Muscle-targeting complex and its use for skipping exon 45 of the DMD gene

Abstract

Aspects of the present disclosure relate to conjugates, and other aspects relate to formulations (e.g., aqueous, lyophilized forms) comprising such conjugates (e.g., each conjugate represented by the exemplary formula below) comprising a phosphorodiamidate morpholino oligomer (e.g., useful for targeting DMD) covalently linked to an antibody (e.g., an anti-TfR1 antibody). Also provided is the use of these formulations for treating subjects with a mutated DMD allele associated with Duchenne muscular dystrophy. JPEG2026503873000028.jpg63163

Description

[Technical Field]

[0001] Related Applications This application claims priority under 35 U.S.C. §119(e) to Provisional Patent Application No. 63 / 381,730, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR SKIPPING EXON 45 OF A DMD GENE," filed October 31, 2022, the entire contents of which are incorporated herein by reference.

[0002] FIELD OF THE INVENTION The present application relates to targeted complexes for delivering oligonucleotide molecular payloads to cells, formulations containing such complexes, and uses thereof, particularly for the treatment of disease.

[0003] Electronic Sequence Listing Reference The contents of the Electronic Sequence Listing (D082470083WO00-SEQ-COB.xml; size: 38,671 bytes; and creation date: October 30, 2023) are incorporated herein by reference in their entirety.

[0004] background Dystrophinopathies are a group of distinct neuromuscular diseases caused by mutations in the dystrophin gene. Dystrophinopathies include Duchenne muscular dystrophy, Becker muscular dystrophy, and X-linked dilated cardiomyopathy. The DMD gene encoding dystrophin is a large gene containing 79 exons totaling approximately 2.6 million base pairs. Numerous mutations in DMD, including exon frameshifts, deletions, substitutions, and duplications, can reduce the expression of functional dystrophin and cause dystrophinopathies.

[0005] overview In some embodiments, the present disclosure provides a muscle-targeting complex useful for promoting the expression or activity of a dystrophin protein (e.g., a truncated dystrophin protein), and / or a method for treating Duchenne muscular dystrophy in a subject. The truncated dystrophin protein is functional (e.g., retains the activity of a wild-type dystrophin protein). In some embodiments, the truncated dystrophin protein retains partial function of a wild-type dystrophin protein.

[0006] According to some embodiments, a compound of formula (I): [R 1 ] n1 -R 2 In some embodiments, each R 1 is represented by formula (Ia): [ka] and a group represented by R in the formula 3 comprises a phosphorodiamidate morpholino oligomer (PMO) comprising the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21); R in the formula 2 comprises an anti-transferrin receptor 1 (anti-TfR1) antibody comprising a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), a heavy chain complementarity determining region 3 (CDR-H3), a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region 3 (CDR-L3) selected from Table 2; R in the formula 1 is R at attachment point A. 2 and wherein n1 is R 1 is an integer of 1 or greater representing the number of instances of 1 Each instance of is covalently linked to a different amino acid residue of the anti-TfR1 antibody.

[0007] In some embodiments, each R1 is represented by formula (Ib): [ka] and a group represented by where -p is a phosphorodiamidate linkage of a phosphorodiamidate morpholino oligomer (PMO), and wherein the PMO comprises the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21); In the formula, R 2 comprises an anti-TfR1 antibody comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 2; In the formula, R 1 is R at attachment point A. 2 and wherein n1 is R 1 is an integer of 1 or greater representing the number of instances of 1 Each instance of is covalently linked to a different amino acid residue of the anti-TfR1 antibody.

[0008] In some embodiments, each R 1 is represented by formula (Ic): [ka] and a group represented by R in the formula 2 comprises an anti-TfR1 antibody comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 2; In the formula, R 1 is R at attachment point A. 2 and wherein n1 is R 1 is an integer of 1 or greater representing the number of instances of 1 Each instance of is covalently linked to a different amino acid residue of the anti-TfR1 antibody.

[0009] In some embodiments, the conjugate has formula (Id): [ka] The structure includes: where -p is a phosphorodiamidate linkage of a phosphorodiamidate morpholino oligomer (PMO), and wherein the PMO comprises the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21); R in the formula 2 comprises an anti-TfR1 antibody comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 2; wherein each instance of the bracketed group in formula (Id) is covalently linked to a different amino acid residue in the anti-TfR1 antibody; and wherein n1 is an integer greater than or equal to 1 representing the number of instances of the bracketed group in formula (Id).

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

[0011] In some embodiments, the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, and a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0012] In some embodiments, R 1 Each instance of is covalently linked to a different lysine residue of the anti-TfR1 antibody.

[0013] In some embodiments, the different amino acid residues include K188 and K190 of the light chain constant region based on Kabat numbering.

[0014] In some embodiments, the different amino acid residue is represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain constant region of the anti-TfR1 antibody.

[0015] According to some aspects, compositions are provided that include the conjugates disclosed herein. In some embodiments, the compositions are in the form of an aqueous solution.

[0016] In some embodiments, the anti-TfR1 antibodies of the conjugate in the composition comprise a light chain constant region, and at least 80% of the light chain constant regions of the anti-TfR1 antibodies of the conjugate in the composition are independently covalently linked to an oligonucleotide at the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of each anti-TfR1 antibody.

[0017] In some embodiments, the anti-TfR1 antibodies of the conjugate in the composition comprise a light chain constant region, and at least 80% of the light chain constant regions of the anti-TfR1 antibodies of the conjugate in the composition are independently covalently linked to an oligonucleotide at a linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain constant region of each antibody.

[0018] According to some aspects, provided herein are methods for promoting dystrophin protein expression or activity in a subject. In some embodiments, the methods comprise administering to the subject a complex or composition disclosed herein.

[0019] In some embodiments, the dystrophin protein is a truncated dystrophin protein.

[0020] According to some aspects, provided herein are methods for treating a subject having a mutated DMD allele associated with Duchenne muscular dystrophy. In some embodiments, the method comprises administering to the subject a complex or composition disclosed herein.

[0021] In some embodiments, the complex promotes the expression or activity of a dystrophin protein in a subject. In some embodiments, the dystrophin protein is a truncated dystrophin protein.

[0022] In some embodiments, the mutated DMD allele comprises a mutation that is amenable to exon 45 skipping.

[0023] In some embodiments, the mutated DMD allele comprises a frameshift mutation in exon 45. [Brief explanation of the drawings]

[0024] [Figure 1] FIG. 1 shows data demonstrating that systemic administration of a conjugate containing an anti-TfR1 Fab covalently linked to an exon 45-targeted oligonucleotide (ASO) achieved higher levels of ASO levels in muscle tissue of non-human primate animals compared to systemic administration of an ASO that was not covalently linked to a Fab.

[0025] [Figure 2] Figure 2 shows exon 45 skipping in human myotubes either wild-type ("WT myotubes") or containing a mutation adaptive for exon 45 skipping introduced by CRISPR / Cas genome editing ("Del46 myotubes").

[0026] [Figure 3] Figure 3 shows tissue exposure (ng / g) in the quadriceps ("Quad"), gastrocnemius ("Gastroc"), heart ("Heart"), and diaphragm ("Dia") of hTfR1 / hDMDWT / mdx mice 7 days after intravenous administration of DMD-targeting oligonucleotide (ASO) contained in an anti-TfR1 Fab-ASO conjugate.

[0027] [Figure 4]Figures 4A-4D show exon 45 skipping in muscle tissues of hTfR1 / hDMDWT / mdx mice intravenously administered with vehicle control ("Veh") or anti-TfR1 Fab-ASO conjugate ("anti-TfR1 Fab-ASO conjugate") 7 days after treatment. Exon skipping was observed in the quadriceps (Figure 4A), gastrocnemius (Figure 4B), heart (Figure 4C), and diaphragm (Figure 4D). DETAILED DESCRIPTION OF THE INVENTION

[0028] DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS According to some aspects, the present disclosure provides a muscle-targeting complex useful for promoting the expression or activity of a dystrophin protein (e.g., a truncated dystrophin protein), and / or a method for treating Duchenne muscular dystrophy in a subject. The truncated dystrophin protein is functional (e.g., retains the activity of a wild-type dystrophin protein). In some embodiments, the truncated dystrophin protein retains partial function of a wild-type dystrophin protein. In some embodiments, the muscle-targeting complex comprises a muscle-targeting agent (e.g., an anti-TfR1 antibody) covalently linked to an oligonucleotide (e.g., a PMO). In some embodiments, the oligonucleotide comprises a region complementary to the DMD sequence.

[0029] According to some aspects, the present disclosure provides a composition comprising a plurality of conjugates. In some embodiments, the conjugate of the composition described herein comprises an antibody (e.g., anti-transferrin receptor 1 (TfR1)) covalently linked to one or more oligonucleotides. In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region. In some embodiments, each of the one or more oligonucleotides is covalently linked to a different amino acid residue of the antibody, such as a lysine residue. In some embodiments, the light chain constant regions of the antibodies in the conjugate in the composition are independently covalently linked to the oligonucleotide at a linkage site represented by K188 (according to Kabat numbering) and / or K190 (according to Kabat numbering) in the light chain constant region of the antibody. In some embodiments, the light chain of the antibody in the conjugate is covalently linked to the oligonucleotide at a linkage site represented by a lysine (K) residue within the light chain sequence motif DYEKHKVYA (SEQ ID NO: 27).

[0030] definition Administering: As used herein, the term "administering" or "administration" means providing a conjugate to a subject in a physiologically and / or (for example, and) pharmacologically useful manner (for example, to treat a disease in a subject).

[0031] As used herein, the term "approximately" or "about," when applied to one or more values ​​of interest, refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "about" refers to a broad range of values ​​that fall within plus or minus (more than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the stated reference value, unless otherwise stated or clear from the context (except where such number would exceed 100% of a feasible value).

[0032] 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 region selected from the group consisting of the constant regions of IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE. In some embodiments, an antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and / or (for example, and) a light (L) chain variable region (abbreviated herein as VL). In some embodiments, an antibody comprises a constant region, for example, an Fc region. An immunoglobulin constant region refers to the constant region of a heavy or light chain. The amino acid sequences of the constant regions of human IgG heavy and light chains and their functional variations are known. With respect to the heavy chain, in some embodiments, the heavy chain of an antibody described herein can be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, the antibody described herein comprises a human gamma 1 CH1 domain, CH2 domain, and / or (for example, 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, such as any known in the art.Non-limiting examples of human constant region sequences are described in the art; see, e.g., U.S. Patent No. 5,693,780, Kabat EA et al., and Kabat EA et al., supra (1991). In some embodiments, the VH domain comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, e.g., via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules comprise a mannose unit, a glucose unit, an N-acetylglucosamine unit, an N-acetylgalactosamine unit, a galactose unit, a fucose unit, or a phospholipid unit. In some embodiments, the antibody is a construct comprising a linker polypeptide or a polypeptide comprising one or more antigen-binding fragments of the present disclosure linked to an immunoglobulin constant region. The linker polypeptide comprises two or more amino acid residues linked by a peptide bond and is used to link one or more antigen-binding moieties. Examples of linker polypeptides have been reported (see, e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, RJ, et al. (1994) Structure 2:1121-1123).Furthermore, an antibody may be part of a larger immunoadhesion molecule formed by covalent or noncovalent association of the 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).

