Muscle targeting complexes and their use for treating facioscapular and brachial muscular dystrophy

Abstract

Aspects of the present disclosure relate to conjugates comprising a muscle targeting agent covalently linked to a molecular payload. In some embodiments, the muscle targeting agent specifically binds to an internalized cell surface receptor on muscle cells. In some embodiments, the molecular payload inhibits the expression or activity of DUX4. In some embodiments, the molecular payload is an oligonucleotide, such as an antisense oligonucleotide or an RNAi oligonucleotide.

Description

[Technical Field]

[0001] This application claims the benefits as of the filing date of U.S. Provisional Application No. 62 / 713,933, titled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY," filed on 2 August 2018; its contents are incorporated herein by reference in whole.

[0002] Field of the present invention This application relates to targeted complexes for delivering molecular payloads (e.g., oligonucleotides) to cells, and their uses, specifically for treating diseases.

[0003] Reference to sequence listings This application has been filed electronically along with a sequence listing. The sequence listing is provided as a file titled D082470001WO00-SEQ.txt, created on July 31, 2019, and measuring 60 kilobytes. The information in the electronic sequence listing is incorporated herein by reference in its entirety. [Background technology]

[0004] Background of the Invention Muscular dystrophy (MD) is a group of diseases characterized by progressive weakness and loss of muscle mass. These diseases are caused by mutations in genes that code for proteins necessary for forming healthy muscle tissue. Facial-scapular-brachial muscular dystrophy (FSHD) is a dominant inherited type of MD that primarily affects the muscles of the face, shoulder blades, and upper arms. Other symptoms of FSHD include weakness of the abdominal muscles, retinal abnormalities, hearing loss, and joint pain and inflammation. FSHD is the most prevalent of the nine types of MD that affect both adults and children, with an incidence of approximately 1 in 8,300 people worldwide. FSHD is caused by the abnormal production of a protein whose function is unknown, double homeobox 4 (DUX4). The DUX4 gene, which encodes the DUX4 protein, is located within the D4Z4 repeat region on chromosome 4 and is typically expressed only during fetal development. Subsequently, the DUX4 gene is suppressed by hypermethylation of the D4Z4 repeats, which surround and compact the DUX4 gene. Two types of FSHD have been described: type I and type II. Type I, which accounts for approximately 95% of cases, is associated with deletions of the D4Z4 repeat on chromosome 4. While unaffected individuals generally have more than 10 repeats arranged in the subtelomeric region of chromosome 4, the most common form of FSHD (FSHD1) is caused by a reduction of the array to fewer than 10 repeats, associated with reduced epigenetic suppression in skeletal muscle and altered expression of DUX4. Type II FSHD, which accounts for approximately 5% of cases, is associated with mutations in the SMCHD1 gene on chromosome 18. Aside from supportive care and treatments to address the symptoms of the disease, there is no effective treatment for FSHD. [Overview of the project]

[0005] Summary of the Invention In some aspects, this disclosure provides complexes that target muscle cells for the purpose of delivering molecular payloads to those cells. In some embodiments, the complexes provided herein are specifically useful for delivering molecular payloads that inhibit the expression or activity of DUX4, for example, in subjects having or suspected of having faculty-scapulohumeral muscular dystrophy (FSHD). Consequently, in some embodiments, the complexes provided herein include a muscle targeting agent (e.g., a muscle-targeting antibody) that specifically binds to receptors on the surface of muscle cells for the purpose of delivering the molecular payload to muscle cells. In some embodiments, the complex is taken up into the cell via receptor-mediated internalization, and the molecular payload can then be released into the cell to perform its function. For example, a complex modified to deliver an oligonucleotide may release the oligonucleotide so that the oligonucleotide can inhibit the expression of the DUX4 gene in muscle cells. In some embodiments, the oligonucleotide is released by endosomal cleavage of a covalent linker connecting the oligonucleotide and the muscle targeting agent of the complex.

[0006] Some aspects of this disclosure include a complex comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit the expression or activity of DUX4, wherein the muscle targeting agent specifically binds to an internalized cell surface receptor on muscle cells.

[0007] In some embodiments, the muscle targeting agent is a muscle targeting antibody. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to an extracellular epitope of the transferrin receptor (for example, an epitope of the apical domain of the transferrin receptor). The muscle targeting antibody may also specifically bind to an epitope of a sequence in the range 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 10 -11 M to 10 -6 It falls within the range up to M.

[0008] In some embodiments, the muscle-targeting antibody of the complex competes with the antibodies listed in Table 1 for specific binding to the transferrin receptor epitope (for example, for specific binding to the transferrin receptor epitope, 10 -6 Kd less than or equal to M, for example, 10 -11 M~10 -6 (The competition lies in Kd, which is within the range of M.)

[0009] In some embodiments, the muscle-targeting antibody of the complex does not specifically bind to the transferrin binding site of the transferrin receptor and / or does not inhibit the binding of transferrin to the transferrin receptor. In some embodiments, the muscle-targeting antibody of the complex cross-reacts with two or more extracellular epitopes of transferrin receptors from humans, non-human primates, and rodents. In some embodiments, the muscle-targeting antibody of the complex is configured to promote the internalization of the transferrin receptor-mediated molecular payload into muscle cells.

[0010] A muscle-targeting antibody (for example, a muscle-targeting antibody is an antibody that specifically binds to the extracellular epitope of the transferrin receptor) is a chimeric antibody, where optionally, a chimeric antibody is a humanized monoclonal antibody. The muscle-targeting antibody may also be in the form of an ScFv, Fab fragment, Fab' fragment, F(ab')2 fragment, or Fv fragment.

[0011] In some embodiments, the molecular payload of the complex is an oligonucleotide (for example, an oligonucleotide targeting DUX4). In some embodiments, the oligonucleotide comprises at least 15 consecutive nucleotides of SEQ ID NO: 45. In some embodiments, the oligonucleotide comprises SEQ ID NO: 45. In some embodiments, the oligonucleotide comprises a sequence complementary to at least 15 consecutive nucleotides of SEQ ID NO: 46.

[0012] In some embodiments, the oligonucleotide comprises a region complementary to the DUX4 gene. In some embodiments, the oligonucleotide comprises an antisense strand that hybridizes in the cell with the wild-type DUX4 mRNA transcript encoded by the allele. In some embodiments, the oligonucleotide comprises an antisense strand that hybridizes in the cell with the mutant DUX4 mRNA transcript encoded by the allele. The oligonucleotide may comprise a strand complementary to the coding sequence of DUX4.

[0013] In some embodiments, the oligonucleotide comprises a strand complementary to the non-coding sequence of DUX4. In some embodiments, the oligonucleotide may comprise a strand complementary to the 5' or 3' UTR sequence of DUX4. In some embodiments, the oligonucleotide mediates epigenetic silencing of DUX4.

[0014] The oligonucleotides of the present disclosure may comprise at least one modified nucleotide internucleoside linkage (e.g., phosphorothioate linkage). In some embodiments, the oligonucleotide comprises phosphorothioate linkages in the Rp stereochemical conformation and in the Sp stereochemical conformation. In some embodiments, the oligonucleotide comprises phosphorothioate linkages all in the Rp stereochemical conformation. In other embodiments, the oligonucleotide comprises phosphorothioate linkages all in the Sp stereochemical conformation.

[0015] The oligonucleotides of the present disclosure may contain one or more modified nucleotides (e.g., 2'-modified nucleotides). In some embodiments, the modified nucleotides are 2'-O-methyl, 2'-fluoro (2'-F), 2'-O-methoxyethyl (2'-MOE), or 2',4'-bridged nucleotides. In some embodiments, the modified nucleotides are bridged nucleotides (e.g., selected from 2',4'-constrained 2'-O-ethyl (cEt) and locked nucleic acid (LNA) nucleotides).

[0016] In some embodiments, the oligonucleotide is a gapmer oligonucleotide that directs RNAse H-mediated cleavage of DUX4 mRNA transcripts in cells. The gapmer oligonucleotide may contain a central portion of 5 to 15 deoxyribonucleotides flanked by wings of 2 to 8 modified nucleotides (e.g., 2'-modified nucleotides) on both sides.

[0017] In some embodiments, the oligonucleotide is a mixmer oligonucleotide (e.g., the mixmer oligonucleotide inhibits the translation of DUX4 mRNA transcripts). The mixmer oligonucleotide may contain two or more different 2'-modified nucleotides.

[0018] In some embodiments, the oligonucleotide is an RNAi oligonucleotide that promotes RNAi-mediated cleavage of DUX4 mRNA transcripts. The RNAi oligonucleotide may be a double-stranded oligonucleotide 19 to 25 nucleotides in length. In some embodiments, the RNAi oligonucleotide contains at least one 2'-modified nucleotide.

[0019] In some embodiments, the oligonucleotide includes a guide sequence for a genome editing nuclease.

[0020] In some embodiments, the oligonucleotide is a phosphorodiamidite tomorpholino oligomer (PMO).

[0021] In other embodiments, the molecular payload is a polypeptide (for example, a polypeptide that inhibits DUX4 expression). In some embodiments, the molecular payload is a polypeptide that binds to a DUX4 enhancer sequence, thereby blocking the recruitment of one or more activators of DUX4 expression.

[0022] In some embodiments, the muscle targeting agent is covalently linked to the molecular payload via a cleavable linker (e.g., a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker). The protease-sensitive linker may contain a sequence that can be cleaved by lysosomal proteases and / or endosomal proteases. In some embodiments, the protease-sensitive linker contains a valine-citrulline dipeptide sequence. The pH-sensitive linker may be cleaved at a pH in the range of 4–6.

[0023] In some embodiments, the muscle targeting agent is covalently linked to the molecular payload via an inclementable linker (e.g., an alkane linker).

[0024] In some embodiments, the muscle-targeting antibody comprises a non-natural amino acid to which the oligonucleotide can be covalently linked. In some embodiments, the muscle-targeting antibody is covalently linked to the oligonucleotide via conjugation to a lysine or cysteine ​​residue of the antibody. In some embodiments, the oligonucleotide is conjugated to the cysteine ​​of the antibody via a maleimide-containing linker, optionally wherein the maleimide linker comprises a maleimide-caproyl or maleimide-methylcyclohexane-1-carboxylate group.

[0025] In some embodiments, the muscle-targeting antibody is a glycosylated antibody containing at least one sugar moiety to which an oligonucleotide is covalently linked. In some embodiments, the glycosylated antibody contains at least one sugar moiety which is branched mannose. In some embodiments, the muscle-targeting antibody is a glycosylated antibody containing 1 to 4 sugar moieties, each of which is covalently linked to a separate oligonucleotide. In some embodiments, the muscle-targeting antibody is a fully glycosylated antibody or a partially glycosylated antibody. Partially glycosylated antibodies may be produced by chemical or enzymatic means. In some embodiments, partially glycosylated antibodies are produced in cells deficient in enzymes in the N- or O-glycosylation pathway.

[0026] Some aspects of this disclosure include a method for delivering a molecular payload to transferrin receptor-expressing cells, the method comprising contacting the cells with a complex comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit the expression or activity of DUX4.

[0027] Some aspects of this disclosure include methods for inhibiting the expression or activity of DUX4 in cells, the methods comprising contacting cells with a complex comprising a muscle targeting agent covalently linked to a molecular payload configured to inhibit the expression or activity of DUX4, in an amount effective to promote the 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.

[0028] Several aspects of this disclosure include methods for treating subjects having one or more deletions of D4Z4 repeats in chromosome 4 associated with facultivic, scapulohumeral, and brachial muscular dystrophy, the methods comprising administering to the subject an effective amount of a complex containing a muscle targeting agent covalently linked to a molecular payload configured to inhibit the expression or activity of DUX4. In some embodiments, the subject has 10 or fewer D4Z4 repeats (for example, the subject has 9, 8, 7, 6, 5, 4, 3, 2, or 1 D4Z4 repeat). In some embodiments, the subject does not have D4Z4 repeats. [Brief explanation of the drawing]

[0029] Simple description of the drawing [Figure 1] Figure 1 shows a non-limiting schematic diagram illustrating the effect of transfecting cells with siRNA.

[0030] [Figure 2] Figure 2 shows a non-restrictive schematic diagram illustrating the activity of a muscle targeting complex containing siRNA.

[0031] [Figure 3] Figures 3A-3B show non-restrictive schematic diagrams illustrating the activity of siRNA-containing muscle targeting complexes in mouse muscle tissue (gastrocnemius and heart) in vivo compared to control experiments. (N=4 C57BL / 6 WT mice)

[0032] [Figure 4A-B] Figures 4A-4E illustrate non-limiting schematic diagrams showing the tissue selectivity of muscle-targeting complexes containing siRNA. [Figure 4C-D] Figures 4C-4D illustrate a non-limiting schematic diagram showing the tissue selectivity of muscle-targeting complexes containing siRNA. [Figure 4E] Figure 4E shows a non-restrictive schematic diagram illustrating the tissue selectivity of muscle-targeting complexes containing siRNA.

[0033] [Figure 5]Figure 5 shows a non-limiting schematic diagram illustrating the expression levels of DUX4 in three cell lines expressing different types of DUX4 (A549, U-2 OS, and HepG2 cell lines) and in immortalized skeletal muscle myoblasts (SkMCs).

[0034] [Figure 6] Figure 6 shows a non-limiting schematic diagram illustrating the ability of phosphorodiamidate morpholino oligomer (PMO) versions of antisense oligonucleotides targeting DUX4 (FM10 PMO) to reduce the expression levels of downstream DUX4 genes (ZSCAN1, MBDL3L2, TRIM43). [Modes for carrying out the invention]

[0035] Detailed description of the invention Aspect of this disclosure concerns the recognition that while certain molecular payloads (e.g., oligonucleotides, peptides, small molecules) may have beneficial effects on muscle cells, effectively targeting such cells is extremely difficult. As described herein, this disclosure provides a complex comprising a muscle targeting agent covalently linked to a molecular payload to overcome this challenge. In some embodiments, the complex is specifically useful for delivering a molecular payload that inhibits the expression or activity of a target gene in muscle cells, for example, in subjects having or suspected of having a rare muscle disease. For example, in some embodiments, the complex is provided for treating subjects having FSHD by targeting DUX4. In some embodiments, the complex provided herein comprises an oligonucleotide that inhibits the expression of DUX4 in subjects having one or more D4Z4 repeat deletions on chromosome 4. In some embodiments, the complex provided herein includes a molecular payload, such as a guide molecule (e.g., guide RNA), which can cause a nucleic acid programmable nuclease (e.g., Cas9) to target the DUX4 gene in order to inactivate the gene in a muscle cell, for example, by removing a portion of the DUX4 gene or by introducing an inactivating mutation or stop codon into the DUX4 gene. In some embodiments, such a nuclear programmable nuclease may be used to inactivate DUX4 that is abnormally expressed in a muscle cell.

[0036] Further aspects of this disclosure, including a description of the terms defined, are provided below.

[0037] I. Definition To administer (give): As used herein, the term “administer” or “give” means to provide a complex to a subject in a physiologically and / or pharmacologically useful manner (for example, to treat a disease in the subject).

[0038] about: When used herein, the terms “approximately” or “about” refer to a value similar to the given reference value when applied to one or more values ​​of interest. In some embodiments, unless otherwise stated or evident from the context (except where such a figure exceeds 100% of a feasible value), the terms “approximately” or “about” refer to a broad range of values ​​that fall within plus or minus (greater than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less than these values.

[0039] antibody: As used herein, the term “antibody” refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, an F(ab')2 fragment, an Fv fragment, or an 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 comprises a framework having a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant region selected from the group consisting of constant regions of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. In some embodiments, the antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody comprises a constant region, e.g., an Fc region. The immunoglobulin constant region refers to the heavy chain or light chain constant region. The amino acid sequences of the human IgG heavy chain and light chain constant region and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of the antibody described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, the antibody described herein comprises 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 the 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, for example, U.S. Patent No. 5,693,780 and Kabat EA et al. (1991) above. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, for example, via glycosylation, phosphorylation, SUMOylation, and / or methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise 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 containing one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues linked by a peptide bond and is used to link one or more antigen-binding sites. Examples of linker polypeptides have been reported (see, for example, Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Furthermore, antibodies may also be part of larger immunoadhesion molecules formed by covalent or noncovalent bonds between the antibody or antibody moiety and one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to construct tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to construct divalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol.Immunol. 31:1047-1058).

[0040] CDR: As used herein, the term "CDR" refers to the complementarity-determining region within an antibody variable sequence. There are three CDRs in each of the heavy and light chain variable regions, designated as CDR1, CDR2, and CDR3 for each variable region. As used herein, the term "CDR set" refers to a group of three CDRs occurring in a single antigen-binding variable region. The precise boundaries of these CDRs are defined differently depending on the system. The system described by Kabat et al., Sequence of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and (1991)) not only provides a unique residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Subportions of the CDRs may be designated as L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries that define CDRs that overlap with Kabat CDRs are described by Padlan (FASEB J.9:133-139 (1995)) and MacCallum (J Mol Biol This is described in 262(5):732-45(1996). Further CDR boundary definitions do not have to strictly adhere to one of the systems above, but may still overlap with Kabat CDRs, and may be shortened or lengthened based on predictions or experimental findings that specific residues, groups of residues, or even the entire CDR do not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems, but preferred embodiments use CDRs defined in Kabat or Chothia.

[0041] CDR-grafted antibodies: The term "CDR-conjugated antibody" refers to an antibody that contains heavy chain and light chain variable region sequences from one species, but in which one or more sequences of the VH and / or VL CDR regions are replaced with CDR sequences from another species. This includes antibodies that have mouse heavy chain and light chain variable regions, but in which one or more of the mouse CDR (e.g., CDR3) are replaced with human CDR sequences.

[0042] Chimeric antibodies: The term "chimeric antibody" refers to an antibody that contains heavy chain and light chain variable region sequences from one species and a constant region sequence from another species, such as an antibody that has mouse heavy chain and light chain variable regions linked to a human constant region.

