Muscle-targeting complexes and uses thereof for treating myotonic dystrophy - Patent Application 20070123333

Abstract

This application relates to oligonucleotides (e.g., antisense oligonucleotides such as gapmers) designed to target DMPK RNA, and targeting complexes for delivering oligonucleotides to cells (e.g., muscle cells), and their uses, particularly for the treatment of disease. In some embodiments, the muscle targeting agent specifically binds to an internalized cell surface receptor on muscle cells. In some embodiments, the molecular payload inhibits expression or activity of DMPK.

Description

[Technical field]

[0001] Related Applications This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 245,262, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY", filed September 17, 2021; of U.S. Provisional Application No. 63 / 179,100, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY", filed April 23, 2021; and of U.S. Provisional Application No. 63 / 133,013, entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING MYOTONIC DYSTROPHY", filed December 31, 2020; the contents of each of which are incorporated herein by reference in their entirety.

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

[0003] Reference to a sequence listing submitted as a text file via EFS-WEB This application contains a Sequence Listing, which has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety. The ASCII copy, created on December 30, 2021, is entitled D082470046WO00-SEQ-ZJG and is 177,748 bytes in size. [Background technology]

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

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

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

[0007] Some aspects of the disclosure provide conjugates comprising a muscle targeting agent covalently linked to an antisense oligonucleotide, wherein the antisense oligonucleotide is 15-20 nucleotides in length and comprises a region of complementarity to at least 15 contiguous nucleosides of any one of SEQ ID NOs: 166, 163, 167, 160, 169, 171, 202, 161, 162, 170, 165, 164, 172, and 168, and comprises a 5'-XYZ-3' configuration, X comprises 3-5 linked nucleosides, where at least one of the nucleosides in X is a 2'-modified nucleoside; Y comprises 6-10 linked 2'-deoxyribonucleosides, where each cytosine in Y is optionally and independently a 5-methyl-cytosine; and Z comprises 3-5 linked nucleosides, where at least one of the nucleosides in Z is a 2'-modified nucleoside.

[0008] In some embodiments, the muscle targeting agent comprises an anti-transferrin receptor 1 (TfR1) antibody.

[0009] In some embodiments, the nucleotide sequence comprises any one of SEQ ID NOs: 179, 187, 180, 185, 189, 182, 191, 184, 174, 186, 190, 188, 177, 192, and 181.

[0010] In some embodiments, each nucleoside in X is a 2'-modified nucleoside and / or each nucleoside in Z is a 2'-modified nucleoside. In some embodiments, each 2'-modified nucleoside is independently a 2'-4' bicyclic nucleoside or a non-bicyclic 2'-modified nucleoside.

[0011] In some embodiments, each nucleoside in X is a non-bicyclic 2'-modified nucleoside and / or each nucleoside in Z is a non-bicyclic 2'-modified nucleoside. In some embodiments, the non-bicyclic 2'-modified nucleoside is a 2'-MOE modified nucleoside.

[0012] In some embodiments, each nucleoside in X is a 2'-4' bicyclic nucleoside and / or each nucleoside in Z is a 2'-4' bicyclic nucleoside. In some embodiments, the 2'-4' bicyclic nucleoside is selected from LNA, cEt, and ENA.

[0013] In some embodiments, X comprises at least one 2'-4' bicyclic nucleoside and at least one non-bicyclic 2'-modified nucleoside, and / or Z comprises at least one 2'-4' bicyclic nucleoside and at least one non-bicyclic 2'-modified nucleoside. In some embodiments, the at least one non-bicyclic 2'-modified nucleoside is a 2'-MOE modified nucleoside, and the at least one 2'-4' bicyclic nucleoside is selected from LNA, cEt, and ENA.

[0014] In some embodiments, the antisense oligonucleotide has the following 5'-XYZ-3' configuration: [Table 1] Including, where "E" is a 2'-MOE modified ribonucleoside; "L" is LNA; "D" is a 2'-deoxyribonucleoside; and "10" or "8" is the number of 2'-deoxyribonucleosides in Y.