[0033] CDR: As used herein, the term "CDR" refers to a complementarity-determining region within an antibody variable sequence. A typical antibody molecule contains a heavy chain variable region (VH) and a light chain variable region (VL), which are primarily responsible for 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 amino-terminus to 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 the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (for example, and) the contact definition, all of which are well known in the art.Also Kabat, EA, et al.(1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication no. 91-3242; IMGTR, the International ImMunoGenetics Information SystemR www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209–212; 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 (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognize. 17:132-143 (2004) available at bioinf.org.uk / abs.As used herein, CDR may refer to a CDR defined by any method known in the art. Two antibodies with the same CDR mean that the two antibodies have the same amino acid sequence of the CDR when determined by the same method, for example, the IMGT definition.

[0034] Each heavy and light chain variable region has three CDRs, which are designated as CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" as used herein refers to a group of three CDRs occurring in a single variable region that can bind to an antigen. The exact boundaries of these CDRs are defined differently according to different systems. The system described by Kabat (Kabat et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries defining the three CDRs. These CDRs are sometimes referred to as Kabat CDRs. Sub-portions of the CDRs are sometimes designated L1, L2, and L3, or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, whose boundaries overlap with the Kabat CDRs. Other boundaries defining CDRs that overlap with the Kabat CDRs are described by Padlan (FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Still other CDR boundary definitions may not strictly adhere to one of the above systems, but may still overlap with the Kabat CDRs, and may be shortened or extended in light of predictions or experimental findings that a particular residue or group of residues, or even an entire CDR, does not significantly affect antigen binding. The methods used herein may utilize CDRs defined according to any of these systems. Examples of CDR definition systems are provided in Table 1. [Table 1]

[0035] As used herein, the term "complementary" refers to the ability for precise pairing between two nucleotides or two pairs of nucleotides. In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in binding between two nucleotides or two pairs of nucleotides. For example, if a base at a certain position in an oligonucleotide can hydrogen bond with a base at a corresponding position in a target nucleic acid (e.g., mRNA), then the bases are considered to be complementary to each other at that position. Base pairing may include both canonical 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, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize to and is considered complementary to any A, C, U, or T. Inosine (I) is also considered a universal base in the art and is considered complementary to any A, C, U, or T.

[0036] Covalently linked: As used herein, the term "covalently linked" refers to the characteristic of two or more molecules being linked together via at least one covalent bond. In some embodiments, two molecules may be covalently linked together by a single bond (e.g., a disulfide bond or disulfide bridge) that acts as an intermolecular linker. However, in some embodiments, two or more molecules may be covalently linked together via a molecule that acts as a linker that connects two or more molecules together through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker.

[0037] DMD allele: As used herein, "DMD allele" refers to any one of the alternative forms (e.g., wild-type or mutant) of the DMD gene. In some embodiments, the DMD allele may encode dystrophin that retains normal and typical function. In some embodiments, the DMD allele may contain one or more mutations that result in muscular dystrophy. Common mutations that lead to Duchenne muscular dystrophy involve frameshifts, deletions, substitutions, and duplications in one or more of the 79 exons present in the dystrophin allele (e.g., exon 8, exon 23, exon 41, exon 44, exon 50, exon 51, exon 52, exon 53, or exon 55). Further examples of DMD mutations are disclosed in Flanigan KM, et al., Mutational spectrum of DMD mutations in dystrophinopathy patients: application of modern diagnostic techniques to a large cohort. Hum Mutat. 2009 Dec; 30 (12):1657-66, the contents of which are incorporated herein by reference in their entirety.

[0038] Duchenne muscular dystrophy: As used herein, the term "Duchenne muscular dystrophy" refers to a muscle disease resulting from a mutation in the DMD allele located on the X chromosome at locus Xp21. Symptoms of Duchenne muscular dystrophy include muscle loss or degeneration, decreased muscle function, pseudohypertrophy of the tongue and calf muscles, increased risk of neurological abnormalities, and shortened lifespan. Duchenne muscular dystrophy is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 310200.

[0039] Dystrophin: As used herein, the term "dystrophin" refers to any of the alternative forms (e.g., wild-type or mutant) of the dystrophin protein. Dystrophin is a rod-shaped cytoplasmic protein that is part of a protein complex that connects the intracellular cytoskeleton of muscle fibers to the extracellular matrix. Deficiency of dystrophin results in Duchenne muscular dystrophy.

[0040] Dystrophinopathy: As used herein, the term "dystrophinopathy" refers to a muscle disease resulting from one or more mutated DMD alleles. Dystrophinopathy encompasses a spectrum of symptoms, including Duchenne muscular dystrophy, Becker muscular dystrophy, and DMD-associated dilated cardiomyopathy (DCM), ranging from mild to severe. In some embodiments, at one end of the spectrum, dystrophinopathy is phenotypically associated with asymptomatic increases in serum creatine phosphokinase (CK) levels and / or muscle spasms accompanied by myoglobinuria (for example, and). In some embodiments, at the other end of the spectrum, dystrophinopathy is phenotypically associated with progressive muscle diseases, generally classified as Duchenne or Becker muscular dystrophy when primarily affecting skeletal muscles, or DMD-associated dilated cardiomyopathy (DCM) when primarily affecting the heart. Symptoms of Duchenne muscular dystrophy include muscle loss or degeneration, decreased muscle function, pseudohypertrophy of the tongue and calf muscles, increased risk of neurological abnormalities, and shortened lifespan. Duchenne muscular dystrophy is associated with Online Mendelian Inheritance in Man (OMIM) Entry # 310200. Becker muscular dystrophy is associated with OMIM Entry # 300376. Dilated cardiomyopathy is associated with OMIM Entry # 300376.

[0041] Exonic splicing enhancer (ESE): As used herein, the term "exonic splicing enhancer" or "ESE" refers to a nucleic acid sequence motif within an exon of a gene, pre-mRNA, or mRNA that induces or enhances splicing from pre-mRNA to mRNA, as described, for example, in Blencowe et al., Trends Biochem Sci 25, 106-10. (2000), which is incorporated herein by reference. ESEs are splicing features. ESEs induce or enhance splicing, for example, to remove one or more introns and / or one or more exons from a gene transcript. ESE motifs are typically 6 to 8 nucleobases in length. SR proteins (e.g., proteins encoded by the SRSF1, SRSF2, SRSF3, SRSF4, SRSF5, SRSF6, SRSF7, SRSF8, SRSF9, SRSF10, SRSF11, SRSF12, TRA2A, or TRA2B genes) bind to ESEs through RNA recognition motif regions and promote splicing. ESE motifs can be identified through a number of methods, including those described in Cartegni et al., Nucleic Acids Research, 2003, Vol. 31, No. 13, 3568-3571, which is incorporated herein by reference.

[0042] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of a variable region minus the CDRs. Because the exact definition of a CDR sequence can be determined by various systems, the meaning of a framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework regions on the light and heavy chains into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. Framework regions, as referred to by others, that do not specify the specific subregion as FR1, FR2, FR3, or FR4, represent the FRs combined within the variable region of a single naturally occurring immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR(s) refers to two or more of the four subregions containing the framework region. Acceptor sequences for human heavy and light chains are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0043] Human antibody: The term "human antibody," as used herein, is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), e.g., in the CDRs, particularly CDR3. However, the term "human antibody," as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0044] Humanized antibody: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but in which at least a portion of the VH and / or (e.g., and) VL sequences have been altered to be more "human-like," i.e., more similar to human germline variable sequences. One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced onto non-human VH and VL sequences, thereby replacing the corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen-binding portions are provided. Such antibodies may be produced by using existing hybridoma technology, followed by humanization using in vitro genetic engineering, such as that disclosed in PCT Publication No. WO 2005 / 123126 A2 to Kasaian et al., to obtain a murine anti-transferrin receptor monoclonal antibody.

[0045] Kabat numbering: The terms "Kabat numbering," "Kabat definition," and "Kabat labeling" are used interchangeably herein. These terms, recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., more hypervariable) than other amino acid residues in the variable regions of the heavy and light chains 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, US Department of Health and Human Services, NIH Publication No. 91-3242). For the heavy chain variable region, the hypervariable region spans amino acid positions 31 to 35 for CDR1, amino acid positions 50 to 65 for CDR2, and amino acid positions 95 to 102 for CDR3. For the light chain variable region, the hypervariable region spans amino acid positions 24 to 34 for CDR1, amino acid positions 50 to 56 for CDR2, and amino acid positions 89 to 97 for CDR3.

[0046] Morpholino: As used herein, the term "morpholino," also known as a "phosphorodiamidate morpholino oligomer," refers to a molecular structure comprising a nucleobase linked to a backbone of methylene morpholine rings linked through phosphorodiamidate groups. In some embodiments, the oligonucleotide may be a morpholino-based compound. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41(14), 4503-4510; Genesis, volume 30, issue 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued July 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein in their entireties).

[0047] 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 may be single-stranded or double-stranded. In some embodiments, oligonucleotides may contain one or more modified nucleosides (e.g., 2'-O-methyl sugar modifications, purine modifications, or pyrimidine modifications). In some embodiments, oligonucleotides may contain one or more modified internucleoside linkages. In some embodiments, oligonucleotides may contain one or more phosphorothioate linkages, which may be in the Rp or Sp stereochemical conformation.

[0048] Region of complementarity: As used herein, the term "region of complementarity" refers to a nucleotide sequence (e.g., of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., of a target nucleic acid) so that the two nucleotide sequences are capable of annealing to each other under physiological conditions (e.g., in a cell). In some embodiments, the region of complementarity is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the region of complementarity is partially complementary (e.g., at least 80%, 90%, 95%, or 99% complementary) to the cognate nucleotide sequence of the target nucleic acid. In some embodiments, the region of complementarity contains 1, 2, 3, or 4 mismatches compared to the cognate nucleotide sequence of the target nucleic acid.

[0049] Specific bind: As used herein, the term "specifically bind" 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. The term "specifically bind" with respect to an antibody refers to the ability of the antibody to bind to a specific antigen with a degree of affinity or avidity that allows the antibody to distinguish the specific antigen from other antigens (e.g., to the extent that allows preferential targeting to certain cells (e.g., muscle cells) through binding to the antigen, as described herein) compared to an appropriate reference antigen(s). In some embodiments, the antibody binds to the target with at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less K D In some embodiments, the antibody specifically binds to a transferrin receptor, e.g., an epitope in the apical domain of the transferrin receptor.