[0043] Complementary: As used herein, the term “complementary” refers to the capacity for accurate pairing between two nucleotides or two sets of nucleotides. In particular, complementarity 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, if a base of an oligonucleotide at a certain position can hydrogen bond with a base of a target nucleic acid (e.g., mRNA) at a corresponding position, then the bases are considered complementary at that position. Base pairing may encompass both standard Watson-Crick base pairings and non-Watson-Crick base pairings (e.g., Wobble base pairings and Hoogsteen base pairings). For example, in some embodiments, as complementary base pairings, 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 complementary to them. Inosine (I) is also considered a universal base in the art and is considered complementary to any of A, C, U, or T.

[0044] Conserved amino acid substitutions: As used herein, “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the protein to which the amino acid substitution is made. Variants may be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, references summarizing such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include substitutions made to amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0045] Covalently linked: As used herein, the term “covalently linked” refers to the characteristic of two or more molecules linked together by at least one covalent bond. In some embodiments, two molecules may be covalently linked together by a single bond (e.g., a disulfide bond or disulfide bridge) acting as an intermolecular linker. However, in some embodiments, two or more molecules may be covalently linked together by a molecule acting as a linker, which binds 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 an incleavable linker.

[0046] Cross-reaction: As used herein, and in the context of targeting agents (e.g., antibodies), the term “cross-reacting” refers to the property of an agent to be able to specifically bind to one or more antigens of a similar type or class (e.g., multiple homologous, paralogous, or orthologous antigens) with similar affinity or binding activity. For example, in some embodiments, an antibody that cross-reacts to antigens of a similar type or class between humans and non-human primates (e.g., human transferrin receptors and non-human primate transferrin receptors) is capable of binding to human antigens and non-human primate antigens with similar affinity or binding activity. In some embodiments, an antibody cross-reacts to human antigens and rodent antigens of a similar type or class. In some embodiments, an antibody cross-reacts to rodent antigens of a similar type or class and non-human primate antigens. In some embodiments, an antibody cross-reacts to human antigens, non-human primate antigens, and rodent antigens of a similar type or class.

[0047] DUX4: As used herein, the term “DUX4” refers to the gene encoding double homeobox 4, a protein expressed during fetal development and in the testes of adult males. In some embodiments, DUX4 may be a gene from humans (Gene ID: 100288687), non-human primates (e.g., Gene ID: 750891, Gene ID: 100405864), or rodents (e.g., Gene ID: 306226). In humans, DUX4 gene expression, excluding fetal development and the testes, is associated with facioscapulohuchimuscular dystrophy. In addition, several human transcript variants encoding various protein isoforms have been characterized (e.g., as annotated in GenBank RefSeq accessions: NM_001293798.2, NM_001306068.2, NM_001363820.1).

[0048] Facial, scapular, and brachial muscular dystrophy (FSHD): As used herein, the term “facial-scapulobrachial muscular dystrophy (FSHD)” refers to a genetic disorder caused by mutations in the DUX4 or SMCHD1 gene, characterized primarily by loss of muscle mass and muscular atrophy in the muscles of the face, scapula, and upper arm. Two types of the disorder are described: type I and type II. Type I is associated with a deletion in the D4Z4 repeat region on chromosome 4 containing the DUX4 gene. Type II is associated with a mutation in the SMCHD1 gene. Both type I and type II FSHD are characterized by abnormal production of the DUX4 protein outside the testis after fetal development. Facioscapulohumeral muscular dystrophy, its genetic basis, and associated symptoms are described in the relevant technical field (see, for example, Campbell, AE, et al., “Facioscapulohumeral dystrophy: Activatin an early embryonic transcriptional program in human skeletal muscle” Human Mol Genet. (2018); Tawil, R. “Facioscapulohumeral muscular dystrophy” Handbook Clin. Neurol. (2018), 148: 541-548). Type I FSHD is associated with Online Mendelian Inheritance in Man (OMIM) entry #158900. Type II FSHD is associated with OMIM entry #158901.

[0049] Framework: As used herein, the term “framework” or “framework sequence” refers to the sequence remaining in the variable region after subtracting the CDRs. Since the precise definition of a CDR sequence can be determined by various systems, the meaning of a framework sequence depends on correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 on the light chain, and CDR-H1, CDR-H2, and CDR-H3 on the heavy chain) also divide the framework regions on the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3 between FR3 and FR4. When referred to elsewhere, framework regions that do not specify a particular sub-region as FR1, FR2, FR3, or FR4 represent the combined FRs within the variable region of a naturally occurring single immunoglobulin chain. When used herein, FR represents one of four subregions, and FR(plural) represents two or more of the four subregions containing the framework region. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0050] Human antibodies: When used herein, the term “human antibody” is intended to encompass antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of this disclosure may include, for example, amino acid residues not encoded by human germline immunoglobulin sequences (mutations introduced, for example, by random mutagenesis or site-directed mutagenesis in vitro, or by somatic mutation in vivo), particularly in CDRs, especially CDR3. However, when used herein, the term “human antibody” is not intended to encompass antibodies in which CDR sequences derived from the germline of another mammalian species, such as mouse, are conjugated onto a human framework sequence.

[0051] Humanized antibodies: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH sequence and / or VL sequence has been modified to be more "human-like," i.e., more similar to the human germline variable sequence. One type of humanized antibody is a CDR-conjugated antibody in which a human CDR sequence is introduced onto a non-human VH and VL sequence and replaced with the corresponding non-human CDR sequence. In one embodiment, a humanized anti-transferrin receptor antibody and antigen-binding moiety are provided. Such an antibody may be produced 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 WO 2005 / 123126 A2 by Kasaian et al.).

[0052] Internalized cell surface receptors: As used herein, the term “internalizing cell surface receptor” refers, for example, to a cell surface receptor that is internalized by a cell in response to an external stimulus (for example, a ligand that binds to the receptor). In some embodiments, the internalizing cell surface receptor is internalized by endocytosis. In some embodiments, the internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalizing cell surface receptor is internalized by clathrin-independent pathways, such as phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or an extracellular domain, which optionally further comprises a ligand-binding domain. In some embodiments, the cell surface receptor becomes internalized by the cell after ligand binding. In some embodiments, the ligand may be a muscle targeting agent or a muscle targeting antibody. In some embodiments, the internalizing cell surface receptor is a transferrin receptor.

[0053] Isolated antibodies: When used herein, "isolated antibody" is intended to refer to an antibody for which there are substantially no other antibodies with different antigen specificities (for example, an isolated antibody that specifically binds to the transferrin receptor has substantially no other antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to the transferrin receptor complex may have cross-reactivity to other antigens, such as transferrin receptor molecules from other species. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0054] Kabat numbering: The terms “Kabat numbering,” “Kabat definition,” and “Kabat labeling” are used interchangeably herein. These terms refer to a system of numbering amino acid residues that are recognized in the art but are more variable (i.e., highly variable) than other amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding moiety (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD Department of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable region extends from amino acid positions 31-35 for CDR1, from amino acid positions 50-65 for CDR2, and from amino acid positions 95-102 for CDR3. In the light chain variable region, the hypervariable region extends to amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

[0055] 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 ligated to or otherwise linked to a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide containing a chain having a region complementary to a target gene.

[0056] Muscle targeting agents: As used herein, the term “muscle targeting agent” refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on the muscle cell may be a membrane protein, e.g., an endogenous membrane protein or a superficial membrane protein. Typically, a muscle targeting agent specifically binds to an antigen on the muscle cell that facilitates the 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 internalization. In some embodiments, the muscle targeting agent is a small molecule, protein, peptide, nucleic acid (e.g., an aptamer), or antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0057] Muscle targeting antibodies: As used herein, the term “muscle-targeting antibody” refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on muscle cells. In some embodiments, a muscle-targeting antibody specifically binds to an antigen on a muscle cell that facilitates the internalization of the muscle-targeting antibody (and any associated molecular payload) into the muscle cell. In some embodiments, a muscle-targeting antibody specifically binds to an internalized cell surface receptor present on the muscle cell. In some embodiments, a muscle-targeting antibody is an antibody that specifically binds to a transferrin receptor.

[0058] Oligonucleotides: As used herein, the term “oligonucleotide” refers to an oligomeric nucleic acid compound with a length of up to 200 nucleotides. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNAs, gapmers, mixmers, phosphorodiamidite morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), etc. Oligonucleotides may be single-stranded or double-stranded. In some embodiments, oligonucleotides may contain one or more modified nucleotides (e.g., 2'-O-methylglycosulfate, purine, or pyrimidine modification). In some embodiments, oligonucleotides may contain one or more modified nucleotide linkages. In some embodiments, oligonucleotides may contain one or more phosphorothioate linkages, which may be in the stereochemical configuration of Rp or Sp.

[0059] Recombinant antibodies: When used herein, the term “recombinant human antibody” refers to all human antibodies prepared, expressed, created, or isolated by recombinant means, such as antibodies expressed using recombinant expression vectors transfected into host cells (as described in detail herein), antibodies isolated from recombinant combinatorial human antibody libraries (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from human immunoglobulin gene transgenic animals (e.g., mice) (e.g., Taylor, LD, et al.) It is intended to include antibodies prepared, expressed, created, or isolated by any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences, as described by al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370). Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, in vivo somatic mutagenesis), and therefore, although the amino acid sequences of the VH and VL regions of the recombinant antibody are derived from and related to the VH and VL sequences of the human germline, they may not be naturally present in the germline repertoire of human antibodies in vivo.One aspect of the present disclosure provides fully human antibodies capable of binding to human transferrin receptors, which may be produced using techniques well known in the art, for example, but not limited to, techniques using a human Ig phage library (e.g., disclosed in PCT publication WO 2005 / 007699 A2 by Jermutus et al.).

[0060] Complementary areas: As used herein, the term “complementary region” refers to a nucleotide sequence (e.g., a nucleotide sequence of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., a nucleotide sequence of a target nucleic acid) such that the two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary to the cognate nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95%, or 99% complementary). In some embodiments, the complementary region contains one, two, three, or four mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0061] Binds specifically: As used herein, the term “specifically binds” refers to the ability of a molecule to bind to a binding partner to a degree of affinity or binding activity that the molecule can be used to distinguish the binding partner from a suitable control in a binding assay or other binding context. With respect to antibodies, the term “specifically binds” refers to the ability of an antibody to bind to a specific antigen to a degree of affinity or binding activity (for example, to the extent that it allows preferential targeting to a cell (e.g., muscle cells) through binding to the antigen, as described herein,) compared to a suitable reference antigen, or an antigen that the antibody can be used to distinguish a particular antigen from other antigens. 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 when having a KD less than this, the antibody binds specifically to the target. In some embodiments, the antibody binds specifically to an epitope of the tip domain of the transferrin receptor, for example, the transferrin receptor.

[0062] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent animal. In some embodiments, the subject is a human. In some embodiments, the subject is a patient suspected of having or having a disease, for example, a human patient. In some embodiments, the subject is a human patient suspected of having or having FSHD.

[0063] Transferrin receptor: As used herein, the term “transferrin receptor” (also known as TFRC, CD71, p90, or TFR1) refers to an internalized cell surface receptor that binds to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may originate from humans (NCBI Gene ID 7037), non-human primates (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodents (e.g., NCBI Gene ID 22042). In addition, several human transcript variants encoding various isoforms of the receptor have been characterized (e.g., those annotated with GenBank RefSeq accessions: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0064] II. Complexes Provided herein are targeting agents, for example, complexes comprising 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 include an antibody that specifically binds to a single antigen site, or an antibody that binds to at least two antigen sites, which may be present on the same antigen or on different antigens.

[0065] 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 the gene, protein, and / or nucleic acid. The molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any other 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 targeting DUX4 in muscle cells.

[0066] In some embodiments, the complex comprises a muscle targeting agent (e.g., an anti-transferrin receptor antibody) covalently linked to a molecular payload (e.g., an antisense oligonucleotide targeting DUX4).

[0067] A. Muscle targeting agents Several aspects of this disclosure provide muscle targeting agents, for example, muscle targeting agents for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents can bind to muscle cells, for example, via specific binding to antigens on muscle cells, and deliver the bound molecular payload to the muscle cells. In some embodiments, the molecular payload is bound to the muscle targeting agent (for example, covalently), and when the muscle targeting agent binds to an antigen on a 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 this disclosure. For example, muscle targeting agents may contain, or consist of, nucleic acids (e.g., DNA or RNA), peptides (e.g., antibodies), lipids (e.g., microvesicles), or sugar moieties (e.g., polysaccharides). Exemplary muscle targeting agents are described in more detail herein, but it should be understood that the exemplary muscle targeting agents provided herein are not intended to be limiting.

[0068] Several aspects of this disclosure provide muscle targeting agents that specifically bind to antigens on muscles, such as skeletal muscle, smooth muscle, or cardiac muscle. In some embodiments, any of the muscle targeting agents provided herein bind (for example, specifically bind) to antigens on skeletal muscle cells, smooth muscle cells, and / or cardiac muscle cells.

[0069] Interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins) can achieve both tissue localization and selective uptake into muscle cells. In some embodiments, molecules that are substrates of muscle uptake transporters are useful for delivering molecular payloads into muscle tissue. Binding to muscle surface recognition elements, followed by endocytosis, can allow even macromolecules such as antibodies to enter muscle cells. As another example, a molecular payload conjugated to transferrin or an anti-transferrin receptor antibody may be taken up by muscle cells via binding to the transferrin receptor and then endocytosed, for example, via clathrin-mediated endocytosis.

[0070] The use of muscle-targeting agents can also be useful for enriching molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with the effect in other tissues. In some embodiments, muscle-targeting agents enrich bound molecular payloads in muscle cells compared to other cell types within the subject. In some embodiments, muscle-targeting agents enrich bound molecular payloads in muscle cells (e.g., skeletal muscle cells, smooth muscle cells, or cardiomyocytes) to at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times more than in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or adipocytes). In some embodiments, the toxicity of the molecular payload in a target 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 target.

[0071] In some embodiments, muscle recognition elements (e.g., muscle cell antigens) may be required to achieve muscle selectivity. For example, the muscle targeter may be a small molecule that is a substrate of a muscle-specific uptake transporter. Another example is that the muscle targeter may be an antibody that enters muscle cells via transporter-mediated endocytosis. Yet another example is that the muscle targeter may be a ligand that binds to cell surface receptors on muscle cells. It should be understood that while transporter-based approaches provide a direct pathway to cell entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0072] i. Muscle targeting antibody In some embodiments, muscle targeting agents are antibodies. Generally, the high specificity of antibodies against 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 this disclosure. For example, antibodies that target the surface of muscle cells are described in Arahata K., et al. “Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide” Nature 1988;333:861-3; Song KS, et al. “Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins” J Biol Chem 1996;271:15160-5; and Weisbart RH et al., “Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb” Mol Immunol. 2003 Mar,39(13):78309; the entire contents of each of these are incorporated herein by reference.

[0073] a. Anti-transferrin receptor antibody Several aspects of this disclosure are based on the recognition that agents that bind to transferrin receptors, such as anti-transferrin receptor antibodies, can target muscle cells. Transferrin receptors are internalized cell surface receptors that transport transferrin across the cell membrane and contribute to the regulation of intracellular iron levels and homeostasis. Several aspects of this disclosure provide transferrin receptor-binding proteins capable of binding to transferrin receptors. Consequently, aspects of this disclosure provide binding proteins (e.g., antibodies) that bind to transferrin receptors. In some embodiments, binding proteins that bind to transferrin receptors are internalized into muscle cells along with any bound molecular payload. As used herein, antibodies that bind to transferrin receptors may also be referred to as anti-transferrin receptor antibodies. Antibodies that bind to transferrin receptors, such as specifically binding antibodies, may be internalized into cells upon binding to the transferrin receptor, for example, through receptor-mediated endocytosis.

[0074] It should be understood that anti-transferrin receptor antibodies may be produced, synthesized, and / or derivatized using several known methodologies, e.g., library design using phage display. Exemplary methodologies 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 MH and Stanley, JR “Antibody Phage Display: Technique and Applications”, J Invest Dermatol. 2014, 134:2.; Engleman, Edgar (Ed.) “Human Hybridomas and Monoclonal Antibodies.” 1985, Springer). In other embodiments, anti-transferrin antibodies have been characterized or disclosed to date.Antibodies that specifically bind to transferrin receptors are known in the art (for example, U.S. Patent No. 4,364,934, filed 12 / 4 / 1979, “Monoclonal antibody to a human early thymocyte antigen and methods for preparing same”; U.S. Patent No. 8,409,573, filed 6 / 14 / 2006, “Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells”; U.S. Patent No. 9,708,406, filed 5 / 20 / 2014, “Anti-transferrin receptor antibodies and methods of use”; U.S. Patent No. 9,611,323, filed 12 / 19 / 2014, “Low affinity blood brain barrier receptor antibodies and uses therefor”; WO 2015 / 098989, filed 12 / 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).

[0075] Any suitable anti-transferrin receptor antibody may be used in the complex disclosed herein. Examples of anti-transferrin receptor antibodies, including the relevant reference and binding epitopes, are listed in Table 1. In some embodiments, the anti-transferrin receptor antibody also includes 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, for example, those listed in Table 1.

[0076] Table 1 - List of anti-transferrin receptor antibody clones, including related reference epitope and binding epitope information. [Table 1-1] [Table 1-2] [Table 1-3]

[0077] 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 the amino acid sequence disclosed herein. In some embodiments, the anti-transferrin receptor antibody may specifically bind to any extracellular epitope of the transferrin receptor or to an epitope (including the tip domain, transferrin-binding domain, and protease-like domain) that will be 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 primate non-human 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 binds to at least about 10 -4 M, 10 -5 M, 10 -6 M, 10-7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 It specifically binds with a binding affinity of M or less. The anti-transferrin receptor antibodies used herein bind to the transferrin receptor with a binding affinity of 10 -3 M, 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 It may be possible to compete with other anti-transferrin receptor antibodies that bind at or below M (e.g., OKT9, 8D3).

[0078] An example of a human transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows: (Sequence ID 1).

[0079] Examples of non-human primate transferrin receptor amino acid sequences corresponding to the NCBI sequence NP_001244232.1 (transferrin receptor protein 1, Macaca mulatta) are as follows: (Sequence ID 2).

[0080] Examples of non-human primate transferrin receptor amino acid sequences corresponding to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) are as follows:

[0081] (Sequence ID 3).