[0015] In some embodiments, the antisense oligonucleotides contain one or more phosphorothioate internucleoside linkages.

[0016] In some embodiments, each internucleoside linkage in the antisense oligonucleotide is a phosphorothioate internucleoside linkage.

[0017] In some embodiments, the antisense oligonucleotide comprises one or more phosphodiester internucleoside linkages. In some embodiments, the phosphodiester internucleoside linkages are in X and / or Z.

[0018] In some embodiments, the antisense oligonucleotide comprises: [Table 2] comprising an oligonucleotide selected from where "xdC" is 5-methyl-deoxycytidine; "dN" is a 2'-deoxyribonucleoside; "+N" is an LNA nucleoside; "oN" is a 2'-MOE modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "+C" is 5-methyl-2'-4'-bicyclic-cytidine (2'-4' methylene bridge); "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl-2'-4'-bicyclic-uridine (2'-4' methylene bridge); "*" indicates a phosphorothioate internucleoside linkage; and the absence of a "*" between nucleosides indicates a phosphodiester internucleoside linkage.

[0019] In some embodiments, the anti-TfR1 antibody comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3) of any of the anti-TfR1 antibodies listed in Table 2.

[0020] In some embodiments, the anti-TfR1 antibody comprises the heavy chain variable region (VH) and light chain variable region (VL) of any of the anti-TfR antibodies listed in Table 3.

[0021] In some embodiments, the anti-TfR1 antibody is a Fab. In some embodiments, the Fab comprises the heavy and light chains of any of the anti-TfR1 Fabs listed in Table 5.

[0022] In some embodiments, the anti-TfR1 antibody is (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32; (ii) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32; or (ii) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 38, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42 Includes.

[0023] In some embodiments, the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:76, and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0024] In some embodiments, the anti-TfR1 antibody is a Fab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0025] In some embodiments, the muscle targeting agent and the antisense oligonucleotide are covalently linked via a linker. In some embodiments, the linker comprises a valine-citrulline sequence.

[0026] Another aspect of the disclosure provides a method of reducing DMPK expression in a muscle cell, the method comprising contacting the muscle cell with a conjugate described herein in an amount effective to promote internalization of an antisense oligonucleotide into the muscle cell.

[0027] In some embodiments, reducing DMPK expression comprises reducing the level of DMPK mRNA in muscle cells. In some embodiments, the DMPK mRNA is a mutant DMPK mRNA.

[0028] Another aspect of the present disclosure provides a method of treating myotonic dystrophy type 1 (DM1), the method comprising administering to a subject in need thereof an effective amount of a conjugate described herein, wherein the subject has a mutant DMPK allele that includes a disease-associated CUG repeat.

[0029] In some embodiments, administration of the complex results in at least a 30% reduction in DMPK mRNA.

[0030] Further provided herein are the following: [Table 3] an antisense oligonucleotide comprising an oligonucleotide selected from where "xdC" is 5-methyl-deoxycytidine; "dN" is a 2'-deoxyribonucleoside; "+N" is an LNA nucleoside; "oN" is a 2'-MOE modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "+C" is 5-methyl-2'-4'-bicyclic-cytidine (2'-4' methylene bridge); "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl-2'-4'-bicyclic-uridine (2'-4' methylene bridge); "*" indicates a phosphorothioate internucleoside linkage; and the absence of a "*" between nucleosides indicates a phosphodiester internucleoside linkage.

[0031] In some embodiments, the antisense oligonucleotide comprises: [Table 4] comprising an oligonucleotide selected from where "xdC" is 5-methyl-deoxycytidine; "dN" is a 2'-deoxyribonucleoside; "+N" is an LNA nucleoside; "oN" is a 2'-MOE modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "+C" is 5-methyl-2'-4'-bicyclic-cytidine (2'-4' methylene bridge); "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl-2'-4'-bicyclic-uridine (2'-4' methylene bridge); "*" indicates a phosphorothioate internucleoside linkage; and the absence of a "*" between nucleosides indicates a phosphodiester internucleoside linkage, and Here, a phosphodiester linkage exists between the 5'-NH2-(CH2)6- and the antisense oligonucleotide.

