. In some embodiments, symptoms related to FOP may include progressive replacement of muscle tissue with bone, restricted movement, loss of mobility, and / or dyspnea and dysphagia.

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

[0339] In some embodiments, a single dose or administration of a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently bound to a molecular payload described herein to a subject is sufficient to inhibit the activity or expression of a target gene 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 a longer period. In some embodiments, a single dose or administration of a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently bound to a molecular payload described herein to a subject is sufficient to inhibit the activity or expression of a target gene 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 of a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently bound to a molecular payload described herein to a subject is sufficient to inhibit the activity or expression of a target gene for at least 1, 2, 3, 4, 5, or 6 months.

[0340] In some embodiments, the pharmaceutical composition may comprise more than one complex, including a muscle targeting agent covalently bound to a molecular payload. In some embodiments, the pharmaceutical composition may further comprise any other suitable therapeutic agent for the treatment of a subject, e.g., a human subject having a muscle disorder (e.g., the muscle disorders provided in Table 1). In some embodiments, the other therapeutic agent may enhance or complement the effectiveness of the complexes described herein. In some embodiments, the other therapeutic agent may function to treat a different symptom or disease than the complexes described herein.

[0341] Example Example 1: Targeting of DMPK with Transfected Antisense Oligonucleotides A gapmer antisense oligonucleotide targeting both wild-type and mutant alleles of DMPK (DTX-P-060) was tested in vitro for its ability to reduce the expression level of DMPK in immortalized cell lines. Briefly, Hepa1-6 cells were transfected with DTX-P-060 (100 nM) formulated with Lipofectamine 2000. The DMPK expression level was evaluated 72 hours after transfection. A control experiment was also performed in which the 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, it was found that DTX-P-060 reduced the DMPK expression level by ~90% compared to the control.

[0342] Example 2: Targeting of DMPK with a Muscle-Targeted Complex A muscle-targeted complex was generated that included the DMPK ASO used in Example 1 (DTX-P-060) covalently linked via a cathepsin-cleavable linker to DTX-A-002 (RI7 217 (Fab)), an anti-transferrin receptor antibody.

[0343] Briefly, the maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate (MC-Val-Cit-PABC-PNP) linker molecule was coupled to NH2-C6-DTX-P-060 using an amide coupling reaction. Excess linker and organic solvents 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 where fraction B7-C2 of the trace (indicated by the vertical lines) contained an ASO to antibody ratio of 1 or 2 as determined by SDS-PAGE. These fractions were pooled to reach the final muscle-targeted conjugate 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] Using the same approach, a control conjugate containing the DMPK ASO used in Example 1 (DTX-P-060) was generated that was covalently linked via a Val-Cit linker to an IgG2a (Fab) antibody (DTX-C-007).

[0346] Next, the purified DTX-C-008 was tested for cellular internalization and inhibition of DMPK. Hepa1-6 cells with a relatively high expression level 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, the cells were isolated and assayed for the expression level of DMPK (Figure 3). Cells treated with DTX-C-008 demonstrated a ~65% reduction in DMPK expression compared to cells treated with vehicle control. In contrast, cells treated with DTX-C-007 had DMPK expression levels comparable to those of the vehicle control (no reduction in DMPK expression). These data suggest that the anti-transferrin receptor antibody of DTX-C-008 enables cellular internalization of the complex, thereby allowing the DMPK ASO to inhibit the expression of DMPK.

[0347] Example 3: Targeting of DMPK in mouse muscle tissue with a muscle-targeted complex DTX-C-008, the muscle-targeted complex described in Example 2, was tested for inhibition of DMPK in mouse tissue. C57BL / 6 wild-type mice were intravenously injected with a single dose of vehicle control, DMPK-1 (3 mg / kg of RNA), DTX-C-008 (3 mg / kg of RNA, equivalent to 20 mg / kg of the antibody conjugate), or DTX-C-007 (3 mg / kg of RNA, equivalent to 20 mg / kg of the antibody conjugate). DTX-P-060, the 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, the mice were euthanized and dissected into isolated tissue types. Subsequently, individual tissue samples were assayed for the expression level of DMPK (Figures 4A - 4E and 5A - 5B).