[0082] An example of a mouse transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_001344227.1 (transferrin receptor protein 1, mus musculus) is as follows: (Sequence ID 4). In some embodiments, the anti-transferrin receptor antibody comprises the following amino acid segments of the receptor: It 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).

[0083] Appropriate methodologies may be used, for example, through the use of recombinant DNA protocols to obtain and / or produce antibodies, antibody fragments, or antigen conjugates. In some embodiments, antibodies may also be produced through the generation of hybridomas (see, for example, Kohler, G and Milstein, C. “Continuous cultures of fused cells secreting antibody of predefined specificity” Nature, 1975, 256:495-497). The antigen of interest may be used as an immunogen of any type or entity, for example, recombinant or naturally occurring type or entity. Hybridomas are screened using standard methods, for example, ELISA screening, to find at least one hybridoma that produces an antibody targeting a specific antigen. Antibodies may also be produced through screening of protein expression libraries expressing the antibody (for example, phage display libraries). Phage display library designs may also be used in several embodiments (see, for example, U.S. Patent No. 5,223,409, 3 / 1 / 1991, “Directed evolution of novel binding proteins”; WO 1992 / 18619, 4 / 10 / 1992, “Heterodimeric receptor libraries using phagemids”; WO 1991 / 17271, 5 / 1 / 1991, “Recombinant library screening methods”; WO 1992 / 20791, 5 / 15 / 1992, “Methods for producing members of specific binding pairs”; and WO 1992 / 15679, 2 / 28 / 1992, “Improved epitope displaying phage”). In some embodiments, antigens of interest may be used to immunize non-human animals, for example, rodents or goats.In some embodiments, once antibodies have been obtained from non-human animals, they may be modified using a number of methodologies, including, for example, recombinant DNA techniques. Examples of antibody production and methodology additions are also known in the art (see, for example, Harlow et al. "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988).

[0084] 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, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glyciation (GPI anchor attachment), and / or phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, the sugar molecules are present in quantities of approximately 1–10, 1–5, 5–10, 1–4, 1–3, or 2. In some embodiments, the glycosylated antibody is glycosylated whole or partially. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in cells (which may optionally be deficient in enzymes in the N- or O-glycosylation pathway, e.g., glycosyltransferase). In some embodiments, the antibody is functionalized with sugar or carbohydrate molecules as described in the international patent application publication WO2014065661, published on 1 May 2014, entitled “Modified antibody, antibody-conjugate and process for the preparation thereof”.

[0085] Several aspects of this disclosure provide proteins that bind to transferrin receptors (e.g., the extracellular portion of the transferrin receptor). In some embodiments, anti-transferrin receptor antibodies provided herein specifically bind to transferrin receptors (e.g., human transferrin receptors). Transferrin receptors are internalized cell surface receptors that transport transferrin across the cell membrane and are involved in the regulation of intracellular iron levels and homeostasis. In some embodiments, anti-transferrin receptor antibodies provided herein specifically bind to transferrin receptors from humans, non-human primate animals, mice, rats, etc. In some embodiments, anti-transferrin receptor antibodies provided herein bind to human transferrin receptors. In some embodiments, anti-transferrin receptor antibodies provided herein specifically bind to human transferrin receptors. In some embodiments, anti-transferrin receptor antibodies provided herein bind to the tip domain of the human transferrin receptor. In some embodiments, anti-transferrin receptor antibodies provided herein specifically bind to the tip domain of the human transferrin receptor.

[0086] In some embodiments, the anti-transferrin receptor antibody of this disclosure contains one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, the anti-transferrin receptor antibody contains CDR-H1, CDR-H2, and CDR-H3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, the anti-transferrin receptor antibody contains CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, the anti-transferrin antibody contains CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-transferrin receptor antibodies selected from Table 1. This disclosure also includes any nucleic acid sequence encoding a molecule containing CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, or CDR-L3, as provided for any one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, the antibody heavy chain and light chain CDR3 domains may play a particularly important role in the antibody's binding specificity / affinity to the antigen. Consequently, the anti-transferrin receptor antibodies of this 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 1.

[0087] In some examples, any of the anti-transferrin receptor antibodies of this disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any of the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3 sequences from one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, the positions 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 may vary by one, two, three, four, five, or six amino acid positions, insofar as immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it originates). For example, in some embodiments, the position defining the CDR of any antibody described herein may vary by one, two, three, four, five, or six amino acid positions compared to any one CDR position of any antibody described herein, by shifting the N-terminal and / or C-terminal boundaries of the CDR, as long as immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (e.g., substantially maintained, e.g., maintained by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to the binding of the original antibody from which it originates).In another embodiment, 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 antibody described herein may vary by 1, 2, 3, 4, 5 amino acids, or more (e.g., shorter or longer), insofar 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% compared to the binding of the original antibody from which it originates).

[0088] Consequently, 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, or more amino acids than one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 1), 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 to the original antibody from which it originates). 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, or more amino acids than one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 1), insofar 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% compared to the binding of the original antibody from which it is derived). In some embodiments, the amino portions of the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3 described herein may be elongated by a difference of 1, 2, 3, 4, 5 amino acids, or more, compared to one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 1), insofar 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% compared to the binding of the original antibody from which it is derived).The carboxyl portions of CDR-H2 and / or CDR-H3 may be elongated by a difference of 1, 2, 3, 4, 5 amino acids, or more, compared to one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 1), insofar as immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained (for example, substantially maintained, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% compared to the binding of the original antibody from which it originates). In some embodiments, the amino portions of the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3 described herein may be shortened by a difference of 1, 2, 3, 4, 5 amino acids, or more, compared to one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 1), insofar 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% compared to the binding of the original antibody from which it is derived). In some embodiments, the carboxyl portions of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3 described herein may be shortened by a difference of 1, 2, 3, 4, 5 amino acids, or more, compared to one or more of the CDRs described herein (for example, CDRs from any of the anti-transferrin receptor antibodies selected from Table 1), insofar 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% compared to the binding of the original antibody from which it is derived).Either method can be used to determine whether immunospecific binding to the transferrin receptor (e.g., human transferrin receptor) is maintained, for example, using binding assays and conditions described in the art.

[0089] In some examples, any of the anti-transferrin receptor antibodies of this disclosure has one or more CDR (e.g., CDR-H or CDR-L) sequences substantially similar to any one of the anti-transferrin receptor antibodies selected from Table 1. For example, an antibody may contain one or more CDR sequences from any of the anti-transferrin receptor antibodies selected from Table 1 that contain up to 5, 4, 3, 2, or 1 amino acid residue variations compared to the corresponding CDR region in any of the CDRs provided herein (e.g., CDRs from any of the anti-transferrin receptor antibodies selected from Table 1), insofar 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%, or at least 95% compared to the binding of the original antibody from which it originates). 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 residues are unlikely to be involved in interaction with the transferrin receptor protein (e.g., human transferrin receptor protein), as determined, for example, based on the crystal structure. Several aspects of this disclosure provide anti-transferrin receptor antibodies comprising one or more heavy-chain variable (VH) and / or light-chain variable (VL) domains provided herein. In some embodiments, any of the VH domains provided herein encompasses one or more 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 1). In some embodiments, any of the VL domains provided herein encompass one or more 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 1).

[0090] In some embodiments, the anti-transferrin receptor antibodies of this disclosure encompass any antibody that includes both the heavy-chain variable domain and / or light-chain variable domain of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, the anti-transferrin receptor antibodies of this disclosure encompass any antibody that includes both the heavy-chain variable and light-chain variable pair of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1.

[0091] Aspects of this disclosure provide anti-transferrin receptor antibodies having heavy-chain variable (VH) and / or light-chain variable (VL) domain amino acid sequences homologous to any of the anti-transferrin receptor antibodies described herein. In some embodiments, an anti-transferrin receptor antibody includes a heavy-chain variable sequence or light-chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy-chain variable sequence and / or any light-chain variable sequence of any anti-transferrin receptor antibody, such as any one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, the homologous heavy-chain variable and / or light-chain variable amino acid sequences do not vary within the range of any of the CDR sequences provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur within the range of heavy-chain variable and / or light-chain variable sequences that exclude any of the CDR sequences provided herein. In some embodiments, any of the anti-transferrin receptor antibodies provided herein includes a heavy-chain variable sequence and a light-chain variable sequence that are 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 1.

[0092] In some embodiments, an anti-transferrin receptor antibody that specifically binds to a transferrin receptor (e.g., the human transferrin receptor) comprises a light chain variable VL domain comprising any of the CDR-L domains (CDR-L1, CDR-L2, and CDR-L3) of any of the anti-transferrin receptor antibodies selected from Table 1, 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., the human transferrin receptor) comprises a light chain variable VL domain comprising CDR-L1, CDR-L2, and CDR-L3 of any of the anti-transferrin receptor antibodies, such as any one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, an anti-transferrin receptor antibody comprises a light chain variable (VL) region sequence comprising one, two, three, or four framework regions of the light chain variable region sequence of any of the anti-transferrin receptor antibodies, such as any one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, an anti-transferrin receptor antibody comprises one, two, three, or four framework regions of a light chain variable region sequence that are at least 75%, 80%, 85%, 90%, 95%, or 100% identical to one, two, three, or four framework regions of a light chain variable region sequence of any anti-transferrin receptor antibody, such as one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence apart from the presence of substitutions, deletions, and / or insertions of up to 10 amino acids, preferably up to 10 substitutions. In some embodiments, the light chain variable 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 corresponding primate non-human or human light chain variable framework region.

[0093] In some embodiments, an anti-transferrin receptor antibody that specifically binds to the transferrin receptor comprises CDR-L1, CDR-L2, and CDR-L3 of any anti-transferrin receptor antibody, such as one of the anti-transferrin receptor antibodies selected from Table 1. In some embodiments, the antibody further comprises one, two, three, or all four VL framework regions derived from the VL of a human or primate antibody. The primate or human light chain framework region of the selected antibody for use with the light chain CDR sequences described herein may have at least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, 98%, or at least 99%) identity with, for example, the light chain framework region of a non-human parent antibody. The selected primate or human antibody may have the same or substantially the same number of amino acids in its light chain complementarity-determining region as any of the amino acids in 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 1). In some embodiments, the amino acid residues of the light chain framework region of a primate or human are from a natural primate or human antibody light chain framework region having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, and 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 1. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable kappa subfamily. In some embodiments, the anti-transferrin receptor antibody further comprises one, two, three, or all four VL framework regions derived from the human light chain variable lambda subfamily.

[0094] In some embodiments, any of the anti-transferrin receptor antibodies provided herein includes a light chain variable domain further comprising 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, for 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 any variant of any of the light chain constant regions provided herein. In some embodiments, the light chain constant region contains 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 1.

[0095] In some embodiments, the anti-transferrin receptor antibody is any of the anti-transferrin receptor antibodies, such as any one of the anti-transferrin receptor antibodies selected from Table 1.

[0096] In some embodiments, the anti-transferrin receptor antibody comprises a VL domain containing the amino acid sequence of any anti-transferrin receptor antibody, such as one of the anti-transferrin receptor antibodies selected from Table 1, wherein the constant region contains the amino acid sequence of the constant region 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, an anti-transferrin receptor antibody comprises either a VL domain or a variant of the VL domain, and either a VH domain or a VH domain variant, where the VL and VH domains, or their variants, are from the same antibody clone, and where the constant region comprises the amino acid sequence of the constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b) immunoglobulin molecule. Non-limiting examples of human constant regions have been described in the art; see, for example, Kabat EA et al. (1991) above.

[0097] In some embodiments, the antibodies of this disclosure have relatively high affinity, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11Antibodies can bind to target antigens (e.g., transferrin receptors) at a KD of M or lower. For example, anti-transferrin receptor antibodies can bind to transferrin receptor proteins (e.g., human transferrin receptors) at affinities between 5 pM and 500 nM, for example between 50 pM and 100 nM, for example between 500 pM and 50 nM. This disclosure also includes antibodies that compete with any of the antibodies described herein for binding to transferrin receptor proteins (e.g., human transferrin receptors) and have affinities of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of anti-transferrin receptor antibodies can be tested using any preferred method, including but not limited to biosensor technologies (e.g., OCTET or BIACORE).

[0098] In some embodiments, the antibodies of this disclosure have relatively high affinity, for example, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 Antibodies can bind to target antigens (e.g., transferrin receptors) at M or lower KD. For example, anti-transferrin receptor antibodies can bind to transferrin receptor proteins (e.g., human transferrin receptors) at affinities between 5 pM and 500 nM, for example between 50 pM and 100 nM, for example between 500 pM and 50 nM. This disclosure also includes antibodies that compete with any of the antibodies described herein for binding to transferrin receptor proteins (e.g., human transferrin receptors) and have affinities of 50 nM or less (e.g., 20 nM or less, 10 nM or less, 500 pM or less, 50 pM or less, or 5 pM or less). The affinity and binding kinetics of anti-transferrin receptor antibodies can be tested using any preferred method, including but not limited to biosensor technologies (e.g., OCTET or BIACORE).

[0099] In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody (for example, an antibody and its variant as described in the international patent publication WO 2016 / 081643 (incorporated herein by reference)).

[0100] 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, Chothia definition, and / or Contact definition, are described. For example, see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USD 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 antibodies [Table 2]

[0101] Heavy chain variable domain (VH) and light chain variable domain sequences are also provided:

[0102] VH QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSS(Sequence ID 33)

[0103] VL DIQMTQSPASLSVSVGETVTITCRASDNLYSNLAWYQQKQGKSPQLLVYDATNLADGVPSRFSGSGSGTQYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELK(Sequence ID 34)

[0104] In some aspects, the anti-transferrin receptor antibodies of this disclosure include the same CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1. Alternatively or in addition, the anti-transferrin receptor antibodies of this disclosure include the same CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1.

[0105] In some aspects, the anti-transferrin receptor antibodies of this disclosure include CDR-H1, CDR-H2, and CDR-H3 containing a combined total of only 5 amino acid variations (e.g., only 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-H1, CDR-H2, and CDR-H3 as shown in Table 1.1. "Combined" means that the total number of amino acid variations in all three heavy-chain CDRs is within the defined range. Alternatively or in addition, the anti-transferrin receptor antibodies of this disclosure may also include CDR-L1, CDR-L2, and CDR-L3 containing a combined total of only 5 amino acid variations (e.g., only 5, 4, 3, 2, or 1 amino acid variation) compared to CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1.

[0106] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure include CDR-H1, CDR-H2, and CDR-H3, at least one of which contains only three amino acid variations (e.g., only three, two, or one amino acid variation) compared to the corresponding heavy-chain CDR as shown in Table 1.1. Alternatively or in addition, the anti-transferrin receptor antibodies of the present disclosure may also include CDR-L1, CDR-L2, and CDR-L3, at least one of which contains only three amino acid variations (e.g., only three, two, or one amino acid variation) compared to the corresponding light-chain CDR as shown in Table 1.1.

[0107] In some embodiments, the anti-transferrin receptor antibody of this disclosure contains CDR-L3, which contains only three amino acid variations (for example, only three, two, or one amino acid variations) compared to CDR-L3 as shown in Table 1.1. In some embodiments, the anti-transferrin receptor antibody of this disclosure contains CDR-L3 containing one amino acid variation compared to CDR-L3 as shown in Table 1.1. In some embodiments, the anti-transferrin receptor antibody of this disclosure contains CDR-L3 of QHFAGTPLT (SEQ ID NO: 31, according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 32, according to the Contact definition system). In some aspects, the anti-transferrin receptor antibodies of this disclosure include the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 as shown in Table 1.1, and also include CDR-L3 of QHFAGTPLT (SEQ ID NO: 31, according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 32, according to the Contact definition system).

[0108] In some embodiments, the anti-transferrin receptor antibodies of the present disclosure together comprise at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) of the heavy chain CDRs shown in Table 1.1. Alternatively or in addition, the anti-transferrin receptor antibodies of the present disclosure together comprise at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) of the light chain CDRs shown in Table 1.1.

[0109] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises VH, which comprises the amino acid sequence of SEQ ID NO: 33. Alternatively or in addition, the anti-transferrin receptor antibody of the present disclosure comprises VL, which comprises the amino acid sequence of SEQ ID NO: 34.

[0110] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises VH containing 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) compared to VH as represented by SEQ ID NO: 33. Alternatively or in addition, the anti-transferrin receptor antibody of the present disclosure comprises VL containing 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) compared to VL as represented by SEQ ID NO: 34.

[0111] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises a VH having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as VH as represented by SEQ ID NO: 33. Alternatively or in addition, the anti-transferrin receptor antibody of the present disclosure comprises a VL having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as VL as represented by SEQ ID NO: 34.

[0112] In some embodiments, the anti-transferrin receptor antibodies of the Disclosure are humanized antibodies (e.g., humanized variants of antibodies). In some embodiments, the anti-transferrin receptor antibodies of the Disclosure include the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as shown in Table 1.1, and include a humanized heavy chain variable region and / or a humanized light chain variable region.

[0113] A humanized antibody is a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity-determining region (CDR) are replaced by residues from the CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capabilities. In some embodiments, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, the humanized antibody may also include residues not found in the recipient antibody or in the imported CDR or framework sequence, but may also include residues that are included to further refine and optimize the performance of the antibody. Generally, a humanized antibody will substantially include all of at least one, typically two, variable domains, in which all or substantially all of the CDR region corresponds to the CDR region of the non-human immunoglobulin, and all or substantially all of the FR region is the FR region of the human immunoglobulin consensus sequence. Humanized antibodies will also, optimally, contain at least a portion of the constant region or domain (Fc) of an immunoglobulin (typically human immunoglobulin). The antibody may have a modified Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (1, 2, 3, 4, 5, 6) that are modified with respect to the original antibody, and these are also called one or more CDRs derived from one or more CDRs from the original antibody. Humanized antibodies may also undergo affinity maturation.

[0114] In some embodiments, humanization is achieved by conjugating CDRs (e.g., as 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 this disclosure are humanized variants comprising one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 45, and 70 (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 (compared to VH as represented by SEQ ID NO: 33). In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a humanized variant comprising amino acid substitutions at all of positions 9, 13, 17, 18, 40, 45, and 70 (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 (compared to VH as represented by SEQ ID NO: 33).