[0050] Splice acceptor site: As used herein, the term "splice acceptor site" or "splice acceptor" refers to a nucleic acid sequence motif at the 3' end of an intron in a gene or pre-mRNA, or across an intron / exon junction, that is involved in pre-mRNA to mRNA splicing (i.e., removal of the intron from the pre-mRNA) and may be referred to as a splicing feature. The splice acceptor site encompasses a terminal AG sequence at the 3' end of the intron and is typically preceded (5') by a pyrimidine (C / U)-rich region. Upstream of the splice acceptor site is the branch point. Transesterification between the branch point and the splice donor site forms a lariat loop intermediate structure, which releases the 3'-OH of the 5' exon, which then reacts with the first nucleotide of the 3' exon, joining the exons and releasing the intron lariat. The AG sequence at the 3' end of the intron in the splice acceptor site is known to be critical for correct splicing, as altering one of these nucleotides results in inhibition of splicing. Rarely, alternative splice acceptor sites have AC at the 3' end of the intron instead of the more common AG. Common splice acceptor site motifs are [Y-rich region]-NCAGG or Y- x It has a sequence of NYAGG or a sequence similar thereto, where Y is a pyrimidine, N is any nucleotide, and x is a number between 4 and 20. The cleavage site is followed by AG, which represents the 3'-terminal nucleotide of the excised intron.

[0051] Subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human animal of the primate order or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, e.g., a human patient having or suspected of having a disease. In some embodiments, the subject is a human patient having or suspected of having a disease resulting from a mutated DMD gene sequence, e.g., a mutation in an exon of the DMD gene sequence. In some embodiments, the subject has a dystrophinopathy, e.g., Duchenne muscular dystrophy.

[0052] Transferrin receptor: As used herein, the term "transferrin receptor" (also known as TFRC, CD71, p90, TFR, or TFR1) refers to an internalized cell surface receptor that binds to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may be of human (NCBI Gene ID 7037) origin, of non-human primate (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007) origin, or of rodent (e.g., NCBI Gene ID 22042) origin. In addition, multiple human transcript variants have been characterized that encode 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).

[0053] Ranges: All ranges provided in this disclosure are inclusive of the endpoints.

[0054] Complex Provided herein is a complex comprising an antibody covalently linked to an oligonucleotide. In some embodiments, the complex comprises a muscle-targeting antibody (for example, an anti-TfR1 antibody) covalently linked to one or more oligonucleotides. In some embodiments, the oligonucleotide is a PMO. In some embodiments, the oligonucleotide is an oligonucleotide that targets a mutated DMD allele to promote exon skipping (for example, promote exon 45 skipping). The complex disclosed herein is useful in a method for promoting the expression or activity of dystrophin protein in a subject and / or a method for treating Duchenne muscular dystrophy, comprising administering an effective amount of the complex to the subject.

[0055] The conjugates disclosed herein generally include a linker that covalently links an antibody disclosed herein (e.g., an anti-TfR1 antibody disclosed herein) to an oligonucleotide (e.g., a PMO). The linker includes at least one covalent bond.

[0056] In some embodiments, the light chain constant region of the antibody of the conjugate is covalently linked to the oligonucleotide at the linkage site represented by K188 (based on Kabat numbering) and / or K190 (based on Kabat numbering) of the light chain constant region. In some embodiments, the light chain of the antibody of the conjugate is covalently linked to the oligonucleotide at the linkage site represented by a lysine (K) residue within the light chain sequence motif DYEKHKVYA (SEQ ID NO: 27).

[0057] In some embodiments, the conjugates disclosed herein have the formula (I): [R 1 ] n1 -R 2 wherein each R 1 independently includes compounds containing oligonucleotides (e.g., PMOs), and R 2comprises an anti-TfR1 antibody, and wherein, in each complex, n1 independently represents the number of TfR1 antibodies in each complex. 1 In some embodiments, each R 1 In some embodiments, each R 1 In some embodiments, R 2 In some embodiments, the R 2 contains an anti-TfR1 Fab.

[0058] R 2In some embodiments, the antibody comprises an anti-TfR1 antibody, the antibody comprises a sequence as set forth in Table 2. For example, in some embodiments, the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as represented in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as represented in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as represented in SEQ ID NO: 3, 9, or 14; and / or a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as represented in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as represented in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as represented in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. In some embodiments, the antibody is a Fab fragment.

[0059] In some embodiments, the value of n1 for each or any conjugate (e.g., any conjugate in any of the compositions or methods disclosed herein) is an integer up to the number of amino acid residues (e.g., the number of lysine residues) of the antibody to which conjugation is desired or targeted. In some embodiments, for each conjugate, the value of n1 is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27. In some embodiments, the value of n1 for each conjugate is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26. In some embodiments, the value of n1 in each conjugate is independently in the range of 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3.

[0060] In some embodiments, the conjugates described herein are presented as compositions (e.g., aqueous solutions) for administration to a subject. In some embodiments, the compositions include multiple conjugates. In some embodiments, the multiple conjugates each include a common targeting agent (e.g., an antibody) and a common oligonucleotide (e.g., a PMO). In such embodiments, different conjugate types are characterized by having different numbers of oligonucleotides covalently linked to the antibody. For example, in some embodiments, the compositions for administration to a subject include multiple conjugate types, wherein each conjugate type has a structure represented by Formula (I): [R 1 ] n1 -R 2 wherein each R 1 independently includes compounds containing oligonucleotides (e.g., PMOs), and R 2 comprises an anti-TfR1 antibody, and wherein, in each conjugate type, n1 independently represents the R 1n1 is an integer equal to or greater than 1 representing the number of instances of n1, and different complex types in the composition are characterized by having different n1 values ​​(e.g., n1 values ​​ranging from 1 to 27, 1 to 26, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3).

[0061] In some embodiments, the light chain constant regions of the antibodies of the conjugate in the composition are independently covalently linked to the oligonucleotide at a linkage site represented by K188 (according to Kabat numbering) and / or K190 (according to Kabat numbering) in the light chain constant region of the antibody. In some embodiments, the light chains of the antibodies of the conjugate in the composition are independently covalently linked to the oligonucleotide at a linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the antibody.

[0062] In some embodiments, the composition for administration to a subject in the methods described herein comprises (e.g., a trace amount of) an unconjugated antibody and an antibody conjugated to one or more oligonucleotides. In some embodiments, the unconjugated antibody is a compound of Formula (I): [R 1 ] n1 -R 2 Accordingly, in some embodiments, the composition for administration to a subject in the methods described herein may be a compound of formula (I): [R 1 ] n1 -R 2 In the formula, each R 1 independently comprises a group comprising an oligonucleotide, and R 2 includes an antibody, and n1 independently represents the R 1 In some embodiments, the composition comprises a compound of formula (I): [R 1 ] n1 -R 2is less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01% compared to all compounds of that structure in which n1 is 1 or greater in the composition.

[0063] In some embodiments, R in the complex 1 Each instance of R is conjugated to a different amino acid residue of the antibody. In some embodiments, each different amino acid comprises an ε-amino group (e.g., lysine, arginine). However, in some embodiments, R 1 In some embodiments, each different amino acid to which R is covalently linked is a cysteine. 1 In some embodiments, each different amino acid to which R is covalently linked is a lysine. 1 is covalently linked directly to an amino acid residue of the antibody. However, in some embodiments, R 1 is indirectly covalently linked to an amino acid of the antibody, e.g., covalently linked to a glycosylation site on the amino acid. 1 is not covalently linked to amino acid residues in the CDR regions of the antibody.

[0064] In some embodiments, the conjugates described herein have the formula (I): [R 1 ] n1 - R 2 wherein each R1 independently represents a group represented by formula (Ia): [ka] wherein R 3 is an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO); where in each complex, n1 is independently R 1 is an integer (e.g., 1 or greater) representing the number of instances of 1 is R at attachment point A. 2In some embodiments, R 2 includes anti-TfR1 antibodies comprising a sequence as set forth in Table 2. For example, in some embodiments, R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as represented in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as represented in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as represented in SEQ ID NO: 3, 9, or 14; and / or a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as represented in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as represented in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as represented in SEQ ID NO: 6 or 16. In some embodiments, R 2 In some embodiments, R comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. 2 In some embodiments, R comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. 2 In some embodiments, R comprises an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. 2 In some embodiments, R comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. 2In some embodiments, R comprises an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. 2 includes an anti-TfR1 antibody that is a Fab fragment. In some embodiments, R3 is an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO) comprising the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21). In some embodiments, each internucleoside linkage of the PMO is a phosphorodiamidate linkage. In some embodiments, R 2 contains Fab, and each R 1 are covalently linked (e.g., indirectly or directly linked, e.g., directly linked) at attachment point A to different amino acid residues of the Fab, and optionally each different amino acid residue is a lysine. In some embodiments, each R 1 is attached to R at attachment point A via a linkage site represented by a lysine (K) residue in the antibody. 2 In some embodiments, the light chain constant region of the antibody of the conjugate is covalently linked to the oligonucleotide at a linkage site represented by K188 (based on Kabat numbering) and / or K190 (based on Kabat numbering) in the light chain constant region. In some embodiments, the light chain of the antibody of the conjugate is covalently linked to the oligonucleotide at a linkage site represented by a lysine (K) residue within the light chain sequence motif DYEKHKVYA (SEQ ID NO: 27). In some embodiments, n1 in each conjugate is independently an integer (e.g., an integer ranging from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3).

[0065] In some embodiments, the conjugates disclosed herein have the formula (I): [R 1 ] n1 -R 2 wherein each R 1 is represented by formula (Ib): [ka] wherein -p is a phosphorodiamidate linkage of a phosphorodiamidate morpholino oligomer (PMO), and wherein the PMO comprises the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21); wherein, in each conjugate, n1 is independently selected from the R 1 is an integer (e.g., 1 or greater) representing the number of instances of 1 is R at attachment point A. 2 In some embodiments, R 2 includes anti-TfR1 antibodies comprising a sequence as set forth in Table 2. For example, in some embodiments, R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as represented in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as represented in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as represented in SEQ ID NO: 3, 9, or 14; and / or a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as represented in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as represented in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as represented in SEQ ID NO: 6 or 16. In some embodiments, R 2 In some embodiments, R comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. 2 In some embodiments, R comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. 2In some embodiments, the RfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. 2 In some embodiments, the RfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. 2 In some embodiments, R comprises an anti-TfR1 antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. 2 comprises an anti-TfR1 antibody that is a Fab fragment. In some embodiments, n1 in each conjugate is independently an integer (e.g., an integer ranging from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, R 2 contains Fab, and each R 1 are covalently linked (e.g., indirectly or directly, e.g., directly) to different amino acid residues of the Fab at attachment point A, optionally where each different amino acid residue is a lysine. In some embodiments, each R 1 is attached to R at attachment point A via a linkage site represented by a lysine (K) residue in the antibody. 2 In some embodiments, the lysine (K) residue linkage site of the anti-TfR1 antibody is represented by K188 (based on Kabat numbering) and / or K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody. In some embodiments, the lysine (K) residue linkage site is represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the anti-TfR1 antibody.