[0348] Mice treated with the DTX-C-008 complex demonstrated reduced DMPK expression in various skeletal, cardiac, and smooth muscle tissues. For example, as shown in FIGS. 4A-4E, DMPK expression levels were significantly reduced in gastrocnemius (50% reduction), heart (30% reduction), esophagus (45% reduction), tibialis anterior (47% reduction), and soleus (31% reduction) tissues compared to mice treated with vehicle control. In contrast, mice treated with the DTX-C-007 complex 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 complex 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 of DTX-C-008 enables cellular internalization of the complex into muscle-specific tissues in an in vivo mouse model, thereby inhibiting the expression of DMPK that is inhibited by DMPK ASO. These data further demonstrate that the DTX-C-008 complex is capable of specifically targeting muscle tissue.

[0351] Example 4: Targeting of DMPK in mouse muscle tissue with a muscle-targeted complex The DTX-C-008, a muscle-targeted complex described in Example 2, was tested for the 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 of RNA), DTX-C-008 (3 mg / kg or 10 mg / kg of RNA, where 3 mg / kg corresponds to 20 mg / kg of antibody conjugate), or DTX-C-007 (3 mg / kg or 10 mg / kg of RNA, where 3 mg / kg corresponds to 20 mg / kg of 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. Seven days after injection, the mice were euthanized and dissected into isolated tissue types. Subsequently, individual tissue samples were assayed for DMPK expression levels (Figures 6A - 6F).

[0352] Mice treated with the DTX-C-008 complex demonstrated reduced DMPK expression in various skeletal muscle tissues. As shown in Figures 6A - 6F, DMPK expression levels were significantly reduced in the vehicle control-treated mice, tibialis anterior muscle (58% and 75% reduction for 3 mg / kg and 10 mg / kg of DTX-C-008, respectively), soleus muscle (55% and 66% reduction for 3 mg / kg and 10 mg / kg of DTX-C-008, respectively), extensor digitorum longus (EDL) muscle (52% and 72% reduction for 3 mg / kg and 10 mg / kg of DTX-C-008, respectively), gastrocnemius muscle (55% and 77% reduction for 3 mg / kg and 10 mg / kg of DTX-C-008, respectively), heart (19% and 35% reduction for 3 mg / kg and 10 mg / kg of DTX-C-008, respectively), and diaphragm muscle (53% and 70% reduction for 3 mg / kg and 10 mg / kg of DTX-C-008, respectively) tissues. Notably, all assayed muscle tissue types 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] On the other hand, mice treated with the control DTX-C-007 complex had DMPK expression levels comparable to those of the vehicle control (no reduction in DMPK expression) for all muscle tissue types assayed. These data suggest that the anti-transferrin receptor antibody of DTX-C-008 enables the intracellular uptake of the complex into the muscle-specific tissues of the in vivo mouse model, thereby allowing the DMPK ASO to inhibit DMPK expression. These data further demonstrate that the DTX-C-008 complex is capable of specifically targeting muscle tissue for dose-dependent inhibition of DMPK.

[0354] Example 5: Targeting of DMPK in Cynomolgus Muscle Tissue with Muscle-Targeted Complexes A muscle-targeted complex containing DTX-P-060 (DTX-C-012) was generated and purified using the method described in Example 2. DTX-C-012 is a complex containing a human anti-transferrin antibody covalently linked to DTX-P-060, an antisense oligonucleotide targeting DMPK, via a cathepsin-cleavable Val-Cit linker. Following HIC-HPLC purification, concentration measurements confirmed that DTX-C-012 had an average ratio of ASO to antibody 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 tissue. Male cynomolgus monkeys (19 - 31 months old; 2 - 3 kg) were injected intravenously on day 0 with a single dose of saline control, DTX-P-060 (naked DMPK ASO) (10 mg / kg of RNA), or DTX-C-012 (10 mg / kg of RNA). Each experimental condition was replicated in three individual male cynomolgus monkeys. On day 7 post-injection, tissue biopsy specimens (containing muscle tissue) were collected. DMPK mRNA expression levels, ASO detection assays, serum clinical chemistry, tissue histology, clinical findings, and body weight were analyzed. The monkeys were euthanized on day 14.