[0115] In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a humanized antibody containing residues at positions 43 and 48 of VL as represented by SEQ ID NO: 34. Alternatively or in addition, the anti-transferrin receptor antibody of the present disclosure is a humanized antibody containing residues at positions 48, 67, 69, 71, and 73 of VH as represented by SEQ ID NO: 33.

[0116] The VH and VL amino acid sequences of examples of humanized antibodies that may be used in accordance with this disclosure are provided below:

[0117] Humanized VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSS(Sequence ID 35)

[0118] Humanized VL DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELK(Sequence ID 36)

[0119] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises VH, which comprises the amino acid sequence of SEQ ID NO: 35. Alternatively or in addition, the anti-transferrin receptor antibody of the present disclosure comprises VL, which comprises the amino acid sequence of SEQ ID NO: 36.

[0120] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises VH containing 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) compared to VH as represented by SEQ ID NO: 35. Alternatively or in addition, the anti-transferrin receptor antibody of the present disclosure comprises VL containing 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) compared to VL as represented by SEQ ID NO: 36.

[0121] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises a VH having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as VH as represented by SEQ ID NO: 35. Alternatively or in addition, the anti-transferrin receptor antibody of the present disclosure comprises a VL having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as VL as represented by SEQ ID NO: 36.

[0122] In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a humanized variant comprising one or more amino acid substitutions at positions 43 and 48 (compared to VL as represented by SEQ ID NO: 34), and / or one or more amino acid substitutions at positions 48, 67, 69, 71, and 73 (compared to VH as represented by SEQ ID NO: 33). In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a humanized variant comprising S43A and / or V48L mutations (compared to VL as represented by SEQ ID NO: 34), and / or one or more A67V, L69I, V71R, and K73T mutations (compared to VH as represented by SEQ ID NO: 33).

[0123] In some embodiments, the anti-transferrin receptor antibody of the present disclosure is a humanized variant comprising one or more amino acid substitutions at positions 9, 13, 17, 18, 40, 43, 48, 45, and 70 (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, 48, 66, 67, 69, 71, 73, 75, 81, 83, 87, and 108 (compared to VH as represented by SEQ ID NO: 33).

[0124] In some embodiments, the anti-transferrin receptor antibodies of this disclosure are chimeric antibodies that may incorporate heavy chain constant regions and light chain constant regions from a human antibody. A chimeric antibody refers to an antibody having a variable region or part of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, both the light chain and heavy chain variable regions mimic the variable regions of antibodies derived from certain mammals (e.g., non-human mammals such as mice, rabbits, and rats), while the constant region is homologous to a sequence in an antibody derived from another mammal, such as a human. In some embodiments, amino acid modifications may be made in the variable region and / or the constant region.

[0125] In some embodiments, the anti-transferrin receptor antibodies described herein are chimeric antibodies that may incorporate the heavy chain constant region and light chain constant region from a human antibody. A chimeric antibody refers to an antibody having a variable region or part of a variable region from a first species and a constant region from a second species. Typically, in these chimeric antibodies, both the light chain and heavy chain variable regions mimic the variable regions of antibodies derived from certain mammals (e.g., non-human mammals such as mice, rabbits, and rats), while the constant region is homologous to the sequence in an antibody derived from another mammal, such as a human. In some embodiments, amino acid modifications may be made in the variable region and / or the constant region.

[0126] In some embodiments, any heavy chain of an anti-transferrin receptor antibody as described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may belong to any preferred source, 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 (Sequence ID 37).

[0127] In some embodiments, any light chain of the anti-transferrin receptor antibody described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP (Sequence ID 38).

[0128] 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.

[0129] Exemplary heavy and light chain amino acid sequences of the described anti-transferrin receptor antibodies are provided below:

[0130] Heavy chain (VH + human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 39)

[0131] Lightweight chain (VL + Kappa lightweight chain) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP(Sequence ID 40)

[0132] Heavy chain (humanized VH + human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVT VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(Sequence ID 41)

[0133] Light chain (humanized VL + kappa light chain) DIQMTQSPSSLSASVGDRVTITCRASDNLYSNLAWYQQKPGKSPKLLVYDATNLADGVPSRFSGSGSGTDYSLKINSLQSEDFGTYYCQHFWGTPLTFGAGTKLELKASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP(Sequence ID 42)

[0134] In some embodiments, the anti-transferrin receptor antibody described herein comprises a heavy chain having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as SEQ ID NO: 39. Alternatively or in addition, the anti-transferrin receptor antibody described herein comprises a light chain having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as SEQ ID NO: 40. In some embodiments, the anti-transferrin receptor antibody described herein comprises a heavy chain having the amino acid sequence of SEQ ID NO: 39. Alternatively or in addition, the anti-transferrin receptor antibody described herein comprises a light chain having the amino acid sequence of SEQ ID NO: 40.

[0135] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises a heavy chain containing 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) compared to the heavy chain as represented by SEQ ID NO: 39. Alternatively or in addition, the anti-transferrin receptor antibody of the present disclosure comprises a light chain containing 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) compared to the light chain as represented by SEQ ID NO: 40.

[0136] In some embodiments, the anti-transferrin receptor antibody described herein comprises a heavy chain having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as SEQ ID NO: 41. Alternatively or in addition, the anti-transferrin receptor antibody described herein comprises a light chain having at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) the same amino acid sequence as SEQ ID NO: 42. In some embodiments, the anti-transferrin receptor antibody described herein comprises a heavy chain having the amino acid sequence of SEQ ID NO: 41. Alternatively or in addition, the anti-transferrin receptor antibody described herein comprises a light chain having the amino acid sequence of SEQ ID NO: 42.

[0137] In some embodiments, the anti-transferrin receptor antibody of the present disclosure comprises a heavy chain containing 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) compared to the heavy chain of a humanized antibody as represented by SEQ ID NO: 39. Alternatively or in addition, the anti-transferrin receptor antibody of the present disclosure comprises a light chain containing 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) compared to the light chain of a humanized antibody as represented by SEQ ID NO: 40.

[0138] In some embodiments, anti-transferrin receptor antibodies are antigen-binding fragments (FABs) of intact antibodies (full-length antibodies). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by standard methods. For example, the F(ab')2 fragment can be produced by pepsin digestion of the antibody molecule, and the Fab fragment can be generated by reducing the disulfide crosslinks of the F(ab')2 fragment. Exemplary FAB amino acid sequences of anti-transferrin receptor antibodies described herein are provided below:

[0139] Heavy chain FAB (VH + some human IgG1 constant region) QVQLQQPGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEINPTNGRTNYIEKFKSKATLTVDKSSSTAYMQLSSLTSEDSAVYYCARGTRAYHYWGQGTSVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP(Sequence ID 43)

[0140] Heavy chain FAB (humanized VH + some human IgG1 constant region) EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWMHWVRQAPGQRLEWIGEINPTNGRTNYIEKFKSRATLTVDKSASTAYMELSSLRSEDTAVYYCARGTRAYHYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCP(Sequence ID 44)

[0141] The anti-transferrin receptor antibodies described herein may be in any form of antibody, but are 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 (e.g., scFv condensed with a portion of the constant region). In some embodiments, the anti-transferrin receptor antibodies described herein are scFv condensed with a constant region (e.g., the human IgG1 constant region as represented by SEQ ID NO: 39).

[0142] b. Other muscle-targeting antibodies In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin Iib, or CD63. In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to myogenic precursor proteins. Exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha-7, integrin alpha-7 beta-1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to skeletal muscle proteins. The exemplified skeletal muscle proteins include, but are not limited to, alpha-sarcoglycans, beta-sarcoglycans, calpain inhibitors, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycans, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha-7, integrin alpha-7 beta-1, integrin beta-1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, muscle-targeted antibodies are antibodies that specifically bind to smooth muscle proteins. The exemplary smooth muscle proteins include, but are not limited to, alpha-smooth muscle actin, VE-cadherin, cardesmon / CALD1, carponin 1, desmin, histamine H2 R, motilin R / GPR38, transgelin / TAGLN, and vimentin. However, it should be understood that antibodies against additional targets are within the scope of this disclosure, and that the list of exemplary targets provided herein is not intended to be limiting.

[0143] c. Antibody characteristics / changes In some embodiments, conservative mutations can be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where, when determined, for example based on the crystal structure, the residue is unlikely to be involved in interaction with the target antigen (e.g., a transferrin receptor). In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or the hinge region, numbered according to the Kabat numbering system (e.g., Kabat's EU index)).

[0144] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased) as described, for example, in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, for example, to facilitate the assembly of the light and heavy chains, or to change the stability of the antibody (e.g., increased or decreased), or to facilitate linker conjugation.

[0145] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1) and / or the CH3 domain (residues 341-447 of human IgG1) and / or the hinge region, numbered according to the Kabat numbering system (e.g., the Kabat EU index)) to increase or decrease the antibody's affinity for Fc receptors. Mutations in the Fc region of antibodies that increase or decrease the antibody's affinity for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof, are known to those skilled in the art. Examples of mutations in the Fc receptor of an antibody that may be made to alter the antibody's affinity for the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and international publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631 (these are incorporated herein by reference).

[0146] 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 an Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. For example, see international publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. patents 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that would alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0147] 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 Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or its FcRn-binding fragment (preferably Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1) in numbering according to the Kabat EU index (Kabat EA et al. (1991) above). In some embodiments, the IgG1 constant region of the antibodies described herein includes a methionine (M) to tyrosine (Y) substitution at position 252, numbered according to the EU index as found in Kabat, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256. See U.S. Patent No. 7,658,921 (which is incorporated herein by reference). This type of mutant IgG, referred to as 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 WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody includes an IgG constant region containing one, two, or three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as found in Kabat.

[0148] In some embodiments, one or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function (one or more) of an anti-transferrin receptor antibody. The effector ligand with altered affinity to itself may be, for example, the Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patents 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) may reduce the binding of the circulating antibody to the Fc receptor, thereby increasing tumor localization. For descriptions of mutations that delete or inactivate the constant region and thereby increase tumor localization, see, for example, U.S. Patents 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 a potential glycosylation site on the Fc region (which may reduce binding to the Fc receptor) (see, for example, Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0149] In some embodiments, one or more amino acid residues in the constant region of the muscle-targeting antibody described herein may be replaced with different amino acid residues so that the antibody may have modified Clq binding and / or reduced or absent complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are modified to alter the complement-binding ability of the antibody. This approach is described in further details in International Publication WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cytotoxicity (ADCC) to cells and / or to increase the antibody's affinity for the Fcγ receptor. This approach is described in further details in International Publication WO 00 / 42072.

[0150] In some embodiments, the heavy chain and / or light chain variable domain(s) sequences(s) of the antibodies provided herein may be used, as described elsewhere herein, for example, to generate CDR-conjugated antibodies, chimeric antibodies, humanized antibodies, or compound human antibodies, or antigen-binding fragments. As will be understood by those skilled in the art, any variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and the variant, CDR-conjugated antibody, chimeric antibody, humanized antibody, or compound antibody will maintain its specific binding ability to the transferrin receptor such that it may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% binding to the transferrin receptor compared to the original antibody from which it is derived.

[0151] In some embodiments, the antibodies provided herein include mutations that confer desired properties to the antibody. For example, to avoid potential complications resulting from Fab-arm exchange, which is known to occur in native IgG4 mAbs, the antibodies provided herein may include a stabilizing "Adair" mutation (Angal S., et al., “A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody”, Mol Immunol 30, 105-108; 1993), where serine 228 (EU numbering; residue 241 Kabat numbering) is converted to proline, resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may contain a stabilizing "Adair" mutation.

[0152] As provided herein, the antibodies of this 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 heavy chain-like IgA, IgD, IgE, IgG, and IgM, and any subclass of any isotype. The antibody may encompass a preferred constant region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, antibodies within the scope of this disclosure may be combined with any preferred constant region to encompass the VH and VL domains, or their antigen-binding regions.

[0153] ii. Muscle-targeting peptides Several aspects of this disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences that bind to specific cell types (for example, peptide sequences with a length of 5 to 20 amino acids) 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 TI, et al., "Elucidation of muscle-binding peptides by phage display screening" Muscle Nerve 1999; 22:460-6; ​​U.S. Patent No. 6,329,501, issued December 11, 2001, titled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE"; and Samoylov AM, et al., "Recognition of cell-specific binding of phage display derived peptides using an acoustic wave Sensors are described in Biomol Eng 2002;18:269-72; the entire contents of each of these are incorporated herein by reference. Selectivity to desired tissues, e.g., muscle, can be achieved by designing peptides to interact with specific cell surface antigens (e.g., receptors). Skeletal muscle targeting has been investigated, and a wide range of molecular payloads can be delivered. These approaches, which do not have many of the practical disadvantages of large antibodies or viral particles, may have high selectivity to muscle tissue. Consequently, in some embodiments, muscle targeting agents are muscle-targeting peptides ranging in length from 4 to 50 amino acids.In some embodiments, muscle-targeting peptides have a length of 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. Muscle-targeting peptides can be generated using one of several methods, such as phage display.

[0154] In some embodiments, muscle-targeting peptides may bind to internalized cell surface receptors (e.g., transferrin receptors) that are overexpressed or relatively highly expressed in muscle cells compared to other cells. In some embodiments, muscle-targeting peptides may target transferrin receptors (e.g., bind to transferrin receptors). In some embodiments, transferrin receptor-targeting peptides may contain naturally occurring ligands, e.g., segments of transferrin. In some embodiments, transferrin receptor-targeting peptides are described in U.S. Patent No. 6,743,893, 11 / 30 / 2000, “RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR”. In some embodiments, the transferrin receptor-targeting peptide is as described in Kawamoto, M. et al, “A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells.” BMC Cancer. 2011 Aug 18;11:359. In some embodiments, the transferrin receptor-targeting peptide is as described in U.S. Patent No. 8,399,653, 5 / 20 / 2011, “TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY.”

[0155] As mentioned above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides have been identified using phage display libraries that present ptapeptides on their surface. As an example, a peptide with the amino acid sequence ASSLNIA (SEQ ID NO: 6) bound to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. Consequently, in some embodiments, muscle-targeting agents contain the amino acid sequence ASSLNIA (SEQ ID NO: 6). This peptide exhibited improved specificity for binding to myocardial and skeletal muscle tissue after intravenous injection into mice, with reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have been identified using phage display. For example, in the context of treatment for DMD, a 12-amino acid peptide was identified by a phage display library for muscle targeting. See Yoshida D., et al., “Targeting of salicylate to skin and muscle following topical injections in rats.” Int J Pharm 2002;231:177-84; this entire content is incorporated by reference thereto. Here, a 12-amino acid peptide with 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.

[0156] Any additional method for identifying peptides selective to muscle (e.g., skeletal muscle) more than other cell types involves 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 this is incorporated herein by reference. Nonspecific cell binders were selected by pre-incubating random 12-mer peptide phage display libraries with a mixture of non-muscle cell types. Following rounds of selection, the 12-amino acid peptide TARGEHKEEELI (SEQ ID NO: 8) appeared most frequently. Consequently, in some embodiments, muscle targeting agents include the amino acid sequence TARGEHKEEELI (SEQ ID NO: 8).

[0157] Muscle targeting agents may be amino acid-containing molecules or peptides. Muscle targeting peptides may correspond to sequences of proteins that preferentially bind to protein receptors found in muscle cells. In some embodiments, muscle targeting peptides contain high propensity of hydrophobic amino acids (e.g., valine) so that the peptides can preferentially target muscle cells. In some embodiments, muscle targeting peptides have not been characterized or disclosed to date. These peptides may be recalled, produced, synthesized, and / or derivatized using one of several methodologies, e.g., phage-dispensed peptide libraries, one-bead-one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. The example methodologies are characterized in the relevant technical field and are incorporated by reference (Gray, BP and Brown, KC “Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides” Chem Rev. 2014, 114:2, 1020-1081.; Samoylova, TI and Smith, BF “Elucidation of muscle-binding peptides by phage display screening.” Muscle Nerve, 1999, 22:4, 460-6).In several aspects, muscle-targeting peptides have been disclosed to date (see, for example, Writer MJet 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, MJet al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004, 342:1, 171-82). The exemplary muscle-targeting peptides include the following amino acid sequences: CQAQGQLVC (SEQ ID NO: 9), CSERSMNFC (SEQ ID NO: 10), CPKTRRVPC (SEQ ID NO: 11), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 12), ASSLNIA (SEQ ID NO: 6), CMQHSMRVC (SEQ ID NO: 13), and DDTRHWG (SEQ ID NO: 14). In some embodiments, the muscle-targeting peptides may contain approximately 2–25 amino acids, approximately 2–20 amino acids, approximately 2–15 amino acids, approximately 2–10 amino acids, or approximately 2–5 amino acids. The muscle-targeting peptides may contain naturally occurring amino acids, such as cysteine, alanine, or amino acids that do not exist naturally, or modified amino acids.Amino acids that do not exist in nature 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, for example, Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

[0158] iii. Muscle-targeting receptor ligands The muscle targeting agent may be a ligand, for example, a ligand that binds to a receptor protein. The muscle targeting ligand may be a protein that binds to an internalized cell surface receptor expressed by muscle cells, for example, transferrin. Consequently, in some embodiments, the muscle targeting agent is transferrin, or a derivative thereof that binds to a transferrin receptor. Alternatively, the muscle targeting ligand may be a small molecule, for example, a lipophilic small molecule that preferentially targets muscle cells compared to other cell types. Exemplary lipophilic small molecules that may target muscle cells include compounds containing cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolenic acid, linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and alkoxy acids.

[0159] iv. Muscle-targeting aptamers Muscle-targeting agents may be aptamers that preferentially target muscle cells compared to other cell types, such as RNA aptamers. In some embodiments, muscle-targeting aptamers have not been characterized or disclosed to date. These aptamers may be recalled, produced, synthesized, and / or derivatized using one of several methodologies, e.g., systematic evolution of ligands by exponential enrichment. The exemplary methodologies are characterized in the art and incorporated by reference (Yan, AC and Levy, M. “Aptamers and aptamer targeted delivery” RNA biology, 2009, 6:3, 316-20.; Germer, K. et al. “RNA aptamers and their therapeutic and diagnostic applications.” Int. J. Biochem. Mol. Biol. 2013; 4:27-40). In some embodiments, muscle-targeted aptamers have been disclosed to date (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, WH et al. “Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation.” Mol Ther. 2016, 24:4, 779-87). Exemplary muscle-targeted 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 approximately 5–15 kDa, approximately 5–10 kDa, approximately 10–15 kDa, approximately 1–5 Daa, approximately 1–3 kDa, or smaller.