[0066] In some embodiments, the conjugates disclosed herein have the formula (I): [R 1 ] n1 -R 2 wherein each R 1 is represented by formula (Ic): [ka] wherein, in each complex, n1 is independently selected from R 1 is an integer (e.g., 1 or greater) representing the number of instances of each R 1 At the attachment point A, R 2 In some embodiments, R 2 includes anti-TfR1 antibodies comprising a sequence as set forth in Table 2. For example, in some embodiments, R 2 comprises an antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as represented in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as represented in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as represented in SEQ ID NO: 3, 9, or 14; and / or a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as represented in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as represented in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as represented in SEQ ID NO: 6 or 16. In some embodiments, R 2 In some embodiments, R comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. 2 In some embodiments, R comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. 2In some embodiments, the RfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. 2 In some embodiments, the RfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. 2 In some embodiments, R comprises an anti-TfR1 antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. 2 comprises an anti-TfR1 antibody that is a Fab fragment. In some embodiments, n1 in each conjugate is independently an integer (e.g., an integer ranging from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, R 2 contains Fab, and each R 1 are covalently linked (e.g., indirectly or directly, e.g., directly) to different amino acid residues of the Fab at attachment point A, optionally where each different amino acid residue is a lysine. In some embodiments, each R 1 is attached to R at attachment point A via a linkage site represented by a lysine (K) residue in the antibody. 2 In some embodiments, the lysine (K) residue linkage site of the anti-TfR1 antibody is represented by K188 (based on Kabat numbering) and / or K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody. In some embodiments, the lysine (K) residue linkage site is represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the anti-TfR1 antibody.

[0067] In some embodiments, the conjugates disclosed herein have the formula (Id): [ka] wherein -p is a phosphorodiamidate linkage of a phosphorodiamidate morpholino oligomer (PMO), and wherein the PMO comprises the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21); R 2 comprises an anti-TfR1 antibody (e.g., Fab) comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 2, optionally wherein the antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and further optionally wherein the antibody (e.g., Fab) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, and wherein in each conjugate, n1 is independently an integer (e.g., 1 or more) representing the number of instances of the bracketed group, wherein each instance of the bracketed group is covalently linked to a different amino acid residue of the antibody (e.g., Fab), and optionally each different amino acid residue is lysine. In some embodiments, R 2 comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as set forth in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as set forth in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as set forth in SEQ ID NO: 3, 9, or 14; and / or an antibody (e.g., a Fab) comprising a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as set forth in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as set forth in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, R 2 In some embodiments, the RfR1 antibody (e.g., Fab) comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. 2In some embodiments, the R comprises an anti-TfR1 antibody (e.g., a Fab) that comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. 2 In some embodiments, the RfR1 antibody (e.g., a Fab) comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. 2 comprises an anti-TfR1 antibody (e.g., Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, n1 in each conjugate is independently an integer (e.g., an integer ranging from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, R 2 includes an anti-TfR1 antibody (e.g., Fab) covalently linked (e.g., indirectly or directly linked, e.g., directly linked) via a different amino acid residue (e.g., Fab), optionally wherein each different amino acid residue is a lysine. In some embodiments, each instance of the group, the bracketed group in the structure represented by formula (Id), is covalently linked to a linkage site represented by a lysine (K) residue of the anti-TfR1 antibody (e.g., Fab). In some embodiments, the lysine (K) residue linkage site of the anti-TfR1 antibody is represented by K188 (according to Kabat numbering) and / or K190 (according to Kabat numbering) of the light chain constant region of the anti-TfR1 antibody. In some embodiments, the lysine (K) residue linkage site is represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the anti-TfR1 antibody.

[0068] In some embodiments, the conjugates described herein have the formula (A): [ka] wherein n is 0-15 (e.g., 3) and m is 0-15 (e.g., 4). In some embodiments, the antibody is an anti-TfR1 antibody (e.g., the anti-TfR1 antibody provided in Table 2). In some embodiments, the oligonucleotide is a PMO and comprises the nucleotide sequence of SEQ ID NO: 21. In some embodiments, each internucleoside linkage of the PMO is a phosphorodiamidate linkage. In some embodiments, the amide shown adjacent to the anti-TfR1 antibody in the structure results from reaction with an amine of the anti-TfR1 antibody, such as a lysine epsilon amine. In some embodiments, the conjugate described herein comprises an anti-TfR1 Fab covalently linked to the 5' end of the PMO via a lysine of the Fab. In some embodiments, the antibody comprises a sequence as set forth in Table 2. For example, in some embodiments, the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as represented in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as represented in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as represented in SEQ ID NO: 3, 9, or 14; and / or a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as represented in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as represented in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as represented in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18.In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv, or an Fv.

[0069] Each of the disclosed conjugates (e.g., those of formula (I): [R 1 ] n1 -R 2 For example, a complex containing a structure represented by each R 1 is a conjugate comprising a group of formula (Ia), (Ib), or (Ic), a conjugate comprising a structure represented by formula (Id), or a conjugate comprising a structure represented by formula (A)) is a conjugate comprising a group of formula (B): [ka] where n is 0 to 15 (e.g., 3) and m is 0 to 15 (e.g., 4). It should be understood that the stereochemistry shown in formula (B) can be applied to the corresponding moiety of any formula or structure provided herein (e.g., formula (Ia), (Ib), (Ic), (Id), or (A)).

[0070] In some embodiments, the conjugates disclosed herein (e.g., those of formula (I): [R 1 ] n1 -R 2 Complexes containing structures represented by R 1In some embodiments, the light chain constant region of an antibody of the present invention (e.g., a conjugate comprising a structure represented by Formula (Ia), (Ib), or (Ic)), a conjugate comprising a structure represented by Formula (Id), or a conjugate comprising a structure represented by Formula (A)) is independently covalently linked to an oligonucleotide by a linkage site represented by K188 (according to Kabat numbering) and / or a linkage site represented by K190 (according to Kabat numbering) in the light chain constant region. 1 ] n1 -R 2 Complexes containing structures represented by R 1 a conjugate comprising a structure represented by Formula (Ia), (Ib), or (Ic), a conjugate comprising a structure represented by Formula (Id), or a conjugate comprising a structure represented by Formula (A)) is independently covalently linked to an oligonucleotide at a linkage site represented by a lysine (K) residue in the light chain sequence motif DYEKHKVYA (SEQ ID NO: 27).

[0071] antibody In some embodiments, the conjugates described herein comprise an antibody that binds to human transferrin receptor 1 (TfR1). An example of the amino acid sequence of human TfR1, corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens), is as follows: MMDQARSAFSNLFGGEDFPAARRLYWDDLKRKLSEKLDSTDFTGTIKLLNENSYVPREAGSQKDENLALYVENQFREFKLSKVWRDQHFVKI QVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNV LKEIKILNIFGVIKGFVEPDHYVVVGAQRDAWGPGAAKSGVGTALLLKLAQMFSDMVLKDGFQPSRSIIFASWSAGDFGSVGATEWLEGYLSSLHLKAFTYINLDKAVLGTSNFKVSASPLLYTLIEKTMQNVKHPVTGQFLYQDSNWASKVEKLTLDNAAFPFLAYSGIPAVSFCFCEDTDYPYLGTTMDTYKELIERIPELNKVARAAAEVAGQFVIKLTHDVELNLDYERYNSQLLSFVRDLNQYRADIKEMGLSLQWLYSARGDFFRATSRLTTDFGNAEKTDRFVMKKLNDRVMRVEYHFLSPYVSPKESPFRHVFWGSGSHTLPALLENLKLRKQNNGAFNETLFRNQLALATWTIQGAANALSGDVWDIDNEF (SEQ ID NO: 22).

[0072] Table 2 provides examples of anti-TfR1 antibody sequences useful in the conjugates provided herein. [Table 2]

[0073] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 1 (according to the IMGT definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 2 (according to the IMGT definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 3 (according to the IMGT definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 4 (according to the IMGT definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 5 (according to the IMGT definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the IMGT definition system).

[0074] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 7 (according to the Kabat definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 8 (according to the Kabat definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 9 (according to the Kabat definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 10 (according to the Kabat definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 11 (according to the Kabat definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 6 (according to the Kabat definition system).

[0075] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain complementarity determining region 1 (CDR-H1) of SEQ ID NO: 12 (according to the Chothia definition system), a heavy chain complementarity determining region 2 (CDR-H2) of SEQ ID NO: 13 (according to the Chothia definition system), a heavy chain complementarity determining region 3 (CDR-H3) of SEQ ID NO: 14 (according to the Chothia definition system), a light chain complementarity determining region 1 (CDR-L1) of SEQ ID NO: 15 (according to the Chothia definition system), a light chain complementarity determining region 2 (CDR-L2) of SEQ ID NO: 5 (according to the Chothia definition system), and a light chain complementarity determining region 3 (CDR-L3) of SEQ ID NO: 16 (according to the Chothia definition system).

[0076] In some embodiments, the anti-TfR1 antibodies of the present disclosure comprise a heavy chain variable region (VH) that contains 25 or fewer amino acid variations (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 variations) in the framework regions compared to a VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or in addition (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 variations) in the framework regions compared to a VL comprising the amino acid sequence of SEQ ID NO: 18.

[0077] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence in a framework region that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to a VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or additionally (e.g., in addition), in some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VL comprising an amino acid sequence in a framework region that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to a VL comprising the amino acid sequence of SEQ ID NO: 18.

[0078] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or additionally (for example, in addition), in some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 18.

[0079] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 19. Alternatively or additionally (e.g., in addition), an anti-TfR1 antibody of the present disclosure comprises a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, an anti-TfR1 antibody of the present disclosure is a Fab comprising a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO: 19. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the present disclosure is a Fab comprising a light chain comprising an amino acid sequence at least 75% (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the amino acid sequence of SEQ ID NO:20.

[0080] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, an anti-TfR1 antibody of the present disclosure is a Fab comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the present disclosure is a Fab comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0081] In some embodiments, the anti-TfR1 antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also referred to as pyroglutamate formation (pyroGlu), may occur at an N-terminal glutamate (Glu) residue and / or glutamine (Gln) residue in the antibody during production. Therefore, it should be understood that an antibody identified as having a sequence containing an N-terminal glutamate or glutamine residue encompasses antibodies that have undergone pyroglutamate formation as a result of post-translational modification. In some embodiments, pyroglutamate formation occurs in the heavy chain sequence. In some embodiments, pyroglutamate formation occurs in the light chain sequence.

[0082] Connection part Provided herein are conjugates comprising an antibody covalently linked to one or more oligonucleotides. In some embodiments, the antibody comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region, and a light chain comprising a light chain variable region (VL) and a light chain constant region. In some embodiments, each of the one or more oligonucleotides is covalently linked at a linkage site represented by a lysine (K) residue of the antibody. In some embodiments, provided herein are compositions comprising a plurality of conjugates, each conjugate comprising an antibody covalently linked to one or more oligonucleotides, each of the one or more oligonucleotides being linked to the antibody via a different linkage site.

[0083] In some embodiments, the linking site is in the light chain of the antibody. In some embodiments, the linking site is in the light chain constant region of the antibody. For example, in some embodiments, the linking site is represented by K188 or K190 in the light chain constant region according to the Kabat numbering system.