[0032] Compositions comprising the antisense oligonucleotides described herein in sodium salt form are also provided. [Brief description of the drawings]

[0033] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Figures 1A-1B show expression of DMPK mRNA in DM1-32F primary cells (expressing DMPK mutant mRNA with 380 CUG repeats) and DM1-CL5 immortalized cells (expressing DMPK mutant mRNA with 2600 CUG repeats) compared to cell lines derived from healthy volunteers. Figure 1A shows the target site of ASO1 in DMPK mRNA in 32F cells. Figure 1B shows the target site of ASO1 in DMPK mRNA in CL5 cells.

[0034] [Figure 2A-B] Figures 2A-2D show the activity of conjugates with a control anti-TfR1 Fab conjugated to ASO1 or ASO32 in reducing DMPK mRNA expression, correcting BIN1 exon 11 splicing defects, and reducing nuclear foci in DM1-32F primary cells. Figure 2A shows that both conjugates reduced DMPK mRNA expression levels in 32F cells. Figure 2B shows that splicing of BIN1 exon 11 was corrected after treatment of the conjugates in 32F cells. [Figure 2C-D] Figures 2C-2D show that both conjugates reduced nuclear foci in 32F cells. In the microscopy images shown in Figure 2D, the bright circles indicate cell nuclei, and the bright spots in the nuclei of DM1 cells (right three microscopy panels) indicate CUG foci.

[0035] [Figure 3A] Figures 3A-3D show the activity of conjugates with a control anti-TfR1 Fab conjugated to ASO1 or ASO32 in reducing DMPK mRNA expression, correcting the BIN1 exon 11 splicing defect, and reducing nuclear foci (measured as the ratio of nuclear foci area to nuclear area) in CL5 cells. Figure 3A shows that both conjugates reduced DMPK mRNA expression levels in CL5 cells. [Figure 3B-D]Figure 3B shows that splicing of BIN1 exon 11 was corrected after treatment of the conjugates in CL5 cells. Figures 3C-3D show that both conjugates reduced nuclear foci in CL5 cells. In the microscopy images shown in Figure 3D, the bright circles indicate cell nuclei, and the bright spots in the nuclei of DM1 cells (right three microscopy panels) indicate CUG foci.

[0036] [Figure 4A-C] Figures 4A-4H show the activity of conjugates with anti-TfR1 Fab conjugated to ASO32, ASO10, ASO8, ASO26, and ASO1 in reducing DMPK mRNA expression, correcting BIN1 exon 11 splicing defects, and reducing nuclear foci (measured as the ratio of nuclear foci area to nuclear area) in 32F cells. All ASOs were conjugated to anti-TfR1 Fab 3M12 - VH4 / VK3. Figure 4A shows the target sites of ASO1, ASO2, and ASO32 in DMPK mRNA with 380 CUG repeats in 32F cells. Figure 4B shows that the tested conjugates reduced DMPK mRNA expression levels in 32F cells. Figure 4C shows that splicing of BIN1 exon 11 was corrected after treatment of the conjugates in 32F cells. [Fig. 4D-F] Figures 4D-4E show that the tested conjugates reduced the area of ​​intranuclear foci in 32F cells. In the microscopic image shown in Figure 4E, the bright circles indicate cell nuclei and the bright spots within the nuclei indicate DMPK foci. Figure 4F shows that the tested conjugates reduced DMPK expression in 32F cells in a dose-dependent manner. [Figure 4G-H] Figure 4G shows that the tested conjugates dose-dependently corrected the mis-splicing of BIN1 in 32F cells, and Figure 4H shows that the tested conjugates dose-dependently reduced the area of ​​nuclear foci in 32F cells.