[0160] 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, muscle targeting agents are substrates of influx transporters specific to muscle tissue. In some embodiments, the influx transporters are specific to skeletal muscle tissue. The two major classes of transporters expressed on the muscle fiber sheath of skeletal muscle are (1) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue, and (2) the solute carrier (SLC) superfamily, which can facilitate the influx of substrates into skeletal muscle. In some embodiments, muscle targeting agents are substrates that bind to the ABC or SLC superfamily of transporters. In some embodiments, the substrates that bind to the ABC or SLC superfamily of transporters are naturally occurring substrates. In some embodiments, the substrates that bind to the ABC or SLC superfamily of transporters are naturally occurring substrates, such as synthetic derivatives that bind to the ABC or SLC superfamily of transporters.

[0161] In some embodiments, muscle targeting agents are substrates of the SLC superfamily of transporters. SLC transporters are either equilibrium-type or utilize a proton or sodium ion gradient created across the membrane to drive substrate transport. Exemplary SLC transporters with high expression in skeletal muscle include, but are not limited to, the SATT transporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7 transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), LAT3 transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-J transporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2 transporter (FLJ46769; SLC22A5), ENT transporter (ENT1; SLC29A1 and ENT2; SLC29A2), PAT2 transporter (SLC36A2), and SAT2 transporter (KIAA1382; SLC38A2). These transporters can provide an opportunity for muscle targeting by facilitating the influx of substrates into skeletal muscle.

[0162] In some embodiments, muscle targeting agents are substrates of the equilibrium nucleoside transporter 2 (ENT2) transporter. Compared to other transporters, ENT2 has one of the highest expression mRNAs in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs, including the brain, heart, placenta, thymus, pancreas, prostate, and kidneys, 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 nucleic acid bases. The hENT2 transporter has low affinity for all nucleosides except inosine (adenosine, guanosine, uridine, thymidine, and cytidine). Consequently, in some embodiments, muscle targeting agents are ENT2 substrates. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and clopharabine. 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 noncovalently linked to the molecular payload.

[0163] In some embodiments, the muscle targeting agent is a substrate of an organic cation / carnitine transporter (OCTN2), which is 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 a molecular payload (e.g., an oligonucleotide payload).

[0164] The muscle targeting agent may be a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be hemoduvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, hemoduvelin may be full-length or fragmentary, 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 with a functional hemoduvelin protein. In some embodiments, the hemoduvelin mutant may be a soluble fragment, may lack N-terminal signaling, and / or may lack a C-terminal anchoring domain. In some embodiments, hemoduberin 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 hemoduberin may originate from humans, non-human primates, or rodents.

[0165] B. Molecular Payload Several aspects of this disclosure provide molecular payloads for modulating biological outcomes, such as the transcription of DNA sequences, protein expression, or protein activity. In some embodiments, the molecular payload is linked to or otherwise conjugated to a muscle targeting agent. In some embodiments, such a molecular payload can target muscle cells, for example, by specific binding to nucleic acids or proteins in muscle cells upon delivery to the muscle cells by the conjugated muscle targeting agent. It should be understood that various types of muscle targeting agents may be used in accordance with this disclosure. For example, the molecular payload may include, or consist of, oligonucleotides (e.g., antisense oligonucleotides), peptides (e.g., peptides that bind to nucleic acids or proteins in disease-related muscle cells), proteins (e.g., proteins that bind to nucleic acids or proteins in disease-related muscle cells), or small molecules (e.g., small molecules that modulate the function of nucleic acids or proteins in disease-related muscle cells). In some embodiments, the molecular payload is an oligonucleotide containing a chain having a region complementary to DUX4. While exemplary molecular payloads are described in more detail herein, it should be understood that the exemplary molecular payloads provided herein are not intended to be limiting.

[0166] i. oligonucleotides Any preferred oligonucleotide may be used as a molecular payload as described herein. In some embodiments, the oligonucleotide may be designed to induce the degradation of mRNA (e.g., the oligonucleotide may be a gapmer, siRNA, ribozyme, or aptamer that induces degradation). In some embodiments, the oligonucleotide may be designed to block the 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 induce the degradation of mRNA and thereby 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., a gene editing enzyme). Other examples of oligonucleotides are provided herein. In some embodiments, it should be understood that by incorporating a functional sequence (e.g., an antisense strand sequence) from one format into the other format, an oligonucleotide of one format (e.g., an antisense oligonucleotide) may be suitably adapted to another format (e.g., an siRNA oligonucleotide).

[0167] As described herein, any suitable oligonucleotide may be used as a molecular payload. Examples of useful oligonucleotides for targeting DUX4 include U.S. Patent No. 9,988,628, published February 2, 2017, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. Patent No. 9,469,851, published October 30, 2014, entitled "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4"; U.S. Patent Application Publication No. 20120225034, published September 6, 2012, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; and "MORPHOLINO TARGETING DUX4 FOR TREATING PCT patent application publication number WO 2013 / 120038, published on August 15, 2013, entitled “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 with the target DUX4 gene or mRNA.

[0168] In some embodiments, the oligonucleotide may have a region complementary to a sequence represented as human DUX4 corresponding to NCBI sequence NM_001293798.1 (SEQ ID NO: 15) and / or mouse DUX4 corresponding to NCBI sequence NM_001081954.1 (SEQ ID NO: 16) as follows. In some embodiments, the oligonucleotide may have a region complementary to a hypomethylated and reduced D4Z4 repeat, as described in Daxinger, et al., “Genetic and Epigenetic Contributors to FSHD,” Lim JW, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1; 24(17): 4817-4828 (the entire contents of each of these are incorporated herein by reference).

[0169] In some embodiments, the oligonucleotide may have a region complementary to a sequence represented as follows, the sequence being an example of the human DUX4 gene sequence (NM_001293798.1) (SEQ ID NO: 15):

[0170] In some embodiments, the oligonucleotide may have a region complementary to a sequence represented as follows, the sequence being an example of the mouse DUX4 gene sequence (SEQ ID NO: 16) (NM_001081954.1):

[0171] In some embodiments, the oligonucleotide may have a region complementary to the DUX4 gene sequence of a species selected from multiple species, such as humans, mice, and non-human species.

[0172] In some embodiments, the oligonucleotide targeting DUX4 is the FM10 sequence. In some embodiments, the oligonucleotide targeting DUX4 is the phosphorodiamidate morpholino version of the FM10 sequence. In some embodiments, the oligonucleotide targeting DUX4 comprises the sequence GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 45). In some embodiments, the oligonucleotide targeting DUX4 comprises a sequence complementary to at least 15 consecutive nucleotides of AGTTCAGAGATATATTAAAATGCCC (SEQ ID NO: 46).

[0173] In some embodiments, muscle-specific E3 ubiquitin ligases are overexpressed in FSHD and play a role in muscular atrophy (see, for example, Vanderplanck, C. et al. “The FSHD Atrophic Myotube Phenotype Is Caused by DUX4 Expression” PLoS One 6,10:e26820, 2011). In some embodiments, downregulation of these ligases presents viable therapeutic strategies. In some embodiments, oligonucleotides may target, for example, MuRF1 (also known as TRIM63) and MAFbx (also known as Fbx032), which inhibit the expression of muscle-specific E3 ubiquitin ligases associated with FSHD. In some embodiments, oligonucleotides may have at least one MuRF1 gene sequence, for example, a region complementary to human MuRF1 (NCBI Gene ID 84676). In some embodiments, the oligonucleotide may have at least one MAFbx gene sequence, for example, a region complementary to human MAFbx (NCBI Gene ID 114907).

[0174] a. Oligonucleotide size / arrangement Oligonucleotides may be of various different lengths, for example, depending on the format. In some embodiments, oligonucleotides are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides or longer. In some embodiments, oligonucleotides are 8-50 nucleotides, 8-40 nucleotides, 8-30 nucleotides, 10-15 nucleotides, 10-20 nucleotides, 15-25 nucleotides, 21-23 nucleotides, and so on.

[0175] In some embodiments, for the purposes of this disclosure, a complementary nucleic acid sequence of an oligonucleotide is specifically hybridizable to or specific to a target nucleic acid when, under conditions where the binding of the sequence to a target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) causing loss of activity (e.g., inhibition of translation) or loss of expression (e.g., degradation of target mRNA), and where avoidance of nonspecific binding is desired, such as under physiological conditions in the case of in vivo assays or therapeutic treatments and in vitro assays, and under conditions where the assay is performed under favorable stringency conditions. Therefore, 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 the consecutive nucleotides of the target nucleic acid. In some embodiments, the complementary nucleotide sequence does not need to be specifically hybridizable to the target nucleic acid or 100% complementary to the target sequence that is specific to the target nucleic acid.

[0176] In some embodiments, the oligonucleotide includes a region complementary to the 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 complementary to the target nucleic acid has a length of 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 some embodiments, the complementary region is complementary to at least 8 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain one, two, or three base mismatches compared to a portion of the target nucleic acid's sequence of nucleotides. In some embodiments, the oligonucleotide may have up to three mismatches over 15 bases, or up to two mismatches over 10 bases.

[0177] b. Oligonucleotide modification: The oligonucleotides described herein may be modified, including, for example, modified sugar moieties, modified nucleoside linkages, modified nucleotides, and / or combinations thereof. In addition, in some embodiments, oligonucleotides may exhibit one or more of the following properties: non-mediating of alternative splicing; non-immunostimulant; nuclease-resistant; having improved cellular uptake compared to unmodified oligonucleotides; non-toxic to cells or mammals; having improved exit into endosomes within cells; minimizing TLR stimulation; or evading pattern recognition receptors. Any 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 contained within the same oligonucleotide.

[0178] In some embodiments, specific nucleotide modifications may be used to make the oligonucleotide into which the modification is incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide or oligoribonucleotide molecule; these modified oligonucleotides remain intact for longer periods than unmodified oligonucleotides. Specific examples of modified oligonucleotides include those with modified backchains, such as phosphorothioates, phosphotriesters, methylphosphonates, short-chain alkyl or cycloalkyl sugar linkages, or modified nucleoside linkages such as short-chain heteroatom or heterocyclic sugar linkages. Consequently, the oligonucleotides of this disclosure can be stabilized against nucleolysis by modification, such as the incorporation of nucleotide modifications.

[0179] In some embodiments, the oligonucleotide may be an oligonucleotide with a length of up to 50 nucleotides or up to 100 nucleotides, where 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30, 2-40, 2-45 nucleotides, or more, are modified nucleotides. The oligonucleotide may be an oligonucleotide with a length of 8-30 nucleotides, where 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30 nucleotides are modified nucleotides. The oligonucleotide may be an oligonucleotide with a length of 8-15 nucleotides, where 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14 nucleotides are modified nucleotides. Optionally, any oligonucleotide may be modified except for nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Oligonucleotide modifications are further described herein.

[0180] c. Modified nucleotides In some embodiments, oligonucleotides include 2'-modified nucleotides, such as 2'-deoxy, 2'-deoxy-2'-fluoro, 2'-O-methyl, 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O--N-methylacetamide (2'-O--NMA).

[0181] In some embodiments, the oligonucleotide may contain at least one 2'-O-methyl-modified nucleotide, and in some embodiments, all nucleotides may contain the 2'-O-methyl modification. In some embodiments, the oligonucleotide contains a modified nucleotide in which the ribose ring includes a bridging portion that connects two atoms in the ring (e.g., a 2'-O atom to a 4'-C atom). In some embodiments, the oligonucleotide is “locked,” and contains a modified nucleotide in which the ribose ring is “locked” by a methylene bridge connecting a 2'-O atom and a 4'-C atom, for example. An example of LNA is described in the international patent application publication WO / 2008 / 043753, published on April 17, 2008, entitled “RNA Antagonist Compounds For The Modulation Of PCSK9” (the contents of which are incorporated herein by reference in their entirety).

[0182] Other modifications that may be used in the oligonucleotides disclosed herein include ethylene-crosslinked nucleic acids (ENAs). ENAs include, but are not limited to, 2'-O,4'-C-ethylene-crosslinked nucleic acids. Examples of ENA are provided in the international patent publication WO 2005 / 042777, published on 12 May 2005, titled "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 entirety of which disclosures are incorporated herein by reference.

[0183] In some embodiments, the oligonucleotide may include cross-linked nucleotides such as locked nucleic acid (LNA) nucleotides, restricted ethyl (cEt) nucleotides, or ethylene cross-linked nucleic acid (ENA) nucleotides. In some embodiments, the oligonucleotides include modified nucleotides 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, currently U.S. Patent 8,957,201, issued February 17, 2015, titled "Oligonucleotide Analogues And Methods Utilizing The Same". For all purposes, the entire contents of each of these are incorporated herein by reference.

[0184] In some embodiments, the oligonucleotide comprises at least one nucleotide modified at the 2' position of the sugar, preferably a 2'-O-alkyl, 2'-O-alkyl-O-alkyl, or 2'-fluoro-modified nucleotide. In other preferred embodiments, the RNA modification comprises a pyrimidine, a debased residue, or a 2'-fluoro, 2'-amino, and 2'O-methyl modification on the ribose of an inverted base at the 3' end of the RNA.

[0185] In some embodiments, an oligonucleotide may have at least one modified nucleotide that results in an increase in the Tm of the oligonucleotide in the range of 1°C, 2°C, 3°C, 4°C, or 5°C compared to an oligonucleotide that has no modified nucleotides. An oligonucleotide may have multiple modified nucleotides that result in an increase in the Tm of the oligonucleotide in the range of 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or above, compared to an oligonucleotide that has no modified nucleotides.

[0186] Oligonucleotides may include alternating arrangements of different types of nucleotides. For example, oligonucleotides may include alternating arrangements of deoxyribonucleotides or ribonucleotides with 2'-fluorodeoxyribonucleotides. Oligonucleotides may include alternating arrangements of deoxyribonucleotides or ribonucleotides with 2'-O-methylnucleotides. Oligonucleotides may include alternating arrangements of 2'-fluoronucleotides with 2'-O-methylnucleotides. Oligonucleotides may include alternating arrangements of cross-linked nucleotides with 2'-fluoro or 2'-O-methylnucleotides.

[0187] d. Internucleotide linkage / backbone In some embodiments, the oligonucleotide may contain phosphorothioate or other modified nucleotide linkages. In some embodiments, the oligonucleotide contains phosphorothioate nucleoside linkages. In some embodiments, the oligonucleotide contains phosphorothioate nucleoside linkages between at least two nucleotides. In some embodiments, the oligonucleotide contains phosphorothioate nucleoside linkages between all nucleotides. For example, in some embodiments, the oligonucleotide contains modified nucleotide linkages at the 5' or 3' end of the nucleotide sequence, at the first, second, and / or third nucleoside linkages.

[0188] Phosphorus-containing linkages that may be used include, but are not limited to, normal 3'-5' linkages, their 2'-5' linkage analogs, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotryesters, aminoalkyl phosphotryesters, methyl and other alkylphosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-aminophosphoramidates and aminoalkylphosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotryesters, and boranophosphorates, and those having opposite polarity (where adjacent pairs of nucleoside units are 3'-5' to 5'-3' or (connected from 2'-5' to 5'-2') and encompassing; US Patent No. 3,687,808; No. 4,469,863; No. 4,476,301; No. 5,023,243; No. 5,177,196; No. 5,188,897; No. 5,264,423; No. 5,276,019; No. 5,278,302; No. 5,286,717; No. 5,321,131; No. 5,39 See issues 9,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0189] 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 (where the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotide is directly or indirectly bonded to the aza nitrogen atom of the polyamide backbone, see Nielsen et al., Science 1991, 254,1497).

[0190] e. Stereospecific oligonucleotides In some embodiments, the phosphorus atoms between nucleotides of the oligonucleotide are chiral, and the properties of the oligonucleotide are tuned based on the stereochemistry of the chiral phosphorus atoms. In some embodiments, suitable methods may be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (as described, for example, 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, comprising nucleoside units linked together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages. In some embodiments, such phosphorothioate oligonucleotides having substantially chiral pure intersugar linkages are prepared by enzymatic or chemical synthesis, for example, as described in U.S. Patent 5,587,261, issued December 12, 1996 (the contents of which are incorporated herein by reference in their entirety). In some embodiments, chiral-controlled oligonucleotides provide selective cleavage patterns for target nucleic acids. For example, in some embodiments, chiral-controlled oligonucleotides provide a single cleavage site within a complementary sequence of nucleic acids, as described, for example, in U.S. Patent Application Publication 20170037399 A1, titled "CHIRAL DESIGN," published on February 2, 2017 (the contents of which are incorporated herein by reference in their entirety).

[0191] f. morpholino In some embodiments, oligonucleotides may be morpholino-based compounds. 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; Naseviius 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, morpholino-based oligomeric compounds are phosphorodiamidate morpholino oligomers (PMOs) (for example, as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; these disclosures are incorporated herein by reference in their entirety).

[0192] g. Peptide nucleic acid (PNA) In some embodiments, both the sugar and nucleoside linkages (back chain) of the nucleotide unit of the oligonucleotide are replaced with novel groups. In some embodiments, the base unit is maintained for hybridization with a suitable nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic shown to have excellent hybridization properties, is called a peptide nucleic acid (PNA). In PNA compounds, the sugar-back chain of the oligonucleotide is replaced with an amide-containing back chain, e.g., an aminoethylglycine back chain. The nucleic acid base is retained and is directly or indirectly bonded to the aza nitrogen atom of the amide portion of the back chain. Representative publications reporting the preparation of PNA compounds include, but are not limited to, U.S. Patents 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.

