Muscle targeting complexes and their use for treating myotonic dystrophy - Patent Application 20070123633

Abstract

This application relates to oligonucleotides (e.g., antisense oligonucleotides, e.g., gapmers) designed to target DMPK RNA, and targeting complexes for delivering oligonucleotides to cells (e.g., muscle cells), and their uses, particularly for the treatment of diseases. 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 expression or activity of DMPK.

Description

[Technical field]

[0001] Related Applications This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63 / 220000, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY," filed on July 9, 2021, and U.S. Provisional Patent Application No. 63 / 316905, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY," filed on March 4, 2022, the entire contents of each of which are incorporated herein by reference.

[0002] FIELD OF THEINVENTION This application relates to oligonucleotides designed to target DMPK RNA and targeting complexes for delivering oligonucleotides to cells (eg, muscle cells) and their uses, particularly for the treatment of disease.

[0003] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (D082470054WO00-SEQ-COB.xml; size: 574,699 bytes; and creation date July 7, 2022) are incorporated herein by reference in their entirety. [Background technology]

[0004] 2. Background of the Invention Myotonic dystrophy (DM) is a dominantly inherited genetic disease characterized by muscle tonia, muscle loss or degeneration, muscle hypofunction, insulin resistance, cardiac arrhythmias, smooth muscle dysfunction, and neurological abnormalities. DM is the most common form of adult-onset muscular dystrophy, with an incidence of approximately 1 in 8000 people worldwide. Two types of the disease have been described: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1, the more common form of the disease, results from a repeat expansion of CTG trinucleotide repeats within the 3' noncoding region of DMPK on chromosome 19, while DM2 results from a repeat expansion of CCTG tetranucleotide repeats in the first intron of ZNF9 on chromosome 3. In DM1 patients, repeat expansions of CTG trinucleotide repeats, which may contain more than about 50 to more than about 3,000 total repeats, result in the generation of toxic RNA repeats that can form hairpin structures that bind with high affinity to essential intracellular proteins, such as muscleblind-like proteins, resulting in protein sequestration and the loss-of-function phenotype characteristic of the disease. Other than supportive care and treatments to address the symptoms of the disease, no effective therapeutic agents for DM1 are currently available. Summary of the Invention

[0005] Summary of the Invention In some aspects, the disclosure provides oligonucleotides designed to target DMPK RNA. In some embodiments, the disclosure provides oligonucleotides complementary to DMPK RNA, useful for reducing the level of toxic DMPK with disease-associated repeat expansion, for example, in subjects with or suspected of having myotonic dystrophy. In some embodiments, the oligonucleotides are designed to direct RNAse H-mediated degradation of the target DMPK RNA. In some embodiments, the oligonucleotides are designed to direct RNAse H-mediated degradation of the target DMPK RNA present in the nucleus of a cell, for example, a muscle cell (e.g., a myotube), or a cell of the nervous system (e.g., a central nervous system (CNS) cell). In some embodiments, the oligonucleotides are designed to have desirable bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desirable binding affinity properties. In some embodiments, the oligonucleotides are designed to have desirable toxicity profiles. In some embodiments, the oligonucleotides are designed to have low complement activation and / or cytokine induction properties.

[0006] In some embodiments, the oligonucleotides provided herein are designed to facilitate conjugation with other molecules, e.g., targeting agents, e.g., muscle targeting agents. Thus, in some aspects, the present disclosure provides complexes that target specific cell types to deliver oligonucleotides to those cells. By way of example, in some embodiments, the present disclosure provides complexes that target muscle cells to deliver oligonucleotides to those cells. In some embodiments, the complexes provided herein are particularly useful for delivering molecular payloads that inhibit the expression or activity of DMPK alleles that contain expanded disease-associated repeats, e.g., to subjects with or suspected of having myotonic dystrophy. Thus, in some embodiments, the complexes provided herein include a muscle targeting agent (e.g., a muscle targeting antibody) that specifically binds to a receptor on the surface of a muscle cell to deliver the molecular payload to the muscle cell. In some embodiments, the complex is taken up into the cell via receptor-mediated internalization, after which the molecular payload may be released to perform a function within the cell. By way of example, a complex engineered to deliver an oligonucleotide may release the oligonucleotide so that the oligonucleotide can inhibit mutant DMPK expression within the muscle cell. In some embodiments, the oligonucleotide is released by endosomal cleavage of a covalent linker that connects the oligonucleotide and the muscle targeting agent of the complex. It is understood that the oligonucleotides and / or complexes provided herein can be useful in multiple tissues and cell types, for example, in muscle tissue (e.g., muscle cells) and in the central nervous system (e.g., CNS cells, e.g., neurons).

[0007] Some aspects of the disclosure provide oligonucleotides that target DMPK RNA.

[0008] According to some aspects, a conjugate is provided that includes an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured to reduce expression or activity of DMPK, wherein the anti-TfR1 antibody includes heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3) of any of the anti-TfR1 antibodies listed in Tables 2-7; and the oligonucleotide comprises a 5'-XYZ-3' configuration, X comprises 3 to 7 linked nucleosides, and at least one of the nucleosides within X is a 2'-modified nucleoside; Y comprises 6 to 15 linked 2'-deoxyribonucleosides, and each cytosine within Y is optionally and independently a 5-methyl-cytosine; and Z comprises 3 to 7 linked nucleosides, and at least one of the nucleosides in Z is a 2'-modified nucleoside; and The oligonucleotide comprises a region of complementarity to at least 15 consecutive nucleosides of any one of SEQ ID NOs: 205, 214, 222, 217, 211, 215, 220, 225, 160-204, 206-210, 212, 213, 216, 218, 219, 221, 223, 224, and 226-230.

[0009] In some embodiments, X comprises 3-5 linked nucleosides, and at least one of the nucleosides within X is a 2'-modified nucleoside; Y comprises 6 to 10 linked 2'-deoxyribonucleosides, and each cytosine within Y is optionally and independently a 5-methyl-cytosine; and Z comprises 3 to 5 linked nucleosides, and at least one of the nucleosides in Z is a 2'-modified nucleoside.

[0010] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76, and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75; Optionally, the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0011] In some embodiments, the anti-TfR1 antibody is a Fab, wherein the Fab comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101, and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90; Optionally, the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

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

[0013] In some embodiments, the oligonucleotides are 15-25 nucleosides in length, and optionally, the oligonucleotides are 15-20 nucleosides in length.

[0014] In some embodiments, the oligonucleotide comprises at least 15 contiguous nucleosides of any one of SEQ ID NOs: 276, 348, 354, 350, 345, 286, 352, 357, 231-275, 277-285, 287-344, 346, 347, 349, 351, 353, 355, 356, and 358-362, wherein each thymine base (T) is independently and optionally replaced by a uracil base (U), and each U is independently and optionally replaced by a T.

[0015] In some embodiments, each nucleoside within X is a 2'-modified nucleoside and / or each nucleoside within Z is a 2'-modified nucleoside, and optionally, each 2'-modified nucleoside is independently a 2'-4' bicyclic nucleoside or a non-bicyclic 2'-modified nucleoside.

[0016] In some embodiments, the oligonucleotide comprises the following 5'-XYZ-3' configuration:

number

[0017] In some embodiments, the oligonucleotide comprises one or more phosphorothioate internucleoside linkages.

[0018] In some embodiments, each internucleoside linkage within the oligonucleotide is a phosphorothioate internucleoside linkage.

[0019] In some embodiments, the oligonucleotide comprises one or more phosphodiester internucleoside linkages, optionally at X and / or Z.

[0020] In some embodiments, the oligonucleotide comprises a structure selected from the following:

number

number

[0021] In some embodiments, the oligonucleotide is conjugated to an amine group at its 5'-end and comprises a structure selected from the following:

number

number

number

[0022] In some embodiments, the oligonucleotide comprises a structure selected from the following:

number

number

[0023] In some embodiments, the oligonucleotide is conjugated to an amine group at its 5'-end and comprises a structure selected from the following:

number

number

[0024] According to some aspects, provided herein is a method for reducing DMPK expression in muscle cells. In some embodiments, the method comprises contacting muscle cells with an effective amount of the complex disclosed herein to reduce DMPK expression in muscle cells.

[0025] In some embodiments, decreasing DMPK expression in the muscle cell comprises decreasing the amount of DMPK RNA in the muscle cell, optionally, the amount of DMPK RNA is decreased in the nucleus of the muscle cell, and optionally, the DMPK RNA is mutant DMPK mRNA.

[0026] In some embodiments, decreasing DMPK expression in the muscle cell comprises decreasing the amount of DMPK protein in the muscle cell.

[0027] According to some aspects, provided herein is a method for treating myotonic dystrophy type 1 (DM1).In some embodiments, the method comprises administering to a subject in need thereof an effective amount of the conjugate disclosed herein.

[0028] In some embodiments, the administration results in at least a 30% reduction in DMPK RNA in muscle cells of the subject, and optionally, the DMPK RNA is DMPK mRNA.

[0029] In some embodiments, the administration results in a decrease in DMPK RNA in the nuclei of the subject's muscle cells, and optionally, the DMPK RNA is DMPK mRNA.

[0030] According to some aspects, provided herein is an oligonucleotide. In some embodiments, the oligonucleotide comprises a structure selected from the following:

number

number

number

[0031] In some embodiments, the oligonucleotide is conjugated to an amine group at its 5'-end and comprises a structure selected from the following:

number

number

[0032] In some embodiments, the oligonucleotide comprises a structure selected from the following:

number

number

number

[0033] In some embodiments, the oligonucleotide is conjugated to an amine group at its 5'-end and comprises a structure selected from the following:

number

number

[0034] According to some aspects, provided herein is a composition comprising an oligonucleotide. In some embodiments, the composition comprises an oligonucleotide disclosed herein in sodium salt form. [Brief description of the drawings]

[0035] [Figure 1A-1D]1A-1H show that a conjugate having an anti-TfR1 Fab conjugated to a DMPK-targeting oligonucleotide delivered the oligonucleotide to various muscle tissues and reduced mouse Dmpk expression in a mouse model expressing human TfR1. The DMPK-targeting oligonucleotide was conjugated to anti-TfR1 Fab 3M12-VH4 / Vk3. FIG. 1A shows that the conjugate reduced mouse wild-type Dmpk in the tibialis anterior muscle by 79%. FIG. 1B shows that the conjugate reduced mouse wild-type Dmpk in the gastrocnemius muscle by 76%. FIG. 1C shows that the conjugate reduced mouse wild-type Dmpk in the heart by 70%. FIG. 1D shows that the conjugate reduced mouse wild-type Dmpk in the diaphragm by 88%. Figures 1E-H show oligonucleotide distribution in the tibialis anterior muscle (Figure 1E), gastrocnemius muscle (Figure 1F), heart (Figure 1G) and diaphragm (Figure 1H). [Fig. 1E-1H] 1A-1H show that a conjugate having an anti-TfR1 Fab conjugated to a DMPK-targeting oligonucleotide delivered the oligonucleotide to various muscle tissues and reduced mouse Dmpk expression in a mouse model expressing human TfR1. The DMPK-targeting oligonucleotide was conjugated to anti-TfR1 Fab 3M12-VH4 / Vk3. FIG. 1A shows that the conjugate reduced mouse wild-type Dmpk in the tibialis anterior muscle by 79%. FIG. 1B shows that the conjugate reduced mouse wild-type Dmpk in the gastrocnemius muscle by 76%. FIG. 1C shows that the conjugate reduced mouse wild-type Dmpk in the heart by 70%. FIG. 1D shows that the conjugate reduced mouse wild-type Dmpk in the diaphragm by 88%. Figures 1E-H show oligonucleotide distribution in the tibialis anterior muscle (Figure 1E), gastrocnemius muscle (Figure 1F), heart (Figure 1G) and diaphragm (Figure 1H).

[0036] [Figure 2A-2B]Figures 2A-D show toxic human DMPK knockdown in heart (Figure 2A), diaphragm (Figure 2B), gastrocnemius (Figure 2C) and tibialis anterior (Figure 2D) muscle tissues of hTfR1 / DMSXL mice following treatment with vehicle control or DMPK-targeting ASOs (ASO58, ASO47, ASO61 or ASO66) conjugated to anti-TfR1 Fab 3M12-VH4 / Vκ3 (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 as analyzed by one-way ANOVA). [Fig. 2C-2D] Figures 2A-D show toxic human DMPK knockdown in heart (Figure 2A), diaphragm (Figure 2B), gastrocnemius (Figure 2C) and tibialis anterior (Figure 2D) muscle tissues of hTfR1 / DMSXL mice following treatment with vehicle control or DMPK-targeting ASOs (ASO58, ASO47, ASO61 or ASO66) conjugated to anti-TfR1 Fab 3M12-VH4 / Vκ3 (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001 as analyzed by one-way ANOVA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] Detailed Description of the Invention Some aspects of the disclosure provide oligonucleotides designed to target DMPK RNA. In some embodiments, the disclosure provides oligonucleotides complementary to DMPK RNA, useful for reducing the level of toxic DMPK with disease-associated repeat expansion, for example, in subjects with or suspected of having myotonic dystrophy. In some embodiments, the oligonucleotides are designed to direct RNAse H-mediated degradation of the target DMPK RNA. In some embodiments, the oligonucleotides are designed to direct RNAse H-mediated degradation of the target DMPK RNA present in the nucleus of a cell, for example, a muscle cell (e.g., myotube) or a central nervous system (CNS) cell. In some embodiments, the oligonucleotides are designed to have desirable bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are designed to have desirable binding affinity properties. In some embodiments, the oligonucleotides are designed to have desirable toxicity profiles. In some embodiments, the oligonucleotides are designed to have low complement activation and / or cytokine induction properties.

[0038] In some aspects, the present disclosure provides a complex comprising a muscle targeting agent covalently linked to the DMPK targeting oligonucleotide described herein for effective delivery of oligonucleotide to muscle cells. In some embodiments, a complex is provided for targeting a DMPK allele comprising an expanded disease-associated repeat for treating a subject with DM1. In some embodiments, the complex provided herein can comprise an oligonucleotide that inhibits the expression of a DMPK allele comprising an expanded disease-associated repeat. As another example, the complex can comprise an oligonucleotide that disrupts the binding of disease-associated DMPK mRNA to muscleblind-like proteins (e.g., MBNL1, 2 and / or (e.g., and) 3), thereby reducing the toxic effect of disease-associated DMPK alleles.

[0039] Further aspects of the disclosure, including explanations of defined terms, are provided below.

[0040] I. Definition Administering: As used herein, the term "administering" or "administration" means providing a conjugate to a subject in a physiologically and / or (e.g., and) pharmacologically useful manner (e.g., treating a condition in the subject).

[0041] Approximately: As used herein, the term "approximately" or "about" refers to a value similar to a specified reference value when applied to one or more values ​​of interest. In certain embodiments, the term "approximately" or "about" refers to a broad range of values ​​that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less in either direction (greater or less than) of the specified reference value, unless otherwise indicated or clear from the context (except when such number exceeds 100% of possible values).

[0042] Antibody: As used herein, the term "antibody" refers to a polypeptide that includes at least one immunoglobulin variable domain or at least one antigenic determinant, e.g., a paratope, that specifically binds to an antigen. In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, an Fv fragment, or an scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelid antibody, or a nanobody derived from a shark antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody comprises a framework with human germline sequences. In another embodiment, the antibody comprises a heavy chain constant domain selected from the group consisting of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, the antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or (e.g., and) a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody comprises a constant domain, e.g., an Fc region. An immunoglobulin constant domain refers to a heavy chain constant domain or a light chain constant domain. Human IgG heavy chain constant domain amino acid sequences and human IgG light chain constant domain amino acid sequences and functional variants thereof are known. With respect to the heavy chain, in some embodiments, the heavy chain of the antibody described herein can be an alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein can comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In certain embodiments, the antibody described herein comprises a human gamma 1 CH1, CH2 and / or (e.g., and) CH3 domain.In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, e.g., any known in the art. Non-limiting examples of human constant region sequences are described in the art, e.g., see U.S. Pat. No. 5,693,780, and Kabat EA et al., (1991), supra. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugars or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment) and / or (e.g., and) phosphoglycosylation. In some embodiments, 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 include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure linked to a linker polypeptide or immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues linked together by peptide bonds and is used to link to one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Furthermore, an antibody may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of an antibody or antibody portion with one or more other proteins or peptides. Examples of such immunoadhesion molecules include the use of streptavidin core regions to generate tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, marker peptides, and C-terminal polyhistidine tags to generate bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0043] CDR: As used herein, the term "CDR" refers to a complementarity determining region in an antibody variable sequence. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL) that are usually involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). Each VH and VL is typically composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methodologies known in the art, for example, by Kabat definition, IMGT definition, Chothia definition, AbM definition, and / or (for example, and) contact definition, all of which are well known in the art.For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; IMGT®, the international ImMunoGeneTics information system (registered trademark) www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids. Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[Epub],5:45-60(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,33:D593-597(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,37:D1006-1012(2009);Lefranc,M.-P.et al.,Nucleic Acids Res.,43:D413-422(2015);Chothia et al.,(1989)Nature 342:877;Chothia,C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. See, e.g., J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also, hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDRs may refer to CDRs defined by any method known in the art.Two antibodies having the same CDR means that the two antibodies have the same amino acid sequence of their CDRs as determined by the same method, e.g., the IMGT definition.

[0044] There are three CDRs in each of the variable regions of heavy and light chains, which are designated as CDR1, CDR2 and CDR3 for each of the variable regions.The term "CDR set" used herein refers to a group of three CDRs that occur in a single variable region that can bind to antigen.The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Sub-portions of the CDRs may be designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries that define CDRs that overlap with the Kabat CDRs have been described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Still other CDR boundary definitions may not strictly adhere to one of the above systems, but may still overlap with the Kabat CDRs, and may be shortened or extended in light of predictions or experimental findings that a particular residue, or group of residues, or even an entire CDR, does not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are provided in Table 1. [Table 1]

[0045] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species, but in which the sequences of one or more of the CDR regions of the VH and / or (e.g., and) VL have been replaced with CDR sequences from another species, such as an antibody having a murine heavy chain variable region and a murine light chain variable region, but in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0046] Chimeric antibody: The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, e.g., an antibody having a murine heavy chain variable region and a murine light chain variable region linked to a human constant region.

[0047] Complementary: As used herein, the term "complementary" refers to the ability for precise pairing between two nucleosides or two sets of nucleosides.In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in binding between two nucleosides or two sets of nucleosides.For example, if the base of an oligonucleotide at a position can hydrogen bond with the base of a target nucleic acid (e.g., mRNA) at the corresponding position, the base is considered to be complementary to each other at that position.Base pairing can include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), cytosine-type bases (C) are complementary to guanosine-type bases (G), and universal bases, such as 3-nitropyrrole or 5-nitroindole, can hybridize to and are considered to be complementary to any A, C, U, or T. Inosine (I) is also considered in the art to be a universal base and is considered to be complementary to any A, C, U, or T.

[0048] Conservative amino acid substitution: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made.Variants can be prepared according to methods for changing polypeptide sequences known to those skilled in the art, and can be found, for example, in references that summarize such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, 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.

[0049] Covalently linked: As used herein, the term "covalently linked" refers to the characteristic of two or more molecules being linked together through at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond, such as a disulfide bond or a disulfide bridge, which acts as a linker between molecules. However, in some embodiments, two or more molecules can be covalently linked together through a molecule that acts as a linker that connects two or more molecules together through multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker.

[0050] Cross-reactivity: As used herein, and in the context of targeting agents (e.g., antibodies), the term "cross-reactivity" refers to the property of an agent that can specifically bind to multiple antigens of the same type or class (e.g., multiple homologues, paralogues, or orthologs of antigens) with similar affinity or avidity. For example, in some embodiments, an antibody that is cross-reactive to similar types or classes of human and non-human primate antigens (e.g., human transferrin receptor and non-human primate transferrin receptor) can bind to human antigens and non-human primate antigens with similar affinity or avidity. In some embodiments, an antibody is cross-reactive to similar types or classes of human antigens and rodent antigens. In some embodiments, an antibody is cross-reactive to similar types or classes of rodent antigens and non-human primate antigens. In some embodiments, an antibody is cross-reactive to similar types or classes of human antigens, non-human primate antigens, and rodent antigens.

[0051] Disease-associated repeats: As used herein, the term "disease-associated repeats" refers to a repeated nucleotide sequence at a genomic location where the number of units of the repeated nucleotide sequence correlates with and / or (for example and) directly or indirectly contributes to or causes a genetic disease, for example DM1. Each repeat unit of the disease-associated repeat can be 2, 3, 4, 5 or more nucleotides long. By way of example, in some embodiments, the disease-associated repeat is a dinucleotide repeat. In some embodiments, the disease-associated repeat is a trinucleotide repeat. In some embodiments, the disease-associated repeat is a tetranucleotide repeat. In some embodiments, the disease-associated repeat is a pentanucleotide repeat. In some embodiments, the disease-associated repeat comprises a CAG repeat, a CTG repeat, a CUG repeat, a CGG repeat, a CCTG repeat, or any nucleotide complement thereof. In some embodiments, the disease-associated repeat is in a non-coding portion of a gene. However, in some embodiments, the disease-associated repeat is in a coding region of a gene. In some embodiments, the disease-associated repeat is extended from a normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, the disease-associated repeat is in RNA (e.g., RNA transcript). In some embodiments, the disease-associated repeat is in DNA (e.g., chromosome, plasmid). In some embodiments, the disease-associated repeat is expanded in a chromosome of a germline cell. In some embodiments, the disease-associated repeat is expanded in a chromosome of a somatic cell. In some embodiments, the disease-associated repeat is expanded to several repeat units associated with congenital onset of the disease. In some embodiments, the disease-associated repeat is expanded to several repeat units associated with childhood onset of the disease. In some embodiments, the disease-associated repeat is expanded to several repeat units associated with adult onset of the disease. In DM1, a trinucleotide repeat region of CTG units in the 3' untranslated region (3'-UTR) of DMPK is disease-associated. Normal DMPK alleles contain about 5 to about 37 CTG repeat units, but in patients with DM1, the length of the CTG repeat region is significantly increased to hundreds or thousands of trinucleotide repeats.