[0084] In some embodiments, the light chain constant region of the antibody of the conjugate in the composition is independently covalently linked to the oligonucleotide at a linkage site represented by K188 (according to Kabat numbering) and / or K190 (according to Kabat numbering) of the light chain constant region of the antibody. For example, in some embodiments, the linkage site is represented by K188 (according to Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K190 (according to Kabat numbering) of the light chain constant region of the antibody. In some embodiments, the linkage site is represented by K188 (according to Kabat numbering) and K190 (according to Kabat numbering) of the light chain constant region of the antibody. In some embodiments, at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the antibody light chain constant region of the conjugate in the composition is independently covalently linked to the oligonucleotide at the linkage site represented by K188 (according to Kabat numbering) and / or the linkage site represented by K190 (according to Kabat numbering) of the antibody light chain constant region. In some embodiments, about 80% to 98%, 80% to 95%, 80% to 90%, 80% to 85%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or 95% to 98% of the antibody light chain constant region of the conjugate in the composition is independently covalently linked to the oligonucleotide at linkage site K188 (based on Kabat numbering) and / or linkage site K190 (based on Kabat numbering) in the antibody light chain constant region.In some embodiments, about 85% to 95% (e.g., 85% to 95%, 85% to 90%, or 90% to 95%) of the antibody light chain constant regions of the conjugate in the composition are independently covalently linked to the oligonucleotide at the linkage site represented by K188 (according to Kabat numbering) and / or the linkage site represented by K190 (according to Kabat numbering) of the antibody light chain constant regions. In some embodiments, about 90% to 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) of the antibody light chain constant regions of the conjugate in the composition are independently covalently linked to the oligonucleotide at the linkage site represented by K188 (according to Kabat numbering) and / or the linkage site represented by K190 (according to Kabat numbering) of the antibody light chain constant regions. A conjugate comprising the light chain constant region of an antibody covalently linked to an oligonucleotide at the linkage site represented by K188 (based on Kabat numbering) and / or the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region should be understood to encompass conjugates comprising an antibody covalently linked to an oligonucleotide at the linkage site represented by K188 (based on Kabat numbering) of the light chain constant region of the antibody; conjugates comprising an antibody covalently linked to an oligonucleotide at the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody; and / or conjugates comprising an antibody covalently linked to an oligonucleotide at the linkage site represented by K188 (based on Kabat numbering) and the linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of the antibody.

[0085] In some embodiments, the numbering of lysine (K) residues referred to herein is based on Kabat numbering (Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)). The variable and constant regions of the heavy and light chains of the antibodies provided herein are numbered separately. Kabat numbering of the light and heavy chain variable regions of an antibody is known in the art, e.g., Kabat et al. (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991). Kabat numbering of the light chain constant regions and heavy chain constant regions of the antibodies provided herein can be found at imgt.org / IMGTScientificChart / Numbering / Hu_IGHGnber.html and imgt.org / IMGTScientificChart / Numbering / Hu_IGKCnber.html (see also Edelman, GM et al., Proc. Natl. Acad. USA, 63, 78-85 (1969). PMID: 5257969; Hieter, PA et al., Cell, 22, 197-207 (1980). PMID: 6775818; Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. 647, 662, 680, 689 (1991)).

[0086] In some embodiments, the antibody light chains of the conjugates in the composition are independently covalently linked to the oligonucleotide at a linkage site represented by a lysine (K) residue in the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27). For example, in some embodiments, the linkage site is represented by a K at position 4 of the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27). In some embodiments, the linkage site is represented by a K at position 6 of the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27). In some embodiments, the linkage site is represented by a K at positions 4 and 6 of the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27). In some embodiments, at least 80% (e.g., at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the antibody light chains of the conjugates in the composition are independently covalently linked to an oligonucleotide at a linkage site represented by a lysine (K) residue in the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27). In some embodiments, 80% to 98%, 80% to 95%, 80% to 90%, 80% to 85%, 85% to 98%, 85% to 95%, 85% to 90%, 90% to 98%, 90% to 95%, 95% to 97%, or 95% to 98% of the antibody light chains of the conjugates in the composition are independently covalently linked to the oligonucleotide at linkage sites represented by lysine (K) residues in the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27). In some embodiments, 85% to 95% (e.g., 85% to 95%, 85% to 90%, or 90% to 95%) of the antibody light chains of the conjugates in the composition are independently covalently linked to the oligonucleotide at linkage sites represented by lysine (K) residues in the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27).In some embodiments, 90% to 95% (e.g., about 90%, about 91%, about 92%, about 93%, about 94%, or about 95%) of the antibody light chains of the conjugates in the composition are independently covalently linked to the oligonucleotide at linkage sites represented by lysine (K) residues in the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27). A conjugate comprising an antibody light chain covalently linked to an oligonucleotide at a linkage site represented by a lysine (K) residue in the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27) should be understood to encompass a conjugate comprising an antibody covalently linked to an oligonucleotide at a linkage site represented by a K at position 4 in the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27); a conjugate comprising an antibody covalently linked to an oligonucleotide at a linkage site represented by a K at position 6 in the antibody light chain sequence motif DYEKHKVYA (SEQ ID NO: 27); and / or a conjugate comprising an antibody covalently linked to an oligonucleotide at a linkage site represented by a K at position 4 and a K at position 6 in the light chain sequence motif DYEKHKVYA (SEQ ID NO: 27).

[0087] As used herein, the term "about" refers to a variation of ±5% to 10% based on the percentage that the term modifies.

[0088] Oligonucleotides In some embodiments, the oligonucleotide of the conjugate described herein is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide is useful for targeting DMD (e.g., for exon skipping). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a DMD allele (e.g., a mutated DMD allele). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target a region of DMD RNA (e.g., the Dp427m transcript of SEQ ID NO: 23, or a DMD RNA such as a pre-mRNA comprising the sequence of SEQ ID NO: 26). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region complementary to DMD RNA (e.g., the Dp427m transcript of SEQ ID NO: 23). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region complementary to an exon (e.g., exon 45) or an intron of DMD RNA. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a region complementary to an exon (e.g., exon 46) or an intron of DMD RNA (e.g., Homo sapiens). The oligonucleotides target splice donor sites, splice acceptor sites, branch points, or exon splicing enhancers (ESEs) of the DMD pre-mRNA encoded by the sapiens dystrophin (DMD) gene (e.g., NCBI accession number NG_012232.1). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target exon splicing enhancer (ESE) sequences within DMD (e.g., the ESE sequence of exon 45). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) target splice acceptor site sequences within DMD (e.g., the splice acceptor site sequence of exon 45).

[0089] Examples of DMD RNA sequences and exon sequences that may be targeted by the oligonucleotides of the complex are provided below.

[0090] Homo sapiens dystrophin (DMD), transcript variant Dp427m, mRNA (NCBI Reference Sequence: NM_004006.2; SEQ ID NO: 23).

[0091] Homo sapiens dystrophin (DMD), locus near exon 45 (nucleotide positions 1376066 to 1376301 of NCBI reference sequence: NG_012232.1, exon 45 is located at Underlined part ): [ka]

[0092] Homo sapiens dystrophin (DMD), transcript variant Dp427m, exon 45 (nucleotide positions 6683-6858 of the NCBI reference sequence: NM_004006.2): [ka]

[0093] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 10-35 (e.g., 10-35, 15-35, 10-30, 15-30, 15-25, 17-27, 18-26, 19-25, 20-24, 20-35, 20-30, 20-25, 20-23, 21-24, 21-23, or 20-22) nucleotides in length. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) are 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, optionally 17-27 or 22 nucleotides in length.

[0094] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a complementary region of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) consecutive nucleotides to DMD RNA. In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a complementary region of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) consecutive nucleotides to an exon of DMD RNA.

[0095] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a complementary region of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30) consecutive nucleotides to a DMD sequence as set forth in any one of SEQ ID NOs: 23-26.

[0096] In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise a complementary region of at least six (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) consecutive nucleotides to the target sequence as set forth in SEQ ID NO: 25 (CAGGAACTCCAGGATGGCATTG). In some embodiments, oligonucleotides useful for targeting DMD (e.g., for exon skipping) comprise at least six (e.g., at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, or 22) consecutive nucleotides of the sequence as set forth in SEQ ID NO: 21 (CAATGCCATCCTGGAGTTCCTG).

[0097] In some embodiments, an oligonucleotide useful for targeting DMD (e.g., for exon skipping) comprises the nucleotide sequence of SEQ ID NO: 21. In some embodiments, any one of the oligonucleotides provided herein is a PMO. In some embodiments, each internucleoside linkage of the PMO is a phosphorodiamidate linkage.

[0098] 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) may be equivalently identified as a thymine nucleotide or nucleoside.

[0099] In some embodiments, any one or more of the thymine bases (T) in any one of the oligonucleotides provided herein may independently and optionally be uracil bases (U), and / or any one or more of the U's in the oligonucleotides provided herein (e.g., the oligonucleotide as set forth in SEQ ID NO: 21) may independently and optionally be T.

[0100] composition In some embodiments, a composition comprising a complex (i.e., multiple complexes) is formulated in a manner suitable for the methods described herein. In some embodiments, a composition comprising a muscle-targeting complex is 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. Thus, in some embodiments, a composition comprising a complex (e.g., multiple complexes comprising a PMO covalently linked to a Fab) is formulated with an appropriate buffer (e.g., a pharmaceutical buffer). In some embodiments, a composition comprising a muscle-targeting complex (e.g., a complex comprising a PMO covalently linked to a Fab) is formulated as an aqueous solution. In some embodiments, a composition comprising multiple complexes can be lyophilized (e.g., for storage). In some embodiments, a lyophilized composition may be reconstituted (e.g., with water) for administration to a subject. The composition (e.g., in an aqueous solution, frozen composition, or lyophilized composition) can be suitably prepared so that a sufficient amount of the complex enters the target muscle cell when administered to a subject, into the environment surrounding the target cell, or systemically.

[0101] In some embodiments, a composition (e.g., in an aqueous solution) for administration to a subject in the methods described herein comprises a conjugate (i.e., a plurality of conjugates), each conjugate comprising a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an antibody. In some embodiments, a composition (e.g., an aqueous solution) for administration to a subject in the methods described herein comprises a conjugate, each conjugate comprising a phosphorodiamidate morpholino oligomer (PMO) covalently linked to an anti-TfR1 antibody, optionally wherein the antibodies in such conjugates comprise CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as described in Table 2, and further optionally wherein the antibodies in such conjugates comprise a VH and / or (e.g., and) a VL as described in Table 2. In some embodiments, the antibody is an anti-TfR1 Fab (e.g., an anti-TfR1 Fab comprising CDRs having the amino acid sequences set forth in Table 2, and / or a VH and / or VL having the amino acid sequences set forth in Table 2).

[0102] In some embodiments, the compositions (e.g., aqueous solutions) for administration to a subject in the methods described herein comprise a composition in which each conjugate is represented by the formula (I): [R 1 ] n1 -R 2 wherein each R 1 independently includes compounds containing oligonucleotides (e.g., PMOs), and R 2 is covalently linked to R 2 comprises an anti-TfR1 antibody, wherein in each conjugate, n1 independently represents the number of TfR1 antibodies in each conjugate. 1 is an integer greater than or equal to 1 that represents the number of instances of

[0103] In some embodiments, the value of n1 for each conjugate in the composition is independently and optionally from 1 to a maximum of 1000 mg / mL of the antibody (R 2In some embodiments, the value of n1 for each conjugate in the composition is independently and optionally selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27. In some embodiments, the value of n1 for each conjugate in the composition is independently and optionally selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26. In some embodiments, the value of n1 for each conjugate in the composition is independently and arbitrarily selected from integers ranging from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3. In some embodiments, the average value of n1 for the conjugates in the composition is within the range of 1 to 3, 1 to 5, 1 to 10, 1 to 26, or 1 to 27.