[0193] h.Gapmer In some embodiments, oligonucleotides are gapmers. Gapmer oligonucleotides generally have the formula 5'-XYZ-3', with X and Z as flanking regions surrounding a gap region Y. In some embodiments, the Y region is a contiguous sequence of nucleotides, e.g., a region of at least 6 DNA nucleotides, capable of recruiting RNAse such as RNAse H. In some embodiments, the gapmer binds to the target nucleic acid at a point where RNAse can be recruited and subsequently cleave the target nucleic acid. In some embodiments, the Y region is flanked from both the 5' and 3' ends by regions X and Z containing high-affinity modified nucleotides, e.g., 1 to 6 modified nucleotides. Examples of modified nucleotides include, but are not limited to, 2'MOE or 2'OMe, or locked nucleobases (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 similar length or not. In some embodiments, the gap segment Y may be a nucleotide sequence having a length of 5 to 20 nucleotides, 6 to 12 nucleotides, or 6 to 10 nucleotides.

[0194] In some embodiments, the gap region of a gapmer oligonucleotide may contain modified nucleotides known to be acceptable for efficient RNase H activity, in addition to DNA nucleotides such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-configured nucleotides. In some embodiments, the gap region contains one or more unmodified nucleosides. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate nucleoside links (e.g., phosphorothioate nucleoside links or other links) between at least two, at least three, at least four, at least five, or more nucleotides. In some embodiments, the gap region and the two flanking regions each independently contain modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, or more nucleotides.

[0195] Gapmers may be produced using appropriate methods. Representative U.S. patents, U.S. patent publications, and PCT publications teaching 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; 5,652,355. This includes Patent Nos. 6; 5,700,922; 5,898,031; 7,432,250; and 7,683,036; U.S. Patent Publications US20090286969, US20100197762, and US20110112170; and PCT Publications WO2008049085 and WO2009090182, each of which is incorporated herein by reference in whole.

[0196] i.Mixmer In some embodiments, the oligonucleotides described herein may be mixmers or may contain mixmer sequence patterns. Generally, a mixmer is an oligonucleotide containing both naturally occurring and non-naturally occurring nucleotides, or an oligonucleotide containing two different types of non-naturally occurring nucleotides, typically in an alternating pattern. Mixmers generally have a higher binding affinity than unmodified oligonucleotides and bind specifically to target molecules; for example, they may be used to block binding sites on target molecules. Generally, mixmers do not recruit RNAse to target molecules and therefore do not promote cleavage of target molecules. Oligonucleotides that are not capable of recruiting RNAse H are described; see, for example, WO2007 / 112754 or WO2007 / 112753.

[0197] In some embodiments, a mixmer includes or consists of repeating patterns of nucleotide analogs and naturally occurring nucleotides, or repeating patterns of one type of nucleotide analog and another type of nucleotide analog. However, a mixmer does not need to include a repeating pattern and instead may 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 repeating pattern may, for example, consist of modified nucleotides 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 nucleotides described herein. It is recognized that repeating patterns of modified nucleotides, such as LNA units, may be combined with modified nucleotides at fixed positions—for example, at the 5' or 3' end.

[0198] In some embodiments, the mixmer does not contain regions of naturally occurring nucleotides, such as DNA nucleotides, that are in a sequence of more than five, more than four, more than three, or more than two consecutive nucleotides. In some embodiments, the mixmer includes at least a region consisting of at least two consecutive modified nucleotides, such as at least two consecutive LNAs. In some embodiments, the mixmer includes at least a region consisting of at least three consecutive modified nucleotides, such as at least three consecutive LNAs.

[0199] In some embodiments, the mixmer does not contain regions of nucleotide analogs such as LNAs that are consecutive in numbers of more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2. In some embodiments, the LNA units may be replaced with other nucleotide analogs, such as the nucleotide analogs referred to herein.

[0200] The mixmer may be designed to contain a mixture of affinity-enhancing modified nucleotides, such as LNA nucleotides and 2'-O-methyl nucleotides, in non-limiting examples. In some embodiments, the mixmer contains modified nucleoside linkages (e.g., phosphorothioate nucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, or more nucleotides.

[0201] The mixmer may be produced using any preferred method. Representative U.S. patents, U.S. patent publications, and PCT publications teaching the preparation of the mixmer include U.S. Patent Publications US20060128646, US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. Patent No. 7687617.

[0202] In some embodiments, the mixmer comprises one or more morpholinonucleotides. For example, in some embodiments, the mixmer may comprise morpholinonucleotides mixed (for example, in alternating ways) with one or more other nucleotides (e.g., DNA, RNA nucleotides) or modified nucleotides (e.g., LNA, 2'-O-methylnucleotides).

[0203] In some embodiments, mixmers are useful for splice correcting or exon skipping, as reported, for example, in Touznik A., et al., "LNA / DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1 SMA fibroblasts," Scientific Reports, volume 7, Article number: 3672 (2017), and Chen S. et al., "Synthesis of a Morpholino Nucleic Acid (MNA)-Uridine Phosphoramidite, and Exon Skipping Using MNA / 2'-O-Methyl Mixmer Antisense Oligonucleotide," Molecules 2016, 21, 1582 (the contents of each of these are incorporated herein by reference).

[0204] j.RNA interference (RNAi) In some embodiments, the oligonucleotides provided herein may be in the form of small interfering RNA (siRNA), also known as small interfering RNA or silencing RNA. siRNA is a type of double-stranded RNA molecule, typically about 20–25 base pairs long, that targets nucleic acids (e.g., mRNA) for degradation via the RNA interference (RNAi) pathway in cells. The specificity of an siRNA molecule may be determined by the binding of the antisense strand molecule to its target RNA. Effective siRNA molecules are generally less than 30–35 base pairs long, although longer siRNAs may also be effective, to prevent the triggering of nonspecific RNA interference pathways in cells via interferon responses.

[0205] Following the selection of an appropriate target RNA sequence, an siRNA molecule containing nucleotide sequences complementary to all or some of the target sequences, i.e., antisense sequences, can be designed and prepared using appropriate methods (see, for example, PCT publication WO2004 / 016735; and U.S. Patent Publications 2004 / 0077574 and 2008 / 0081791).

[0206] siRNA molecules can be double-stranded (i.e., dsRNA molecules containing an antisense strand and a complementary sense strand) or single-stranded (i.e., ssRNA molecules containing only an antisense strand). siRNA molecules may include double-stranded, asymmetric double-stranded, hairpin, or asymmetric hairpin secondary structures having self-complementary sense and antisense strands.

[0207] Double-stranded siRNAs may contain RNA strands of the same or different lengths. Double-stranded siRNA molecules can also be associated with single oligonucleotides in a stem-loop structure (where the self-complementary sense and antisense regions of the siRNA molecule are linked using one or more nucleic acid-based or non-nucleic acid-based linkers), as well as circular single-stranded RNAs 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 produce an active siRNA molecule 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, typically separated by a spacer or loop sequence. Breaking of a spacer or loop provides a single-stranded RNA molecule and its reverse complement (which may optionally result in the addition or removal of one, two, three or more nucleotides from the 3' and / or 5' ends of one or both strands) so that they can anneal to form a dsRNA molecule. The spacer may be long enough to anneal the antisense and sense sequences to form a double-stranded structure (or stem) prior to the break of the spacer (and optionally the subsequent processing step which may result in the addition or removal of one, two, three, four or more nucleotides from the 3' and / or 5' ends of one or both strands). The spacer sequence may be an unrelated nucleotide sequence placed between two complementary nucleotide sequence regions, which will contain shRNA once annealed to form a double-stranded nucleic acid.

[0208] The overall length of an siRNA molecule can vary from approximately 14 to 100 nucleotides, depending on the type of siRNA molecule designed. Generally, between approximately 14 and 50 of these nucleotides, they constitute the complementary RNA target sequence, i.e., the specific antisense sequence of the siRNA molecule. For example, when the siRNA is double-stranded or single-stranded, the length can vary from approximately 14 to 50 nucleotides, while when the siRNA is shRNA or a circular molecule, the length can vary from approximately 40 to 100 nucleotides.

[0209] An siRNA molecule may have a 3' overhang at one end of the molecule, and the other end may be blunt or also have an overhang (5' or 3'). When an siRNA molecule has overhangs at both ends of the molecule, the lengths of the overhangs may be the same or different. In one embodiment, the siRNA molecule of this disclosure has 3' overhangs of about 1 to about 3 nucleotides on both ends of the molecule.

[0210] k. microRNA (miRNA) In some embodiments, oligonucleotides may be microRNAs (miRNAs). MicroRNAs (referred to as "miRNAs") are small, non-coding RNAs belonging to a class of regulatory molecules that control gene expression by binding to complementary sites on target RNA transcripts. Typically, miRNAs are generated from large RNA precursors (called pri-miRNAs), which are processed in the nucleus to become approximately 70-nucleotide pre-miRNAs, which fold into incomplete stem-loop structures. These pre-miRNAs typically undergo additional processing steps in the cytoplasm, but mature miRNAs in the cytoplasm, which are 18-25 nucleotides in length, are excised from one end of the pre-miRNA hairpin by the RNase III enzyme Dicer.

[0211] When used herein, miRNA encompasses pri-miRNA, pre-miRNA, mature miRNA, or fragments of its variants that retain the biological activity of mature miRNA. In one embodiment, the size range of miRNA may be from 21 to 170 nucleotides. In one embodiment, the size range of miRNA is from 70 to 170 nucleotides in length. In another embodiment, mature miRNA with a length of 21 to 25 nucleotides may be used.

[0212] l. aptamer In some embodiments, the oligonucleotides provided herein may be in the form of aptamers. Generally, in the context of molecular payloads, an aptamer is any nucleic acid that specifically binds to a target such as a small molecule in a cell, a protein, or a nucleic acid. 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, naturally occurring nucleotides with a hydrocarbon linker (e.g., alkylene) or polyether linker (e.g., PEG linker) inserted between one or more nucleotides, modified nucleotides with a hydrocarbon or PEG linker inserted between one or more nucleotides, or a combination thereof. Exemplary publications and patents describing 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.

[0213] m. Ribozyme In some embodiments, the oligonucleotides provided herein may be in the form of ribozymes. Ribozymes (ribonucleic acid enzymes) are molecules, typically RNA molecules, that are capable of carrying out specific biochemical reactions similar to those of protein enzymes. Ribozymes are molecules with enzymatic activity that encompasses the ability to cleave specific phosphodiester links in RNA molecules (such as mRNA, RNA-containing substrates, lncRNA, and the ribozyme itself) with which they hybridize.

[0214] Ribozymes can take on one of several physical structures, one of which is called a "hammerhead." A hammerhead ribozyme consists of a catalytic core containing nine conserved bases, a double-stranded stem and loop structure (stem-loop II), and two regions complementary to the target RNA region surrounding the catalytic core. These flanking regions allow the ribozyme to specifically bind to the target RNA by forming double-stranded stems I and III. Cleavage occurs either cis (i.e., cleavage of the same RNA molecule containing the hammerhead motif) or trans (cleavage of an RNA substrate other than the ribozyme) following a specific ribonucleotide triplet from a transesterification reaction of 3',5'-phosphate diester to 2',3'-cyclic phosphate diester. While we do not wish to be constrained by theory, this catalytic activity is thought to require the presence of a highly conserved specific sequence within the catalytic region of the ribozyme.

[0215] Modifications in ribozyme structures also include substitution or replacement of non-core portions of various molecules with non-nucleotide molecules. For example, Benseller et al. (J.Am.Chem.Soc.(1993)115:8483-8484) disclosed a hammerhead-like molecule in which all two base pairs of stem II and all four nucleotides of loop II were replaced with non-nucleoside linkers based on hexaethylene glycol, propanediol, bis(triethylene glycol) phosphate, tris(propanediol)bisphosphate, or bis(propanediol) phosphate. Ma et al. (Biochem.(1993)32:1751-1758; Nucleic Acids Res.(1993)21:2585-2589) replaced the six-nucleotide loop of the TAR ribozyme hairpin with a non-nucleotide linker related to ethylene glycol. Thomson et al. (Nucleic Acids Res. (1993) 21:5600-5603) replaced loop II with linear non-nucleotide linkers with lengths of 13, 17, and 19 atoms.

[0216] Ribozyme oligonucleotides can be prepared using well-known methods (see, for example, PCT publications WO9118624; WO9413688; WO9201806; and WO92 / 07065; and U.S. Patents 5436143 and 5650502) or purchased from commercial sources (e.g., U.S. Biochemicals), and, if desired, nucleotide analogs may be incorporated to increase the oligonucleotide's resistance to degradation by cellular nucleases. Ribozymes may be synthesized by any known method, for example, using commercially available synthesizers (e.g., manufactured by Applied Biosystems, Inc. or Milligen). Ribozymes may also be produced in recombinant vectors by conventional means. See Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (Current edition). Ribozyme RNA sequences may be synthesized by conventional methods, for example, using RNA polymerases such as T7 or SP6.

[0217] n. Guide nucleic acid In some embodiments, oligonucleotides are guide nucleic acids, e.g., guide RNA (gRNA) molecules. Generally, guide RNA is a small synthetic RNA consisting 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 closer to the DNA target sequence. In some embodiments, napDNAbp is a nucleic acid-programmable protein that forms a complex with (e.g., by binding to or linking to) one or more RNAs that cause the nucleic acid-programmable protein to target a target DNA sequence (e.g., a target genomic DNA sequence). In some embodiments, a nucleic acid-programmable nuclease is sometimes referred to as a nuclease:RNA complex when it is in complex with RNA. Guide RNA can exist as a complex of two or more RNAs or as a single RNA molecule.

[0218] Guide RNAs (gRNAs) existing as a single RNA molecule are sometimes referred to as single guide RNAs (sgRNAs), but the term gRNA is also used to refer to guide RNAs existing as either a single molecule or a complex of two or more molecules. Typically, gRNAs existing as a single RNA species contain two domains: (1) a domain that shares homology with the target nucleic acid (i.e., directed to the binding of the Cas9 complex to its target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence known as tracrRNA and contains a stem-loop structure. In some embodiments, domain (2) is identical 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).

[0219] In some embodiments, the gRNA may include two or more domains (1) and (2) and may be referred to as an extended gRNA. For example, an extended gRNA may bind to two or more Cas9 proteins and to target nucleic acids in two or more distinct regions, as described herein. The gRNA contains a nucleotide sequence complementary to the target site that mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity for the nuclease:RNA complex.In this case, an RNA scaffold was used (CRIS PR vaccine) Cas9 strain Streptococcus pyogenes and Cas9(Csn1) and “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate [ PMC free article ] [ PubMed ] [ Cross Ref ] 5. Suvorov S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW., Roe BA., McLaughlin RE,Proc.Natl.Acad.Sci.USA98:4658-4663(2001);“CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski K., Sharma CM, Gonzales K., Chao Y., Pirzada ZA, Eckert MR, Vogel J., Charpentier E.,Nature 471:602-607(2011);および“A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.” Jinek M.,Chylinski K.,Fonfara I.,Hauer M.,Doudna JA,Charpentier E.Science 337:816-821(2012)(This skeletal muscle is specifically designed to be used as a solvent).

[0220] o.Share In some embodiments, the molecular payload may include a multimer (e.g., a concatemer) of two or more oligonucleotides linked by a linker. Thus, in some embodiments, the oligonucleotide loading of the complex / conjugate may be increased beyond the available linking sites on the targeting agent (e.g., available thiol sites on the antibody) or otherwise arranged to reach a specific payload load. The oligonucleotides in the multimer may be the same or different (e.g., targeting different genes, different sites on the same gene, or their products).

[0221] In some embodiments, the multimer comprises two or more oligonucleotides linked together by a cleavable linker. However, in some embodiments, the multimer comprises two or more oligonucleotides linked together by an incleavable linker. In some embodiments, the multimer comprises two, three, four, five, six, seven, eight, nine, ten, or more oligonucleotides linked together. In some embodiments, the multimer comprises two to five, two to ten, or four to twenty oligonucleotides linked together.

[0222] In some embodiments, a multimer comprises two or more oligonucleotides linked end-to-end (in a linear arrangement). In some embodiments, a multimer comprises two or more oligonucleotides linked end-to-end via an oligonucleotide-based linker (e.g., a poly-dT linker, a basic linker). In some embodiments, a multimer comprises the 5' end of one oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, a multimer comprises the 3' end of one oligonucleotide linked to the 3' end of another oligonucleotide. In some embodiments, a multimer comprises the 5' end of one oligonucleotide linked to the 5' end of another oligonucleotide. Furthermore, in some embodiments, a multimer may comprise a branched structure comprising multiple oligonucleotides linked together by a branched linker.

[0223] Further examples of multimers that may be used in the complexes provided herein include, for example, U.S. Patent Application No. 2015 / 0315588 A1, published on 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 on September 3, 2015, entitled "Multimeric Oligonucleotide Compounds"; U.S. Patent Application No. US 2011 / 0158937 A1, published on June 30, 2011, entitled "Immunostimulatory Oligonucleotide Multimers"; and Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers Targeting Nucleic Acids Comprising Mixed Sequences Of Purines And This is disclosed in U.S. Patent No. 5,693,773, titled Pyrimidines, the contents of which are incorporated herein by reference to the whole.

[0224] ii.Small molecules: Any suitable small molecule may be used as a molecular payload as described herein. In some embodiments, the small molecules are as described in U.S. Patent Application Publication 20170340606, published November 30, 2017, entitled "METHODS OF TREATING MUSCULAR DYSTROPHY," or as described in U.S. Patent Application Publication 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 transcription inhibitor such as SHC351, SHC540, or SHC572. In some embodiments, the small molecule is STR00316, which increases the production or activity of another protein such as an integrin. In some embodiments, the small molecule is a bromodomain inhibitor (BETi) such as JQ1, PF1-1, I-BET-762, I-BET-151, RVX-208, or CPI-0610.

[0225] iii. Peptides Any suitable peptide or protein may be used as a molecular payload as described herein. In some embodiments, the protein is an enzyme. These peptides or proteins may be produced, synthesized, and / or derivatized using several methodologies, e.g., phage-dispenseed peptide libraries, one-bead-one-compound peptide libraries, or site-scanning synthetic peptide combinatorial libraries. In some embodiments, the peptide or protein may inhibit DUX4 expression by binding to a DME1 or DME2 enhancer, e.g., by blocking activator binding.