[0052] DMPK: As used herein, the term "DMPK" refers to the gene encoding myotonin protein kinase (also known as myotonic dystrophy protein kinase or myotonic dystrophy protein kinase), a serine / threonine protein kinase. The substrates of this enzyme can include myogenin, the beta subunit of the L-type calcium channel, and phospholemman. In some embodiments, DMPK can be a human (gene ID: 1760), non-human primate (e.g., gene ID: 456139, gene ID: 715328), or rodent gene (e.g., gene ID: 13400). In humans, a CTG repeat expansion in the 3' non-coding untranslated region of DMPK is associated with myotonic dystrophy type I (DM1). In addition, multiple human transcript variants encoding distinct protein isoforms (e.g., as annotated under GenBank RefSeq accession numbers: NM_001081563.2, NM_004409.4, NM_001081560.2, NM_001081562.2, NM_001288764.1, NM_001288765.1 and NM_001288766.1) have been characterized.

[0053] DMPK allele: As used herein, the term "DMPK allele" refers to any one of the alternative forms (e.g., wild-type or mutant forms) of the DMPK gene. In some embodiments, the DMPK allele may encode a wild-type myotonin protein kinase that retains its normal and typical function. In some embodiments, the DMPK allele may contain one or more disease-associated repeat expansions. In some embodiments, a normal subject has two DMPK alleles that contain repeat units within the range of 5-37. In some embodiments, the number of CTG repeat units in a subject with DM1 is within the range of about 50 to about 3,000 or more, with the higher the number of repeats being associated with a greater severity of the disease. In some embodiments, a mildly affected DM1 subject has at least one DMPK allele that has repeat units within the range of 50-150. In some embodiments, a subject with classical DM1 has at least one DMPK allele that has repeat units within the range of 100-1,000 or more. In some embodiments, a subject with congenital-onset DM1 can have at least one DMPK allele that contains more than 2,000 repeat units.

[0054] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of the CDR sequence can be determined by different systems, the meaning of the framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2 and CDR-L3 of the light chain, and CDR-H1, CDR-H2 and CDR-H3 of the heavy chain) also divide the framework region on the light chain and the heavy chain into four subregions (FR1, FR2, FR3 and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Without specifying the specific subregion as FR1, FR2, FR3 or FR4, the framework region, when referred to by others, represents the combined FRs in the variable region of a naturally occurring single immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FRs refers to two or more of the four subregions that make up the framework region.Human heavy chain acceptor sequences and human light chain acceptor sequences are known in the art.In one embodiment, acceptor sequences known in the art can be used in the antibody disclosed herein.

[0055] Human antibody: The term "human antibody" as used herein is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. The human antibody of the present disclosure may, for example, include amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, for example, mouse, are grafted onto human framework sequences.

[0056] Humanized antibody: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but at least a portion of the VH and / or (e.g., and) VL sequences have been altered to be more "human-like", i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody, in which human CDR sequences are introduced into non-human VH and non-human VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-TfR1 antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining mouse anti-TfR1 monoclonal antibodies using existing hybridoma technology, followed by humanization using in vitro genetic engineering, e.g., as disclosed by Kasaian et al PCT Publication No. WO 2005 / 123126.

[0057] Internalized cell surface receptor: As used herein, the term "internalized cell surface receptor" refers to a cell surface receptor that is internalized by a cell, for example, upon an external stimulus, such as ligand binding to the receptor. In some embodiments, the internalized cell surface receptor is internalized by endocytosis. In some embodiments, the internalized cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalized cell surface receptor is internalized by a clathrin-independent pathway, such as, for example, phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, the internalized cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or (e.g., and) an extracellular domain, which may optionally further comprise a ligand binding domain. In some embodiments, the cell surface receptor becomes internalized by a cell after ligand binding. In some embodiments, the ligand may be a muscle targeting agent or a muscle targeting antibody. In some embodiments, the internalizing cell surface receptor is a transferrin receptor.

[0058] Isolated antibody: "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to the transferrin receptor is substantially free of antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to the transferrin receptor complex may have cross-reactivity to other antigens, including transferrin receptor molecules from other species. Additionally, an isolated antibody may be substantially free of other cellular material and / or (e.g., and) chemicals.

[0059] Kabat numbering: The terms "Kabat numbering", "Kabat definition" and "Kabat labeling" are used interchangeably herein. These terms, recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody or its antigen-binding portion (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region spans amino acid positions 31-35 for CDR1, amino acid positions 50-65 for CDR2, and amino acid positions 95-102 for CDR3. For the light chain variable region, the hypervariable region spans amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

[0060] Molecular payload: As used herein, the term "molecular payload" refers to a molecule or species that functions to regulate a biological outcome. In some embodiments, the molecular payload is linked to or otherwise associated with a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, a protein, a peptide, a nucleic acid, or an oligonucleotide. In some embodiments, the molecular payload functions to regulate the transcription of a DNA sequence, to regulate the expression of a protein, or to regulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide that includes a strand having a region of complementarity to a target gene.

[0061] Muscle targeting agent: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on a muscle cell can be a membrane protein, for example an integral membrane protein or a peripheral membrane protein. Typically, the muscle targeting agent specifically binds to an antigen on a muscle cell, thereby facilitating internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle and can be internalized into the muscle cell through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0062] Muscle-targeting antibody: As used herein, the term "muscle-targeting antibody" refers to a muscle-targeting agent that is an antibody that specifically binds to an antigen found in or on a muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an antigen on a muscle cell, thereby facilitating internalization of the muscle-targeting antibody (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an internalizing cell surface receptor present on a muscle cell. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0063] Myotonic dystrophy (DM): As used herein, the term "myotonic dystrophy (DM)" refers to a genetic disease caused by mutations in the DMPK gene or the CNBP (ZNF9) gene, characterized by muscle loss, muscle weakness and muscle function. Two types of this disease have been described: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is associated with the expansion of a CTG trinucleotide repeat in the 3' non-coding region of DMPK. DM2 is associated with the expansion of a CCTG tetranucleotide repeat in the first intron of ZNF9. In both DM1 and DM2, the nucleotide expansion results in a toxic RNA repeat that can form a hairpin structure that binds with high affinity to important intracellular proteins, such as muscleblind-like protein. Myotonic dystrophy, the genetic basis of the disease, and associated symptoms have been described in the art (see, e.g., Thornton, CA, "Myotonic Dystrophy" Neurol Clin. (2014), 32(3):705-719.; and Konieczny et al. "Myotonic dystrophy: candidate small molecule therapeutics" Drug Discovery Today (2017), 22:11.). In some embodiments, a subject is born with a mutation in DM1, referred to as congenital myotonic dystrophy. Symptoms of congenital myotonic dystrophy are present from birth and include any muscle weakness, breathing problems, clubfoot, developmental delay, and intellectual disability. DM1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry #160900. DM2 is associated with OMIM Entry #602668.

[0064] Oligonucleotide: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, gapmers, mixmers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), and the like. Oligonucleotides can be single-stranded or double-stranded. In some embodiments, oligonucleotides can include one or more modified nucleosides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, oligonucleotides can include one or more modified internucleoside linkages. In some embodiments, oligonucleotides can include one or more phosphorothioate linkages that can be in Rp or Sp stereochemical configuration.

[0065] Recombinant antibody: The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, generated or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell (described in further detail in this disclosure), antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from human immunoglobulin gene transgenic animals (e.g., mice) (see, e.g., Taylor, LD, et al., J. Immunology 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), and antibodies isolated from human immunoglobulin gene transgenic animals (e.g., mice) (see, e.g., Taylor, LD, et al., J. Immunology 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378). al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or any other means involving splicing of human immunoglobulin gene sequences into other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur within the germline repertoire of human antibodies in vivo.One aspect of the present disclosure provides fully human antibodies capable of binding to the human transferrin receptor that can be generated using techniques well known in the art, including, but not limited to, human Ig phage libraries, including, but not limited to, those disclosed in Jermutus et al., PCT Publication No. WO 2005 / 007699.

[0066] Complementary region: As used herein, the term "complementary region" refers to a nucleotide sequence, e.g., of an oligonucleotide, that is sufficiently complementary to the cognate 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 (e.g., at least 80%, 90%, 95% or 99% complementary) to the cognate nucleotide sequence of the target nucleic acid. In some embodiments, the complementary region contains 1, 2, 3 or 4 mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0067] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that allows the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context. With respect to an antibody, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or avidity that allows the antibody to be used to distinguish the specific antigen from others, as described herein, compared to one or more appropriate reference antigens, to the extent that it allows preferential targeting to a specific cell, e.g., a muscle cell, through binding to the antigen. In some embodiments, an antibody has at least about 10% affinity or avidity to bind to a target. -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10-8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or lower K D In some embodiments, the antibody specifically binds to a target if it has the following structure: In some embodiments, the antibody specifically binds to a transferrin receptor, e.g., an epitope of the apical domain of the transferrin receptor.

[0068] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate or rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, e.g., a human patient, having or suspected of having a disease. In some embodiments, the subject is a human patient, having or suspected of having a disease resulting from, e.g., a disease-associated repeat expansion in a DMPK allele.

[0069] 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 can be of human (NCBI gene ID 7037), non-human primate (e.g., NCBI gene ID 711568 or NCBI gene ID 102136007), or rodent (e.g., NCBI gene ID 22042) origin. In addition, multiple human transcript variants that code for different isoforms of the receptor (e.g., as annotated under GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1 and NP_001300895.1) have been characterized.

[0070] 2'-modified nucleoside: As used herein, the terms "2'-modified nucleoside" and "2'-modified ribonucleoside" are used interchangeably and refer to a nucleoside having a modified sugar moiety at the 2' position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, in which the 2' and 4' positions of the sugar are bridged (e.g., via a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, for example, in which the 2' position of the sugar moiety is substituted. Non-limiting examples of 2'-modified nucleosides include 2'deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA, methylene bridged nucleic acid), ethylene bridged nucleic acid (ENA), and (S)-constrained ethyl bridged nucleic acid (cEt). In some embodiments, the 2'-modified nucleosides described herein are high affinity modified nucleosides, and oligonucleotides comprising the 2'-modified nucleosides have increased affinity for target sequences compared to unmodified oligonucleotides. Examples of structures of 2'-modified nucleosides are provided below: [ka] Although the examples are shown with a phosphate group, any internucleoside linkage is contemplated between the 2'-modified nucleosides.

[0071] Provided herein is a complex that comprises an antibody covalently linked to a targeting agent, such as a molecular payload.In some embodiments, the complex comprises a muscle-targeting antibody covalently linked to an oligonucleotide.The complex may comprise an antibody that specifically binds to a single antigen site, or an antibody that binds to at least two antigen sites that may be present on the same antigen or on different antigens.

[0072] The complex can be used to regulate the activity or function of at least one gene, protein and / or (e.g., and) nucleic acid. In some embodiments, the molecular payload present in the complex is responsible for regulating the gene, protein and / or (e.g., and) nucleic acid. The molecular payload can be a small molecule, protein, nucleic acid, oligonucleotide or any molecular entity that can regulate the activity or function of gene, protein and / or (e.g., and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets disease-related repeats in cells, such as muscle cells or CNS cells.

[0073] In some embodiments, the conjugate comprises a muscle targeting agent, e.g., an anti-TfR1 antibody, covalently linked to a molecular payload, e.g., DMPK, e.g., an antisense oligonucleotide that targets a nucleic acid comprising a disease-associated repeat, e.g., a DMPK allele.

[0074] A. Muscle-targeting Agents Some aspects of the present disclosure provide muscle targeting agents for, e.g., delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents are capable of binding to muscle cells, e.g., via specific binding to an antigen on the muscle cell, and delivering associated molecular payloads to the muscle cell. In some embodiments, the molecular payload is bound (e.g., covalently bound) to the muscle targeting agent and is internalized into the muscle cell upon binding of the muscle targeting agent to an antigen on the muscle cell, e.g., via endocytosis. It should be understood that various types of muscle targeting agents can be used in accordance with the present disclosure, and that any muscle target (e.g., muscle surface protein) can be targeted by any type of muscle targeting agent described herein. By way of example, the muscle targeting agent can include or consist of a small molecule, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide). Exemplary muscle targeting agents are described in further detail herein, but it should be understood that the exemplary muscle targeting agents provided herein are not meant to be limiting.

[0075] Some aspects of the present disclosure provide muscle targeting agents that specifically bind to antigens on muscle, for example, skeletal muscle, smooth muscle, or cardiac muscle. In some embodiments, any of the muscle targeting agents provided herein bind (e.g., specifically bind) to antigens on skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells.

[0076] Through interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, molecules that are substrates for muscle uptake transporters are useful for delivering molecular payloads into muscle tissue. Binding to muscle surface recognition elements followed by endocytosis can allow large molecules, even antibodies, to enter muscle cells. As another example, molecular payloads conjugated to transferrin or anti-TfR1 antibodies can be taken up by muscle cells via binding to transferrin receptors and then ingested into the plasma membrane, for example, via clathrin-mediated endocytosis.

[0077] The use of muscle targeting agents can be useful to concentrate molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells compared to other cell types in a subject. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells (e.g., skeletal muscle cells, smooth muscle cells, or cardiomyocytes) to at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than the amount in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or fat cells). In some embodiments, the toxicity of the molecular payload in a subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90% or 95% when delivered to a subject when conjugated to a muscle targeting agent.

[0078] In some embodiments, a muscle recognition element (e.g., muscle cell antigen) may be required to achieve muscle selectivity. As an example, the muscle targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, the muscle targeting agent may be an antibody that enters muscle cells via transporter-mediated endocytosis. As another example, the muscle targeting agent may be a ligand that binds to a cell surface receptor on muscle cells. It should be understood that while the transporter-based approach provides a direct pathway to cell entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0079] i. Muscle targeting antibody In some embodiments, the muscle targeting agent is an antibody.In general, the high specificity of antibodies to their target antigens provides the possibility of selectively targeting muscle cells (e.g., skeletal muscle cells, smooth muscle cells and / or (e.g., and) cardiomyocytes).This specificity can also limit off-target toxicity.Examples of antibodies that can target the surface antigens of muscle cells have been reported and are within the scope of the present disclosure. By way of 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 contents of each of which are incorporated herein by reference in their entirety.

[0080] a. Anti-transferrin receptor (TfR) antibody Some aspects of the present disclosure are based on the recognition that agents that bind to transferrin receptors, e.g., anti-transferrin receptor antibodies, can target muscle cells. Transferrin receptors are internalized cell surface receptors that transport transferrin across cell membranes and participate in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins that can bind to transferrin receptors. Thus, aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to transferrin receptors. In some embodiments, the binding proteins that bind to transferrin receptors are internalized into muscle cells along with any attached molecular payload. As used herein, antibodies that bind to transferrin receptors can be interchangeably referred to as transferrin receptor antibodies, anti-transferrin receptor antibodies, or anti-TfR1 antibodies. Antibodies that bind, e.g., specifically bind, to transferrin receptors can be internalized into cells, e.g., through receptor-mediated endocytosis, upon binding to transferrin receptors.

[0081] It should be understood that anti-TfR1 antibodies can be produced, synthesized and / or (e.g., and) derivatized using several known methodologies, such as library design using phage display. Exemplary methodologies have been characterized in the art and are incorporated by reference (Diez, P. et al. "High-throughput phage-display screening in array format", Enzyme and microbial technology, 2015, 79, 34-41.; Christoph MH and Stanley, JR "Antibody Phage Display: Technique and Applications" J Invest Dermatol. 2014, 134:2.; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies" 1985, Springer.). In other embodiments, anti-TfR1 antibodies have been previously characterized or disclosed.Antibodies that specifically bind to the transferrin receptor are known in the art (see, e.g., U.S. Pat. No. 4,364,934, filed Dec. 4, 1979, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Pat. No. 8,409,573, filed Jun. 14, 2006, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Pat. No. 9,708,406, filed May 20, 2014, "Anti-transferrin receptor antibodies and methods of use"; U.S. Pat. No. 9,611,323, filed Dec. 19, 2014, "Low affinity blood brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed Dec. 24, 2014, "Novel anti-Transferrin receptor antibody that passes through blood-brain barrier"; Schneider C. et al. al.“Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9.”J Biol Chem.1982,257:14,8516-8522.;Lee et al.“Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse”2000,J See Pharmacol. Exp. Ther., 292:1048-1052. ).

[0082] In some embodiments, the anti-TfR1 antibodies described herein bind to the transferrin receptor with high specificity and affinity. In some embodiments, the anti-TfR1 antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor or an epitope that becomes exposed to the antibody. In some embodiments, the anti-TfR1 antibodies provided herein specifically bind to transferrin receptor from human, non-human primate, mouse, rat, etc. In some embodiments, the anti-TfR1 antibodies provided herein bind to the human transferrin receptor. In some embodiments, the anti-TfR1 antibodies described herein bind to an amino acid segment of the human or non-human primate transferrin receptor provided in SEQ ID NOs: 105-108. In some embodiments, the anti-TfR1 antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor set forth in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor.

[0083] In some embodiments, the anti-TfR1 antibodies described herein (e.g., anti-TfR clone 8 in Table 2 below) bind to an epitope of TfR1, the epitope comprising residues of amino acids 214-241 and / or amino acids 354-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope comprising residues of amino acids 214-241 and amino acids 354-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope comprising one or more of residues Y222, T227, K231, H234, T367, S368, S370, T376 and S378 of human TfR1 set forth in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope that includes residues Y222, T227, K231, H234, T367, S368, S370, T376 and S378 of human TfR1 as set forth in SEQ ID NO:105.

[0084] In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 in Table 2 below, and variants thereof) bind to an epitope of TfR1 that includes residues of amino acids 258-291 and / or amino acids 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 in Table 2 below, and variants thereof) bind to an epitope that includes residues of amino acids 258-291 and amino acids 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 in Table 2 below, and variants thereof) bind to an epitope that includes one or more of residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as set forth in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 in Table 2 below, and variants thereof) bind to an epitope that includes residues K261, S273, Y282, T362, S368, S370 and K371 of human TfR1 as set forth in SEQ ID NO:105.

[0085] An example of a human transferrin receptor amino acid sequence corresponding to the NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows: (Sequence number 105).

[0086] An example of a non-human primate transferrin receptor amino acid sequence that corresponds to the NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Macaca mulatta) is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLGVDEEENTDNNTKPNGTKPKRCGGNICYGTIAVIIFFLIGFMIGYLGYCKGVEPKTECERLAGTESPAREEPEEDFPAAPRLYWDDLKRKLSEKLDTTDFTSTIKLLNENLYVPREAGSQKDENLALYIENQFREFKLSKVWRDQHFVKI QVKDSAQNSVIIVDKNGGLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLDSPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVKADLSFFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCKMVTSENKSVKLTVSNV LKETKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSSVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQDVKHPVTGRSLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTM DTYKELVERIPELNKVARAAAEVAGQFVIKLTHDTELNLDYERYNSQLLLFLRDLNQYRADVKEMGLSLQWLYSARGDFFRATSRLTTDFRNAEKRDKFVMKKLNDRVMRVEYYFLSPYVSPKESPFRHVFWGSGSHTLSALLESLKLRRQNNSAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO:106)

[0087] An example of a non-human primate transferrin receptor amino acid sequence that corresponds to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: (SEQ ID NO:107)

[0088] 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: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYV KIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKN VLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTR LDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF (SEQ ID NO:108)

[0089] In some embodiments, the anti-TfR1 antibody binds to a receptor such as: FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and does not inhibit the binding interaction between the transferrin receptor and transferrin and / or (e.g., and) human hemochromatosis protein (also known as HFE). In some embodiments, the anti-TfR1 antibodies described herein do not bind to the epitope of SEQ ID NO: 109.

[0090] Suitable methodologies can be used to obtain and / or (e.g., and) produce antibodies, antibody fragments, or antigen-binding agents, for example, through the use of recombinant DNA protocols. In some embodiments, antibodies can 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 can be used as an immunogen in any form or entity, for example, recombinant or naturally occurring form or entity. Hybridomas are screened using standard methods, for example, ELISA screening, to find at least one hybridoma that produces an antibody that targets a particular antigen. Antibodies can also be produced through screening of protein expression libraries, for example, phage display libraries, that express antibodies. Phage display library design may also be used in some embodiments (see, e.g., U.S. Pat. No. 5,223,409, filed 3 / 1 / 1991, "Directed evolution of novel binding proteins"; WO 1992 / 18619, filed 4 / 10 / 1992, "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, filed 5 / 1 / 1991, "Recombinant library screening methods"; WO 1992 / 20791, filed 5 / 15 / 1992, "Methods for producing members of specific binding pairs"; and WO 1992 / 15679, filed 2 / 28 / 1992, "Improved epitope displaying phage"). In some embodiments, the antigen of interest may be used to immunize a non-human animal, e.g., a rodent or goat.In some embodiments, antibodies can then be obtained from the non-human animal and optionally modified using several methodologies, for example, using recombinant DNA technology. Additional examples of antibody production and methodologies are known in the art (see, for example, Harlow et al. "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988).

[0091] In some embodiments, the antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules are present. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or intracellularly, and optionally may be deficient in an enzyme, e.g., a glycosyltransferase, in the N- or O-glycosylation pathway. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0092] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VL domain and / or (e.g., and) a VH domain of any one of the anti-TfR1 antibodies selected from any one of Tables 2-7, and comprises a constant region comprising the amino acid sequence of the constant region of an 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) of an immunoglobulin molecule. Non-limiting examples of human constant regions are described in the art, see, e.g., Kabat EA et al., (1991), supra.

[0093] In some embodiments, an agent that binds to transferrin receptor, e.g., an anti-TfR1 antibody, can target muscle cells and / or mediate transport of the agent across the blood-brain barrier (e.g., toward CNS cells). Transferrin receptor is an internalized cell surface receptor that transports transferrin across cell membranes and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins that can bind to transferrin receptor. An antibody that binds, e.g., specifically binds, to transferrin receptor can be internalized into the cell, e.g., through receptor-mediated endocytosis, when bound to transferrin receptor.

[0094] In some aspects, humanized antibodies are provided herein that bind to the transferrin receptor with high specificity and affinity. In some embodiments, the humanized anti-TfR1 antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor, or an epitope that becomes exposed to the antibody. In some embodiments, the humanized anti-TfR1 antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the humanized anti-TfR1 antibodies provided herein bind to the human transferrin receptor. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to an amino acid segment of the human or non-human primate transferrin receptor provided in SEQ ID NOs: 105-108. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor set forth in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to TfR1 but not to TfR2.