[0356] Compared to the saline control, significant knockdown (KD) of DMPK mRNA expression using DTX-C-012 was observed in the soleus muscle, deep digital flexor muscle, and masseter muscle at 39% KD, 62% KD, and 41% KD, respectively (Figs. 7A - 7C). Robust knockdown of DMPK mRNA expression by DTX-C-012 was further observed in the gastrocnemius muscle (62% KD; Fig. 7D), EDL (29% KD; Fig. 7E), anterior tibialis muscle (23% KD; Fig. 7F), diaphragm (54% KD; Fig. 7G), tongue (43% KD; Fig. 7H), cardiac muscle (36% KD; Fig. 7I), quadriceps muscle (58% KD; Fig. 7J), biceps brachii muscle (51% KD; Fig. 7K), and deltoid muscle (47% KD; Fig. 7L). Knockdown of DMPK mRNA expression by DTX-C-012 in smooth muscle was also observed in the intestine, with 63% KD at the jejunal duodenal end (Fig. 8A) and 70% KD in the ileum (Fig. 8B). Notably, DTX-P-060, a naked DMPK ASO (i.e., not linked to a muscle targeting agent), had minimal effect on DMPK expression levels compared to the vehicle control (i.e., DMPK expression was minimally or not reduced at all) for all muscle tissue types assayed. Monkeys treated with the DTX-C-012 complex demonstrated no change in DMPK expression in non-muscle tissues such as liver, kidney, brain, and spleen tissues (Figs. 9A - 9D). Additional tissues were examined as depicted in Fig. 10 showing 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 complex had normal reticulocyte levels, platelet levels, hemoglobin expression, alanine aminotransferase (ALT) expression, aspartate aminotransferase (AST) expression, and blood urea nitrogen (BUN) levels throughout the entire experimental period. These data indicate that a single dose of the complex containing DTX-P-060 is safe and tolerable in cynomolgus monkeys.

[0358] These data indicate that the anti-transferrin receptor antibody of the DTX-C-012 complex enables intracellular uptake of the complex into muscle-specific tissues in an in vivo cynomolgus monkey model, thereby suggesting that DMPK ASO (DTX-P-060) can inhibit DMPK expression. These data further demonstrate that the DTX-C-012 complex is capable of specifically targeting muscle tissue without substantially affecting non-muscle tissues for dose-dependent inhibition of DMPK. This is a direct comparison with the limited ability of naked DMPK ASO (i.e., not linked to a muscle targeting agent), DTX-P-060, to inhibit DMPK expression in muscle tissue of an in vivo cynomolgus monkey model.

[0359] Example 6: Targeting of DMPK in Mouse Muscle Tissue with a Muscle-Targeted Complex The muscle-targeting complex described in Example 2, DTX-C-008, was tested for the 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 of RNA), or DTX-C-008 (10 mg / kg of RNA) as described in Table 2, and euthanized after a predetermined period. Following euthanasia, the mice were dissected into isolated tissue types, and subsequently tissue samples were assayed for DMPK expression levels (Figures 11A - 11B). Table 2 - Experimental Conditions

Table 4

[0360] Equivalents and Technical Terms The disclosure described herein in a suitable manner can be practiced without any element(s) or limitation(s) not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising", "consisting essentially of", and "consisting of" may be replaced by either of the other two terms. The terms and expressions employed are used as terms of description and not of limitation, and there is no intention to exclude any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure. Accordingly, although the disclosure is specifically disclosed by preferred embodiments, it should be understood that any feature, conceptual modification, and variation of the disclosure disclosed herein may be reclassified by those skilled in the art, and such modifications and variations are considered to be within the scope of the disclosure.

[0361] In addition, when features or aspects of the disclosure are described from the perspective of a Markush group or other alternative group, those skilled in the art will recognize that the disclosure is thereby also described from the perspective of any individual member or sub-group member of the Markush group or other group.

[0362] In some embodiments, it should be understood that the sequences presented in the sequence listing may be referred to when describing the structure of oligonucleotides or other nucleic acids. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (by way of example, the RNA equivalent of a DNA nucleotide or the DNA equivalent of an RNA nucleotide), 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 specific sequence when compared to the specific sequence.

[0363] The use of the terms "a", "an", and "the" and similar referents, in the context of describing the present invention (especially in the context of the following claims), unless otherwise indicated herein or unless the context clearly dictates otherwise, should be construed to cover both the singular and the plural forms. The terms "comprising", "having", "including", and "containing", unless otherwise noted, should be construed as open-ended terms (i.e., meaning "including but not limited to"). The recitation of a range of values herein is merely intended to serve as a convenient method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually recited herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or unless the context clearly dictates otherwise. The use of any and all examples or exemplary language (e.g., "such as") provided herein is merely intended to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. Words in this specification should be construed so that no element is indicated as essential to the practice of the invention unless it is specifically claimed as such.