[0037] [Figure 5A]Figures 5A-5H show the activity of conjugates with anti-TfR1 Fab conjugated to one of ASO32, ASO10, ASO8, ASO26, and ASO1 in reducing DMPK mRNA expression, correcting BIN1 exon 11 splicing defects, and reducing nuclear foci (measured as the ratio of nuclear foci area to nuclear area) in CL5 cells. All ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Figure 5A shows the target sites of ASO1, ASO2, and ASO32 in DMPK mRNA with 2600 CUG repeats in CL5 cells. [Figure 5B-C] Figure 5B shows that the tested conjugates reduced DMPK mRNA expression level in CL5 cells. Figure 5C shows that splicing of BIN1 exon 11 was corrected after treatment of the conjugates in CL5 cells. [Fig. 5D-F] Figures 5D-5E show that the conjugates tested reduced the area of ​​intranuclear foci in CL5 cells. In the microscopy image shown in Figure 5E, the bright circles indicate cell nuclei and the bright spots within the nuclei indicate DMPK foci. Figure 5F shows that conjugates with anti-TfR1 Fab conjugated to ASO10 or ASO8 reduced DMPK expression in CL5 cells in a dose-dependent manner. [Figure 5G-H] Figure 5G shows that the tested conjugates corrected BIN1 missplicing in CL5 cells in a dose-dependent manner, and Figure 5H shows that the tested conjugates reduced the area of ​​nuclear foci in CL5 cells to similar levels at all doses tested.

[0038] [Figure 6] Figure 6 shows that conjugates with anti-TfR1 Fab conjugated to ASO10, ASO8, and ASO26 were able to knock down DMPK expression in rhabdomyosarcoma (RD) cells in a dose-dependent manner, and the ASO1-conjugate was able to knock down DMPK in non-human primate (NHP) cells in a dose-dependent manner. All ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3.

[0039] [Figure 7] Figure 7 shows that various chemical modifications of the same nucleobase sequence can affect the efficacy of DMPK-targeting oligonucleotides. All ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. At an oligo concentration of 500 nM, ASO32-conjugates were able to reduce DMPK expression by 88%, ASO31-conjugates were able to reduce DMPK expression by 70%, and ASO30-conjugates were able to reduce DMPK expression by 39%.

[0040] [Figure 8] Figures 8A-8B show that different lengths and chemical modifications of the parent nucleobase sequence can affect the efficacy of DMPK-targeting oligonucleotides. All ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Figure 8A shows the activity of ASO32-conjugates, ASO10-conjugates, ASO8-conjugates, and ASO9-conjugates in knocking down DMPK in human RD cells. Figure 8B shows the activity of ASO32-conjugates, ASO11-conjugates, ASO20-conjugates, ASO26-conjugates, and ASO2-conjugates in knocking down DMPK in human RD cells.

[0041] [Figure 9A-C]Figures 9A-9C show that conjugates with anti-TfR1 Fab conjugated to ASO32 reduced human mutant DMPK expression in various muscle tissues of a mouse model expressing human TfR1 and a human DMPK mutant harboring an expanded CUG repeat. Figures 9D-9K show that conjugates with anti-TfR1 Fab conjugated to ASO32 reduced mouse DMPK expression in various muscle tissues of a mouse model expressing human TfR1. In Figures 9A-9C, ASO32 was conjugated to a control anti-TfR1 Fab. In Figures 9D-9K, ASO32 was conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Figure 9A shows that ASO32-conjugates reduced DMPK mRNA levels in the tibialis anterior muscle by 36%. Figure 9B shows that ASO32-conjugates reduced DMPK mRNA levels in the diaphragm by 46%. Figure 9C shows that the ASO32-conjugate reduced human mutant DMPK in the heart by 42%. [Fig. 9D-G] Figure 9D shows that ASO32-conjugates reduced mouse wild-type Dmpk by 79% in tibialis anterior muscle. Figure 9E shows that ASO32-conjugates reduced mouse wild-type Dmpk by 76% in gastrocnemius muscle. Figure 9F shows that ASO32-conjugates reduced mouse wild-type Dmpk by 70% in heart. Figure 9G shows that ASO32-conjugates reduced mouse wild-type Dmpk by 88% in diaphragm. [Fig. 9H-K] Figures 9H-9K show ASO32 distribution in tibialis anterior, gastrocnemius, heart, and diaphragm. All tissues showed increased ASO32 levels compared to vehicle controls.