[0095] In some embodiments, the anti-TFR1 antibody is at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 (e.g., human or non-human primate TfR1) with a binding affinity (e.g., as indicated by Kd) of 1 M or less. In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 with a Kd in the sub-nanomolar range. In some embodiments, the anti-TfR1 antibodies described herein selectively bind to transferrin receptor 1 (TfR1) but not transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR1 antibodies described herein bind to human TfR1 and cynomolgus monkey TfR1 but not (e.g., 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less) and does not bind to mouse TfR1. The affinity and binding kinetics of anti-TfR1 antibodies can be tested using any suitable method, including but not limited to biosensor technology (e.g., OCTET or BIACORE). In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit transferrin binding to TfR1. In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit HFE-beta2-microglobulin binding to TfR1.

[0096] Non-limiting examples of anti-TfR1 antibodies are provided in Table 2. [Table 2-1] [Table 2-2] [Table 2-3]

[0097] In some embodiments, an anti-TfR1 antibody of the disclosure is a humanized variant of any one of the anti-TfR1 antibodies provided in Table 2. In some embodiments, an anti-TfR1 antibody of the disclosure comprises the same CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as the CDR-H1, CDR-H2 and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 2, and comprises a humanized heavy chain variable region and / or (e.g., and) a humanized light chain variable region.

[0098] Examples of amino acid sequences of the anti-TfR1 antibodies described herein are provided in Table 3. [Table 3-1] [Table 3-2]

[0099] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising a CDR-H1, CDR-H2 and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid mutations in the framework regions compared to the respective VHs provided in Table 3. Alternatively, or additionally (e.g., in addition), an anti-TfR1 antibody of the disclosure comprises a VL comprising a CDR-L1, CDR-L2 and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid mutations in the framework regions compared to the respective VLs provided in Table 3. In some embodiments, the VH of the anti-TfR1 antibody is a humanized VH and / or the VL of the anti-TfR1 antibody is a humanized VL.

[0100] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH that includes CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 3 and comprises an amino acid sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identical in the framework regions compared to the respective VHs provided in Table 3. Alternatively, or in addition (e.g., in addition), an anti-TfR1 antibody of the present disclosure comprises a VL that includes CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 3 and comprises an amino acid sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identical in the framework regions compared to the respective VLs provided in Table 3. In some embodiments, the VH of the anti-TfR1 antibody is a humanized VH and / or the VL of the anti-TfR1 antibody is a humanized VL.

[0101] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:69, and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0102] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:71, and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0103] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:72, and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0104] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73, and a VL comprising the amino acid sequence of SEQ ID NO:74.

[0105] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73, and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0106] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76, and a VL comprising the amino acid sequence of SEQ ID NO:74.

[0107] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76, and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0108] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77, and a VL comprising the amino acid sequence of SEQ ID NO:78.

[0109] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:79, and a VL comprising the amino acid sequence of SEQ ID NO:80.

[0110] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77, and a VL comprising the amino acid sequence of SEQ ID NO:80.

[0111] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:154, and a VL comprising the amino acid sequence of SEQ ID NO:155.

[0112] In some embodiments, the anti-TfR1 antibodies described herein are full-length IgGs that can include heavy and light chain constant regions from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein can include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can be of any suitable origin, e.g., human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is derived from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81)

[0113] In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein comprises a mutant human IgG1 constant region. For example, the introduction of LALA mutations in the CH2 domain of human IgG1 (a mutant derived from mAb b12 mutated to replace the lower hinge residues Leu234 Leu235 with Ala234 and Ala235) is known to reduce Fcγ receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The mutant human IgG1 constant region is provided below (mutations are bolded and underlined):

number

[0114] In some embodiments, the light chain of any of the anti-TfR1 antibodies described herein may further comprise a light chain constant region (CL), which can be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)

[0115] Heavy and light chain constant regions of other antibodies are known in the art, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php, both of which are incorporated herein by reference.

[0116] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region that contains no more than 25 amino acid mutations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof, and a heavy chain constant region set forth in SEQ ID NO: 81. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof, and a heavy chain constant region set forth in SEQ ID NO: 82.

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

[0118] Examples of IgG heavy and light chain amino acid sequences of the described anti-TfR1 antibodies are provided in Table 4 below. [Table 4-1] [Table 4-2] [Table 4-3] [Table 4-4]

[0119] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain that contains 25 or fewer amino acid mutations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 or fewer amino acid mutations) compared to a heavy chain set forth in any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively, or additionally (e.g., in addition), an anti-TfR1 antibody of the disclosure includes a light chain that contains 25 or fewer amino acid mutations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 or fewer amino acid mutations) compared to a light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0120] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94 and 156. Alternatively, or in addition (e.g., in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94 and 156. Alternatively, or additionally (eg, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157.

[0121] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:84, and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0122] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:86, and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0123] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:87, and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0124] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0125] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0126] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91, and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0127] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91, and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0128] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92, and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0129] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:94, and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0130] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92, and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0131] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:156 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0132] In some embodiments, the anti-TfR1 antibody is a Fab fragment, a Fab' fragment, or a F(ab')2 fragment of an intact antibody (full-length antibody). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared via routine methods (e.g., recombinantly or by digesting the heavy chain constant region of a full-length IgG using an enzyme, e.g., papain). By way of example, F(ab')2 fragments can be produced by pepsin or papain digestion of an antibody molecule, and Fab fragments can be generated by reducing disulfide bridges of the F(ab')2 fragment. In some embodiments, the heavy chain constant region within the Fab fragment of the anti-TfR1 antibodies described herein is It contains the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96).

[0133] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region that contains 25 or less amino acid mutations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less amino acid mutations) compared to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region set forth in SEQ ID NO: 96.

[0134] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any variants thereof listed in Table 3, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any variants thereof listed in Table 3, and a light chain constant region that contains 25 or less amino acid mutations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or less amino acid mutations) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any variants thereof listed in Table 3, and a light chain constant region set forth in SEQ ID NO: 83.

[0135] Examples of Fab heavy and light chain amino acid sequences of the described anti-TfR1 antibodies are provided in Table 5 below. [Table 5-1] [Table 5-2] [Table 5-3] [Table 5-4]

[0136] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain that contains 25 or fewer amino acid mutations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid mutations) compared to a heavy chain set forth in any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively, or additionally (e.g., in addition), an anti-TfR1 antibody of the disclosure includes a light chain that contains 25 or fewer amino acid mutations (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 or fewer amino acid mutations) compared to a light chain set forth in any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0137] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to any one of SEQ ID NOs: 97-103, 158 and 159. Alternatively, or in addition (e.g., in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to any one of SEQ ID NOs: 97-103, 158 and 159. Alternatively, or additionally (eg, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157.

[0138] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:97, and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0139] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:98 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0140] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0141] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100, and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0142] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100, and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0143] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101, and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0144] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101, and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0145] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102, and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0146] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:103, and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0147] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102, and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0148] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:158 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0149] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:159, and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0150] Other known anti-TfR1 antibodies Any other suitable anti-TfR1 antibody known in the art can be used as muscle targeting agent in the complex disclosed herein. Examples of known anti-TfR1 antibodies, including related references and binding epitopes, are listed in Table 6. In some embodiments, the anti-TfR1 antibody comprises the complementarity determining region (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) of any of the anti-TfR1 antibodies provided herein, for example, the anti-TfR1 antibodies listed in Table 6. [Table 6-1] [Table 6-2]

[0151] In some embodiments, an anti-TfR1 antibody of the disclosure includes one or more of the CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, an anti-TfR1 antibody includes CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, an anti-TfR1 antibody includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 provided for any one of the anti-TfR1 antibodies selected from Table 6.

[0152] In some embodiments, the anti-TfR1 antibodies of the disclosure include any antibody that includes a heavy chain variable domain and / or (e.g., and) a light chain variable domain of any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the anti-TfR1 antibodies of the disclosure include any antibody that includes a heavy chain variable pair and a light chain variable pair of any one of the anti-TfR1 antibodies selected from Table 6.

[0153] Aspects of the present disclosure provide anti-TfR1 antibodies having heavy chain variable (VH) and / or (e.g., and) light chain variable (VL) domain amino acid sequences homologous to any of those described herein. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable sequence or a light chain variable sequence that is at least 75% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identical to the heavy chain variable sequence and / or any light chain variable sequence of any anti-TfR1 antibody, e.g., any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the homologous heavy chain variable and / or (e.g., and) light chain variable amino acid sequences do not vary in any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) can occur within the heavy and / or (e.g., and) light chain variable sequences excluding any of the CDR sequences provided herein. In some embodiments, any of the anti-TfR1 antibodies provided herein comprises heavy and light chain variable sequences that comprise framework sequences that are at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the framework sequences of any anti-TfR1 antibody, e.g., any one of the anti-TfR1 antibodies selected from Table 6.

[0154] An example of a transferrin receptor antibody that may be used in accordance with the present disclosure is described in International Application Publication WO 2016 / 081643, which is incorporated herein by reference. The amino acid sequence of this antibody is provided in Table 7. [Table 7-1] [Table 7-2]

[0155] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-H1, CDR-H2, and CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. Alternatively, or additionally (e.g., in addition), an anti-TfR1 antibody of the disclosure comprises a CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

[0156] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 that contains no more than three amino acid mutations (e.g., no more than three, two or one amino acid mutations) compared to the CDR-L3 shown in Table 7. In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 that contains one amino acid mutation compared to the CDR-L3 shown in Table 7. In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system). In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1 and CDR-L2 that are the same as the CDR-H1, CDR-H2 and CDR-H3 shown in Table 7, and comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system).

[0157] In some embodiments, anti-TfR1 antibodies of the disclosure comprise heavy chain CDRs that, together, are at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identical to the heavy chain CDRs shown in Table 7. Alternatively, or additionally (e.g., in addition), anti-TfR1 antibodies of the disclosure comprise light chain CDRs that, together, are at least 80% (e.g., 80%, 85%, 90%, 95% or 98%) identical to the light chain CDRs shown in Table 7.

[0158] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 124. Alternatively, or additionally (e.g., in addition), an anti-TfR1 antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 125.

[0159] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 128. Alternatively, or additionally (e.g., in addition), an anti-TfR1 antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 129.

[0160] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH that contains no more than 25 amino acid mutations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VH set forth in SEQ ID NO: 128. Alternatively, or in addition (e.g., in addition), an anti-TfR1 antibody of the disclosure comprises a VL that contains no more than 15 amino acid mutations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid mutations) compared to the VL set forth in SEQ ID NO: 129.

[0161] In some embodiments, the anti-TfR1 antibodies of the present disclosure are full-length IgG1 antibodies that can include heavy and light chain constant regions from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein can include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can be of any suitable origin, e.g., human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is derived from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 81)

[0162] In some embodiments, the light chain of any of the anti-TfR1 antibodies described herein may further comprise a light chain constant region (CL), which can be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 83)

[0163] In some embodiments, the anti-TfR1 antibodies described herein are chimeric antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 132. Alternatively, or in addition (e.g., in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0164] In some embodiments, the anti-TfR1 antibodies described herein are fully human antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 134. Alternatively, or additionally (e.g., in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0165] In some embodiments, the anti-TfR1 antibody is an antigen-binding fragment (Fab) of an intact antibody (full-length antibody). In some embodiments, the anti-TfR1 Fab described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 136. Alternatively, or in addition (e.g., in addition), the anti-TfR1 Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-TfR1 Fab described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 137. Alternatively, or in addition (e.g., in addition), the anti-TfR1 Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0166] The anti-TfR1 antibodies described herein can be any antibody form, including, but not limited to, an intact (i.e., full-length) antibody, an antigen-binding fragment thereof (e.g., Fab, Fab', F(ab')2, Fv), a single-chain antibody, a bispecific antibody, or a nanobody. In some embodiments, the anti-TfR1 antibodies described herein are scFv. In some embodiments, the anti-TfR1 antibodies described herein are scFv-Fab (e.g., scFv fused to a portion of a constant region). In some embodiments, the anti-TfR1 antibodies described herein are scFv fused to a constant region (e.g., human IgG1 constant region set forth in SEQ ID NO:81).

[0167] In some embodiments, conservative mutations can be introduced into an antibody sequence (e.g., CDR or framework sequences) at positions where the residues are unlikely to be involved in interactions with the target antigen (e.g., transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., within the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) within the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) within the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) of an anti-TfR1 antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or (e.g., and) antigen-dependent cellular cytotoxicity.

[0168] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region (CH1 domain) of the Fc region such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), e.g., as described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate conjugation of a linker.

[0169] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., within the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) within the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) within the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody 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 Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0170] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn-binding fragment (preferably, Fc or hinge-Fc domain fragment) to change (e.g., increase or decrease) the half-life of the antibody in vivo. For example, see International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutations that change (e.g., increase or decrease) the half-life of the antibody in vivo.

[0171] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-TfR1 antibody in vivo. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al., (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, incorporated herein by reference. This type of mutant IgG, referred to as a "YTE mutant", has been shown to exhibit a 4-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.

[0172] In some embodiments, one, two or more amino acid substitutions are introduced into the IgG constant domain Fc region to change the effector function of anti-TfR1 antibody. The effector ligand to which the affinity is changed can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent No. 5,624,821 and U.S. Patent No. 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, for example, U.S. Patent No. 5,585,097 and U.S. Patent No. 8,591,886 for a description of mutations that delete or inactivate constant domains, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced in the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0173] In some embodiments, one or more amino acids in the constant region of the anti-TfR1 antibody described herein can be replaced with different amino acid residues so that the antibody has altered C1q binding and / or (e.g., and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or (e.g., and) increase the affinity of the antibody for Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0174] In some embodiments, the heavy and / or (e.g., and) light chain variable domain sequences of the antibodies provided herein can be used to generate, by way of example, CDR-grafted, chimeric, humanized or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be appreciated by one of skill in the art, any variant, CDR-grafted, chimeric, humanized or composite antibody derived from any of the antibodies provided herein can be useful in the compositions and methods described herein, where the variant, CDR-grafted, chimeric, humanized or composite antibody maintains the ability to specifically bind to the transferrin receptor, such that it has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it is derived.

[0175] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibodies. For example, to avoid potential complications due to Fab arm exchange that is known to occur in natural IgG4 mAbs, the antibodies provided herein may contain a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline resulting in an IgG1-like hinge sequence. Thus, any of the antibodies may contain a stabilizing "Adair" mutation.

[0176] In some embodiments, the antibody is modified, e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules are present. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or intracellularly, and optionally may be deficient in an enzyme, e.g., a glycosyltransferase, in the N- or O-glycosylation pathway. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0177] In some embodiments, any one of the anti-TfR1 antibodies described herein may include a signal peptide (e.g., an N-terminal signal peptide) in the heavy and / or (e.g., and) light chain sequence. In some embodiments, the anti-TfR1 antibodies described herein include any one of the VH and VL sequences, any one of the IgG heavy chain and IgG light chain sequences, or any one of the F(ab') heavy chain and F(ab') light chain sequences described herein, and further include a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide includes the amino acid sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0178] In some embodiments, the antibody provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also called pyroglutamate formation (pyroGlu), may occur on the antibody at N-terminal glutamate (Glu) and / or glutamine (Gln) residue during production. Thus, it should be understood that an antibody identified as having a sequence that includes an N-terminal glutamate or glutamine residue encompasses an antibody that has undergone pyroglutamate formation resulting from post-translational modification. In some embodiments, pyroglutamate formation occurs within the heavy chain sequence. In some embodiments, pyroglutamate formation occurs within the light chain sequence.

[0179] b. Other muscle-targeting antibodies In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin IIb, or CD63. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a myogenic precursor protein. Exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha7, integrin alpha7 beta1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a skeletal muscle protein. Exemplary skeletal muscle proteins include, but are not limited to, alpha-sarcoglycan, beta-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha 7, integrin alpha 7 beta 1, integrin beta 1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a smooth muscle protein. Exemplary smooth muscle proteins include, but are not limited to, alpha-smooth muscle actin, VE-cadherin, caldesmon / CALD1, calponin 1, desmin, histamine H2 R, motilin R / GPR38, Transgelin / TAGLN, and vimentin, although it is understood that antibodies to additional targets are within the scope of the present disclosure and that the exemplary list of targets provided herein is not meant to be limiting.

[0180] C. Antibody characteristics / changes In some embodiments, conservative mutations can be introduced into an antibody sequence (e.g., CDR or framework sequences) at positions where the residues are unlikely to be involved in interactions with the target antigen (e.g., transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., within the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) within the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) within the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) of a muscle-targeting antibody described herein to alter one or more functional properties of the antibody, for example, serum half-life, complement fixation, Fc receptor binding and / or (e.g., and) antigen-dependent cellular cytotoxicity.

[0181] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region (CH1 domain) of the Fc region such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), e.g., as described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate conjugation of a linker.

[0182] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., within the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) within the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) within the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody 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 Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0183] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn-binding fragment (preferably, Fc or hinge-Fc domain fragment) to change (e.g., increase or decrease) the half-life of the antibody in vivo. For example, see International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745 for examples of mutations that change (e.g., increase or decrease) the half-life of the antibody in vivo.

[0184] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody can have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al., (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, incorporated herein by reference. This type of mutant IgG, referred to as a "YTE mutant", has been shown to exhibit a 4-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.

[0185] In some embodiments, one, two or more amino acid substitutions are introduced into the IgG constant domain Fc region to change the effector function of the anti-transferrin receptor antibody. The effector ligand to which the affinity is changed can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Patent No. 5,624,821 and U.S. Patent No. 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce Fc receptor binding of circulating antibodies, thereby increasing tumor localization. See, for example, U.S. Patent No. 5,585,097 and U.S. Patent No. 8,591,886 for a description of mutations that delete or inactivate constant domains, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced in the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0186] In some embodiments, one or more amino acids in the constant region of the muscle-targeting antibody described herein can be replaced with different amino acid residues so that the antibody has altered Clq binding and / or (e.g., and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are altered to thereby alter the ability of the antibody to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or (e.g., and) increase the affinity of the antibody for Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0187] In some embodiments, the heavy and / or (e.g., and) light chain variable domain sequences of the antibodies provided herein can be used to generate, by way of example, CDR-grafted, chimeric, humanized or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be appreciated by one of skill in the art, any variant, CDR-grafted, chimeric, humanized or composite antibody derived from any of the antibodies provided herein can be useful in the compositions and methods described herein, where the variant, CDR-grafted, chimeric, humanized or composite antibody maintains the ability to specifically bind to the transferrin receptor, such that it has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it is derived.

[0188] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibodies. For example, to avoid potential complications due to Fab arm exchange that is known to occur in natural IgG4 mAbs, the antibodies provided herein may contain a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline resulting in an IgG1-like hinge sequence. Thus, any of the antibodies may contain a stabilizing "Adair" mutation.

[0189] As provided herein, the antibody of the present disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be attached at its C-terminus to a light chain constant domain like Cκ or Cλ. Similarly, the VH domain or a portion thereof may be attached to a full or partial heavy chain like IgA, IgD, IgE, IgG and IgM, and any subclass of isotype. The antibody may include a suitable constant region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, the antibody within the scope of the present disclosure may include a VH domain and a VL domain, or an antigen-binding portion thereof, combined with any suitable constant region.

[0190] ii. Muscle-targeting peptides Some aspects of the present disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences (e.g., peptide sequences between 5 and 20 amino acids in length) that bind to specific cell types are described. By way of example, cell-targeting peptides can be found in Vines e., et al., A. “Cell-penetrating and cell-targeting peptides in drug delivery” Biochim Biophys Acta 2008,1786:126-38; Jarver P., et al., “In vivo biodistribution and efficacy of peptide mediated delivery” Trends Pharmacol Sci 2010;31:528-35; Samoylova TI, et al., “Elucidation of muscle-binding peptides by phage display screening” Muscle Nerve 1999;22:460-6; ​​U.S. Patent No. 6,329,501, issued December 11, 2001, entitled “METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE”; and Samoylov AM, et al., “Recognition of cell-specific binding of "Phage display derived peptides using an acoustic wave sensor." Biomol Eng 2002;18:269-72. By designing peptides to interact with specific cell surface antigens (e.g., receptors), selectivity for a desired tissue, e.g., muscle, can be obtained. Skeletal muscle targeting has been explored, and a wide range of molecular payloads can be delivered. These approaches, which lack many of the practical disadvantages associated with large antibodies or viral particles, can be highly selective for muscle tissue. Thus, in some embodiments, the muscle targeting agent is a muscle-targeting peptide of 4-50 amino acids in length.In some embodiments, the muscle targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Muscle targeting peptides can be generated using any of a number of methods, including, for example, phage display.

[0191] In some embodiments, the muscle targeting peptide may bind to an internalized cell surface receptor that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells, e.g., the transferrin receptor. In some embodiments, the muscle targeting peptide may target, e.g., bind to, the transferrin receptor. In some embodiments, the transferrin receptor targeting peptide may include a segment of a naturally occurring ligand, e.g., transferrin. In some embodiments, the transferrin receptor targeting peptide is as described in U.S. Patent No. 6,743,893, 11 / 30 / 2000 application, "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR." In some embodiments, the transferrin receptor targeting peptide is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells." BMC Cancer. 2011 Aug 18; 11:359. In some embodiments, the transferrin receptor targeting peptide is as described in U.S. Patent No. 8,399,653, filed 5 / 20 / 2011, entitled "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY."

[0192] As mentioned above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides have been identified using phage display libraries that display surface heptapeptides. As an example, a peptide with amino acid sequence ASSLNIA (SEQ ID NO: 363) bound to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. Thus, in some embodiments, the muscle targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 363). This peptide showed improved specificity for binding to cardiac and skeletal muscle tissues after intravenous injection into mice, and reduced binding to 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, the entire contents of which are incorporated herein by reference, in which a 12 amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO:364) was identified, and this muscle-targeting peptide showed improved binding to C2C12 cells compared to the ASSLNIA (SEQ ID NO:363) peptide.

[0193] Additional methods for identifying peptides selective for muscle (e.g., skeletal muscle) over other cell types include 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 contents of which are incorporated herein by reference. Non-specific cell binders were selected by pre-incubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After repeated selection, the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 365) emerged most frequently. Thus, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 365).