[0104] In some embodiments, the composition for administration to a subject in the methods described herein comprises (e.g., a trace amount of) an unconjugated antibody and an antibody conjugated to one or more oligonucleotides. In some embodiments, the unconjugated antibody has the formula (I): [R 1 ] n1 -R 2 (wherein n1 is 0). Accordingly, in some embodiments, the composition for administration to a subject in the methods described herein may be referred to as a compound having a structure represented by formula (I): [R 1 ] n1 -R 2 In the formula, each R 1 independently comprises a group comprising an oligonucleotide, and R 2 includes an antibody, and n1 independently represents the R 1 In some embodiments, the compound of formula (I): [R 1 ] n1 -R 2is less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05%, or less than 0.01% compared to all compounds of that structure in which n1 is 1 or greater in the composition.

[0105] In some embodiments, R in the conjugates herein (e.g., conjugates of the compositions provided herein) 1 Each instance of R is conjugated to a different amino acid residue of the antibody. In some embodiments, each different amino acid comprises an ε-amino group (e.g., lysine, arginine). However, in some embodiments, R 1 In some embodiments, each different amino acid to which R is covalently linked is a cysteine. 1 Each different amino acid to which R is covalently linked is a lysine. 1 is covalently linked directly to an amino acid residue of the antibody. 1 is covalently linked directly to an amino acid residue of the antibody. 1 In some embodiments, the R is present in trace amounts, undetectable amounts, or is completely absent. 1 is covalently linked to amino acid residues in the CDR regions of the antibody, the conjugate is undetectable in the preparation using standard detection techniques.

[0106] In some embodiments, the light chain constant regions of the antibodies of the conjugate in the composition are independently covalently linked to the oligonucleotide at linkage sites represented by K188 (according to Kabat numbering) and / or K190 (according to Kabat numbering) of the light chain constant regions of the antibodies. In some embodiments, the light chains of the antibodies of the conjugate in the composition are independently covalently linked to the oligonucleotide at linkage sites represented by the lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the antibodies.

[0107] In some embodiments, the compositions (e.g., aqueous solutions) described herein comprise a compound of formula (I): [R 1 ] n1 -R 2 wherein each R in the complex of the composition provided herein comprises a complex comprising a structure represented by 1 are independently represented by formula (Ia): [ka] wherein R 3 is an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO); where in each complex, n1 is independently R 1 is an integer (e.g., 1 or greater) representing the number of instances of each R 1 is R at attachment point A. 2 In some embodiments, R 2 includes anti-TfR1 antibodies comprising a sequence as set forth in Table 2. For example, in some embodiments, R 2comprises an anti-TfR1 antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as set forth in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as set forth in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as set forth in SEQ ID NO: 3, 9, or 14; and / or a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as set forth in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as set forth in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, R 2 In some embodiments, the R comprises an anti-TfR1 antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. 2 In some embodiments, the R comprises an anti-TfR1 antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. 2 In some embodiments, the RfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. 2 In some embodiments, the RfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. 2 In some embodiments, R comprises an anti-TfR1 antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. 2includes an anti-TfR1 antibody that is a Fab fragment. In some embodiments, R3 is an oligonucleotide, e.g., a phosphorodiamidate morpholino oligomer (PMO) comprising the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21). In some embodiments, each internucleoside linkage of the PMO is a phosphorodiamidate linkage. In some embodiments, R 2 contains an anti-TfR1 antibody (e.g., Fab), and each R 1 are covalently linked (e.g., indirectly or directly, e.g., directly) to different amino acid residues of an antibody (e.g., a Fab) at attachment point A, optionally where each different amino acid residue is a lysine. In some embodiments, each R 1 is attached to R at the attachment point A via a linkage site represented by a lysine (K) residue in the antibody. 2 In some embodiments, the lysine (K) residue linkage site of the anti-TfR1 antibody is represented by K188 (based on Kabat numbering) and / or K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody. In some embodiments, the lysine (K) residue linkage site is represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the anti-TfR1 antibody. In some embodiments, n1 in each conjugate is independently an integer (e.g., an integer ranging from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, the composition (e.g., in an aqueous solution) for administration to a subject in the methods described herein further comprises a lysine (K) residue of formula (I): [R 1 ] n1 -R 2 wherein n1 is 0.

[0108] In some embodiments, the compositions (e.g., aqueous solutions) described herein comprise a compound of formula (I): [R 1 ] n1 -R 2 wherein R in the complex of the composition described herein comprises a complex comprising the structure 1Each example has formula (Ib): [ka] wherein -p is a phosphorodiamidate linkage of a phosphorodiamidate morpholino oligomer (PMO), and wherein the PMO comprises the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21); in each conjugate, n1 is independently selected from the R 1 is an integer (e.g., 1 or greater) representing the number of instances of 1 is R at attachment point A. 2 In some embodiments, R 2 includes anti-TfR1 antibodies comprising a sequence as set forth in Table 2. For example, in some embodiments, R 2 comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as set forth in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as set forth in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as set forth in SEQ ID NO: 3, 9, or 14; and / or an anti-TfR1 antibody comprising a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as set forth in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as set forth in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, R 2 In some embodiments, the R comprises an anti-TfR1 antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. 2 In some embodiments, the R comprises an anti-TfR1 antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18.2 In some embodiments, the RfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. 2 In some embodiments, the RfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. 2 In some embodiments, R comprises an anti-TfR1 antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. 2 comprises an anti-TfR1 antibody that is a Fab fragment. In some embodiments, n1 in each conjugate is independently an integer (e.g., an integer ranging from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, R 2 contains an anti-TfR1 antibody (e.g., Fab), and each R 1 are covalently linked (e.g., indirectly or directly, e.g., directly) to different amino acid residues of an antibody (e.g., a Fab) at attachment point A, optionally where each different amino acid residue is a lysine. In some embodiments, each R 1 is attached to R at the attachment point A via a linkage site represented by a lysine (K) residue in the antibody. 2 In some embodiments, the lysine (K) residue linkage site of the anti-TfR1 antibody is represented by K188 (based on Kabat numbering) and / or K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody. In some embodiments, the lysine (K) residue linkage site is represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the anti-TfR1 antibody. In some embodiments, the composition (e.g., in an aqueous solution) for administration to a subject in the methods described herein further comprises a lysine (K) residue of formula (I): [R 1 ] n1 -R 2wherein n1 is 0.

[0109] In some embodiments, the compositions (e.g., aqueous solutions) described herein comprise a compound of formula (I): [R 1 ] n1 -R 2 wherein each example R in the complex of the composition described herein comprises a complex comprising a structure represented by 1 is represented by formula (Ic): [ka] wherein, in each complex, n1 independently represents a group represented by R 1 An integer representing the number of instances of (e.g., 1 (or more), and each R 1 is R at attachment point A. 2 In some embodiments, R 2 includes anti-TfR1 antibodies comprising a sequence as set forth in Table 2. For example, in some embodiments, R 2 comprises an anti-TfR1 antibody comprising a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as set forth in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as set forth in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as set forth in SEQ ID NO: 3, 9, or 14; and / or a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as set forth in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as set forth in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as set forth in SEQ ID NO: 6 or 16. In some embodiments, R 2In some embodiments, the R comprises an anti-TfR1 antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. 2 In some embodiments, the R comprises an anti-TfR1 antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. 2 In some embodiments, the RfR1 antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. 2 In some embodiments, the RfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. 2 In some embodiments, R comprises an anti-TfR1 antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv. 2 comprises an anti-TfR1 antibody that is a Fab fragment. In some embodiments, n1 in each conjugate is independently an integer (e.g., an integer ranging from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, R 2 contains an anti-TfR1 antibody (e.g., Fab), and each R 1 are covalently linked (e.g., indirectly or directly, e.g., directly) to different amino acid residues of an antibody (e.g., a Fab) at attachment point A, optionally where each different amino acid residue is a lysine. In some embodiments, each R 1 is attached to R at the attachment point A via a linkage site represented by a lysine (K) residue in the antibody. 2In some embodiments, the lysine (K) residue linkage site of the anti-TfR1 antibody is represented by K188 (based on Kabat numbering) and / or K190 (based on Kabat numbering) of the light chain constant region of the anti-TfR1 antibody. In some embodiments, the lysine (K) residue linkage site is represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the anti-TfR1 antibody. In some embodiments, the composition (e.g., in an aqueous solution) for administration to a subject in the methods described herein further comprises a lysine (K) residue of formula (I): [R 1 ] n1 -R 2 wherein n1 is 0.

[0110] In some embodiments, the compositions (e.g., aqueous solutions) described herein comprise a compound of Formula (Id): [ka] wherein -p is a phosphorodiamidate linkage of a phosphorodiamidate morpholino oligomer (PMO), the PMO having the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21), and R 2 comprises an anti-TfR1 antibody (e.g., Fab) comprising CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 2, optionally wherein the antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and further optionally wherein the antibody (e.g., Fab) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20, and wherein in each conjugate, n1 is independently an integer (e.g., 1 or more) representing the number of instances of the bracketed group, each instance of the bracketed group being covalently linked to a different amino acid residue of the antibody (e.g., Fab), and optionally each different amino acid residue is lysine. In some embodiments, R 2includes an anti-TfR1 antibody (e.g., a Fab) comprising a sequence as set forth in Table 2. For example, in some embodiments, R 2 comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as represented in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as represented in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as represented in SEQ ID NO: 3, 9, or 14; and / or an anti-TfR1 antibody (e.g., a Fab) comprising a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as represented in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as represented in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as represented in SEQ ID NO: 6 or 16. In some embodiments, R 2 In some embodiments, the RfR1 antibody (e.g., Fab) comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. 2 In some embodiments, the R comprises an anti-TfR1 antibody (e.g., a Fab) that comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. 2 In some embodiments, the RfR1 antibody (e.g., a Fab) comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. 2comprises an anti-TfR1 antibody (e.g., Fab) comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, n1 in each conjugate is independently an integer (e.g., an integer ranging from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, n1 in each conjugate is independently an integer of 1 or greater. In some embodiments, R 2 includes an anti-TfR1 antibody (e.g., Fab) covalently linked (e.g., indirectly or directly linked, e.g., directly linked) via a different amino acid residue (e.g., Fab), optionally wherein each different amino acid residue is a lysine. In some embodiments, each instance of a group enclosed in square brackets in the structure represented by formula (Id) is covalently linked to a linkage site represented by a lysine (K) residue in an anti-TfR1 antibody (e.g., Fab). In some embodiments, the lysine (K) residue linkage site in an anti-TfR1 antibody is represented by K188 (according to Kabat numbering) and / or K190 (according to Kabat numbering) in the light chain constant region of the anti-TfR1 antibody. In some embodiments, the lysine (K) residue linkage site is represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain of the anti-TfR1 antibody. In some embodiments, the composition (eg, in an aqueous solution) for administration to a subject in the methods described herein further comprises a conjugate wherein n1 is 0.