[0226] iv. Nucleic acid constructs Any preferred 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, for example, as 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 poly(A) tail, optionally up to 160 nucleotides in length. In some embodiments, the gene expression construct may be expressed in the nucleus of a muscle cell, or, for example, overexpressed. In some embodiments, the gene expression construct encodes an oligonucleotide (e.g., shRNA-targeted DUX4) or a protein that downregulates DUX4 expression (e.g., a peptide or protein that inhibits DUX4 expression by binding to a DME1 or DME2 enhancer, e.g., by blocking activator binding). In some embodiments, the gene expression construct encodes an oligonucleotide (e.g., shRNA-targeted MuRF1 or MAFbx) that downregulates MuRF1 or MAFbx expression, respectively. In some embodiments, the gene expression construct encodes a protein containing at least one zinc finger. 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 the international patent application publication WO2017152149A1, titled "CLOSED-ENDED LINEAR DUPLEX DNA FOR NON-VIRAL GENE TRANSFER," published on 19 September 2017; U.S. Patent 8,853,377B2, titled "MRNA FOR USE IN TREATMENT OF HUMAN GENETIC DISEASES," issued on 7 October 2014; and U.S. Patent US8822663B2, "ENGINEERED NUCLEIC ACIDS AND METHODS OF USE THEREOF," issued on 2 September 2014, the contents of each of these are incorporated herein by reference in their entirety.

[0227] C. Linker The complexes 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, e.g., a disulfide bond or disulfide bridge, connecting the muscle targeting agent to the molecular payload. However, in some embodiments, the linker may connect the muscle targeting agent to the molecular payload through multiple covalent bonds. In some embodiments, the linker may be cleavable. However, in some embodiments, the linker may be incleavable. The linker is generally stable in vitro and in vivo, and may be stable in certain cellular environments. In addition, the linker generally 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, JR and Owen, SC “Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry” AAPS J. 2015, 17:2, 339-351.).

[0228] The linker precursor will typically contain two different highly reactive species capable of attaching to both the muscle targeting agent and the molecular payload. In some embodiments, the two different highly reactive species may be a nucleophile and / or an electrophile. In some embodiments, the linker is linked to the muscle targeting agent via conjugation to a lysine or cysteine ​​residue of the muscle targeting agent. In some embodiments, the linker is linked to a cysteine ​​residue of the muscle targeting agent via a maleimide-containing linker, where optionally the maleimide-containing linker contains a maleimide-caproyl or maleimide-methylcyclohexane-1-carboxylate group. In some embodiments, the linker is linked to a cysteine ​​residue or a thiol-functionalized molecular payload of the muscle targeting agent via a 3-arylpropionitrile functional group. In some embodiments, the linker is linked to the muscle targeting agent and / or the molecular payload via an amide bond, hydrazide, triazole, thioether, or disulfide bond.

[0229] i. Cuttable 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, outside the cells of muscle cells.

[0230] Protease-sensitive linkers are cleavable by protease enzyme activity. These linkers typically contain peptide sequences, which may be 2–10 amino acids, about 2–5 amino acids, about 5–10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length. In some embodiments, the peptide sequences may contain naturally occurring amino acids, e.g., cysteine, alanine, or amino acids that are not naturally occurring or are modified. Amino acids that are not naturally occurring 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 linkers contain valine-citrulline or alanine-citrulline dipeptide sequences. In some embodiments, the protease-sensitive linkers can be cleaved by lysosomal proteases, e.g., cathepsin B, and / or endosomal proteases.

[0231] A pH-sensitive linker is a covalent linkage that is readily degraded in a high or low pH environment. In some embodiments, the pH-sensitive linker may be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved within an endosome or lysosome.

[0232] In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with a glutathione species inside the cell. In some embodiments, the disulfide moiety further comprises at least one amino acid, for example, a cysteine ​​residue.

[0233] In some embodiments, the linker is a Val-cit linker (as described, for example, in U.S. Patent 6,214,345, incorporated herein by reference). In some embodiments, the pre-conjugation Val-cit linker has the following structure: [ka]

[0234] In some embodiments, the conjugated val-cit linker has the following structure: [ka]

[0235] ii. Non-cuttable linker In some embodiments, an uncleavable linker may be used. Generally, an uncleavable linker cannot be readily degraded in the cellular or physiological environment. In some embodiments, the uncleavable linker comprises an optionally substituted alkyl group, where substitution may include halogens, hydroxyl groups, oxygen species, and other common substitutions. In some embodiments, the linker may comprise an optionally substituted alkyl group, an optionally substituted alkylene, an optionally substituted arylene, a optionally substituted heteroarylene, a peptide sequence comprising at least one non-natural amino acid, a truncated glycan, an enzymatically undegradable sugar(s), an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, biotin, biphenyl, polyethylene glycol or equivalent compounds of repeating units, an acid ester, an acid amide, a sulfamide, and / or an alkoxy-amine linker. In some embodiments, sortase-mediated ligation may be used to ligate muscle targeting agents containing LPXT sequences to molecular payloads containing (G)n sequences (see, for example, Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1):1-10).

[0236] In some embodiments, the linker may include a substituted alkylene, optionally substituted alkenylene, optionally substituted alkylene, optionally substituted cycloalkylene, optionally substituted cycloalkenylene, optionally substituted arylene, optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; imino, optionally substituted nitrogen species, optionally substituted oxygen species O, optionally substituted sulfur species, or poly(alkylene oxide), for example, polyethylene oxide or polypropylene oxide.

[0237] iii. Linker conjugation In some embodiments, the linker is linked to the muscle targeting agent and / or molecular payload via a phosphate, thioether, ether, carbon-carbon, or amide bond. In some embodiments, the linker is linked to the oligonucleotide through a phosphate or phosphorothioate group, e.g., a phosphate at the terminal of the oligonucleotide backbone. In some embodiments, the linker is linked to the muscle targeting agent, e.g., an antibody, through a lysine residue or cysteine ​​residue present on the muscle targeting agent.

[0238] In some embodiments, the linker is linked to a muscle targeting agent and / or molecular payload by a cycloaddition reaction between an azide and an alkyne forming a triazole, where the azide and alkyne may be positioned on the muscle targeting agent, molecular payload, or linker. In some embodiments, the alkyne may be a cyclic alkyne, for example, cyclooctane. In some embodiments, the alkyne may be bicyclononine (also known as bicyclo[6.1.0]nonine or BCN) or a substituted bicyclononine. In some embodiments, cyclooctane is as described in the international patent application publication WO2011136645, published on November 3, 2011, titled "Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions". In some embodiments, the azide may be a sugar or carbohydrate molecule containing the azide. In some embodiments, the azide may be 6-azide-6-deoxygalactose or 6-azide-N-acetylgalactosamine. In some embodiments, sugar or carbohydrate molecules containing azides are as described in the international patent application publication WO2016170186, published on 27 October 2016, titled "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 on May 1, 2014, titled "Modified antibody, antibody - conjugate and process for the preparation thereof"; or International Patent Application Publication WO2016170186, published on 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".

[0239] In some embodiments, the linker further comprises a spacer, such as a polyethylene glycol spacer or an acyl / carbamoyl sulfamide spacer, such as a HydraSpace™ spacer. In some embodiments, the spacer is as described in Verkade, J.M.M. et al., “A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody - Drug Conjugates”, Antibodies, 2018, 7, 12.

[0240] In some embodiments, the linker is linked to the muscle targeting agent and / or molecular payload by a Diels-Alder reaction between the dienephile and the diene / heterodiene, where the dienephile and the diene / heterodiene may be located on the muscle targeting agent, molecular payload, or linker. In some embodiments, the linker is linked to the muscle targeting agent and / or molecular payload by other pericyclic reactions, e.g., ene reactions. In some embodiments, the linker is linked to the muscle targeting agent and / or molecular payload by amide, thioamide, or sulfonamide bonding reactions. In some embodiments, the linker is linked to the muscle targeting agent and / or molecular payload by a condensation reaction that forms an oxime group, hydrazone group, or semicarbazide group present between the linker and the muscle targeting agent and / or molecular payload.

[0241] 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.

[0242] D. Examples of Antibody-Molecular Payload Complexes Other aspects of this disclosure provide a complex comprising one of the muscle targeting agents described herein (e.g., an anti-transferrin receptor antibody) covalently linked to one of the molecular payloads described herein (e.g., an oligonucleotide). In some embodiments, the muscle targeting agent (e.g., an anti-transferrin receptor antibody) is covalently linked to the molecular payload (e.g., an oligonucleotide) via a linker. Any of the linkers described herein may be used. In some embodiments, the linker is linked to the 5' end, 3' end, or internally to the oligonucleotide. In some embodiments, the linker is linked to the antibody via thiol-reactive linkage (e.g., via cysteine ​​in the antibody).

[0243] An exemplary structure of a complex containing an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker is provided below: [ka] Here, the linker is linked to the 5' end, 3' end, or internally to the oligonucleotide, and here the linker is linked to the antibody via thiol-reactive linkage (e.g., via cysteine ​​in the antibody).

[0244] It should be understood that antibodies can be conjugated to oligonucleotides in various stoichiometric ways, and this property is sometimes referred to as the drug-antibody ratio (DAR), where "drug" is oligonucleotide. In some embodiments, one oligonucleotide is conjugated to the antibody (DAR=1). In some embodiments, two oligonucleotides are conjugated to the antibody (DAR=2). In some embodiments, three oligonucleotides are conjugated to the antibody (DAR=3). In some embodiments, four oligonucleotides are conjugated to the antibody (DAR=4). In some embodiments, a mixture of different complexes is provided, each having a different DAR. In some embodiments, the average DAR of the complexes in such a mixture may be in the range of 1-3, 1-4, or 1-5 or more. The DAR may be increased by conjugating oligonucleotides to various sites on the antibody and / or by conjugating multimers 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 dimeric oligonucleotide to a single site on the antibody.

[0245] In some embodiments, the complex described herein comprises an anti-transferrin receptor antibody (e.g., the antibody as described herein or any variant thereof) covalently linked to an oligonucleotide. In some embodiments, the complex described herein comprises an anti-transferrin receptor antibody (e.g., the antibody as described herein or any variant thereof) covalently linked to an oligonucleotide via a linker (e.g., a Val-cit linker). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the 5' end, 3' end, or internally to the oligonucleotide. In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., the antibody as described herein or any variant thereof) via thiol-reactive linkage (e.g., via cysteine ​​in the antibody).

[0246] In some embodiments, the complexes described herein include an anti-transferrin receptor antibody covalently linked to an oligonucleotide, wherein the anti-transferrin receptor antibody includes the same CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1; and the same CDR-L1, CDR-L2, and CDR-L3 shown in Table 1.1.

[0247] In some embodiments, the complex described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide, wherein the anti-transferrin receptor antibody comprises VH having the amino acid sequence of SEQ ID NO: 33 and VL having the amino acid sequence of SEQ ID NO: 34. In some embodiments, the complex described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide, wherein the anti-transferrin receptor antibody comprises VH having the amino acid sequence of SEQ ID NO: 35 and VL having the amino acid sequence of SEQ ID NO: 36.

[0248] In some embodiments, the complex 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 complex 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.

[0249] In some embodiments, the complexes described herein include 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 includes the same CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1; and the same CDR-L1, CDR-L2, and CDR-L3 shown in Table 1.1.

[0250] In some embodiments, the complex 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 VH having the amino acid sequence of SEQ ID NO: 33 and VL having the amino acid sequence of SEQ ID NO: 34. In some embodiments, the complex 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 VH having the amino acid sequence of SEQ ID NO: 35 and VL having the amino acid sequence of SEQ ID NO: 36.

[0251] In some embodiments, the complex 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 complex 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.

[0252] In some aspects, the complex described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the anti-transferrin receptor antibody comprises the same CDR-H1, CDR-H2, and CDR-H3 shown in Table 1.1; and the same CDR-L1, CDR-L2, and CDR-L3 shown in Table 1.1; wherein the complex comprises the following structure: [ka] Here, the Val-cit linker is ligated to the 5' end, 3' end, or internally to the oligonucleotide, and here the Val-cit linker is ligated to the antibody (e.g., the antibody as described herein or any variant thereof) via thiol-reactive linkage (e.g., via cysteine ​​in the antibody).

[0253] In some embodiments, the complex described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the anti-transferrin receptor antibody comprises VH having the amino acid sequence of SEQ ID NO: 33 and VL having the amino acid sequence of SEQ ID NO: 34, and wherein the complex comprises the following structure: [ka] Here, the Val-cit linker is ligated to the 5' end, 3' end, or internally to the oligonucleotide, and here the Val-cit linker is ligated to the antibody (e.g., the antibody as described herein or any variant thereof) via thiol-reactive linkage (e.g., via cysteine ​​in the antibody).

[0254] 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

[0255] 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

[0256] In some embodiments, the complex described herein comprises an anti-transferrin receptor antibody covalently linked to an oligonucleotide via a Val-cit linker, wherein the anti-transferrin receptor antibody comprises a heavy chain having the amino acid sequence of SEQ ID NO: 41 and a light chain having the amino acid sequence of SEQ ID NO: 42, and wherein the complex comprises the following structure: [ka] Here, the Val-cit linker is ligated to the 5' end, 3' end, or internally to the oligonucleotide, and here the Val-cit linker is ligated to the antibody (e.g., the antibody as described herein or any variant thereof) via thiol-reactive linkage (e.g., via cysteine ​​in the antibody).

[0257] Tour. Pharmaceuticals The complexes provided herein may be formulated in any preferred manner. Generally, the complexes provided herein are formulated in a manner suitable for pharmaceutical use. For example, the complex may be delivered to a target using a formulation that minimizes degradation and facilitates delivery and / or uptake, or that provides the complex in the formulation with other beneficial properties. In some embodiments, provided herein are compositions comprising the complex and a pharmaceutically acceptable carrier. Such compositions may be suitably formulated so that a sufficient amount of the complex can enter target muscle cells when administered either into the environment surrounding target cells of a subject or into the subject's system. In some embodiments, the complex may be formulated in a buffer solution such as phosphate-buffered saline, in liposomes, in micelle structures, and in capsids.

[0258] In some embodiments, it should be understood that the composition may individually encompass one or more components of the complex provided herein (e.g., a muscle targeting agent, a linker, a molecular payload, or a precursor molecule of any one thereof).

[0259] In some embodiments, the complex is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the complex is formulated in a basic buffered aqueous solution (e.g., PBS). In some embodiments, the formulation as disclosed herein includes an excipient. In some embodiments, the excipient imparts to the composition improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient. 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).

[0260] In some embodiments, the complex or its components (e.g., oligonucleotides or antibodies) are lyophilized to extend their shelf life and then dissolved before use (e.g., administration to a subject). Consequently, the excipients in compositions comprising the complex or its components as described herein may be cryoprotectants (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or decay temperature modifiers (e.g., dextran, ficol, or gelatin).

[0261] In some embodiments, pharmaceutical compositions are formulated to be compatible with their intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous, intradermal, and subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous.

[0262] Pharmaceutical compositions suitable for use in injections include sterile aqueous solutions (wherein soluble in water) or dispersions, and sterile powders for the immediate preparation of sterile injection solutions or dispersions. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. In some embodiments, the formulation may include isotonic agents in the composition, such as sugars, polyalcohols such as mannitol and sorbitol, and sodium chloride. Sterile injection solutions may be prepared by incorporating the required amount of the complex, along with one or a combination thereof of the components listed above, into a selected solvent, and subsequently by filtration sterilization, as required.

[0263] In some embodiments, the composition may contain a percentage of the active ingredient(s) (one or more) between approximately 1% and approximately 80% or more of the total weight or volume of the composition, but may also contain at least approximately 0.1% of the complex or its components. Factors such as solubility, bioavailability, biological half-life, route of administration, and product shelf life, as well as other pharmacological considerations, will be considered by those skilled in the art preparing such pharmaceutical formulations. Therefore, various dosages and treatment plans may be desired.

[0264] IV. Method of Use / Procedure A complex containing a muscle targeting agent covalently bound to a molecular payload as described herein is effective in treating FSHD. In some embodiments, the complex is effective in treating type I FSHD. In some embodiments, the complex is effective in treating type II FSHD. In some embodiments, FSHD is associated with a deletion in the D4Z4 repeat region on chromosome 4 containing the DUX4 gene. In some embodiments, FSHD is associated with a mutation in the SMCHD1 gene.

[0265] In some embodiments, the subject may be a human subject, a non-human primate subject, a rodent subject, or any preferred mammalian subject. In some embodiments, the subject may have myotonic dystrophy. In some embodiments, the subject may have elevated expression of the DUX4 gene, excluding fetal development and testes. In some embodiments, the subject may have type I or type II facioscapulohumeral muscular dystrophy. In some embodiments, the subject with FSHD has a mutation in the SMCHD1 gene. In some embodiments, the subject with FSHD has a deletion mutation in the D4Z4 repeat region on chromosome 4.

[0266] Aspects of this disclosure include methods involving the administration of an effective amount of the complex as described herein to a subject. In some embodiments, an effective amount of a pharmaceutical composition comprising a complex containing a muscle targeting agent covalently bound to a molecular payload can be administered to a subject in need of treatment. In some embodiments, the pharmaceutical composition comprising the complex as described herein may be administered by a preferred route, which may include intravenous administration, for example, as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration may be carried out by an intramuscular, intraperitoneal, intracerebral, subcutaneous, intraarticular, intrabursal, 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 sprayed or lyophilized form may be reconstituted with an aqueous or liquid solution.

[0267] Compositions for intravenous administration may contain various carriers such as vegetable oil, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). Water-soluble antibodies for intravenous injection may be administered by drip infusion, thereby injecting a pharmaceutical preparation containing the antibody and pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Preparations for intramuscular use, such as sterile preparations in a suitable soluble salt form of the antibody, may be administered dissolved in pharmaceutical excipients such as water for injection, 0.9% saline, or 5% glucose solution.

[0268] In some embodiments, pharmaceutical compositions comprising a complex containing a muscle-targeting agent covalently bound to a molecular payload are administered via site-specific or local delivery techniques. Examples of these techniques include implantable depot sources of the complex, local delivery catheters, site-specific carriers, direct injection, or direct application.