[0194] The muscle targeting agent can be an amino acid-containing molecule or peptide. The muscle targeting peptide can correspond to a sequence of a protein that preferentially binds to a protein receptor found in muscle cells. In some embodiments, the muscle targeting peptide contains a strong hydrophobic amino acid, e.g., valine, nature, such that the peptide preferentially targets muscle cells. In some embodiments, the muscle targeting peptide has not been previously characterized or disclosed. These peptides can be conceived, produced, synthesized, and / or derivatized (e.g., and) using any of several methodologies, e.g., phage-displayed peptide libraries, one-bead one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. Exemplary methodologies are characterized in the art and are incorporated by reference (Gray, BP and Brown, KC "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides" Chem Rev. 2014, 114:2, 1020-1081.; Samoylova, TI and Smith, BF "Elucidation of muscle-binding peptides by phage display screening." Muscle Nerve, 1999, 22:4.460-6.).In some embodiments, muscle-targeting peptides have been previously disclosed (see, e.g., Writer MJ et al. "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display." J. Drug Targeting. 2004;12:185; Cai, D. "BDNF-mediated enhancement of inflammation and injury in the aging heart." Physiol Genomics. 2006,24:3,191-7.; Zhang, L. "Molecular profiling of heart endothelial cells." Circulation, 2005,112:11,1601-11.; McGuire, MJ et al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004,342:1,171-82). Exemplary muscle-targeting peptides include the following groups of amino acid sequences: CQAQGQLVC (SEQ ID NO: 366), CSERSMNFC (SEQ ID NO: 367), CPKTRRVPC (SEQ ID NO: 368), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 369), ASSLNIA (SEQ ID NO: 363), CMQHSMRVC (SEQ ID NO: 370), and DDTRHWG (SEQ ID NO: 371). In some embodiments, muscle-targeting peptides can include about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. Muscle-targeting peptides can include naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art.In some embodiments, the muscle-targeting peptide can be linear, while in other embodiments, the muscle-targeting peptide can be cyclic, e.g., bicyclic (see, e.g., Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

[0195] iii. Muscle-targeted receptor ligands The muscle targeting agent can be a ligand, for example, a ligand that binds to a receptor protein. The muscle targeting ligand can be a protein, for example, transferrin, that binds to an internalized cell surface receptor expressed by muscle cells. Thus, in some embodiments, the muscle targeting agent is transferrin or a derivative thereof that binds to the transferrin receptor. Alternatively, the muscle targeting ligand can 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 can target muscle cells include compounds that include cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linoleic acid, linoleic acid, myristic acid, sterol, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and alkoxy acids.

[0196] iv. Muscle-targeting aptamers The muscle targeting agent can be an aptamer, e.g., an RNA aptamer, that preferentially targets muscle cells over other cell types. In some embodiments, the muscle targeting aptamer has not been previously characterized or disclosed. These aptamers can be conceived, produced, synthesized, and / or (e.g., and) derivatized using any of several methodologies, e.g., Systematic Evolution of Ligands by Exponential Enrichment. Exemplary methodologies have been characterized in the art and are incorporated by reference (Yan, A C and Levy, M. "Aptamers and aptamer targeted delivery" RNA biology, 2009, 6:3, 316-20.; Germer, K. et al. "RNA aptamers and their therapeutic and diagnostic applications" Int. J. Biochem. Mol. Biol. 2013; 4:27-40.). In some embodiments, muscle-targeting aptamers have been previously disclosed (see, e.g., Phillippou, S. et al. "Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers." Mol Ther Nucleic Acids. 2018, 10:199-214.; Thiel, W H et al. "Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation." Mol Ther. 2016, 24:4,779-87.). Exemplary muscle-targeting aptamers include the A01B RNA aptamer and RNA Apt 14. In some embodiments, the aptamer is a nucleic acid-based aptamer, an oligonucleotide aptamer, or a peptide aptamer. In some embodiments, the aptamer can be about 5-15 kDa, about 5-10 kDa, about 10-15 kDa, about 1-5 Da, about 1-3 kDa or less.

[0197] v. Other muscle-targeting agents One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use a substrate of a muscle transporter protein, e.g., a transporter protein expressed on the sarcolemma. In some embodiments, the muscle targeting agent is a substrate of an influx transporter specific to muscle tissue. In some embodiments, the influx transporter is specific to skeletal muscle tissue. The two major classes of transporters expressed on the sarcolemma of skeletal muscle are (1) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, which promotes efflux from skeletal muscle tissue, and (2) the solute carrier (SLC) superfamily, which can promote the influx of substrates into skeletal muscle. In some embodiments, the muscle targeting agent is a substrate that binds to the ABC or SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a naturally occurring substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-naturally occurring substrate, e.g., a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0198] In some embodiments, the muscle targeting agent is any of the muscle targeting agents described herein that target the SLC superfamily of transporters (e.g., antibodies, nucleic acids, small molecules, peptides, aptamers, lipids, sugar moieties).In some embodiments, the muscle targeting agent is a substrate of the SLC superfamily of transporters.SLC transporters are equilibria or use the proton or sodium ion gradient created across the membrane to drive the transport of substrates. Exemplary SLC transporters with high skeletal muscle expression include, but are not limited to, the SATT transporter (ASCT1; SLC1A4), the GLUT4 transporter (SLC2A4), the GLUT7 transporter (GLUT7; SLC2A7), the ATRC2 transporter (CAT-2; SLC7A2), the LAT3 transporter (KIAA0245; SLC7A6), the PHT1 transporter (PTR4; SLC15A4), the OATP-J transporter (OATP5A1; SLC21A15), the OCT3 transporter (EMT; SLC22A3), the OCTN2 transporter (FLJ46769; SLC22A5), the ENT transporters (ENT1; SLC29A1 and ENT2; SLC29A2), the PAT2 transporter (SLC36A2) and the SAT2 transporter (KIAA1382; SLC38A2). These transporters may facilitate the influx of substrates into skeletal muscle and provide opportunities for muscle targeting.

[0199] In some embodiments, the muscle targeting agent is a substrate for equilibrative nucleoside transporter 2 (ENT2) transporter. Compared to other transporters, ENT2 has one of the highest mRNA expression in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs, such as brain, heart, placenta, thymus, pancreas, prostate and kidney, but is particularly abundant in skeletal muscle. Human ENT2 promotes the uptake of its substrates according to their concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. hENT2 transporter has low affinity for all nucleosides except inosine (adenosine, guanosine, uridine, thymidine and cytidine). Thus, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and clofarabine. In some embodiments, any of the muscle targeting agents provided herein are associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular payload.

[0200] In some embodiments, the muscle targeting agent is a substrate of the organic cation / carnitine transporter (OCTN2), a sodium ion-dependent high affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, mildronate, acetylcarnitine, or any derivative thereof that binds to OCTN2. In some embodiments, carnitine, mildronate, acetylcarnitine, or a derivative thereof is covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0201] The muscle targeting agent can be a protein that is a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein can be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, hemojuvelin can be full-length or a fragment, or a variant that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to a functional hemojuvelin protein. In some embodiments, the hemojuvelin variant can be a soluble fragment, can lack N-terminal signaling, and / or can lack (e.g., and) a C-terminal anchoring domain. In some embodiments, hemojuvelin may be annotated under GenBank RefSeq accession numbers NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3 or NM_213653.3. It is understood that hemojuvelin may be of human, non-human primate or rodent origin.

[0202] B. Molecular Payload Some aspects of the disclosure provide oligonucleotides designed to target molecular payloads, e.g., DMPK RNA, to modulate DMPK expression or activity. In some embodiments, modulating DMPK expression or activity includes reducing the level of DMPK RNA and / or (e.g., and) protein. In some embodiments, the DMPK RNA is disease-associated, e.g., has a disease-associated repeat expansion or is encoded from an allele that has a disease-associated repeat expansion. In some embodiments, the DMPK RNA includes a CUG repeat expansion or the allele by which it is encoded includes a CTG repeat expansion. In some embodiments, the disclosure provides oligonucleotides complementary to DMPK RNA, useful for reducing the level of toxic DMPK with a disease-associated repeat expansion, e.g., in subjects with or suspected of having myotonic dystrophy. In some embodiments, the oligonucleotides are designed to direct RNAse H-mediated degradation of the target DMPK RNA. In some embodiments, the oligonucleotides are designed to direct RNAse H-mediated degradation of the target DMPK RNA present in the nucleus of a cell, e.g., a muscle cell (e.g., a myotube) or a CNS cell (e.g., a neuron). In some embodiments, the oligonucleotide is designed to have desirable bioavailability and / or serum stability properties. In some embodiments, the oligonucleotide is designed to have desirable binding affinity properties. In some embodiments, the oligonucleotide is designed to have desirable toxicity profile. In some embodiments, the oligonucleotide is designed to have low complement activation and / or cytokine induction properties.

[0203] In some embodiments, the oligonucleotide is linked or otherwise associated with the muscle targeting agent described herein. In some embodiments, such oligonucleotides can be targeted in muscle cells, for example, via specific binding to DMPK sequences in muscle cells after delivery to muscle cells by the associated muscle targeting agent. It should be understood that various types of muscle targeting agents can be used in accordance with the present disclosure. In some embodiments, the oligonucleotide comprises a region of complementarity to a DMPK allele that contains a disease-associated repeat expansion. Although exemplary oligonucleotides targeting DMPK RNA are described in further detail herein, it should be understood that the exemplary molecular payloads provided herein are not meant to be limiting.

[0204] i. Oligonucleotides In some embodiments, the DMPK-targeting oligonucleotides described herein are designed to trigger RNase H-mediated degradation of DMPK mRNA. It should be understood that in some embodiments, an oligonucleotide of one format (e.g., an antisense oligonucleotide) can be conveniently adapted to another format (e.g., an siRNA oligonucleotide) by incorporating a functional sequence (e.g., an antisense strand sequence) from one format into the other format.

[0205] Examples of oligonucleotides useful for targeting DMPK include U.S. Patent Application Publication No. 20100016215, published January 1, 2010, entitled Compound And Method For Treating Myotonic Dystrophy; U.S. Patent Application Publication No. 20130237585, published July 19, 2010, entitled Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; U.S. Patent Application Publication No. 20150064181, published March 5, 2015, entitled "Antisense Conjugates For Decreasing Expression Of Dmpk; U.S. Patent Application Publication No. 20150064181, published August 27, 2015, entitled "Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy," the contents of each of which are incorporated herein in their entirety. No. 20150238627, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression," published on October 20, 2016.

[0206] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence set forth below, which is an exemplary human DMPK gene sequence (Gene ID 1760; NM_001081560.2):

[0207] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence set forth below, which is an exemplary mouse DMPK gene sequence (Gene ID 13400; NM_001190490.1).

[0208] In some embodiments, the oligonucleotide may have a region of complementarity to mutant forms of DMPK, such as those reported in Botta A. et al. "The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients" J Med Genet. 2008 Oct; 45(10): 639-46.; and Machuca-Tzili L. et al. "Clinical and molecular aspects of the myotonic dystrophies: a review" Muscle Nerve. 2005 Jul; 32(1): 1-18., the contents of each of which are incorporated herein in their entirety.

[0209] In some embodiments, the oligonucleotide provided herein is an antisense oligonucleotide targeting DMPK.In some embodiments, the oligonucleotide targeting is any one of the antisense oligonucleotides (e.g., gapmers) targeting DMPK as described in US Patent Application Publication No. 20160304877, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression", published on October 20, 2016, which is incorporated herein by reference.In some embodiments, the DMPK targeting oligonucleotide targets the region of the DMPK gene sequence described in Genbank Accession No. NM_001081560.2 (SEQ ID NO: 130) or described in Genbank Accession No. NG_009784.1 (SEQ ID NO: 395).

[0210] In some embodiments, the DMPK targeting oligonucleotide comprises a nucleotide sequence that includes a region complementary to a target region that is at least 10 consecutive nucleotides (e.g., at least 10, at least 12, at least 14, at least 16, at least 18, at least 20 or more consecutive nucleotides) of SEQ ID NO:130.

[0211] In some embodiments, the DMPK-targeted oligonucleotide comprises a gapmer motif. By "gapmer" is meant a chimeric antisense compound in which an internal region having multiple nucleosides that support RNase H cleavage is disposed between external regions having one or more nucleotides, and the nucleosides that comprise the internal region are chemically distinct from the nucleosides or nucleosides that comprise the external region. The internal region can be referred to as a "gap segment" and the external region can be referred to as a "wing segment". In some embodiments, the DMPK-targeted oligonucleotide comprises one or more modified nucleosides and / or one or more modified internucleoside linkages (e.g., and). In some embodiments, the internucleoside linkages are phosphorothioate linkages. In some embodiments, the oligonucleotide comprises a complete phosphorothioate backbone. In some embodiments, the oligonucleotide is a DNA gapmer with a cET end (e.g., 3-10-3; cET-DNA-cET). In some embodiments, a DMPK targeted oligonucleotide comprises one or more 6'-(S)-CH3 bicyclic nucleosides, one or more β-D-2'-deoxyribonucleotides, and / or (eg, and) one or more 5-methylcytosine nucleosides.

[0212] a. Oligonucleotide size / sequence Oligonucleotides can be of a variety of different lengths, for example, depending on the format. In some embodiments, the oligonucleotides are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides long. In some embodiments, the oligonucleotides are 8-50 nucleotides long, 8-40 nucleotides long, 8-30 nucleotides long, 10-15 nucleotides long, 10-20 nucleotides long, 15-25 nucleotides long, 21-23 nucleotides long, 15-20 nucleotides long, 20-25 nucleotides long, etc.

[0213] In some embodiments, the nucleic acid sequence of an oligonucleotide for the purposes of this disclosure is "complementary" to a target nucleic acid if it can specifically hybridize to the target nucleic acid. In some embodiments, an oligonucleotide that hybridizes to a target nucleic acid (e.g., an mRNA or pre-mRNA molecule) results in the modulation of the activity or expression of the target (e.g., reduced mRNA translation, altered pre-mRNA splicing, exon skipping, target mRNA degradation, etc.). In some embodiments, the nucleic acid sequence of an oligonucleotide has a sufficient degree of complementarity to its target nucleic acid so that it does not hybridize to non-target sequences under conditions where it is desired to avoid non-specific binding, e.g., physiological conditions. Thus, in some embodiments, an oligonucleotide can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to consecutive nucleotides of a target nucleic acid. In some embodiments, complementary nucleotide sequence does not need to be 100% complementary to the sequence of its target that can specifically hybridize to target nucleic acid or be specific to target nucleic acid.In certain embodiments, oligonucleotide comprises one or more mismatched nucleobases with target nucleic acid.In certain embodiments, such mismatch reduces target-related activity, but reduces non-target-related activity to a greater extent (i.e., increases selectivity for target nucleic acid and reduces off-target effect).

[0214] In some embodiments, the oligonucleotide comprises a region of complementarity to a target nucleic acid that is within the range of 8-15, 8-30, 8-40, or 10-50, or 5-50, 15-20, 20-25, or 5-40 nucleotides in length. In some embodiments, the region of complementarity of the oligonucleotide to the target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 8 consecutive nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may contain 1, 2 or 3 base mismatches compared to a portion of the contiguous nucleotides of the target nucleic acid. In some embodiments, the oligonucleotide may have up to 3 mismatches for 15 bases or up to 2 mismatches for 10 bases.

[0215] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 contiguous nucleotides of a sequence comprising any one of SEQ ID NOs: 231-362. In some embodiments, the oligonucleotide comprises a sequence comprising any one of SEQ ID NOs: 231-362. In some embodiments, the oligonucleotide comprises a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95% or 97% sequence identity with at least 12 or at least 15 contiguous nucleotides of any one of SEQ ID NOs: 231-362.

[0216] In some embodiments, the oligonucleotide comprises a region of complementarity to a nucleotide sequence set forth in any one of SEQ ID NOs: 160-230. In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides (e.g., contiguous nucleotides) that are complementary to a nucleotide sequence set forth in any one of SEQ ID NOs: 160-230. In some embodiments, the oligonucleotide comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% complementary to at least 12 or at least 15 contiguous nucleotides of any one of SEQ ID NOs: 160-230.

[0217] In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to the target sequence of any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 8, Table 9, and Table 10). In some embodiments, such target sequences are 100% complementary to the oligonucleotides listed in Table 8, Table 9, or Table 10.

[0218] It should be understood that in some embodiments, methylation of the nucleobase uracil at the C5 position forms thymine. Thus, in some embodiments, a nucleotide or nucleoside having a C5 methylated uracil (or 5-methyl-uracil) can be equivalently identified as a thymine nucleotide or nucleoside.

[0219] In some embodiments, any one or more of the thymine bases (T) of any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Tables 8, 9, and 10) can independently and optionally be uracil bases (U), and / or any one or more of the U can independently and optionally be T.

[0220] b. Oligonucleotide Modification: The oligonucleotides described herein may be modified, for example, include modified sugar moieties, modified internucleoside linkages, modified nucleotides or modified nucleosides, and / or (for example, and) combinations thereof.In addition, in some embodiments, oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immunostimulatory; are nuclease resistant; have improved cellular uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; have improved endosomal internal exit in cells; minimize TLR stimulation; or avoid pattern recognition receptors.Any of the modified chemical natures or forms of oligonucleotides described herein can be combined with each other.For example, one, two, three, four, five or more different types of modifications can be included in the same oligonucleotide.

[0221] In some embodiments, specific nucleotide or nucleoside modifications can be used that make the oligonucleotides they are incorporated into more resistant to nuclease digestion than natural oligodeoxynucleotide or oligoribonucleotide molecules, and these modified oligonucleotides remain intact for longer periods than unmodified oligonucleotides.Specific examples of modified oligonucleotides include those that include modified backbones, for example modified internucleoside linkages, for example phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or short heteroatom or heterocyclic intersugar linkages.Thus, the oligonucleotides of the present disclosure can be stabilized against nucleic acid degradation by, for example, incorporating modifications, for example nucleotide or nucleoside modifications.

[0222] In some embodiments, the oligonucleotide may be up to 50 nucleotides in length, or up to 100 nucleotides in length, and 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30, 2-40, 2-45 or more nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The oligonucleotide may be 8-30 nucleotides in length, and 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The oligonucleotide may be 8-15 nucleotides in length, and 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. Optionally, the oligonucleotide may have every nucleotide or nucleoside modified except for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides / nucleosides. Oligonucleotide modifications are described in more detail herein.

[0223] C modified nucleosides In some embodiments, the oligonucleotide described herein comprises at least one nucleoside modified at the 2' position of the sugar.In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleoside.In some embodiments, all of the nucleosides in the oligonucleotide are 2'-modified nucleosides.

[0224] In some embodiments, the oligonucleotides described herein contain one or more non-bicyclic 2'-modified nucleosides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA) modified nucleosides.

[0225] In some embodiments, the oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleosides in which the ribose ring comprises a bridging moiety that connects the 2'-O atom to the 4'-C atom via, for example, a methylene (LNA) bridge, an ethylene (ENA) bridge, or an (S)-constrained ethyl (cEt) bridge, connecting two atoms within the ring. Examples of LNA are described in International Patent Application Publication WO / 2008 / 043753, entitled "RNA Antagonist Compounds For The Modulation Of PCSK9," published on April 17, 2008, the entire contents of which are incorporated herein by reference. Examples of ENAs are provided in International Patent Publication WO 2005 / 042777, published May 12, 2005, entitled "APP / ENA Antisense," the disclosures of which are incorporated herein by reference in their entireties; 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. Examples of cEt are provided in US Pat. No. 7,101,993, US Pat. No. 7,399,845, and US Pat. No. 7,569,686, each of which is incorporated by reference herein in its entirety.

[0226] In some embodiments, the oligonucleotides are disclosed in any of the following U.S. patents or published U.S. patent applications: U.S. Patent No. 7,399,845, entitled "6-Modified Bicyclic Nucleic Acid Analogs," issued on July 15, 2008; U.S. Patent No. 7,741,457, entitled "6-Modified Bicyclic Nucleic Acid Analogs," issued on June 22, 2010; U.S. Patent No. 8,022,193, entitled "6-Modified Bicyclic Nucleic Acid Analogs," issued on September 20, 2011; U.S. Patent No. 7,569,686, entitled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs," issued on August 4, 2009; U.S. Patent No. 7,569,686, entitled "Novel Nucleoside And Oligonucleotide Analogs," issued on February 26, 2008, the contents of each of which are incorporated herein by reference in their entirety for all purposes. No. 7,335,765, entitled "Novel Nucleoside And Oligonucleotide Analogues," issued on January 1, 2008; U.S. Patent No. 7,314,923, entitled "Novel Nucleoside And Oligonucleotide Analogues," issued on October 19, 2010; and U.S. Patent Publication No. 2011 / 0009471, entitled "Oligonucleotide Analogues And Methods Utilizing The Same," issued on February 17, 2015, now U.S. Patent No. 8,957,201.

[0227] In some embodiments, an oligonucleotide comprises at least one modified nucleoside that results in an increase in the Tm of the oligonucleotide in the range of 1° C., 2° C., 3° ​​C., 4° C., or 5° C., compared to an oligonucleotide that does not have at least one modified nucleoside. An oligonucleotide can have multiple modified nucleosides that, in total, result in an increase in the Tm of the oligonucleotide in the range of 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., or more, compared to an oligonucleotide that does not have the modified nucleoside.

[0228] An oligonucleotide may contain a mixture of different kinds of nucleosides. For example, an oligonucleotide may contain a mixture of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. An oligonucleotide may contain a mixture of deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may contain a mixture of 2'-fluoro modified nucleosides and 2'-O-methyl modified nucleosides. An oligonucleotide may contain a mixture of bridged nucleosides and 2'-fluoro or 2'-O-methyl modified nucleosides. An oligonucleotide may contain a mixture of non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). The oligonucleotide may comprise a mixture of 2'-fluoro and 2'-O-Me modified nucleosides. The oligonucleotide may comprise a mixture of 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro or 2'-O-Me modified nucleosides. The oligonucleotide may comprise a mixture of non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0229] Oligonucleotides may contain different types of alternating nucleosides. For example, oligonucleotides may contain alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. Oligonucleotides may contain alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. Oligonucleotides may contain alternating 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. Oligonucleotides may contain alternating bridged nucleosides and 2'-fluoro or 2'-O-methyl modified nucleosides. Oligonucleotides may contain alternating non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). The oligonucleotides may contain alternating 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro or 2'-O-Me modified nucleosides. The oligonucleotides may contain alternating non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, 2'-fluoro or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0230] In some embodiments, the oligonucleotides described herein contain a 5'-vinylphosphonate modification, one or more abasic residues, and / or one or more reverse abasic residues.