[0111] In some embodiments, the compositions (e.g., aqueous solutions) described herein comprise a compound of Formula (A): [ka] wherein n is 0 to 15 (e.g., 3), and m is 0 to 15 (e.g., 4). In some embodiments, the antibody is an anti-TfR1 antibody (e.g., the anti-TfR1 antibody provided in Table 2). In some embodiments, the oligonucleotide is a PMO and comprises the base sequence of SEQ ID NO: 21. In some embodiments, each internucleoside linkage of the PMO is a phosphorodiamidate linkage. In some embodiments, the amide shown adjacent to the antibody in the structure results from reaction with an amine of the antibody, such as the lysine epsilon amine. In some embodiments, the antibody comprises a sequence as set forth in Table 2. For example, in some embodiments, the antibody comprises a heavy chain complementarity determining region 1 (CDR-H1) comprising the sequence as represented in SEQ ID NO: 1, 7, or 12, a heavy chain complementarity determining region 2 (CDR-H2) comprising the sequence as represented in SEQ ID NO: 2, 8, or 13, a heavy chain complementarity determining region 3 (CDR-H3) comprising the sequence as represented in SEQ ID NO: 3, 9, or 14; and / or a light chain complementarity determining region 1 (CDR-L1) comprising the sequence as represented in SEQ ID NO: 4, 10, or 15, a light chain complementarity determining region 2 (CDR-L2) comprising the sequence as represented in SEQ ID NO: 5 or 11, and a light chain complementarity determining region 3 (CDR-L3) comprising the sequence as represented in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20.In some embodiments, the antibody is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, an scFv, or an Fv.

[0112] In each of the compositions disclosed herein, the complex of the composition (e.g., each R 1 a conjugate of formula (I): [R 1 ] n1 -R 2 a complex comprising a structure represented by formula (Id); a complex comprising a structure represented by formula (A) is provided by the method of claim 1, wherein the complex comprises a compound represented by formula (B): [ka] wherein n is 0 to 15 (e.g., 3) and m is 0 to 15 (e.g., 4). It should be understood that the stereochemistry shown in formula (B) can be applied to the corresponding moiety of any formula or structure provided herein (e.g., formula (Ia), (Ib), (Ic), (Id), or (A)).

[0113] In some embodiments, the conjugates (e.g., each R 1 a conjugate of formula (I): [R 1 ] n1 -R 2の In some embodiments, the light chain constant regions of the antibodies of the conjugate (e.g., a conjugate comprising a structure represented by Formula (Id); a conjugate comprising a structure represented by Formula (Id); or a conjugate comprising a structure represented by Formula (A)) are independently covalently linked to an oligonucleotide at linkage sites represented by K188 (based on Kabat numbering) and / or K190 (based on Kabat numbering) of the light chain constant region of the antibody of the conjugate. 1 a conjugate of formula (I): [R 1 ] n1 -R 2a conjugate comprising a structure represented by Formula (Id); or a conjugate comprising a structure represented by Formula (A)) is independently covalently linked to an oligonucleotide at a linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the antibody light chain of the conjugate.

[0114] In some embodiments, provided is a product (e.g., a lyophilized composition described herein) produced by a process comprising lyophilizing an aqueous solution of the composition (e.g., an aqueous form).

[0115] In some embodiments, the composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral, e.g., intravenous, intradermal, and subcutaneous. Typically, the route of administration is intravenous or subcutaneous.

[0116] Method of Use / Treatment A conjugate comprising an anti-TfR1 antibody (e.g., a Fab) covalently linked to an oligonucleotide (e.g., a phosphorodiamidate morpholino oligomer (PMO)) described herein is effective in treating a subject with a dystrophinopathy, e.g., Duchenne muscular dystrophy. In some embodiments, the conjugate comprises an oligonucleotide that promotes exon skipping of mRNA expressed from a mutated DMD allele.

[0117] 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 suffer from Duchenne muscular dystrophy or other dystrophinopathy. In some embodiments, the subject has a mutated DMD allele, which may optionally include at least one mutation in a DMD exon that causes a frameshift mutation and leads to inappropriate RNA splicing / processing. In some embodiments, the subject suffers from severe dystrophinopathy, for example, muscle atrophy or muscle loss. In some embodiments, the subject has asymptomatic increases in serum creatine phosphokinase (CK) levels and / or muscle spasms accompanied by myoglobinuria (for example, and). In some embodiments, the subject has a progressive muscle disease, such as Duchenne or Becker muscular dystrophy, or DMD-related dilated cardiomyopathy (DCM). In some embodiments, the subject does not suffer from dystrophinopathy symptoms.

[0118] In some embodiments, the subject has a mutation in the DMD gene that is amenable to exon 45 skipping. In some embodiments, the complexes described herein are effective in treating a subject with a mutation in the DMD gene that is amenable to exon 45 skipping. In some embodiments, the complexes comprise an oligonucleotide, e.g., an antisense oligonucleotide, that promotes exon 45 skipping of a pre-mRNA in a pre-mRNA encoded from a mutated DMD gene (e.g., a mutated DMD gene that is amenable to exon 45 skipping).

[0119] In some embodiments, pharmaceutical compositions comprising the conjugates described herein may be administered by a suitable route, which may include intravenous administration, for example, as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration may be performed by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial, or intrathecal routes. In some embodiments, the pharmaceutical composition may be in solid, aqueous, or liquid form. In some embodiments, the aqueous or liquid form may be sprayed or lyophilized. In some embodiments, the sprayed or lyophilized form may be reconstituted with an aqueous or liquid solution.

[0120] Compositions for intravenous administration may contain various carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by infusion, whereby a pharmaceutical formulation containing the conjugate and a pharmaceutically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the conjugate may be dissolved and administered in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0121] In some embodiments, a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent (e.g., an anti-TfR1 antibody, e.g., an anti-TfR1 Fab) covalently linked to an oligonucleotide (e.g., a PMO) is administered by site-specific or local delivery techniques. Examples of these techniques include an implantable depot source of the conjugate, a local delivery catheter, a site-specific carrier, direct injection, or direct application.

[0122] In some embodiments, a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent (e.g., an anti-TfR1 antibody, e.g., an anti-TfR1 Fab) covalently linked to an oligonucleotide (e.g., a PMO) is administered to a subject at an effective concentration to confer a therapeutic effect. As will be appreciated by those skilled in the art, the effective amount will vary depending on the severity of the disease, the specific characteristics of the subject being treated, such as age, physical condition, health, or weight, the duration of the treatment, the nature of any concomitant therapy, the route of administration, and related factors. These related factors are known to those skilled in the art and can be addressed with minimal routine experimentation. In some embodiments, the effective concentration is the maximum dose deemed safe for the patient. In some embodiments, the effective concentration will be the lowest feasible concentration that provides maximum efficacy.

[0123] Empirical considerations, such as the half-life of the conjugate in the subject, will generally contribute to determining the concentration of pharmaceutical composition used for treatment. The frequency of administration may be empirically determined and adjusted to maximize the efficacy of treatment.

[0124] The efficacy of treatment can be assessed using any suitable method. In some embodiments, the efficacy of treatment can be assessed by observing the symptoms associated with dystrophinopathy, such as muscle atrophy or muscle weakness, by subject self-reported outcomes, such as mobility, self-care, usual activities, pain / discomfort, and anxiety / depression, or by quality of life indicators, such as lifespan.

[0125] In some embodiments, a pharmaceutical composition comprising a conjugate comprising a muscle-targeting agent (e.g., an anti-TfR1 antibody, e.g., an anti-TfR1 Fab) covalently linked to an oligonucleotide (e.g., a PMO) described herein is administered to a subject at an effective concentration sufficient to modulate target gene activity or expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% compared to a control, e.g., a baseline level of gene expression before treatment.

[0126] example Example 1. Delivery of DMD-targeted oligonucleotides to muscle tissue after systemic administration to non-human primates This study evaluated the delivery of a conjugate (referred to in this example as the "anti-TfR1 Fab-ASO conjugate") comprising an anti-TfR1 Fab having the heavy and light chain sequences shown in Table 2, covalently linked (by lysine conjugation) to a DMD exon 45 skipping oligonucleotide (the oligonucleotide is referred to in this example as the "ASO") via a cleavable linker containing a valine-citrulline sequence. The ASO is a PMO and comprises the base sequence of SEQ ID NO: 21. The anti-TfR1 Fab-ASO conjugate has the following structure: [ka] where -p is a phosphorodiamidate linkage of the ASO, and where R 2 contains an anti-TfR1 Fab.

[0127] The distribution of ASOs in muscle tissue of healthy non-human primates was examined in vivo. Naive male cynomolgus monkeys (n=4 per group) were administered two doses of 30 mg / kg of ASO not covalently linked to an antibody ("naked") or an anti-TfR1 Fab-ASO conjugate (a dose equivalent to 30 mg / kg of ASO) by intravenous infusion at weeks 0 and 4. Animals were sacrificed, and tissues were harvested 4 weeks after the second dose (i.e., 8 weeks after the first dose).

[0128] Tissue ASO accumulation was quantified using a hybridization ELISA with a probe complementary to the ASO sequence. A standard curve was generated, and ASO levels (in ng / g) were derived from linear regression of the standard curve. Following administration of the anti-TfR1 Fab-ASO conjugate, ASOs were distributed at higher levels in all tissues evaluated compared to administration of naked ASOs. Intravenous administration of naked ASOs resulted in ASO levels at the time points evaluated that were near background levels in the heart and diaphragm ("Dia") and extremely low in the quadriceps ("Quad") and biceps ("Biceps") muscles. Administration of the anti-TfR1 Fab-ASO conjugate resulted in greater accumulation of ASOs in the measured tissues at the time points evaluated, in the following order: heart > diaphragm ("Dia") > quadriceps ("Quad") > biceps ("Biceps") muscle (Figure 1).

[0129] The results demonstrated that a complex comprising an anti-TfR1 antibody (e.g., a Fab) covalently linked to an oligonucleotide (e.g., a PMO) achieved enhanced delivery of the oligonucleotide to muscle tissue following systemic administration compared to administration of an ASO that was not included within the complex (i.e., not covalently linked to the anti-TfR1 antibody).

[0130] Example 2. Exon 45 skipping in human skeletal myoblasts This study evaluated exon 45 skipping in DMD transcripts after treatment with the conjugate described in Example 1 (referred to in this example as the "anti-TfR1 Fab-ASO conjugate"), which comprises an anti-TfR1 Fab covalently linked to a DMD exon 45 skipping oligonucleotide ("ASO").

[0131] Human skeletal myoblasts (obtained from the Association Institut de Myologie) were modified using CRISPR / Cas to introduce a deletion of exon 46 of the DMD gene to reflect a common mutation in Duchenne muscular dystrophy patients amenable to exon 45 skipping, generating a mutant myoblast cell line ("Del46"). Wild-type and mutant myoblasts were used to generate myotubes ("WT myotubes" and "Del46 myotubes," respectively).