[0269] In some embodiments, a pharmaceutical composition comprising a complex containing a muscle-targeting agent covalently bound to a molecular payload is administered at an effective concentration that imparts a therapeutic effect to the target. The effective dose varies, as is recognized by those skilled in the art, depending on the severity of the disease, the specific characteristics of the target being treated, e.g., age, physical condition, health status, or weight, treatment time, the nature of any concomitant therapy, the route of administration, and related factors. These related factors are known to those skilled in the art and can be addressed with a few standard 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 minimum feasible concentration that provides maximum efficacy.

[0270] Empirically, for example, the half-life of the complex in the subject will generally contribute to determining the concentration of the pharmaceutical composition used for treatment. The frequency of administration may be determined and adjusted empirically to maximize the efficacy of the treatment.

[0271] In general, for administration of any of the complexes described herein, the initial candidate dose may be approximately 1 to 100 mg / kg or more, depending on the factors described above, e.g., safety or efficacy. 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 maximum efficacy to the subject while minimizing the risk to safety. In general, efficacy and risks to the treatment and safety may be monitored throughout the course of treatment.

[0272] The effectiveness of the treatment may be assessed using any preferred method. In some embodiments, the effectiveness of the treatment may be assessed by evaluating findings of symptoms associated with FSHD, including muscle loss and muscular atrophy, primarily in the muscles of the shoulder, scapula, and upper arm.

[0273] In some embodiments, a pharmaceutical composition comprising a conjugate containing 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., baseline level of gene expression prior to treatment).

[0274] In some embodiments, a single dose or administration to a subject of a pharmaceutical composition comprising a complex containing a muscle targeting agent covalently bound to a molecular payload as described herein is sufficient to inhibit the activity or expression of a target gene for at least 1 to 5 days, 1 to 10 days, 5 to 15 days, 10 to 20 days, 15 to 30 days, 20 to 40 days, 25 to 50 days, or longer. In some embodiments, a single dose or administration to a subject of a pharmaceutical composition comprising a complex containing a muscle targeting agent covalently bound to a molecular payload as described herein 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 to a subject of a pharmaceutical composition comprising a complex containing a muscle targeting agent covalently bound to a molecular payload as described herein is sufficient to inhibit the activity or expression of a target gene for at least 1, 2, 3, 4, 5, or 6 months.

[0275] In some embodiments, the pharmaceutical composition may comprise one or more complexes containing a muscle targeting agent covalently bonded to a molecular payload. In some embodiments, the pharmaceutical composition may further comprise any other suitable therapeutic agent for the treatment of a subject, for example, a human subject having FSHD. In some embodiments, the other therapeutic agent may enhance or complement the efficacy of the complex described herein. In some embodiments, the other therapeutic agent may function to treat a different symptom or disease than that of the complex described herein.

[0276] example Example 1: Targeting gene expression with transfected antisense oligonucleotides We in vitro tested the ability of hypoxanthine phosphoribosyltransferase (HPRT)-targeting siRNAs to reduce HPRT expression levels in immortalized cell lines. In short, Hepa1-6 cells were transfected with either a control siRNA formulated with lipofectamine 2000 (siCTRL; 100nM) or an HPRT-targeting siRNA (siHPRT; 100nM). HPRT expression levels were evaluated 48 hours after transfection. A control experiment was also conducted. In the control experiment, a vehicle (phosphate-buffered saline) was delivered to cultured Hepa1-6 cells, maintaining the cells for 48 hours. As shown in Figure 1, HPRT siRNA reduced HPRT expression levels by approximately 90% compared to the control. Table 2. Sequences of siHPRT and siCTRL [Table 3]

[0277] Example 2: Targeting of HPRT in muscle targeting complexes A muscle targeting complex containing the HPRT siRNA used in Example 1 (siHPRT), which was covalently linked to the anti-transferrin receptor antibody DTX-A-002 via an incleavable N-gamma-maleimidobutyryl-oxysuccinimide (GMBS) linker, was generated.

[0278] In short, the GMBS linker was dissolved in dry DMSO and coupled to the 3' end of the siHPRT sense chain via amide bond formation under aqueous conditions. Completion of the reaction was verified by the Kaiser test. Excess linker and organic solvent were removed by gel permeation chromatography. The purified maleimide-functionalized sense chain of siHPRT was then coupled to the DTX-A-002 antibody using the Michael addition reaction.

[0279] Next, the antibody coupling reaction product was subjected to hydrophobic interaction chromatography (HIC-HPLC). The anti-TfR-siHPRT complex, containing one or two siHPRT molecules covalently attached to the DTX-A-002 antibody, was purified. Concentration measurements confirmed that the purified complex sample had an average siHPRT antibody ratio of 1.46. SDS-PAGE analysis demonstrated that >90% of the purified complex sample contained DTX-A-002 linked to one or two siHPRT molecules.

[0280] Using the same method described above, a control IgG2a-siHPRT complex containing the HPRT siRNA used in Example 1 (siHPRT), covalently linked to the IgG2a(Fab) antibody (DTX-A-003) via a GMBS linker, was generated. Concentration measurements confirmed that DTX-C-001 had an average siHPRT antibody ratio of 1.46, and SDS-PAGE demonstrated that >90% of the purified control complex samples contained DTX-A-003 linked to one or two siHPRT molecules.

[0281] Next, the anti-TfR-siHPRT complex was tested for cellular internalization and in-cellulo inhibition of HPRT. Hepa1-6 cells with relatively high levels of transferrin receptor expression were incubated for 72 hours in the presence of a vehicle (phosphate-buffered saline), IgG2a-siHPRT (100 nM), anti-TfR-siCTRL (100 nM), or anti-TfR-siHPRT (100 nM). After 72 hours of incubation, cells were isolated and assayed for HPRT expression levels (Figure 2). Cells treated with anti-TfR-siHPRT showed a ~50% reduction in HPRT expression compared to cells treated with the vehicle control. On the other hand, cells treated with either IgG2a-siHPRT or anti-TfR-siCTRL had HPRT expression levels comparable to the vehicle control (no reduction in HPRT expression). These data suggest that anti-transferrin receptor antibodies against TfR-siHPRT enable the cellular internalization of the complex, thereby allowing siHPRT to inhibit HPRT expression.

[0282] Example 3: Targeting of HPRT in mouse muscle tissue using a muscle targeting complex. The muscle-targeting complex and anti-TfR-siHPRT described in Example 2 were tested for inhibition of HPRT in mouse tissues. C57BL / 6 wild-type mice were intravenously injected with a single dose of vehicle control (phosphate-buffered saline); siHPRT (2 mg / kg RNA); IgG2a-siHPRT (2 mg / kg RNA, equivalent to 9 mg / kg antibody complex); or anti-TfR-siHPRT (2 mg / kg RNA, equivalent to 9 mg / kg antibody complex). Each experimental condition was reproduced in four individual C57BL / 6 wild-type mice. Three days after injection, the mice were euthanized and subdivided to isolate tissue types. Subsequently, individual tissue samples were assayed for HPRT expression levels (Figures 3A-3B and 4A-4E).

[0283] Mice treated with the anti-TfR-siHPRT complex demonstrated reduced HPRT expression in the gastrocnemius muscle (31% reduction; p<0.05) and heart (30% reduction; p<0.05) compared to mice treated with the siHPRT control (Figures 3A-3B). On the other hand, mice treated with the IgG2a-siHPRT complex had HPRT expression levels comparable to the siHPRT control (little to no reduction in HPRT expression) in all muscle tissue types assayed.

[0284] Mice treated with the anti-TfR-siHPRT complex demonstrated no alteration in HPRT expression in non-muscle tissues such as the brain, liver, lungs, kidneys, and spleen (Figures 4A-4E).

[0285] These data suggest that an anti-transferrin receptor antibody against the anti-TfR-siHPRT complex enables the intracellular location of the complex into muscle-specific tissue in an in vivo mouse model, thereby allowing siHPRT to inhibit HPRT expression. These data further demonstrate that the anti-TfR oligonucleotide complex of this disclosure can specifically target muscle tissue.

[0286] Example 4: Targeting DUX4 with transfected antisense oligonucleotides Three cell lines expressing DUX4 (A549, U-2 OS, and HepG2 cell lines) and immortalized skeletal muscle myoblasts (SkMCs) were screened for DUX4 mRNA expression (Figure 5). Cells were seeded at a density of 10,000 cells / well, and total RNA was recovered. cDNA was synthesized from the total RNA extract, and the concentration of DUX4 was determined by technical quadruplicate compared to the control gene (PPIB) using qPCR. These data, useful for selecting the U-2 OS cell line, were used for downstream development of DUX4-targeted oligonucleotides.

[0287] Following the selection of U-2 OS cells for the development of DUX4-targeted oligonucleotides, a phosphorodiamidate morpholino oligomer (PMO) version of an antisense oligonucleotide targeting DUX4 (FM10 PMO) was evaluated for its in vitro DUX4 targeting ability. FM10 PMO contains the sequence GGGCATTTTAATATATCTCTGAACT (SEQ ID NO: 45). A control phosphorodiamidate morpholino oligomer (PMO) containing the sequence CCTCTTACCTCAGTTACAATTTATA was used as a negative control.

[0288] In short, U-2 OS cells were seeded at a density of 10k cells / well before being allowed to recover overnight. The cells were then treated with either control PMO (100nM) or FM10 PMO (10μM). The cells were incubated for 72 hours before total RNA was harvested. cDNA was then synthesized from the total RNA extract, and qPCR was performed to determine the expression of downstream DUX4 genes (ZSCAN1, MBDL3L2, TRIM43) in technical quadruple derivation. All qPCR data were analyzed using a standard ΔΔCT method and normalized to a negative control based on a plate containing untreated cells (i.e., none of the oligonucleotides). The results were then converted to magnification changes to assess efficacy.

[0289] As shown in Figure 6, ZSCAN1, MBDL3L2, and TRIM43 all showed decreased expression in the presence of FM10 PMO compared to the control PMO (42%, 34%, and 32%, respectively). These data demonstrate that FM10 PMO can target DUX4 in vitro.

[0290] Example 5: Targeting of DUX4 in muscle targeting complexes A muscle targeting complex was generated containing an antisense oligonucleotide targeting a mutant allele of DUX4 (DUX4 ASO), which was covalently linked to the anti-transferrin receptor antibody DTX-A-002 (RI7 217(Fab)) via a cathepsin-cleavable linker.

[0291] In short, the maleimidocaproyl-L-valine-L-citrulline-p-aminobenzyl alcohol p-nitrophenyl carbonate (MC-Val-Cit-PABC-PNP) linker molecule is coupled to NH2-C6-DUX4 ASO using an amide coupling reaction. Excess linker and organic solvent are removed by gel permeation chromatography. The purified Val-Cit-linker-DUX4 ASO is then coupled to a highly thiol-reactive anti-transferrin receptor antibody (DTX-A-002).

[0292] Next, the product of the antibody coupling reaction is subjected to hydrophobic interaction chromatography (HIC-HPLC) to purify the muscle-targeting complex. Concentration measurement and SDS-PAGE analysis of the purified complex determine the average ratio of ASO to antibody and the total purity, respectively.

[0293] Using the same method as described above, a control complex containing DUX4 ASO covalently linked to the IgG2a(Fab) antibody via a Val-Cit linker is generated.

[0294] Next, the purified muscle targeting complex containing DTX-A-002 covalently linked to DUX4 ASO is tested for cellular internalization and DUX4 inhibition. Disease-related muscle cells with relatively high levels of transferrin receptor expression are incubated for 72 hours in the presence of a vehicle control (saline), the muscle targeting complex (100 nM), or the control complex (100 nM). After 72 hours of incubation, the cells are isolated and the DUX4 expression level is assayed.

[0295] Equivalents and technical terms The disclosures described herein as preferably described herein can be practiced even without any elements (singular or plural) or limitations (singular or plural) that are not specifically disclosed herein. For example, in each case herein, any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with any of the other two terms. The terms and expressions adopted are used as descriptive terms, but not as limiting terms, and there is no intention to use such terms and expressions to exclude any equivalent of the features or parts thereof shown and described, although it is recognized that various modifications are possible within the scope of the disclosure. Therefore, although the disclosures are specifically disclosed in preferred embodiments, it should be understood that any modifications and variations of any features, concepts disclosed herein may be reclassified by those skilled in the art, and that such modifications and variations are considered to be within the scope of the disclosure.

[0296] In addition, if any feature or aspect of this disclosure is described in terms of the Markush group or other alternative groups, a person skilled in the art will recognize that this disclosure is also described in terms of any individual member or subgroup of the Markush group or other groups.

[0297] In some embodiments, it should be understood that sequences presented in a sequence listing may be referred to when describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., an RNA equivalent of a DNA nucleotide or a DNA equivalent of an RNA nucleotide), and / or one or more modified nucleotides, and / or one or more modified nucleotide linkages, and / or one or more other modifications, while retaining essentially the same or similar complementary properties as a particular sequence.

[0298] The use of the terms “a,” “an,” and “the,” as well as similar referents, in the context of describing the invention (particularly in the context of the following claims), should be interpreted to cover both singular and plural forms, unless otherwise indicated herein or unless explicitly contradicted by the context. The terms “including,” “having,” “including,” and “containing,” should be interpreted as open-ended terms (i.e., “including, but not limited to,”) unless otherwise noted herein. The enumeration of value ranges herein is merely intended to serve as a simple way to individually refer to each separate value that falls within the range, unless otherwise indicated herein, and each separate value is incorporated herein even if it is individually enumerated herein. All methods described herein may be carried out in any preferred order, unless otherwise indicated herein or unless explicitly contradicted by the context. The use of any example or illustrative language provided herein (for example, "such as") is intended solely to better illustrate the invention and does not limit the scope of the invention unless otherwise asserted. The language herein should be construed as not indicating any element not asserted as essential to the practice of the invention.

[0299] Aspects of the present invention are described herein. Variations of these aspects may become apparent to those skilled in the art after reading the above description.

[0300] The inventors expect those skilled in the art to adopt such variations as appropriate, and they also intend that the invention may be carried out in ways different from those specifically described herein. Consequently, the invention encompasses all modifications and equivalents of the subject matter described in the claims appended herein, as permitted by applicable law. Furthermore, any combination of the above elements is covered by the invention in all their viable variations, unless otherwise indicated herein or unless explicitly contradicted by context. Those skilled in the art will be able to recognize or verify many equivalents to specific aspects of the invention described herein using a few standard experimental methods. Such equivalents are intended to be covered by the following claims.

Claims

1. A complex comprising an anti-transferrin receptor antibody covalently linked to an oligonucleotide targeting DUX4, The oligonucleotide is 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length and contains a region complementary to the DUX4 sequence represented by Genbank accession number NM_001306068.2, the complementary region being at least 12 nucleotides in length; Oligonucleotides mediate the degradation of DUX4 RNA; The oligonucleotide is covalently linked to the anti-transferrin receptor antibody via a cleavable linker, the cleavable linker containing a valine-citrulline sequence; and The anti-transferrin receptor antibody binds to the C89-F760 region of human transferrin receptor protein 1 (TfR1), which has the amino acid sequence represented by SEQ ID NO: 1, and the anti-transferrin receptor antibody does not specifically bind to the transferrin binding site of TfR1; The aforementioned composite.

2. Anti-transferrin receptor antibodies: (a) ScFv, Fab fragment, Fab' fragment, F(ab') ２ It is in the form of a fragment, or Fv fragment; (b) 10 －１１ M-10 －６ M's K Ｄ Binds to human TfR1; and / or (c) containing humanized antibodies; The composite according to claim 1.

3. The complex according to claim 1 or 2, wherein the oligonucleotide comprises a chain complementary to the 5' or 3' UTR sequence of DUX4.

4. The complex according to any one of claims 1 to 3, wherein the oligonucleotide comprises one or more modified nucleosides, wherein optionally, one or more modified nucleosides are one or more 2'-modified nucleosides selected from the group consisting of 2'-O-methyl, 2'-fluoro, 2'-O-methoxyethyl, and 2',4'-bridged nucleosides.

5. The complex according to any one of claims 1 to 4, wherein the oligonucleotide comprises one or more modified nucleoside linkages, and optionally wherein the oligonucleotide comprises one or more phosphorothioate linkages.

6. The complex according to any one of claims 1 to 5, wherein the oligonucleotide comprises a phosphorodiamidite tomorpholino oligomer (PMO).

7. Oligonucleotides: (a) comprising a region complementary to at least 15 consecutive nucleotides of Sequence ID No. 46; or (b) comprising at least 15 consecutive nucleotides of sequence number 45; The composite according to any one of claims 1 to 6.

8. The complex according to any one of claims 1 to 7, wherein an anti-transferrin receptor antibody is covalently linked to an oligonucleotide via conjugation to a lysine residue or cysteine ​​residue of the anti-transferrin receptor antibody.

9. The complex according to any one of claims 1 to 8, wherein the complex is configured to promote the internalization of oligonucleotides into muscle cells mediated by transferrin receptors.

10. The complex according to any one of claims 1 to 9, wherein the oligonucleotide results in a reduction in the expression level of DUX4 in muscle cells.

11. The complex according to any one of claims 1 to 10, wherein the oligonucleotide comprises or consists of an antisense oligonucleotide.

12. The complex according to any one of claims 1 to 10, wherein the oligonucleotide comprises siRNA.

13. The complex according to claim 12, wherein the siRNA comprises one or more 2'-modified nucleosides selected from the group consisting of 2'-O-methyl and 2'-fluoro.

14. The complex according to claim 12 or 13, wherein the siRNA comprises a sense strand and an antisense strand.

15. The composite according to claim 14, wherein the sense chain and the antisense chain are of different lengths.

16. A complex according to any one of claims 1 to 15 for use in a method of treating a disease or illness that can be improved or prevented by reducing the expression of DUX4 in muscle cells, wherein the method comprises bringing muscle cells into contact with the complex.

17. A complex according to any one of claims 1 to 15 for use in a method for treating facultivic, scapulohumeral, or brachial muscular dystrophy (FSHD) in subjects having one or more deletions of the D4Z4 repeat in chromosome 4, wherein the method comprises administering an effective amount of the complex to the subject.

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