[0231] d. Internucleoside linkage / backbone In some embodiments, the oligonucleotide may contain phosphorothioate or other modified internucleoside linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between at least two nucleosides. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleosides. By way of example, in some embodiments, the oligonucleotide comprises modified internucleoside linkages at the first, second and / or (for example and) third internucleoside linkages at the 5' or 3' end of the nucleotide sequence.

[0232] Phosphorus-containing linkages that may be used include, but are not limited to, normal 3'-5' linkages, phosphorothioates having their 2'-5' linked analogs, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3' alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidates and aminoalkyl phosphoramidates, thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters and boranophosphates, as well as those having opposite polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2', see U.S. Pat. No. 3,687,808; U.S. Pat. No. 4,469,863; No. 5,476,301, U.S. Patent No. 5,023,243, U.S. Patent No. 5,177,196, U.S. Patent No. 5,188,897, U.S. Patent No. 5,264,423, U.S. Patent No. 5,276,019, U.S. Patent No. 5,278,302, U.S. Patent No. 5,286,717, U.S. Patent No. 5,321,131, U.S. Patent No. 5,399,676, U.S. Patent No. 5,405,939, U.S. Patent No. 5,453, 496, U.S. Patent No. 5,455,233, U.S. Patent No. 5,466,677, U.S. Patent No. 5,476,925, U.S. Patent No. 5,519,126, U.S. Patent No. 5,536,821, U.S. Patent No. 5,541,306, U.S. Patent No. 5,550,111, U.S. Patent No. 5,563,253, U.S. Patent No. 5,571,799, U.S. Patent No. 5,587,361 and U.S. Patent No. 5,625,050.

[0233] In some embodiments, oligonucleotides may have heteroatom backbones, e.g., methylene (methylimino) or MMI backbones, amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374), morpholino backbones (see Summerton and Weller, U.S. Pat. No. 5,034,506), or peptide nucleic acid (PNA) backbones (in which the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone and the nucleotides are attached directly or indirectly to aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

[0234] e. Stereospecific oligonucleotides In some embodiments, the phosphorus atom between oligonucleotides is chiral, and the properties of the oligonucleotide are adapted based on the configuration of the chiral phosphorus atom. In some embodiments, suitable methods can be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms. Chem Soc Rev. 2011 Dec; 40(12): 5829-43.). In some embodiments, phosphorothioate-containing oligonucleotides are provided that include nucleoside units linked together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages. In some embodiments, such phosphorothioate oligonucleotides with substantially chiral pure intersugar linkages are prepared by enzymatic synthesis or chemical synthesis, for example, as described in U.S. Patent No. 5,587,261, issued December 12, 1996, the entire contents of which are incorporated herein by reference. In some embodiments, chiral controlled oligonucleotides can produce selective cleavage patterns of target nucleic acid.For example, in some embodiments, chiral controlled oligonucleotides can produce single-site cleavage within the complementary sequence of nucleic acid, as described, for example, in U.S. Patent Application Publication No. 20170037399, entitled "CHIRAL DESIGN," published on February 2, 2017, the entire contents of which are incorporated herein by reference.

[0235] h. gapmer In some embodiments, the oligonucleotides described herein are gapmers. Gapmer oligonucleotides generally have the formula 5'-XYZ-3' with X and Z as flanking regions around the gap region Y. In some embodiments, the flanking region X of the formula 5'-XYZ-3' is also referred to as the X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, the flanking region Z of the formula 5'-XYZ-3' is also referred to as the Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, the gap region Y of the formula 5'-XYZ-3' is also referred to as the Y region, Y segment, or gap segment Y. In some embodiments, each nucleoside in the gap region Y is a 2'-deoxyribonucleoside, and neither the 5' wing region X nor the 3' wing region Z contains a 2'-deoxyribonucleoside. In some embodiments, the gapmer oligonucleotide comprises a region of complementarity to at least 15 contiguous nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleosides) of a target sequence provided in Table 8 (e.g., any one of SEQ ID NOs: 160-230), and / or comprises at least 15 contiguous nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleosides) of an antisense sequence of Table 8, 9, or 10, or an ASO structure provided in Table 9 or 10 (e.g., any one of SEQ ID NOs: 231-362), wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by a T.

[0236] In some embodiments, the Y region is a contiguous stretch of nucleotides, e.g., a region of six or more DNA nucleotides, capable of recruiting an RNAse, e.g., RNAse H. In some embodiments, the gapmer binds to the target nucleic acid, at which point the RNAse can be recruited and then cleave the target nucleic acid. In some embodiments, the Y region is flanked on the 5' and 3' by regions X and Z that contain high affinity modified nucleosides, e.g., 1-6 high affinity modified nucleosides. Examples of high affinity modified nucleosides include, but are not limited to, 2'-modified nucleosides (e.g., 2'-MOE, 2'O-Me, 2'-F) or 2'-4' bicyclic nucleosides (e.g., LNA, cEt, ENA). In some embodiments, the flanking sequences X and Z can be 1-20 nucleotides long, 1-8 nucleotides long, or 1-5 nucleotides long. The flanking sequences X and Z can be of similar or dissimilar lengths. In some embodiments, the gap segment Y can be a nucleotide sequence that is 5 to 20 nucleotides in length, 5 to 15 nucleotides in length, 5 to 12 nucleotides in length, or 6 to 10 nucleotides in length.

[0237] In some embodiments, the gap region of a gapmer oligonucleotide may contain, in addition to DNA nucleotides, modified nucleosides known to be permissive for efficient RNase H action, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-type nucleotides. In some embodiments, the gap region contains one or more unmodified internucleosides. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least 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.

[0238] Gapmers may be produced using any suitable method. Representative U.S. patents, U.S. patent publications, and PCT publications which teach the preparation of gapmers include, but are not limited to, U.S. Pat. No. 5,013,830, U.S. Pat. No. 5,149,797, U.S. Pat. No. 5,220,007, U.S. Pat. No. 5,256,775, U.S. Pat. No. 5,366,878, U.S. Pat. No. 5,403,711, U.S. Pat. No. 5,491,133, U.S. Pat. No. 5,565,350, U.S. Pat. No. 5,623,065, U.S. Pat. No. 5,652,355, U.S. Pat. No. 5,652,356, U.S. Pat. No. 5,700,922, U.S. Pat. No. 5,898,031, U.S. Pat. No. 7,015,315, U.S. Pat. No. 7,101,993, U.S. Pat. No. 7,399,845, U.S. Pat. No. 7,432,250, U.S. Pat. No. 7,56 No. 9,686, U.S. Patent No. 7,683,036, U.S. Patent No. 7,750,131, U.S. Patent No. 8,580,756, U.S. Patent No. 9,045,754, U.S. Patent No. 9,428,534, U.S. Patent No. 9,695,418, U.S. Patent No. 10,017,764, U.S. Patent No. 10,260,069, U.S. Patent No. 9,428,534, U.S. Patent No. 8,580,756, U.S. Patent Publication No. 20 No. 050074801, U.S. Patent Publication No. 20090221685, U.S. Patent Publication No. 20090286969, U.S. Patent Publication No. 20100197762 and U.S. Patent Publication No. 20110112170, PCT Publication Nos. WO2004069991, WO2005023825, WO2008049085 and WO2009090182, and European Patent No. 2,149,605.

[0239] In some embodiments, gapmers are 10-40 nucleosides in length. By way of example, gapmers can be 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35, or 35-40 nucleosides in length. In some embodiments, the gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleosides in length.

[0240] In some embodiments, the gap region Y of the gapmer is 5-20 nucleosides in length. By way of example, the gap region Y can be 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 nucleosides in length. In some embodiments, the gap region Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, each nucleoside in the gap region Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides in the gap region Y are 2'-deoxyribonucleosides. In some embodiments, one or more of the nucleosides in the gap region Y are modified nucleosides (e.g., 2'-modified nucleosides, e.g., those described herein). In some embodiments, one or more cytosines in gap region Y are optionally 5-methyl-cytosines. In some embodiments, each cytosine in gap region Y is a 5-methyl-cytosine.

[0241] In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are independently 1 to 20 nucleosides in length. By way of example, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') can independently be 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, 2 to 5, 2 to 7, 3 to 5, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleosides in length. In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleosides in length. In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are the same length. In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are different lengths. In some embodiments, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) is longer than the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula). In some embodiments, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) is shorter than the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula).

[0242] In some embodiments, the gapmer is 5-10-5, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3, 2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1, 2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12- 5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14 -1, 2-14-3, 3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-1 1-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1- 13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4 -11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4,1-19-1、1-18-2、2-18-1、1-17-3、3-17-1、2-17-2、1-16-4、4-16-1、2-16-3、3-16-2、1-15-5、2-15-4、4-15-2、3-15-3、1-14-6、6-14-1、2-14-5、5-14-2、3-14-4、4-14-3、1-13-7、7-13-1、2-13-6、6-13-2、3-13-5、5-13-3、4-13-4、1-12-8、8-12-1、2-12-7、7-12-2、3-12-6、6-12-3、4-12-5、5-12-4、2-11-8、8-11-2、3-11-7、7-11-3、4-11-6、6-11-4、5-11-5、1-20-1、1-19-2、2-19-1、1-18-3、3-18-1、2-18-2、1-17-4、4-17-1、2-17-3、3-17-2、1-16-5、2-16-4、4-16-2、3-16-3、1-15-6、6-15-1、2-15-5、5-15-2、3-15-4、4-15-3、1-14-7、7-14-1、2-14-6、6-14-2、3-14-5、5-14-3、4-14-4、1-13-8、8-13-1、2-13-7、7-13-2、3-13-6、6-13-3、4-13-5、5-13-4、2-12-8、8-12-2、3-12-7、7-12-3、4-12-6、6-12-4、5-12-5、3-11-8、8-11-3、4-11-7、7-11-4、5-11-6、6-11-5、1-21-1、1-20-2、2-20-1、1-20-3、3-19-1、2-19-2、1-18-4、4-18-1、2-18-3、3-18-2、1-17-5、2-17-4、4-17-2、3-17-3、1-16-6、6-16-1、2-16-5、5-16-2、3-16-4、4-16-3、1-15-7、7-15-1、2-15-6、6-15-2、3-15-5、5-15-3、4-15-4、1-14-8、8-14-1、2-14-7、7-14-2、3-14-6、6-14-3、4-14-5、5-14-4、2-13-8、8-13-2、3-13-7、7-13-3、4-13-6、6-13-4、5-13-5、1-12-10、10-12-1、2-12-9、9-12-2、3-12-8、8-12-3、4-12-7、7-12-4、5-12-6、6-12-5、4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, and 5'-XYZ-3' of 1-15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7 or 7-11-6. The numbers indicate the number of nucleosides in the X, Y and Z regions in the 5'-XYZ-3' gapmer.

[0243] In some embodiments, one or more nucleosides in the 5' wing region of a gapmer (X in the 5'-XYZ-3' formula) or the 3' wing region of a gapmer (Z in the 5'-XYZ-3' formula) are modified nucleotides (e.g., high affinity modified nucleosides). In some embodiments, the modified nucleoside (e.g., high affinity modified nucleoside) is a 2'-modified nucleoside. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside or a non-bicyclic 2'-modified nucleoside. In some embodiments, the high affinity modified nucleoside is a 2'-4' bicyclic nucleoside (e.g., LNA, cEt or ENA) or a non-bicyclic 2'-modified nucleoside (e.g., 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA)).

[0244] In some embodiments, one or more nucleosides in the 5' wing region of a gapmer (X of the formula 5'-XYZ-3') are high affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of a gapmer (X of the formula 5'-XYZ-3') is a high affinity modified nucleoside. In some embodiments, one or more nucleosides in the 3' wing region of a gapmer (Z of the formula 5'-XYZ-3') are high affinity modified nucleosides. In some embodiments, each nucleoside in the 3' wing region of a gapmer (Z of the formula 5'-XYZ-3') is a high affinity modified nucleoside. In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') are high affinity modified nucleosides and one or more nucleosides in the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are high affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') is a high affinity modified nucleoside and each nucleoside in the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') is a high affinity modified nucleoside.

[0245] In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') comprises the same high affinity nucleosides as the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3'). By way of example, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') may comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, or 2'-O-Me). In another example, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') may comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE, or 2'-O-Me). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt).

[0246] In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9 or 10) nucleosides in length, each nucleoside of X and Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE, or 2'-O-Me), and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9 or 10) nucleosides in length, each nucleoside of X and Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) comprises a different high affinity nucleoside than the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula). By way of example, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) may comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, or 2'-O-Me) and the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) may comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) may comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, or 2'-O-Me) and the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) may comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0247] In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9 or 10) nucleosides in length, each nucleoside of X is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), each nucleoside of Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6 or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9 or 10) nucleosides in length, each nucleoside of X is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), each nucleoside of Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE, or 2'-O-Me), and each nucleoside of Y is a 2'-deoxyribonucleoside.

[0248] In some embodiments, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0249] In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6 or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9 or 10) nucleosides in length, at least one but not all (e.g., 1, 2, 3, 4, 5 or 6) of positions 1, 2, 3, 4, 5, 6 or 7 of X (the 5'-most position is position 1) is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE, or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside of Y is a 2' deoxyribonucleoside. In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6 or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9 or 10) nucleosides in length, at least one but not all (e.g., 1, 2, 3, 4, 5 or 6) of positions 1, 2, 3, 4, 5, 6 or 7 of Z (the 5'-most position is position 1) is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE, or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside of Y is a 2' deoxyribonucleoside. In some embodiments, a gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6 or 7) nucleosides in length and Y is 6 to 10 (e.g., 6, 7, 8, 9 or 10) nucleosides in length, and at least one but not all (e.g., 1, 2, 3, 4, 5 or 6) of positions 1, 2, 3, 4, 5, 6 or 7 of X and Z (the 5'-most position is position 1) are 5'-XYZ-3'. At least one of positions 1, 2, 3, 4, 5, 6 or 7, but not all (e.g., 1, 2, 3, 4, 5 or 6) of X, Z, Y ...

[0250] Non-limiting examples of gapmer configurations having mixtures of non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA or cEt) in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and / or the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) include: BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE ;BBB-(D)n-BBBAA;KKK-(D)n-KKKAA;LLL-(D)n-LLLAA;BBB-(D)n-BBBEE;KKK-(D)n-KKKEE;LLL-(D)n-LLLEE;BBB-(D)n-BBBAAA;KKK-(D)n-KKKAAA;L LL-(D)n-LLLAAA;BBB-(D)n-BBBEEE;KKK-(D)n-KKKEEE;LLL-(D)n-LLLEEE;BBB-(D)n-BBBAAA;KKK-(D)n-KKKAAA;LLL-(D)n-LLLAAA;BBB-(D)n-BBBEE E;KKK-(D)n-KKKEEE;LLL-(D)n-LLLEEE;BABA-(D)n-ABAB;KAKA-(D)n-AKAK;LALA-(D)n-ALAL;BEBE-(D)n-EBEB;KEKE-(D)n-EKEK;LELE-(D)n-ELEL; BABA-(D)n-ABAB;KAKA-(D)n-AKAK;LALA-(D)n-ALAL;BEBE-(D)n-EBEB;KEKE-(D)n-EKEK;LELE-(D)n-ELEL;ABAB-(D)n-ABAB;AKAK-(D)n-AKAK;ALAL- (D)n-ALAL;EBEB-(D)n-EBEB;EKEK-(D)n-EKEK;ELEL-(D)n-ELEL;ABAB-(D)n-ABAB;AKAK-(D)n-AKAK;ALAL-(D)n-ALAL;EBEB-(D)n-EBEB;EKEK-(D)n- EKEK;ELEL-(D)n-ELEL;AABB-(D)n-BBAA;BBAA-(D)n-AABB;AAKK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;AABB-(D)n-BBAA;AACK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;BBB-(D)n-BBA;CCK-(D)n-KKA;LLL-(D) n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LL-B-D)n-LLE; BB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;ABBB-(D)n-BBBA; BBB-(D)n-BBBE;ECC-(D)n-CCKE;ELLL-(D)n-LLLE;ABBB-(D)n-BBBA;ACCC-(D)n-CCKA;ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;ECC-(D)n-CCKE;ELLL-LLLE; (D)n-LLLE;ABBB-(D)n-BBBAA;ACC-(D)n-CCKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;ECCC-(D)n-CKKEE;ELLL-(D)n-LLLEE;ABBB-(D)n-BBBAA;ALL-(D)n-LLLAA;EBBB-(D)n-BBBEE; CCC-(D)n-CCKAA;ALL-(D)n-LLLAA;EBBB-(D)n-BBBEE;CCK-(D)n-CCKEE;ELLL-(D)n-LLLEE;AABBB-(D)n-BBB;AACCC-(D)n-CCK;AALLL-(D)n-LLLL; EEBBB-(D)n-BBB;EECCC-(D)n-CCK;EELLL-(D)n-LLL;AABBB-(D)n-BBB;AACCC-(D)n-CCK;AALLL-(D)n-LLL;EEBBB-(D)n-BBB;EECCC-(D)n-KKK; LL-(D)n-LLL;AABBB-(D)n-BBBA;AACCC-(D)n-CCKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKCC-(D)n-CCKE;EELLL-(D)n-LLLE;AABBB-(D)n-BBBA;AAKKK-(D)n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE;ABBAABB-(D)n-BB;AKKAAKK-(D)n-KK;ALL AALLL-(D)n-LL;EBBEEBB-(D)n-BB;EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL;ABBAAABB-(D)n-BB;AKKAAKK-(D)n-KK;ALLAALL-(D)n-LL;EBBEE BB-(D)n-BB;EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALLL-(D)n-LLL;EBBEBB-(D)n-BBB;EKKEKK- (D)n-KKK;ELLELL-(D)n-LLL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALL-(D)n-LLL;EBBEBB-(D)n-BBB;EKKEKK-(D)n-KKK;ELLELL-(D) n-LLL;EEEK-(D)n-EEEEEEEE;EEK-(D)n-EEEEEEEEEE;EK-(D)n-EEEEEEEEEE;EK-(D)n-EEEKK;K-(D)n-EEEKEKE;K-(D)n-EEEKEKEE;K-(D)n- EEKEK;EK-(D)n-EEEEKEKE;EK-(D)n-EEEKEK;EEK-(D)n-KEEKE;EK-(D)n-EEKEK;EK-(D)n-KEEK;EEK-(D)n-EEEEKEK;EK-(D)n-KEEEKEE;EK- (D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK, where "A" represents a 2'-modified nucleoside, "B" represents a 2'-4' bicyclic nucleoside, "K" represents a constrained ethyl nucleoside (cEt), "L" represents an LNA nucleoside, "E" represents a 2'-MOE modified ribonucleoside, "D" represents a 2'-deoxyribonucleoside, and "n" represents the length of the gap segment (Y in the 5'-XYZ-3' configuration) and is an integer from 1 to 20;

[0251] In some embodiments, any one of the gapmers described herein comprises one or more modified nucleoside linkages (e.g., phosphorothioate linkages) in each of the X, Y and Z regions. In some embodiments, each internucleoside linkage in any one of the gapmers described herein is a phosphorothioate linkage. In some embodiments, each of the X, Y and Z regions independently comprises a mixture of phosphorothioate and phosphodiester linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate linkage, 5' wing region X comprises a mixture of phosphorothioate and phosphodiester linkages, and 3' wing region Z comprises a mixture of phosphorothioate and phosphodiester linkages.

[0252] Non-limiting examples of DMPK targeting oligonucleotides are provided in Tables 8, 9 and 10. [Table 8-1] [Table 8-2] [Table 9-1] [Table 9-2] [Table 9-3] [Table 10-1] [Table 10-2] [Table 10-3]

[0253] In some embodiments, the DMPK-targeting oligonucleotides described herein are 15-25 nucleosides (e.g., 15-20, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and contain a region of complementarity to at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleosides) of any one of SEQ ID NOs: 160-230. In some embodiments, the DMPK-targeting oligonucleotide comprises a 5'-XYZ-3' configuration, where X comprises 3-7 (e.g., 3-5, 3, 4, 5, 6, or 7) linked nucleosides, where at least one of the nucleosides within X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), and Y comprises 6-15 (e.g., 6-10, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 and Z comprises 3 to 7 (e.g., 3 to 5, 3, 4, 5, 6, or 7) linked 2'-deoxyribonucleosides, each cytosine in Y is optionally and independently a 5-methyl-cytosine, and Z comprises 3 to 7 (e.g., 3 to 5, 3, 4, 5, 6, or 7) linked nucleosides, and at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA).

[0254] In some embodiments, the DMPK-targeting oligonucleotides described herein comprise at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleosides) of the nucleotide sequence of any one of SEQ ID NOs: 231-362, wherein each thymine base (T) is independently and optionally replaced by a uracil base (U), and each U is independently and optionally replaced by a T. In some embodiments, the DMPK-targeting oligonucleotide comprises a 5'-XYZ-3' configuration, where X comprises 3-7 (e.g., 3-5, 3, 4, 5, 6, or 7) linked nucleosides, where at least one of the nucleosides within X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), and Y comprises 6-15 (e.g., 6-10, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94 and Z comprises 3 to 7 (e.g., 3 to 5, 3, 4, 5, 6, or 7) linked 2'-deoxyribonucleosides, each cytosine in Y is optionally and independently a 5-methyl-cytosine, and Z comprises 3 to 7 (e.g., 3 to 5, 3, 4, 5, 6, or 7) linked nucleosides, and at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA).

[0255] In some embodiments, the DMPK-targeted oligonucleotide described herein comprises the nucleotide sequence of any one of SEQ ID NOs: 231-362, wherein each thymine base (T) may be independently and optionally replaced with a uracil base (U), and each U may be independently and optionally replaced with a T. In some embodiments, the DMPK-targeted oligonucleotide comprises a 5'-XYZ-3' configuration, wherein X comprises 3-7 (e.g., 3-5, 3, 4, 5, 6, or 7) linked nucleosides, at least one of the nucleosides within X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), and Y comprises 6-15 (e.g., 6-10, 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, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, and Z comprises 3 to 7 (e.g., 3 to 5, 3, 4, 5, 6, or 7) linked 2'-deoxyribonucleosides, each cytosine in Y is optionally and independently a 5-methyl-cytosine, and Z comprises 3 to 7 (e.g., 3 to 5, 3, 4, 5, 6, or 7) linked nucleosides, and at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA).