[0132] WT and Del46 myotubes were treated with anti-TfR1 Fab-ASO conjugates at an ASO-equivalent concentration of 10 μM. After incubation with the conjugate, total RNA was collected and cDNA synthesis was performed. To assess the extent of exon 45 skipping in the cells, end-point PCR was performed. PCR products were quantified using capillary electrophoresis, and the relative amounts of skipped and non-skipped amplicons were calculated:

number

[0133] The results demonstrate that the anti-TfR1 Fab-ASO conjugate induces greater exon 45 skipping in myotubes containing DMD mutations amenable to exon 45 skipping than in wild-type myotubes (Figure 2). After treatment with the anti-TfR1 Fab-ASO conjugate, exon 45 skipping measured in wild-type myotubes was approximately 18%, compared with approximately 70% in Del46 myotubes. This indicates that the anti-TfR1 Fab-ASO conjugate enables exon 45 skipping in myotubes, and that such skipping is more pronounced in myotubes containing DMD mutations amenable to exon 45 skipping.

[0134] Example 3. Delivery of DMD-targeted oligonucleotides to muscle tissue after systemic administration and resulting skipping of exon 45 in a humanized mouse model of Duchenne muscular dystrophy This study evaluated the delivery to muscle tissue and subsequent exon 45 skipping following a single intravenous administration of the conjugate described in Example 1 (referred to in this example as the "anti-TfR1 Fab-ASO conjugate"), which comprises an anti-TfR1 Fab covalently linked to a DMD exon 45 skipping oligonucleotide ("ASO")

[0135] hDMD WT hTfR1 / hDMD WT / mdx mice (obtained from Academisch Ziekenhuis Leiden) were crossed with mice expressing human TfR1 to generate hTfR1 / hDMD WT / mdx mice. WT Anti-TfR1 Fab-ASO conjugates were intravenously administered to / mdx mice at an ASO equivalent dose of 30 mg / kg. Seven days after a single dose of the conjugate, the concentrations of ASO and exon 45 skipping were measured in various muscle tissues. ASO tissue exposure (ng ASO per gram of tissue) was measured by hybridization ELISA (Nucleic Acid Ther. 2015 Oct;25(5):275-84, incorporated herein by reference). Exon 45 skipping was measured using end-point PCR as described in Example 2 above.

[0136] The results demonstrate that the ASO accumulated in various muscle tissues after a single dose of the conjugate. The measured tissue exposures in muscle tissues were approximately 2,000 ng / g in the quadriceps ("Quad"), 2,400 ng / g in the gastrocnemius ("Gastroc"), 14,000 ng / g in the heart ("Heart"), and 7,300 ng / g in the diaphragm ("Dia") (Figure 3). Furthermore, enhanced exon 45 skipping was observed in each muscle tissue examined in mice treated with the conjugate compared to mice administered the vehicle control ("Veh"). Exon 45 skipping was approximately 0.4% vs. 1.0% in quadriceps muscle (Figure 4A); 0.2% vs. 0.75% in gastrocnemius muscle (Figure 4B); 0.7% vs. 1.5% in heart (Figure 4C); and 0.2% vs. 1.2% in diaphragm (Figure 4D) in vehicle-treated vs. conjugate-treated mice.

[0137] These results indicate that a conjugate comprising an anti-TfR1 antibody (e.g., a Fab) covalently linked to an oligonucleotide (e.g., a PMO such as an exon-skipping PMO) achieves delivery of the oligonucleotide to muscle tissue after systemic administration, and that such a conjugate comprising an exon-skipping oligonucleotide (e.g., an exon 45-skipping PMO oligonucleotide) induces exon skipping in muscle tissue after systemic delivery.

[0138] Equivalents and Terminology The present disclosure as illustrated and described herein may suitably be practiced in the absence of any element(s), limitation(s) not specifically disclosed herein. Thus, for example, in each instance of this application, any of the terms "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms. The terms and expressions employed are used as terms of description and not of limitation, and the use of such terms and expressions is not intended to exclude any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the present disclosure. Thus, although the present disclosure has been specifically disclosed by preferred embodiments, it should be understood that any features, modifications, and variations of the concepts disclosed herein may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of the present disclosure.

[0139] Additionally, where features or aspects of the present disclosure are described as a Markush group or other grouping of alternatives, those skilled in the art will recognize that the present disclosure is also described thereby as any individual member or subgroup of members of the Markush group or other group.

[0140] It should be understood that in some embodiments, reference may be made to the sequences presented in the sequence listing when describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides (e.g., RNA counterparts of DNA nucleotides, or DNA counterparts of RNA nucleotides) and / or (for example, and) one or more modified nucleotides and / or (for example, and) one or more modified internucleotide linkages and / or (for example, and) one or more other modifications compared to the designated sequence, while retaining essentially the same or similar complementary properties as the designated sequence.

[0141] The use of the terms "a," "an," and "the" and similar referents in the context of describing the present invention (particularly in the context of the claims below) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms "comprise," "have," "include," and "contain" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise indicated herein. The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated herein as if it were individually set forth herein. All methods described herein can be performed in any suitable order, unless otherwise indicated herein or clearly contradicted by context. Unless otherwise claimed, the use of any and all examples or exemplary language (e.g., "such as") provided herein is intended merely to better illuminate the invention and does not impose limitations on the scope of the invention. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0142] Aspects of the invention have been described herein. Variations of those aspects may become apparent to those of skill in the art upon reading the foregoing description.

[0143] The inventors anticipate that those of ordinary skill in the art will employ such variations as appropriate, and the inventors intend that the invention may be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

1. Formula (I): [R １ ] ｎ１ -R ２ wherein each R １ is represented by formula (Ia): 【Chemistry 1】 and a group represented by In the formula R ３ comprises a phosphorodiamidate morpholino oligomer (PMO) comprising the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21); In the formula R ２ comprises an anti-transferrin receptor 1 (anti-TfR1) antibody comprising a heavy chain complementarity determining region 1 (CDR-H1), a heavy chain complementarity determining region 2 (CDR-H2), a heavy chain complementarity determining region 3 (CDR-H3), a light chain complementarity determining region 1 (CDR-L1), a light chain complementarity determining region 2 (CDR-L2), and a light chain complementarity determining region (CDR-L3) selected from Table 2; In the formula R １ is attached at point A, R ２ and wherein n1 is covalently linked to R １ is an integer of 1 or more representing the number of instances of １ Each instance of the conjugate is covalently linked to a different amino acid residue of the anti-TfR1 antibody.

2. Formula (I): [R １ ] ｎ１ -R ２ wherein each R １ is represented by formula (Ib): 【Chemistry 2】 and a group represented by where -p is a phosphorodiamidate linkage of a phosphorodiamidate morpholino oligomer (PMO), and wherein the PMO comprises the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21); In the formula R ２ comprises an anti-TfR1 antibody comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 2; In the formula R １ is R at attachment point A ２ and wherein n1 is R １ is an integer of 1 or more representing the number of instances of １ Each instance of the conjugate is covalently linked to a different amino acid residue of the anti-TfR1 antibody.

3. Formula (I): [R １ ] ｎ１ -R ２ wherein each R １ is represented by formula (Ic): 【Transformation 3】 and a group represented by In the formula R ２ comprises an anti-TfR1 antibody comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 2; In the formula R １ is R at attachment point A ２ and wherein n1 is covalently linked to R １ and R is an integer of 1 or greater representing the number of instances of １ Each instance of the conjugate is covalently linked to a different amino acid residue of the anti-TfR1 antibody.

4. Formula (Id): 【Chemistry 4】 A complex comprising a structure represented by where -p is a phosphorodiamidate linkage of a phosphorodiamidate morpholino oligomer (PMO), and wherein the PMO comprises the base sequence CAATGCCATCCTGGAGTTCCTG (SEQ ID NO: 21); In the formula R ２ comprises an anti-TfR1 antibody comprising a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 selected from Table 2; wherein each instance of the bracketed group in formula (Id) is covalently linked to a different amino acid residue of an anti-TfR1 antibody; and wherein n1 is an integer greater than or equal to 1 representing the number of instances of the bracketed group in formula (Id).

5. The anti-TfR1 antibody may be a Fab fragment, a full-length IgG, a Fab' fragment, or a F(ab') ２ The conjugate according to any one of claims 1 to 4, which is a fragment.

6. The composition according to any one of claims 1 to 5, wherein the anti-TfR1 antibody is a Fab fragment.

7. The conjugate of any one of claims 1 to 6, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO:

18.

8. The conjugate of any one of claims 1 to 7, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO:

20.

9. R １ The conjugate of any one of claims 1 to 8, wherein each instance of is covalently linked to a different lysine residue of the anti-TfR1 antibody.

10. The conjugate according to any one of claims 1 to 9, wherein the different amino acid residues include K188 and K190 in the light chain constant region based on Kabat numbering.

11. The conjugate of any one of claims 1 to 10, wherein the different amino acid residue is represented by a lysine (K) residue in the sequence motif DYEKHKVYA (SEQ ID NO: 27) of the light chain constant region of the anti-TfR1 antibody.

12. A composition comprising a complex according to any one of claims 1 to 11, optionally in the form of an aqueous solution.

13. The composition of claim 12, wherein the anti-TfR1 antibodies of the conjugate in the composition comprise light chain constant regions, and wherein at least 80% of the light chain constant regions of the anti-TfR1 antibodies of the conjugate in the composition are independently covalently linked to an oligonucleotide at a linkage site represented by K188 (based on Kabat numbering) and / or a linkage site represented by K190 (based on Kabat numbering) of the light chain constant region of each anti-TfR1 antibody.

14. The composition of claim 12, wherein the anti-TfR1 antibodies of the conjugate in the composition comprise light chain constant regions, and wherein at least 80% of the light chain constant regions of the anti-TfR1 antibodies of the conjugate in the composition are independently covalently linked to an oligonucleotide at a linkage site represented by a lysine (K) residue in the sequence motif DYEKHKVYA (sequence number 27) of the light chain constant region of each antibody.

15. A method for promoting the expression or activity of a dystrophin protein in a subject, the method comprising administering to the subject a complex according to any one of claims 1 to 11 or a composition according to any one of claims 12 to 14.

16. 16. The method of claim 15, wherein the dystrophin protein is a truncated dystrophin protein.

17. 15. A method of treating a subject having a mutated DMD allele associated with Duchenne muscular dystrophy, the method comprising administering to the subject a conjugate of any one of claims 1 to 11 or a composition of any one of claims 12 to 14.

18. 18. The method of claim 17, wherein the complex promotes the expression or activity of a dystrophin protein in a subject.

19. 19. The method of claim 18, wherein the dystrophin protein is a truncated dystrophin protein.

20. 20. The method of any one of claims 17 to 19, wherein the mutated DMD allele comprises a mutation that is adaptive for exon 45 skipping.

21. 21. The method of any one of claims 17 to 20, wherein the mutated DMD allele comprises a frameshift mutation in exon 45.

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