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

[0365] The inventors expect those skilled in the art to adopt such variations as necessary, and the inventors also intend that the present invention be practiced otherwise than as specifically described herein. Consequently, the present invention encompasses 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 elements is covered by the present invention in all its feasible variations, unless otherwise indicated herein or clearly contradicted by context. Those skilled in the art will be able to recognize or verify many equivalents of the specific embodiments of the present invention described herein using only routine experimental techniques. Such equivalents are intended to be covered by the following claims.

Claims

1. A complex comprising a muscle targeting agent covalently linked via a cleavable linker to an oligonucleotide containing a region complementary to a muscle disease gene, wherein the muscle disease gene is DMPK, and wherein the complementary region is complementary to a part of the sequence of NM_001081560.2, and wherein the muscle targeting agent is an anti-transferrin receptor antibody that specifically binds in the range of C89 to F760 of transferrin receptor protein 1 (TfR1) having an amino acid sequence as represented by any one of SEQ ID NOs: 1 to 3, and wherein the cleavable linker comprises a protease-sensitive linker containing a valine-citrulline sequence and is covalently linked to the anti-transferrin receptor antibody and / or the oligonucleotide by a cycloaddition reaction between an azide and an alkyne that forms a triazole, wherein the azide or the alkyne is positioned on the anti-transferrin receptor antibody, the oligonucleotide or the cleavable linker, and wherein the alkyne is bicyclononine or a substituted bicyclononine, said complex.

2. The complex according to claim 1, wherein the oligonucleotide targets a muscle disease gene in muscle cells.

3. The complex according to claim 1 or 2, wherein the oligonucleotide is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or 35 nucleotides in length.

4. The complex according to any one of claims 1 to 3, wherein the complementary region is at least 12 nucleotides in length.

5. The anti-transferrin receptor antibody is (a) in the form of a ScFv, Fab fragment, Fab' fragment, F(ab')2 fragment, or Fv fragment; (b) 10 －１１ M to 10 －６ K of M Ｄ binds to human TfR1; and / or (c) comprises a humanized antibody, The complex according to any one of claims 1 to 4.

6. The complex according to any one of claims 1 to 5, wherein the oligonucleotide comprises or consists of an antisense oligonucleotide.

7. The complex according to any one of claims 1 to 5, wherein the oligonucleotide comprises or consists of siRNA.

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

9. The complex according to any one of claims 1 to 8, wherein the oligonucleotide contains a central portion of 5 to 15 deoxyribonucleosides, which is sandwiched between 2 to 8 nucleosides on each side thereof, and each of the two sides contains one or more modified nucleosides.

10. The complex according to claim 9, wherein each of the 2 to 8 nucleosides on each of the two sides is a modified nucleoside.

11. The complex according to any one of claims 8 to 10, wherein the one or more modified nucleosides are 2'-modified nucleosides selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, and 2',4'-bridged nucleosides.

12. The complex according to any one of claims 1 to 11, wherein the oligonucleotide is directed to RNase H-mediated cleavage of DMPK mRNA transcripts in cells.

13. The complex according to any one of claims 1 to 12, wherein the oligonucleotide contains one or more modified nucleoside linkages.

14. The complex according to any one of claims 1 to 13, wherein the oligonucleotide contains one or more phosphorothioate linkages.

15. The complex according to any one of claims 1 to 14, wherein the oligonucleotide contains phosphorothioate nucleoside linkages between all nucleosides.

16. The complex according to any one of claims 1 to 15, wherein the oligonucleotide has a length of 16 to 30 nucleotides.

17. The complex according to any one of claims 1 to 15, wherein the complementary region has a length of 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34 or 35 nucleotides.

18. The complex according to any one of claims 1 to 17, wherein the anti-transferrin receptor antibody is covalently linked to the oligonucleotide via conjugation to a lysine residue or a cysteine residue of the anti-transferrin receptor antibody.

19. The complex according to any one of claims 1 to 18, which is configured to promote the internalization of the oligonucleotide into muscle cells mediated by the transferrin receptor.

20. A composition comprising the complex according to any one of claims 1 to 19 for use in a method of treating a disease or disorder that can be ameliorated or prevented by modulating the expression or activity of DMPK in muscle cells, said method comprising contacting the muscle cells with the complex, said composition.

21. A composition comprising the complex according to any one of claims 1 to 19 for use in a method of treating myotonic dystrophy type 1 (DM1) in a subject, said method comprising administering to the subject an effective amount of the complex, said composition.

22. The composition for use according to claim 21, wherein the complex is administered to the subject by injection and the oligonucleotide is released into muscle cells after cleavage of the cleavable linker.

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