[0256] In some embodiments, the DMPK-targeting oligonucleotides described herein are 15-25 nucleosides (e.g., 15-20, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and include a region of complementarity to at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleosides) of any one of SEQ ID NOs: 160-230. and 5'-XYZ-3' configuration, where at least one of the nucleosides in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), each cytosine in Y is optionally and independently a 5-methyl-cytosine, and at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each nucleoside in X is a 2'-modified nucleoside and / or (e.g., and) each nucleoside in Z is a 2'-modified nucleoside. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside (eg, LNA, cEt or ENA) or a non-bicyclic 2'-modified nucleoside (eg, a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside).

[0257] In some embodiments, the DMPK-targeting oligonucleotides described herein are 15-25 nucleosides (e.g., 15-20, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and include a region of complementarity to at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleosides) of any one of SEQ ID NOs: 160-230. and at least one of the nucleosides in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), each cytosine in Y is optionally and independently a 5-methyl-cytosine, and at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each nucleoside in X is a non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside), and / or (e.g., and) each nucleoside in Z is a non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside). In some embodiments, each nucleoside in X is a 2'-4' bicyclic nucleoside (e.g., an LNA, cEt or ENA), and / or (e.g., and) each nucleoside in Z is a 2'-4' bicyclic nucleoside (e.g., an LNA, cEt or ENA).

[0258] In some embodiments, the DMPK-targeting oligonucleotides described herein are 15-25 nucleosides (e.g., 15-20, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length and include a region of complementarity to at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleosides) of any one of SEQ ID NOs: 160-230. and at least one of the nucleosides in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), each cytosine in Y is optionally and independently a 5-methyl-cytosine, and at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, X comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside), and / or (e.g., and) Z comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside).

[0259] In some embodiments, the DMPK-targeting oligonucleotides described herein comprise at least 15 contiguous nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleosides) of the nucleotide sequence of any one of SEQ ID NOs:231-362, wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by a T. In some embodiments, the nucleosides include a 5'-XYZ-3' configuration, where at least one of the nucleosides in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), each cytosine in Y is optionally and independently a 5-methyl-cytosine, and at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each nucleoside in X is a 2'-modified nucleoside, and / or (e.g., and) each nucleoside in Z is a 2'-modified nucleoside. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside (eg, LNA, cEt or ENA) or a non-bicyclic 2'-modified nucleoside (eg, a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside).

[0260] In some embodiments, the DMPK-targeting oligonucleotides described herein comprise at least 15 contiguous nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleosides) of the nucleotide sequence of any one of SEQ ID NOs:231-362, wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by a T. and 5'-XYZ-3' configuration, where at least one of the nucleosides in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), each cytosine in Y is optionally and independently a 5-methyl-cytosine, and at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each nucleoside in X is a non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside), and / or (e.g., and) each nucleoside in Z is a non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside). In some embodiments, each nucleoside in X is a 2'-4' bicyclic nucleoside (e.g., an LNA, cEt or ENA), and / or (e.g., and) each nucleoside in Z is a 2'-4' bicyclic nucleoside (e.g., an LNA, cEt or ENA).

[0261] In some embodiments, the DMPK-targeting oligonucleotides described herein comprise at least 15 contiguous nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleosides) of the nucleotide sequence of any one of SEQ ID NOs:231-362, wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by a T. and 5'-XYZ-3' configuration, where at least one of the nucleosides in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), each cytosine in Y is optionally and independently a 5-methyl-cytosine, and at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, X comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside), and / or (e.g., and) Z comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside).

[0262] In some embodiments, the DMPK-targeting oligonucleotide described herein comprises the nucleotide sequence of any one of SEQ ID NOs: 231-362 (wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by a T), and comprises a 5'-XYZ-3' configuration, wherein at least one of the nucleosides within X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), each cytosine within Y is optionally and independently a 5-methyl-cytosine, and at least one of the nucleosides within Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each nucleoside in X is a 2'-modified nucleoside and / or (e.g., and) each nucleoside in Z is a 2'-modified nucleoside. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside (e.g., LNA, cEt or ENA) or a non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside).

[0263] In some embodiments, the DMPK-targeting oligonucleotide described herein comprises the nucleotide sequence of any one of SEQ ID NOs: 231-362 (wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by a T), and comprises a 5'-XYZ-3' configuration, wherein at least one of the nucleosides within X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), each cytosine within Y is optionally and independently a 5-methyl-cytosine, and at least one of the nucleosides within Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each nucleoside in X is a non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside), and / or (e.g., and) each nucleoside in Z is a non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside). In some embodiments, each nucleoside in X is a 2'-4' bicyclic nucleoside (e.g., an LNA, cEt or ENA), and / or (e.g., and) each nucleoside in Z is a 2'-4' bicyclic nucleoside (e.g., an LNA, cEt or ENA).

[0264] In some embodiments, the DMPK-targeting oligonucleotide described herein comprises the nucleotide sequence of any one of SEQ ID NOs: 231-362 (wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by a T), and comprises a 5'-XYZ-3' configuration, wherein at least one of the nucleosides within X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA), each cytosine within Y is optionally and independently a 5-methyl-cytosine, and at least one of the nucleosides within Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, X comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside), and / or (e.g., and) Z comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside).

[0265] In some embodiments, the DMPK targeted oligonucleotide comprises one or more phosphorothioate internucleoside linkages. In some embodiments, each internucleoside linkage in the DMPK targeted oligonucleotide is a phosphorothioate internucleoside linkage. In some embodiments, the DMPK targeted oligonucleotide comprises one or more phosphodiester internucleoside linkages, optionally, the phosphodiester internucleoside linkages are at X and / or Z. In some embodiments, the DMPK targeted oligonucleotide comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages. In some embodiments, the DMPK targeted oligonucleotide comprises 1 phosphodiester internucleoside linkage (PO), 2 PO, 3 PO, 4 PO, 5 PO, 6 PO, 7 PO, 8 PO, 9 PO, 10 PO, 11 PO, 12 PO, 13 PO, 14 PO, 15 PO, 16 PO, 17 PO, 18 PO, 19 PO, 20 PO, 21 PO, 22 PO, 23 PO, 24 PO, 25 PO, 26 PO, 27 PO, 28 PO, or 29 PO, with the remaining internucleoside linkages being phosphorothioate internucleoside linkages (PS). By way of example, a 20 nucleotide DMPK-targeting oligonucleotide may contain 1 PO and 18 PS, 2 PO and 17 PS, 3 PO and 16 PS, 4 PO and 15 PS, 5 PO and 14 PS, 6 PO and 13 PS, 7 PO and 12 PS, 8 PO and 11 PS, 9 PO and 10 PS, 10 PO and 9 PS, 11 PO and 8 PS, 12 PO and 7 PS, 13 PO and 6 PS, 14 PO and 5 PS, 15 PO and 4 PS, 16 PO and 3 PS, 17 PO and 2 PS, or 18 PO and 1 PS.In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate internucleoside linkage, X comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages, and Z comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate internucleoside linkage, and each internucleoside linkage in X is a phosphorothioate internucleoside linkage, and Z comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate internucleoside linkage, X comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages, and each internucleoside linkage in Z is a phosphorothioate internucleoside linkage. By way of example, a DMPK-targeted oligonucleotide may comprise wing regions X and Z with mixed phosphodiester / phosphorothioate backbones and gap region Y with a complete phosphorothioate backbone, or it may comprise one wing region (i.e., X or Z) with a mixed phosphodiester / phosphorothioate backbone, the other wing region with a complete phosphorothioate backbone, and gap region Y with a complete phosphorothioate backbone. In some embodiments, gap region Y comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages, and wing regions X and Y each independently have a complete phosphorothioate backbone or comprise one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages. By way of example, a DMPK-targeted oligonucleotide may comprise wing regions X and Z with a mixed phosphodiester / phosphorothioate backbone and gap region Y with a mixed phosphodiester / phosphorothioate backbone.

[0266] In some embodiments, the formula: X1(E) X2 (L) X3 (D) X4 (L) X5 (E) X6 (L) X7 Antisense oligonucleotides are provided which are wherein each (L) is a 2'-4' bicyclic nucleoside; wherein each (E) is a non-bicyclic 2'-modified nucleoside; wherein each (D) is a 2'-deoxyribonucleoside; In the formula, X1 is independently an integer from 0 to 5 that represents the number of corresponding instances of L; In the formula, X2 is independently an integer from 0 to 5 that represents the number of corresponding instances of E; In the formula, X3 is independently an integer from 0 to 5 that represents the number of corresponding instances of L; In the formula, X4 is independently an integer from 5 to 12 representing the number of instances of D; In the formula, X5 is independently an integer from 0 to 5 that represents the number of corresponding instances of L; In the formula, X6 is independently an integer of 0 to 5 that represents the number of corresponding instances of E; In the formula, X7 is independently an integer of 0 to 5 that represents the number of corresponding instances of L; In the formula, at least one of X1, X2, and X3 is within the range of 1 to 5, and at least one of X5, X6, and X7 is within the range of 1 to 5.

[0267] In some embodiments, X1, X3, X5 and X7 are each 0, and X2 and X6 are independently 1, 2, 3, 4 or 5.

[0268] In some embodiments, X1, X2, X5 and X6 are each 0, and X3 and X7 are independently 1, 2, 3, 4 or 5.

[0269] In some embodiments, X3 and X5 are each 0, and X1, X2, X6 and X7 are independently 1, 2, 3, 4 or 5.

[0270] In some embodiments, X1 and X7 are each 0, and X2, X3, X5 and X6 are independently 1, 2, 3, 4 or 5.

[0271] In some embodiments, X4 is 5, 6, 7, 8, 9 or 10.

[0272] In some embodiments, the 2'-4' bicyclic nucleoside is selected from an LNA, cEt, and ENA nucleoside. In some embodiments, the non-bicyclic 2'-modified nucleoside is a 2'-MOE modified nucleoside or a 2'-OMe modified nucleoside.

[0273] In some embodiments, the nucleosides of the oligonucleotide are linked together by phosphorothioate internucleoside linkages, phosphodiester internucleoside linkages, or a combination thereof. In some embodiments, the oligonucleotide contains only phosphorothioate internucleoside linkages linking each nucleoside. In some embodiments, the oligonucleotide contains at least one phosphorothioate internucleoside linkage. In some embodiments, the oligonucleotide contains a mixture of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In some embodiments, the oligonucleotide contains only phosphorothioate internucleoside linkages linking each pair of 2'-deoxyribonucleosides, and a mixture of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages linking the remaining nucleosides.

[0274] In some embodiments, the oligonucleotide comprises the following 5'-XYZ-3' configuration:

number

[0275] wherein "E" is a 2'-MOE modified ribonucleoside; "L" is LNA; "D" is a 2'-deoxyribonucleoside; and "10" or "8" is the number of 2'-deoxyribonucleosides in Y, and the oligonucleotide comprises phosphorothioate internucleoside linkages, phosphodiester internucleoside linkages, or a combination thereof.

[0276] In some embodiments, in any one of the DMPK-targeting oligonucleotides described herein, each cytidine (e.g., a 2'-modified cytidine) within X and / or Z is optionally and independently a 5-methyl-cytidine, and / or each uridine (e.g., a 2'-modified uridine) within X and / or Z is optionally and independently a 5-methyl-uridine.

[0277] In some embodiments, the DMPK targeting oligonucleotide is selected from the ASOs listed in Tables 8, 9 and 10. In some embodiments, the DMPK targeting oligonucleotide is complementary to a target sequence listed in Table 8.

[0278] In some embodiments, the DMPK targeting oligonucleotide is complementary to any one of SEQ ID NOs: 205, 211, 214, 217, 222, 215, 220 and 225. In some embodiments, the DMPK targeting oligonucleotide is complementary to any one of SEQ ID NOs: 205, 214, 215 and 220. In some embodiments, the DMPK targeting oligonucleotide is complementary to any one of SEQ ID NOs: 211, 217, 222 and 225. In some embodiments, the DMPK targeting oligonucleotide is complementary to any one of SEQ ID NOs: 205, 214, 217 and 222. In some embodiments, the DMPK targeting oligonucleotide is complementary to any one of SEQ ID NOs: 211, 215, 220 and 225.

[0279] In some embodiments, the DMPK targeting oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 276, 282, 285, 286, 288, 291, 293, 296, 345, 348, 350, 352, 354, and 357. In some embodiments, the DMPK targeting oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 276, 285, 286, 291, 348, and 352. In some embodiments, the DMPK targeting oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 282, 288, 293, 296, 345, 350, 354, and 357. In some embodiments, the DMPK targeting oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 276, 285, 288, 293, 348, 350, and 354. In some embodiments, the DMPK targeted oligonucleotide comprises the nucleobase sequence of any one of SEQ ID NOs: 282, 286, 291, 296, 345, 352, and 357. In some embodiments, each thymine base (T) in the DMPK targeted oligonucleotide may be independently and optionally replaced with a uracil base (U), and each U may be independently and optionally replaced with a T.

[0280] In some embodiments, the DMPK targeting oligonucleotide comprises a structure selected from the following:

number

[0281] In some embodiments, the DMPK targeting oligonucleotide comprises a structure selected from the following:

number

[0282] In some embodiments, the DMPK targeting oligonucleotide comprises a structure selected from the following:

number

[0283] In some embodiments, the DMPK targeting oligonucleotide comprises a structure selected from the following:

number

[0284] In some embodiments, the DMPK targeting oligonucleotide comprises a structure selected from the following:

number

[0285] In some embodiments, any one of the DMPK targeting oligonucleotides can be in a salt form, for example, as a sodium salt, a potassium salt, or a magnesium salt.

[0286] In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8, Table 9, and Table 10) is conjugated to an amine group, optionally via a spacer. In some embodiments, the spacer comprises an aliphatic moiety. In some embodiments, the spacer comprises a polyethylene glycol moiety. In some embodiments, a phosphodiester linkage is present between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8, Table 9, and Table 10) is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A-, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)NR A -, -NR A S(O)-, or a combination thereof, A is independently hydrogen or substituted or unsubstituted alkyl. In certain embodiments, the spacer is a substituted or unsubstituted alkylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )- or -C(=O)N(R A )2, or a combination thereof.

[0287] In some embodiments, the 5' or 3' nucleoside of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Tables 8, 9 and 10) has the formula -NH2-(CH2) n -, where n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the phosphodiester linkage is of the formula NH2-(CH2) n - is present between the compound of formula NH2-(CH2)6- and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to the oligonucleotide by reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide.

[0288] In some embodiments, the oligonucleotide is conjugated to a targeting agent, e.g., a muscle targeting agent, such as an anti-TfR1 antibody, e.g., via an amine group.

[0289] C. Linker The conjugates described herein generally comprise a linker that covalently connects any one of the anti-TfR1 antibodies described herein to a molecular payload. The linker comprises at least one covalent bond. In some embodiments, the linker can be a single bond, such as a disulfide bond or a disulfide bridge, that covalently connects the anti-TfR1 antibody to the molecular payload. However, in some embodiments, the linker can covalently connect any one of the anti-TfR1 antibodies described herein to the molecular payload through multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker. The linker is typically stable in vitro and in vivo, and can be stable in a particular cellular environment. In addition, the linker typically does not negatively affect the functional properties of either the anti-TfR1 antibody or the molecular payload. Examples and methods for the synthesis of linkers 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.).

[0290] The linker typically contains two different reactive species that allow attachment to both the anti-TfR1 antibody and the molecular payload. In some embodiments, the two different reactive species can be nucleophiles and / or electrophiles. In some embodiments, the linker contains two different electrophiles or nucleophiles that are specific for two different nucleophiles or electrophiles. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody via conjugation to a lysine or cysteine ​​residue of the anti-TfR1 antibody. In some embodiments, the linker is covalently linked to a cysteine ​​residue of the anti-TfR1 antibody via a maleimide-containing linker, optionally comprising a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some embodiments, the linker is covalently linked to a cysteine ​​residue of the anti-TfR1 antibody or a thiol-functionalized molecular payload via a 3-arylpropionitrile functional group. In some embodiments, the linker is covalently linked to a lysine residue of the anti-TfR1 antibody. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) the molecular payload, independently, via an amide bond, a carbamate bond, a hydrazide, a triazole, a thioether, and / or a disulfide bond.

[0291] i. Cleavable Linker The cleavable linker may be a protease-, pH- or glutathione-sensitive linker. These linkers are typically only cleavable intracellularly and are preferably stable in the extracellular environment, e.g., outside of muscle cells or CNS cells.

[0292] Protease-sensitive linkers are cleavable by protease enzyme activity. These linkers typically comprise peptide sequences and can be 2-10 amino acids long, about 2-5 amino acids long, about 5-10 amino acids long, about 10 amino acids long, about 5 amino acids long, about 3 amino acids long, or about 2 amino acids long. In some embodiments, the peptide sequences can comprise naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the protease-sensitive linkers comprise a valine-citrulline or alanine-citrulline sequence. In some embodiments, the protease-sensitive linkers can be cleaved by lysosomal proteases, such as cathepsin B, and / or endosomal proteases (e.g., and).

[0293] A pH-sensitive linker is a covalent linkage that easily degrades in a high or low pH environment. In some embodiments, the pH-sensitive linker can be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved in an endosome or lysosome.

[0294] In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by disulfide exchange reaction with glutathione species inside the cell. In some embodiments, the disulfide moiety further comprises at least one amino acid, for example, a cysteine ​​residue.

[0295] In some embodiments, the linker comprises a valine-citrulline sequence (e.g., as described in U.S. Patent No. 6,214,345, incorporated herein by reference). In some embodiments, prior to conjugation, the linker comprises the following structure: [ka]

[0296] In some embodiments, after conjugation, the linker comprises the following structure: [ka]

[0297] In some embodiments, prior to conjugation, the linker comprises the following structure: [ka] In the formula, n is any number from 0 to 10. In some embodiments, n is 3.

[0298] In some embodiments, the linker comprises the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3 and / or (e.g., and) m is 4.

[0299] In some embodiments, the linker comprises the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3 and / or (e.g., and) m is 4.

[0300] ii. Non-cleavable linkers In some embodiments, a non-cleavable linker may be used. In general, a non-cleavable linker cannot be easily degraded in a cellular or physiological environment. In some embodiments, a non-cleavable linker comprises an optionally substituted alkyl group, where the substitution may include halogen, hydroxyl group, oxygen species, and other common substitutions. In some embodiments, the linker may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one unnatural amino acid, a cleavable glycan, a sugar or sugars that cannot be enzymatically degraded, an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, a biotin, a biphenyl, a repeat unit of polyethylene glycol or an equivalent compound, an acid ester, an acid amide, a sulfamide, and / or an alkoxy-amine linker. In some embodiments, a sortase-mediated ligation is carried out to link an anti-TfR1 antibody comprising an LPXT sequence to a (G) n It can be used to covalently link molecular payloads containing sequences (see, e.g., Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1):1-10).

[0301] In some embodiments, the linker may comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O and S, an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O and S, an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), such as polyethylene oxide or polypropylene oxide. In some embodiments, the linker may be a non-cleavable N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS) linker.

[0302] iii. Linker conjugation In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) the molecular payload via phosphate, thioether, ether, carbon-carbon, carbamate, or amide bond. In some embodiments, the linker is covalently linked to the oligonucleotide via a phosphate group or a phosphorothioate group, e.g., the terminal phosphate of the oligonucleotide backbone. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody via a lysine or cysteine ​​residue present on the anti-TfR1 antibody.

[0303] In some embodiments, the linker or a portion thereof is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload by a cycloaddition reaction between an azide and an alkyne to form a triazole, and the azide or alkyne can be located on the anti-TfR1 antibody, the molecular payload, or the linker. In some embodiments, the alkyne can be a cyclic alkyne, e.g., a cyclooctyne. In some embodiments, the alkyne can be a bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctyne is as described in International Patent Application Publication WO2011136645, entitled "Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions," published on November 3, 2011. In some embodiments, the azide can be an azide-containing sugar or carbohydrate molecule. In some embodiments, the azide can be 6-azido-6-deoxygalactose, or 6-azido-N-acetylgalactosamine. In some embodiments, the sugar or carbohydrate molecule containing the azide is as described in International Patent Application Publication WO2016170186, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase," published on October 27, 2016.In some embodiments, the cycloaddition reaction between an azide and an alkyne (wherein the azide and alkyne can be disposed on an anti-TfR1 antibody, a molecular payload, or a linker) to form a triazole is as described in International Patent Application Publication No. WO2014065661, published on May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or International Patent Application Publication No. WO2016170186, published on October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase."

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

[0305] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload by a Diels-Alder reaction between a dienophile and a diene / hetero-diene, where the dienophile or diene / hetero-diene can be disposed on the anti-TfR1 antibody, the molecular payload or the linker. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload by other pericyclic reactions, e.g., ene reactions. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload by an amide, thioamide or sulfonamide coupling reaction. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload by a condensation reaction forming an oxime group, a hydrazone group or a semicarbazide group present between the linker and the anti-TfR1 antibody and / or (e.g., and) molecular payload.

[0306] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) the molecular payload by the conjugate addition reaction between a nucleophile, such as an amine group or a hydroxyl group, and an electrophile, such as a carboxylic acid, carbonate, or aldehyde. In some embodiments, the nucleophile can be present on the linker and the electrophile can be present on the anti-TfR1 antibody or the molecular payload prior to the reaction between the linker and the anti-TfR1 antibody or the molecular payload. In some embodiments, the electrophile can be present on the linker and the nucleophile can be present on the anti-TfR1 antibody or the molecular payload prior to the reaction between the linker and the anti-TfR1 antibody or the molecular payload. In some embodiments, the electrophile can be an azide, pentafluorophenyl, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimidyl ester, sulfosuccinimidyl ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center, and / or activated sulfur center. In some embodiments, the nucleophile can be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilide group, and / or a thiol group.

[0307] In some embodiments, the linker comprises a valine-citrulline sequence covalently linked to a reactive chemical moiety (e.g., an azide or BCN moiety for click chemistry). In some embodiments, a linker comprising a valine-citrulline sequence covalently linked to a reactive chemical moiety (e.g., an azide moiety for click chemistry) comprises the following structure: [ka] In the formula, n is any number from 0 to 10. In some embodiments, n is 3.

[0308] In some embodiments, a linker comprising the structure of formula (A) is covalently linked (e.g., optionally via an additional chemical moiety) to a molecular payload (e.g., an oligonucleotide). In some embodiments, a linker comprising the structure of formula (A) is covalently linked to an oligonucleotide, e.g., through nucleophilic displacement by an amine-L1-oligonucleotide forming a carbamate bond, resulting in a compound comprising the following structure: [ka] In the formula, n is any number from 0 to 10. In some embodiments, n is 3.

[0309] In some embodiments, the compound of formula (B) is further covalently linked to an additional moiety via a triazole, which is formed by a click reaction between an azide of formula (A) or formula (B) and an alkyne provided on the bicyclononyne. In some embodiments, the bicyclononyne-containing compound comprises the following structure: [ka] In the formula, m is any number from 0 to 10. In some embodiments, m is 4.

[0310] In some embodiments, the azide of the compound of structure (B) forms a triazole via a click reaction with an alkyne of the compound of structure (C) to form a compound comprising the following structure: [ka] In the formula, n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3 and m is 4.

[0311] In some embodiments, the compound of structure (D) is further covalently linked to a lysine of an anti-TfR1 antibody to form a conjugate comprising the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3 and / or (e.g., and) m is 4. It is understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine, e.g., lysine epsilon amine, of the anti-TfR1 antibody.

[0312] In some embodiments, the compound of formula (C) is further covalently linked to a lysine of an anti-TfR1 antibody to form a compound comprising the following structure: [ka] wherein m is 0 to 15 (e.g., 4). It is understood that the amide shown adjacent to the anti-TfR1 antibody in formula (F) results from reaction with an amine, e.g., lysine epsilon amine, of the anti-TfR1 antibody.

[0313] In some embodiments, the azide of the compound of structure (B) forms a triazole via a click reaction with an alkyne of the compound of structure (F) to form a conjugate comprising the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3 and / or (e.g., and) m is 4. It is understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine, e.g., lysine epsilon amine, of the anti-TfR1 antibody.

[0314] In some embodiments, an azide of a compound of structure (A) forms a triazole via a click reaction with an alkyne of a compound of structure (F) to form a compound comprising the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3, and / or (for example, and) m is 4. In some embodiments, the oligonucleotide is covalently linked to a compound comprising the structure of formula (G), thereby forming a complex comprising the structure of formula (E). It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (G) results from reaction with an amine, e.g., lysine epsilon amine, of the anti-TfR1 antibody.

[0315] In some embodiments, in any one of the conjugates described herein, the anti-TfR1 antibody is covalently linked through a lysine of the anti-TfR1 antibody to a molecular payload (e.g., an oligonucleotide) via a linker comprising the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3 and / or (e.g., and) m is 4.

[0316] In some embodiments, in any one of the conjugates described herein, the anti-TfR1 antibody is covalently linked through a lysine of the anti-TfR1 antibody to a molecular payload (e.g., an oligonucleotide) via a linker comprising the following structure: [ka] wherein n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3 and / or (e.g., and) m is 4.

[0317] In some embodiments, in formulas (B), (D), (E) and (I), L is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)NR A -, -NR A S(O)-, or a combination thereof, where R A is independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L is [ka] and In the formula, L2 is [ka] or [ka] where a represents a moiety directly linked to the carbamate moiety of formulas (B), (D), (E) and (I) and b represents a moiety covalently linked (directly or via an additional chemical moiety) to an oligonucleotide.

[0318] In some embodiments, L1 is [ka] and wherein a represents a moiety directly linked to the carbamate moiety of formulas (B), (D), (E) and (I), and b represents a moiety covalently linked (directly or via an additional chemical moiety) to an oligonucleotide.

[0319] In some embodiments, L1 is [ka] It is.

[0320] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide. In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide. In some embodiments, the linkage of L1 to the 5' phosphate of the oligonucleotide forms a phosphodiester bond between L1 and the oligonucleotide.

[0321] In some embodiments, L1 is optional (eg, does not have to be present).

[0322] In some embodiments, any one of the conjugates described herein has the following structure: [ka] wherein n is 0 to 15 (e.g., 3) and m is 0 to 15 (e.g., 4). It is understood that the amide shown adjacent to the anti-TfR1 antibody in formula (J) results from reaction with an amine, e.g., lysine epsilon amine, of the anti-TfR1 antibody.

[0323] In some embodiments, any one of the conjugates described herein has the following structure: [ka] In the formula, n is 0 to 15 (eg, 3), and m is 0 to 15 (eg, 4).

[0324] In some embodiments, oligonucleotides are modified to contain an amine group at the 5' or 3' end, or internally (e.g., as an amine-functionalized nucleobase), prior to linking to a compound, e.g., a compound of Formula (A) or Formula (G).

[0325] Although the linker conjugations are described in relation to anti-TfR1 antibodies and oligonucleotide molecular payloads, it should be understood that the use of such linker conjugations to other muscle targeting agents, including other muscle targeting antibodies, and / or other molecular payloads, is contemplated.

[0326] D. Examples of Antibody-Molecular Payload Conjugates Further provided herein are non-limiting examples of conjugates comprising any of the anti-TfR1 antibodies described herein covalently linked to any of the molecular payloads (e.g., oligonucleotides) described herein. In some embodiments, the anti-TfR1 antibody (e.g., any one of the anti-TfR1 antibodies provided in Tables 2-7) is covalently linked to the molecular payload (e.g., an oligonucleotide, e.g., the oligonucleotides provided in Tables 8, 9, and 10) via a linker. Any of the linkers described herein may be used. In some embodiments, when the molecular payload is an oligonucleotide, the linker is linked to the 5' end of the oligonucleotide, the 3' end of the oligonucleotide, or an internal site of the oligonucleotide. In some embodiments, the linker is linked to the anti-TfR1 antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the anti-TfR1 antibody). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is linked to the antibody (e.g., the anti-TfR1 antibody described herein) via an amine group (e.g., via a lysine in the antibody). In some embodiments, the molecular payload is a DMPK-targeting oligonucleotide (eg, a DMPK-targeting oligonucleotide listed in Table 8, Table 9, or Table 10).

[0327] An example structure of a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload via a linker is provided below: [ka] wherein the linker is linked to the antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody). In some embodiments, the molecular payload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8, Table 9, or Table 10).

[0328] Another example of the structure of a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload via a linker is provided below: [ka] wherein n is a number from 0 to 10, m is a number from 0 to 10, and the linker is linked to the antibody via an amine group (e.g., on a lysine residue) and / or (e.g., and) the linker is linked to the oligonucleotide (e.g., at the 5' end, 3' end, or internally). In some embodiments, the linker is linked to the antibody via a lysine, the linker is linked to the oligonucleotide at the 5' end, n is 3, and m is 4. In some embodiments, the molecular payload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8, Table 9, or Table 10).

[0329] In some embodiments, L1 is [ka] It is understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine, for example the lysine epsilon amine, of the anti-TfR1 antibody.

[0330] Antibodies can be linked to molecular payloads with various stoichiometries, and this property can be referred to as the drug-antibody ratio (DAR), with the understanding that the "drug" is the molecular payload. In some embodiments, one molecular payload is linked to the antibody (DAR=1). In some embodiments, two molecular payloads are linked to the antibody (DAR=2). In some embodiments, three molecular payloads are linked to the antibody (DAR=3). In some embodiments, four molecular payloads are linked to the antibody (DAR=4). In some embodiments, a mixture of different conjugates, each having a different DAR, is provided. In some embodiments, the average DAR of the conjugates in such a mixture can range from 1 to 3, 1 to 4, 1 to 5, or more. The DAR can be increased by conjugating the molecular payloads to various sites on the antibody and / or by conjugating (e.g., and) multimers to one or more sites on the antibody. By way of example, a DAR of 2 can be achieved by conjugating a single molecular payload to two different sites on the antibody, or by conjugating a dimeric molecular payload to a single site on the antibody.

[0331] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody described herein (e.g., an antibody provided in Tables 2-7) covalently linked to a molecular payload. In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody described herein (e.g., an antibody provided in Tables 2-7) covalently linked to a molecular payload via a linker (e.g., a linker comprising a valine-citrulline sequence). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is linked to an antibody (e.g., an anti-TfR1 antibody described herein) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is linked to an antibody (e.g., an anti-TfR1 antibody described herein) via an amine group (e.g., via a lysine in the antibody). In some embodiments, the molecular payload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Tables 8, 9, or 10).

[0332] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of any one of the antibodies listed in Table 2. In some embodiments, the molecular payload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8, Table 9 or Table 10).

[0333] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 71, or SEQ ID NO: 72, and a VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0334] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0335] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the molecular payload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8, Table 9, or Table 10).

[0336] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77, and a VL comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the molecular payload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8, Table 9, or Table 10).

[0337] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 79, and a VL comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0338] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 154, and a VL comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0339] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 86, or SEQ ID NO: 87, and a light chain comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0340] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0341] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91, and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0342] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 94, and a light chain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0343] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 92 and a light chain comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0344] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 156, and a light chain comprising the amino acid sequence of SEQ ID NO: 157. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0345] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99, and a light chain comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0346] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101, and a light chain comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0347] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101, and a light chain comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0348] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102, and a light chain comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0349] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 102 or SEQ ID NO: 103, and a light chain comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0350] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 158 or SEQ ID NO: 159, and a light chain comprising the amino acid sequence of SEQ ID NO: 157. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8, Table 9, or Table 10).

[0351] In any of the exemplary conjugates described herein, in some embodiments, the anti-TfR1 antibody is covalently linked to the molecular payload via a linker comprising the following structure: [ka] In the formula, n is 3, m is 4, and L1 is [ka] It is.

[0352] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to the 5' end of a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8, Table 9 or Table 10) via a lysine in the anti-TfR1 antibody, wherein the anti-TfR1 antibody comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 of any one of the antibodies listed in Table 2, and the conjugate has the following structure: [ka] In the formula, n is 3, m is 4, and L1 is [ka] It is understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine, for example, the lysine epsilon amine, of the anti-TfR1 antibody.

[0353] In some embodiments, the conjugate described herein comprises an anti-TfR1 antibody covalently linked to the 5' end of a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8, Table 9 or Table 10) via a lysine in the anti-TfR1 antibody, wherein the anti-TfR1 antibody comprises a VH and VL of any one of the antibodies listed in Table 3, and the conjugate has the following structure: [ka] In the formula, n is 3, m is 4, and L1 is [ka] It is understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine, for example, the lysine epsilon amine, of the anti-TfR1 antibody.

[0354] In some embodiments, the conjugate described herein comprises an anti-TfR1 antibody covalently linked to the 5' end of a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8, Table 9 or Table 10) via a lysine in the anti-TfR1 antibody, wherein the anti-TfR1 antibody comprises the heavy and light chains of any one of the antibodies listed in Table 4, and the conjugate has the following structure: [ka] In the formula, n is 3, m is 4, and L1 is [ka] It is understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine, for example, the lysine epsilon amine, of the anti-TfR1 antibody.

[0355] In some embodiments, the conjugates described herein comprise an anti-TfR1 Fab covalently linked to the 5' end of a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8, Table 9 or Table 10) via a lysine in the anti-TfR1 antibody, wherein the anti-TfR1 Fab comprises the heavy and light chains of any one of the antibodies listed in Table 5, and the conjugate has the following structure: [ka] In the formula, n is 3, m is 4, and L1 is [ka] It is understood that the amide shown adjacent to the anti-TfR1 antibody in formula (E) results from reaction with an amine, for example, the lysine epsilon amine, of the anti-TfR1 antibody.

[0356] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide. In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide. In some embodiments, the linkage of L1 to the 5' phosphate of the oligonucleotide forms a phosphodiester bond between L1 and the oligonucleotide.

[0357] In some embodiments, L1 is optional (eg, does not have to be present).

[0358] In some embodiments, the DMPK-targeting oligonucleotide of the conjugate described herein comprises a structure selected from the following:

number

[0359] III. Preparations The complexes provided herein may be formulated in any suitable manner. In general, the complexes provided herein are formulated in a manner suitable for pharmaceutical use. For example, the complexes can be delivered to a subject using a formulation that minimizes degradation, facilitates delivery and / or (e.g., and) uptake, or provides another beneficial property to the complex in the formulation. In some embodiments, a composition is provided herein that includes a complex and a pharma- ceutically acceptable carrier. Such a composition can be suitably formulated so that, upon administration to a subject, a sufficient amount of the complex enters a target cell (e.g., a muscle cell or a CNS cell), either in the environment surrounding the target cell, or in the entire body. In some embodiments, the complexes are formulated in a buffer solution, for example, a phosphate buffered saline solution, a liposome, a micellar structure, and a capsid.

[0360] It should be understood that in some embodiments, a composition may separately include one or more components of the conjugates provided herein (e.g., a muscle targeting agent, a linker, a molecular payload, or a precursor molecule of any one of these).

[0361] In some embodiments, the complex is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the complex is formulated in a basic buffered aqueous solution (e.g., PBS). In some embodiments, the formulations disclosed herein include an excipient. In some embodiments, the excipient confers improved stability, improved absorption, improved solubility, and / or therapeutic enhancement of the active ingredient (e.g., and) to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or a vehicle (e.g., a buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil).

[0362] In some embodiments, the complex or its components (e.g., oligonucleotides or antibodies) are lyophilized to extend their shelf life and then brought into solution prior to use (e.g., administration to a subject). Thus, the excipient in the composition comprising the complex or its components described herein can be a cryoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0363] In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, e.g., intravenous administration, intradermal administration, subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous.

[0364] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (if water soluble) or dispersions, and sterile powders for extemporaneous preparation of sterile injection solutions or dispersions.Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and suitable mixtures thereof.In some embodiments, the formulations include isotonic agents in the composition, for example, sugars, polyalcohols, for example, mannitol, sorbitol, and sodium chloride.Sterile injection solutions can be prepared by incorporating the required amount of the complex into the selected solvent with one or a combination of the above-listed components, and then sterilizing by filtration if necessary.

[0365] In some embodiments, the composition may contain at least about 0.1% of the complex or its components, or more, but the percentage of active ingredient may be about 1% to about 80% or more by weight or volume of the total composition. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations will be taken into account by those skilled in the art of preparing such pharmaceutical formulations. Therefore, various dosages and treatment regimes may be desirable.

[0366] IV. Methods of Use / Treatment The complex comprising the muscle targeting agent covalently linked to the molecular payload described herein is effective for treating myotonic dystrophy.In some embodiments, the complex is effective for treating myotonic dystrophy type 1 (DM1).In some embodiments, DM1 is associated with the expansion of CTG / CUG trinucleotide repeats in the 3' non-coding region of DMPK.In some embodiments, the nucleotide expansion results in a toxic RNA repeat that can form a hairpin structure that binds with high affinity to important intracellular proteins, such as muscleblind-like proteins.

[0367] In some embodiments, the subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, the subject may have myotonic dystrophy. In some embodiments, the subject may have a DMPK allele that may optionally contain a disease-associated repeat sequence. In some embodiments, the subject may have a DMPK allele that has an expanded disease-associated repeat comprising about 2-10 repeat units, about 2-50 repeat units, about 2-100 repeat units, about 50-1,000 repeat units, about 50-500 repeat units, about 50-250 repeat units, about 50-100 repeat units, about 500-10,000 repeat units, about 500-5,000 repeat units, about 500-2,500 repeat units, about 500-1,000 repeat units, or about 1,000-10,000 repeat units. In some embodiments, the subject suffers from symptoms of DM1, such as muscle atrophy or muscle loss. In some embodiments, the subject does not suffer from symptoms of DM1. In some embodiments, the subject has congenital myotonic dystrophy.

[0368] Aspects of the present disclosure include methods involving administering an effective amount of a conjugate as described herein to a subject. In some embodiments, an effective amount of a pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload can be administered to a subject in need of treatment. In some embodiments, a pharmaceutical composition comprising a conjugate as described herein can be administered by a suitable route, which can include, for example, intravenous administration as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration can be by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, or intrathecal routes. In some embodiments, the pharmaceutical composition can be in solid, aqueous, or liquid form. In some embodiments, the aqueous or liquid form can be sprayed or lyophilized. In some embodiments, the sprayed or lyophilized form can be reconstituted with an aqueous or liquid solution.

[0369] Compositions for intravenous administration may contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (such as glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by drip infusion, whereby a pharmaceutical formulation containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0370] In some embodiments, pharmaceutical compositions comprising a conjugate comprising a muscle-targeting agent covalently linked to a molecular payload are administered via site-specific or localized delivery techniques. Examples of these techniques include an implanted depot source of the conjugate, a localized delivery catheter, a site-specific carrier, direct injection, or direct application.

[0371] In some embodiments, the pharmaceutical composition comprising the complex comprising the muscle targeting agent covalently linked to the molecular payload is administered at an effective concentration that provides a therapeutic effect to the subject. The effective amount will vary according to the severity of the disease, the specific characteristics of the subject being treated, such as age, physical condition, health status or weight, duration of treatment, the nature of any concomitant treatment, route of administration, and related factors, as recognized by those skilled in the art. These related factors are known to those skilled in the art and can be addressed with a degree of routine experimentation. In some embodiments, the effective concentration is the maximum dose that is considered safe for the patient. In some embodiments, the effective concentration is the lowest possible concentration that provides maximum efficacy.

[0372] Empirical considerations, such as the half-life of the conjugate in the subject, will generally help determine the concentration of pharmaceutical composition used for treatment. The frequency of administration can be empirically determined and adjusted to maximize the effectiveness of the treatment.

[0373] The effectiveness of treatment can be assessed using any suitable method.In some embodiments, the effectiveness of treatment can be assessed by observing the symptoms associated with DM1, such as muscle atrophy or muscle weakness, through the subject's self-reported outcomes, such as mobility, self-care, normal activity, pain / discomfort, and anxiety / depression, or by quality of life indicators, such as lifespan.

[0374] In some embodiments, a pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload described herein is administered to a subject at an effective concentration sufficient to inhibit target gene activity or expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to a control, e.g., a baseline level of gene expression prior to treatment. Additional Aspects 1. A conjugate comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured to reduce expression or activity of DMPK, wherein the anti-TfR1 antibody comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3) of any of the anti-TfR1 antibodies listed in Tables 2-7; and the oligonucleotide comprises a 5'-XYZ-3' configuration, X comprises 3 to 7 linked nucleosides, and at least one of the nucleosides within X is a 2'-modified nucleoside; Y comprises 6 to 15 linked 2'-deoxyribonucleosides, and each cytosine within Y is optionally and independently a 5-methyl-cytosine; and A conjugate wherein Z comprises 3 to 7 linked nucleosides, and at least one of the nucleosides in Z is a 2'-modified nucleoside. 2. The conjugate of embodiment 1, X comprises 3 to 5 linked nucleosides, and at least one of the nucleosides within X is a 2'-modified nucleoside; Y comprises 6 to 10 linked 2'-deoxyribonucleosides, and each cytosine within Y is optionally and independently a 5-methyl-cytosine; and A conjugate wherein Z comprises 3 to 5 linked nucleosides, and at least one of the nucleosides in Z is a 2'-modified nucleoside. 3. The conjugate of embodiment 1 or embodiment 2, wherein the anti-TfR1 antibody comprises the heavy chain variable region (VH) and the light chain variable region (VL) of any of the anti-TfR1 antibodies listed in Table 3. 4. The anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 76, and / or a light chain variable region (VL) comprising an amino acid sequence at least 95% identical to SEQ ID NO: 75; 4. The conjugate of any one of aspects 1 to 3, optionally wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO: 75. 5. The conjugate of any one of embodiments 1-3, wherein the anti-TfR1 antibody is a Fab, and optionally the Fab comprises the heavy and light chains of any of the anti-TfR1 Fabs listed in Table 5. 6. the Fab comprises a heavy chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 101, and / or a light chain comprising an amino acid sequence at least 85% identical to SEQ ID NO: 90; The conjugate of embodiment 5, optionally wherein the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90. 7. The conjugate of any one of aspects 1 to 6, wherein the antibody and the oligonucleotide are covalently linked via a linker. 8. The conjugate of embodiment 7, wherein the linker is a cleavable linker. 9. The conjugate of embodiment 7 or embodiment 8, wherein the linker comprises a valine-citrulline sequence. 10. The conjugate of any one of aspects 1-9, wherein the oligonucleotide is 15-25 nucleosides in length and comprises a region of complementarity to at least 15 consecutive nucleosides of any one of SEQ ID NOs: 160-230, and optionally, the oligonucleotide is 15-20 nucleosides in length. 11. The conjugate of any one of aspects 1-10, wherein the oligonucleotide comprises at least 15 contiguous nucleosides of any one of SEQ ID NOs: 231-362, wherein each thymine base (T) is independently and optionally replaced by a uracil base (U), and each U is independently and optionally replaced by a T. 12. The conjugate of any one of embodiments 1-11, wherein each nucleoside within X is a 2'-modified nucleoside and / or each nucleoside within Z is a 2'-modified nucleoside, and optionally each 2'-modified nucleoside is independently a 2'-4' bicyclic nucleoside or a non-bicyclic 2'-modified nucleoside. 13. The conjugate of any one of embodiments 1-12, wherein each nucleoside within X is a non-bicyclic 2'-modified nucleoside and / or each nucleoside within Z is a non-bicyclic 2'-modified nucleoside, and optionally, the non-bicyclic 2'-modified nucleoside is a 2'-MOE modified nucleoside. 14. The oligonucleotide comprises the following 5'-XYZ-3' configuration:

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

number

[0375] example Example 1. In vitro activity of DMPK-targeting oligonucleotides (ASOs) Gapmer antisense oligonucleotides (ASOs) were generated to target DMPK. Each individual oligonucleotide was evaluated for its ability to target DMPK in cells at two doses: 500 pM (low dose) and 50 nM (high dose).

[0376] Briefly, DM1 Cl5 immortalized myoblasts were cultured to near confluence (approximately 80% confluence) in T-75 flasks. Myoblasts were then detached by trypsin and seeded into 96-well microplates at a density of 50,000 cells / well. To induce differentiation into myotubes, cells were allowed to recover overnight, after which the growth medium was washed off and replaced with serum-free medium. Differentiation proceeded for 7 days prior to treatment with DMPK-targeting oligonucleotides.

[0377] On day 7 after induction of differentiation, DM1 Cl5 myotubes were transfected with individual oligonucleotides using 0.3 μL Lipofectamine MessengerMax per well. All oligonucleotides were tested at both 500 pM and 50 nM final concentrations in biological triplicates. After treatment with oligonucleotides, cells were incubated for 72 hours before being harvested for total RNA. cDNA was synthesized from total RNA extracts and qPCR was performed to determine expression levels of DMPK in technical quadruplicates. All qPCR data were analyzed using conventional 2 -ΔΔCT The results were analyzed using the ELISA method and normalized to plate-based negative controls, including cells treated with a vehicle control (0.3 μL / well Lipofectamine MessengerMax without oligonucleotide). The results of these experiments are shown in Table 11. The "remaining transcript" for each antisense oligonucleotide in Table 11 refers to the expression level of DMPK in cells treated with the ASO compared to expression in the negative control vehicle-treated cells (expression level of the negative control was normalized to 1.00).

[0378] The majority of the DMPK-targeting gapmer ASOs tested demonstrated a reduction in DMPK expression in differentiated myotubes at both the low and high dose concentrations tested. These data demonstrate that the ASOs shown in Table 9 are capable of targeting DMPK in cells, and suggest that muscle-targeting complexes comprising antisense oligonucleotides (e.g., DMPK-targeting oligonucleotides provided herein) are capable of targeting DMPK in muscle tissue in vivo. [Table 11]

[0379] Example 2. In vivo activity of conjugates containing anti-TfR1 Fab conjugated to DMPK-targeting oligonucleotides in mice expressing human TfR1 A conjugate containing anti-TfR1 Fab 3M12-VH4 / Vκ3 conjugated to a DMPK-targeting oligonucleotide was tested in a mouse model expressing human TfR1. Anti-TfR1 Fab 3M12-VH4 / Vκ3 was covalently linked to a DMPK-targeting oligonucleotide via a cleavable linker having the structure of formula (I). The conjugate was administered to mice at a dose equivalent to 10 mg / kg oligonucleotide on days 0 and 7. Mice were sacrificed on day 14, and various muscle tissues were collected and analyzed for mouse Dmpk mRNA levels and oligonucleotide concentrations in the tissues. The conjugate reduced mouse wild-type Dmpk by 79% in tibialis anterior (Figure 1A), 76% in gastrocnemius (Figure 1B), 70% in heart (Figure 1C), and 88% in diaphragm (Figure 1D). Oligonucleotide distribution in tibialis anterior, gastrocnemius, heart and diaphragm is shown in Figures 1E-H.

[0380] These data show that anti-TfR1 Fab 3M12-VH4 / Vκ3 enabled the cellular internalization of the conjugate to muscle tissue in an in vivo mouse model, thereby enabling the DMPK-targeting oligonucleotide to reduce the expression of DMPK. Similarly, an anti-TfR1 antibody (e.g., anti-TfR1 Fab 3M12-VH4 / Vκ3) can enable the cellular internalization of a conjugate containing an anti-TfR1 antibody conjugated to another DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide provided herein) to reduce the expression of DMPK.

[0381] Example 3. In vitro activity of conjugates containing anti-TfR1 Fab covalently linked to DMPK-targeting antisense oligonucleotides (ASOs) In vitro experiments were performed to determine the activity of DMPK-targeted antisense oligonucleotides (ASOs) listed in Table 9 in reducing DMPK mRNA expression in rhabdomyosarcoma cells (RD; ATCC, Manassas, VA) and primary DM1-32F cells expressing mutant DMPK mRNA containing 380 CUG repeats (32F cells; Cook MyoSite, Pittsburg, PA) and correcting the BIN1 exon 11 splicing defect in DM1-32F cells. All ASOs were covalently linked to an anti-TfR1 Fab antibody (3M12-VH4 / Vκ3) to form a complex containing the structure of formula (E).

[0382] RD cells were expanded and seeded in 384-well plates at a density of 10,000 cells / well. Cells were allowed to recover overnight at 37°C. The next day, medium was changed and cells were treated with Fab-ASO complexes at the equivalent of 1,000 nM ASO and incubated for 72 h. After 72 h, total RNA was extracted and cDNA was generated using the TaqMan Fast-Advanced Cells-to-Ct kit (ThermoFisher Scientific, Waltham, MA). cDNA was used to assess total DMPK knockdown using a specific TaqMan PCR assay (ThermoFisher Scientific). Data were normalized to PPIB expression and 2 μg / mL was used to determine residual DMPK expression compared to vehicle-treated control cells. -ΔΔCt The methods used were (Table 12).

[0383] DM1 32F primary cells were thawed, allowed to recover, and then seeded in 384-well plates at a density of 10,000 cells / well in growth medium. The next day, the growth medium was replaced with low-serum differentiation medium, and the cells were treated with Fab-ASO complexes at 10, 100, or 1,000 nM ASO equivalents. Cells were incubated with the complexes for 10 days, then total RNA was extracted and cDNA was generated using the TaqMan Fast-Advanced Cells-to-Ct kit.

[0384] cDNA was used to assess total DMPK knockdown using a specific TaqMan PCR assay. Data were normalized to PPIB expression and were then compared to vehicle-only controls to determine DMPK knockdown. -ΔΔCtThe DMPK expression method was used. Data are presented as residual DMPK expression compared to vehicle-treated control cells (Table 12). In addition, DM1-mediated aberrant splicing modifications were evaluated using a multiplex TaqMan qPCR assay (ThermoFisher Scientific) to assess aberrantly spliced ​​and normal transcripts. Since the exclusion of exon 11 from BIN1 is associated with DM1, BIN1 transcripts encompassing exon 11 were measured. These data are presented as the average ratio of aberrantly spliced ​​cells to normal cells compared to vehicle-treated cells (Table 13). A ratio of 1 indicates that no aberrant splicing changes were observed when compared to DM1 patient myotubes treated with vehicle control. A ratio greater than 1 indicates that more transcripts had wild-type splicing patterns. A ratio less than 1 indicates that more transcripts had DM1-associated splicing patterns.

[0385] These data indicate that anti-TfR1 Fab 3M12-VH4 / Vκ3 enabled cellular internalization of the Fab-ASO complex, thereby enabling the DMPK-targeted ASO to reduce DMPK mRNA expression and promote correction of the BIN1 exon 11 splicing defect. Similarly, an anti-TfR1 antibody (e.g., anti-TfR1 Fab 3M12-VH4 / Vκ3) can enable cellular internalization of a conjugate containing an anti-TfR1 antibody conjugated to another DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide provided herein) to reduce DMPK expression and promote its downstream effects (e.g., correction of the DM1-associated splicing defect). [Table 12-1] [Table 12-2] [Table 13-1] [Table 13-2]

[0386] Example 4. In vitro activity of conjugates containing anti-TfR1 Fab covalently linked to DMPK-targeting antisense oligonucleotides (ASOs) In vitro experiments were performed to determine the activity of conjugates containing the DMPK-targeted antisense oligonucleotides (ASOs) listed in Table 10 covalently linked to anti-TfR1 Fab (3M12-VH4 / Vκ3) in reducing DMPK mRNA expression in rhabdomyosarcoma cells (RD; ATCC, Manassas, VA) and primary DM1-32F cells expressing mutant DMPK mRNA containing 380 CUG repeats (32F cells; Cook MyoSite, Pittsburg, PA) and correcting the BIN1 exon 11 splicing defect in DM1-32F cells. All ASOs were covalently linked to anti-TfR1 Fab antibody (3M12-VH4 / Vκ3) to form a complex containing the structure of formula (E).

[0387] RD cells were expanded and seeded in 384-well plates at a density of 10,000 cells / well. Cells were allowed to recover overnight at 37°C. The next day, medium was changed and cells were treated with Fab-ASO complexes at the equivalent of 100 nM ASO and incubated for 72 h. After 72 h, total RNA was extracted and cDNA was generated using the TaqMan Fast-Advanced Cells-to-Ct kit (ThermoFisher Scientific, Waltham, MA). cDNA was used to assess total DMPK knockdown using a specific TaqMan PCR assay (ThermoFisher Scientific). Data were normalized to PPIB expression and 2 μg / mL was used to determine DMPK expression in conjugate-treated cells compared to vehicle-treated control cells. -ΔΔCtThe method was used (Table 14). Data are presented as percent knockdown, with more positive values ​​indicating greater knockdown of DMPK expression.

[0388] DM1 32F primary cells were thawed, allowed to recover, and then seeded in 384-well plates at a density of 10,000 cells / well in growth medium. The next day, the growth medium was replaced with low-serum differentiation medium, and the cells were treated with Fab-ASO complexes at 10, 100, or 1,000 nM ASO equivalents. Cells were incubated with the complexes for 10 days, then total RNA was extracted and cDNA was generated using the TaqMan Fast-Advanced Cells-to-Ct kit.

[0389] cDNA was used to assess total DMPK knockdown using a specific TaqMan PCR assay. Data were normalized to PPIB expression and were then compared 2× to determine DMPK expression in conjugate-treated cells compared to vehicle-treated control cells. -ΔΔCt The DMPK knockdown method was used (Table 14). Data are presented as percent knockdown, with more positive values ​​indicating greater knockdown of DMPK expression and negative values ​​indicating no detectable DMPK knockdown in conjugate-treated cells compared to corresponding vehicle-treated control cells.

[0390] In addition, DM1-mediated aberrant splicing modification was evaluated using a multiplex TaqMan qPCR assay (ThermoFisher Scientific) to evaluate aberrantly spliced ​​and normal BIN1 transcripts in DM1 32F primary cells treated with 100 nM ASO equivalent Fab-ASO complexes. These data are presented as the average ratio of aberrantly spliced ​​BIN1 to normal BIN1 in Fab-ASO complex-treated cells compared to vehicle-treated cells (Table 14). A ratio of 1 indicates that no aberrant splicing change was observed when compared to DM1 patient myotubes treated with vehicle control. A ratio greater than 1 indicates that more transcripts had wild-type splicing patterns in cells treated with Fab-ASO complexes compared to cells treated with vehicle control. A ratio less than 1 may indicate that more transcripts had DM1-associated splicing patterns.

[0391] All of the conjugates tested achieved DMPK knockdown in at least one of the cell types tested, and all promoted some degree of correction of DM1-mediated aberrant splicing. Conjugates containing ASO47, ASO55, ASO58, ASO61, ASO66, ASO71, ASO76, and ASO81 performed best.

[0392] These data indicate that anti-TfR1 Fab 3M12-VH4 / Vκ3 enabled cellular internalization of the Fab-ASO complex, thereby enabling the DMPK-targeted ASO to reduce DMPK mRNA expression and promote correction of the BIN1 exon 11 splicing defect. Similarly, an anti-TfR1 antibody (e.g., anti-TfR1 Fab 3M12-VH4 / Vκ3) can enable cellular internalization of a conjugate containing an anti-TfR1 antibody conjugated to another DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide provided herein) to reduce DMPK expression and promote its downstream effects (e.g., correction of the DM1-associated splicing defect). [Table 14-1] [Table 14-2]

[0393] Example 5. Knockdown activity of DMPK-targeting oligonucleotides (ASOs) in hTfR1 / DMSXL hemizygous mice Conjugates containing anti-TfR1 Fab 3M12-VH4 / Vκ3 covalently linked to DMPK-targeting oligonucleotides (ASO58, ASO47, ASO61 or ASO66) were tested in mice expressing both human TfR1 and a mutant human DMPK transgene with an expanded CTG repeat (hTfR1 / DMSXL mice). The anti-TfR1 Fab was covalently linked to each ASO via a cleavable linker having the structure of formula (I). Mice were administered vehicle control (PBS) or an ASO-equivalent dose of 7.5 mg / kg ASO 58 conjugate), 8.8 mg / kg (ASO47 conjugate), 8.1 mg / kg (ASO61 conjugate) or 5.6 mg / kg (ASO66 conjugate) of the anti-TfR1 Fab-ASO conjugate on days 0 and ...

Claims

**Claim 1** A complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide configured to reduce the expression or activity of DMPK, wherein the anti-TfR1 antibody comprises any of the heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3) of the anti-TfR1 antibodies listed in Tables 2-7, and the oligonucleotide comprises a 5'-X-Y-Z-3' configuration, where X comprises 3 to 7 linked nucleosides, at least one of the nucleosides in X being a 2'-modified nucleoside; Y comprises 6 to 15 linked 2'-deoxyribonucleosides, each cytosine in Y being optionally and independently 5-methyl-cytosine; and Z comprises 3 to 7 linked nucleosides, at least one of the nucleosides in Z being a 2'-modified nucleoside; and the oligonucleotide comprises a complementary region to at least 15 consecutive nucleosides of any one of SEQ ID NOs: 222, 205, 214, 217, 211, 215, 220, 225, 160-204, 206-210, 212, 213, 216, 218, 219, 221, 223, 224, and 226-230, optionally, the anti-TfR1 antibody comprises CDR-H1, CDR-H2, and CDR-H3 of the heavy chain variable region (VH) described in SEQ ID NO: 76, and / or CDR-L1, CDR-L2, and CDR-L3 of the light chain variable region (VL) described in SEQ ID NO: 75, said complex. **Claim 2** The complex according to claim 1, wherein X comprises 3 to 5 linked nucleosides, at least one of the nucleosides in X being a 2'-modified nucleoside; Y comprises 6 to 10 linked 2'-deoxyribonucleosides, each cytosine in Y being optionally and independently 5-methyl-cytosine; and Z comprises 3 to 5 linked nucleosides, at least one of the nucleosides in Z being a 2'-modified nucleoside, said complex. **Claim 3** The complex according to claim 1, wherein the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO: 76, and / or a light chain variable region (VL) comprising an amino acid sequence that is at least 95% identical to SEQ ID NO:

75.

4. The complex according to claim 3, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO:

75.

5. The complex according to claim 1, wherein the anti-TfR1 antibody is a Fab.

6. The complex according to claim 5, wherein the Fab comprises a heavy chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 101, and / or a light chain comprising an amino acid sequence that is at least 85% identical to SEQ ID NO:

90.

7. The complex according to claim 5, wherein the Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO:

90.

8. The complex according to claim 1, wherein the anti-TfR1 antibody comprises a heavy chain comprising an N-terminal pyroglutamate.

9. The complex according to claim 1, wherein the anti-TfR1 antibody and the oligonucleotide are covalently linked via a cleavable linker.

10. The complex according to claim 9, wherein the cleavable linker comprises a valine-citrulline sequence.

11. The complex according to claim 1, wherein the anti-TfR1 antibody is covalently linked to the oligonucleotide via a non-cleavable linker.

12. The complex according to claim 11, wherein the non-cleavable linker comprises an optionally substituted alkyl group.

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

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

15. The complex according to any one of claims 1 to 14, wherein the oligonucleotide is 15 to 25 nucleoside in length, and optionally, the oligonucleotide is 15 to 20 nucleoside in length.

16. The complex according to any one of claims 1 to 14, wherein the oligonucleotide comprises at least 15 consecutive nucleosides of any one of SEQ ID NOs: 354, 276, 348, 350, 345, 286, 352, 357, 231 to 275, 277 to 285, 287 to 344, 346, 347, 349, 351, 353, 355, 356, and 358 to 362, and each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by T.

17. The complex according to any one of claims 1 to 14, wherein each nucleoside in X is a 2'-modified nucleoside and / or each nucleoside in Z is a 2'-modified nucleoside.

18. The complex according to claim 17, wherein each 2'-modified nucleoside is independently a 2'-4' bicyclic nucleoside or an acyclic 2'-modified nucleoside.

19. The oligonucleotide comprises the following 5'-X-Y-Z-3' configuration: 【Number 1】 wherein "E" is a 2'-MOE modified ribonucleoside; "L" is LNA; "D" is a 2'-deoxyribonucleoside; and "10" or "8" is the number of 2'-deoxyribonucleosides in Y. The complex according to any one of claims 1 to 14.

20. The complex according to any one of claims 1 to 14, wherein the oligonucleotide comprises one or more phosphorothioate internucleoside linkages.

21. The complex according to any one of claims 1 to 14, wherein each internucleoside linkage in the oligonucleotide is a phosphorothioate internucleoside linkage.

22. The complex according to any one of claims 1 to 14, wherein the oligonucleotide comprises one or more phosphodiester internucleoside linkages.

23. The complex according to any one of claims 1 to 14, wherein one or more phosphodiester internucleoside linkages are in X and / or Z.

24. The complex according to any one of claims 1 to 14, wherein the oligonucleotide is in a salt form, and the salt form is a sodium salt, a potassium salt, or a magnesium salt.

25. The oligonucleotide comprises a structure selected from the following: 【Number 2-1】 【Number 2-2】 Here, "xdC" is 5-methyl-deoxycytidine; "dN" is 2'-deoxyribonucleoside; "oN" is 2'-MOE-modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "oU" is 5-methyl-2'-MOE-uridine; "xoG" is 7-methyl-2'-MOE-guanosine; and "*" indicates a phosphorothioate (PS) internucleoside linkage, the complex according to any one of claims 1 to 14.

26. The oligonucleotide is conjugated to an amine group at its 5'-end and contains a structure selected from the following: [Number 3-1] [Number 3-2] Here, "xdC" is 5-methyl-deoxycytidine; "dN" is 2'-deoxyribonucleoside; "oN" is 2'-MOE-modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "oU" is 5-methyl-2'-MOE-uridine; "xoG" is 7-methyl-2'-MOE-guanosine; and "*" indicates a phosphorothioate (PS) internucleoside linkage, the complex according to claim 25.

27. A phosphodiester linkage or other moiety is present between 5'-NH 2 -(CH 2 ) 6 - and the oligonucleotide, the complex according to claim 26.

28. The oligonucleotide contains a structure selected from the following: [Number 4-1] 【Number 4-2】 Here, "xdC" is 5-methyl-deoxycytidine; "dN" is 2'-deoxyribonucleoside; "oN" is 2'-MOE-modified ribonucleoside; "xC" is 5-methyl-2'-MOE-cytidine; "x+C" is 5-methyl LNA cytidine; "+N" is LNA nucleoside; "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl LNA uridine; and "*" indicates a phosphorothioate (PS) internucleoside linkage, the complex according to any one of claims 1 to 14.

29. The oligonucleotide is conjugated to an amine group at its 5'-end and contains a structure selected from the following: 【Number 5-1】 【Number 5-2】 Here, "xdC" is 5-methyl-deoxycytidine; "dN" is a 2'-deoxyribonucleoside; "oN" is a 2'-MOE-modified ribonucleoside; "xoC" is 5-methyl-2'-MOE-cytidine; "x + C" is 5-methyl LNA cytidine; "+ N" is an LNA nucleoside; "oU" is 5-methyl-2'-MOE-uridine; "+ U" is 5-methyl LNA uridine; and "*" indicates a phosphorothioate (PS) internucleoside linkage, the complex according to claim 28.

30. A phosphodiester linkage or other moiety is between 5'-NH 2 -(CH 2 ) 6 - and the oligonucleotide, the complex according to claim 29.

31. A pharmaceutical composition comprising the complex according to any one of claims 1 to 14 for use in a method of treating a disease or condition that can be improved by reducing DMPK expression in muscle cells, the method comprising contacting the muscle cells with the complex.

32. The pharmaceutical composition according to claim 31, wherein reducing DMPK expression in muscle cells comprises reducing the amount of DMPK RNA in muscle cells.

33. The pharmaceutical composition according to claim 31, wherein the amount of DMPK RNA is reduced in the nucleus of muscle cells.

34. The pharmaceutical composition according to claim 31, wherein the DMPK RNA is mutant DMPK mRNA.

35. The pharmaceutical composition according to claim 31, wherein reducing DMPK expression in muscle cells comprises reducing the amount of DMPK protein in muscle cells.

36. A pharmaceutical composition comprising the complex according to any one of claims 1 to 14 for use in a method of treating myotonic dystrophy type 1 (DM1), the method comprising administering the complex to a subject in need thereof.

37. The pharmaceutical composition according to claim 36, wherein the administration results in at least a 30% reduction in DMPK RNA in the muscle cells of the subject.

38. The pharmaceutical composition according to claim 37, wherein the DMPK RNA is DMPK mRNA.

39. The pharmaceutical composition according to claim 36, wherein the administration results in a reduction in DMPK RNA in the nucleus of the muscle cells of the subject.

40. The pharmaceutical composition according to claim 39, wherein the subject is human.

41. An oligonucleotide comprising a structure selected from the following: 【Figure 6-1】 [Number 6-2] (Here, "xdC" is 5-methyl-deoxycytidine; "dN" is 2'-deoxyribonucleoside; "oN" is 2'-MOE-modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "oU" is 5-methyl-2'-MOE-uridine; "xoG" is 7-methyl-2'-MOE-guanosine; and "*" indicates a phosphorothioate (PS) internucleoside linkage). **Claim 42** The oligonucleotide is conjugated to an amine group at its 5'-end and comprises a structure selected from the following: 【Figure 7-1】 【Figure 7-2】 【Figure 7-3】 Here, "xdC" is 5-methyl-deoxycytidine; "dN" is 2'-deoxyribonucleoside; "oN" is 2'-MOE-modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "oU" is 5-methyl-2'-MOE-uridine; "xoG" is 7-methyl-2'-MOE-guanosine; and "*" indicates a phosphorothioate (PS) internucleoside linkage. The oligonucleotide according to Claim 41. **Claim 43** A phosphodiester linkage or other moiety is between 5'-NH 2 -(CH 2 ) 6 - and the oligonucleotide according to claim 42. **Claim 44** An oligonucleotide comprising a structure selected from the following: 【Figure 8-1】 【Number 8-2】 (Here, "xdC" is 5-methyl-deoxycytidine; "dN" is 2'-deoxyribonucleoside; "oN" is 2'-MOE-modified ribonucleoside; "xoC" is 5-methyl-2'-MOE-cytidine; "x + C" is 5-methyl LNA cytidine; "+N" is LNA nucleoside; "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl LNA uridine; and "*" indicates a phosphorothioate (PS) internucleoside linkage). **Claim 45** The oligonucleotide is conjugated to an amine group at its 5'-end and comprises a structure selected from the following: 【Number 9-1】 【Number 9-2】 [Number 9-3] Here, "xdC" is 5-methyl-deoxycytidine; "dN" is 2'-deoxyribonucleoside; "oN" is 2'-MOE-modified ribonucleoside; "xC" is 5-methyl-2'-MOE-cytidine; "x+C" is 5-methyl LNA cytidine; "+N" is LNA nucleoside; "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl LNA uridine; "*" indicates a phosphorothioate (PS) internucleoside linkage, the oligonucleotide according to claim 44.

46. A phosphodiester linkage or other moiety is 5'-NH 2 -(CH 2 ) 6 - The oligonucleotide according to claim 45, which is present between the oligonucleotide and the oligonucleotide.

47. A composition comprising the oligonucleotide according to any one of claims 41 to 46 in salt form.

48. The composition according to claim 47, wherein the salt form is a sodium salt.

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