[0042] [Figure 10A-B]Figures 10A-10E show that in a mouse model expressing human TfR1 and a human DMPK mutant harboring an expanded CUG repeat, conjugates with anti-TfR1 Fab conjugated to ASO32, ASO10, ASO8, ASO26, and ASO1 reduced human mutant DMPK expression in various muscle tissues, and the ASO10-conjugate reduced nuclear foci in the heart. ASO32 was conjugated to a control anti-TfR1 Fab. All other ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. The conjugates reduced human DMPK mRNA levels in the heart (Figure 10A), diaphragm (Figure 10B), gastrocnemius (Figure 10C), and tibialis anterior (Figure 10D). [Figure 10C-D] Figures 10A-10E show that in a mouse model expressing human TfR1 and a human DMPK mutant harboring an expanded CUG repeat, conjugates with anti-TfR1 Fab conjugated to ASO32, ASO10, ASO8, ASO26, and ASO1 reduced human mutant DMPK expression in various muscle tissues, and the ASO10-conjugate reduced nuclear foci in the heart. ASO32 was conjugated to a control anti-TfR1 Fab. All other ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. The conjugates reduced human DMPK mRNA levels in the heart (Figure 10A), diaphragm (Figure 10B), gastrocnemius (Figure 10C), and tibialis anterior (Figure 10D). [Figure 10E] Figure 10E shows that mice injected with the ASO10-conjugate at a dose equivalent to 10 mg / kg of ASO10 had reduced intranuclear foci in the heart. In the microscopic image shown in Figure 10E, the circles represent cell nuclei and the dark dots within the nuclei represent DMPK foci.

[0043] [Figure 11A-B]Figures 11A-11D show conjugates with anti-TfR1 Fab conjugated to ASO32, ASO10, ASO8. ASO26 and ASO1 reduced mouse Dmpk expression in various muscle tissues of a mouse model expressing human TfR1 and a human DMPK mutant harboring an expanded CUG repeat, despite a single nucleotide mismatch in the target sequence. ASO32 was conjugated to a control anti-TfR1 Fab. All other ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. The conjugates reduced mouse DMPK mRNA levels in the heart (Figure 11A), diaphragm (Figure 11B), gastrocnemius (Figure 11C), and tibialis anterior (Figure 11D). [Figure 11C-D] Figures 11A-11D show conjugates with anti-TfR1 Fab conjugated to ASO32, ASO10, ASO8. ASO26 and ASO1 reduced mouse Dmpk expression in various muscle tissues of a mouse model expressing human TfR1 and a human DMPK mutant harboring an expanded CUG repeat, despite a single nucleotide mismatch in the target sequence. ASO32 was conjugated to a control anti-TfR1 Fab. All other ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. The conjugates reduced mouse DMPK mRNA levels in the heart (Figure 11A), diaphragm (Figure 11B), gastrocnemius (Figure 11C), and tibialis anterior (Figure 11D).

[0044] [Figure 12A-B] Figures 12A-12D show the amounts of ASO10, ASO8, ASO26, and ASO1 in the heart (Figure 12A), diaphragm (Figure 12B), gastrocnemius (Figure 12C), or tibialis anterior (Figure 12D), respectively, following administration of conjugates containing anti-TfR1 Fab conjugated to the indicated oligonucleotides. All ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. [Fig. 12C-D]Figures 12A-12D show the amounts of ASO10, ASO8, ASO26, and ASO1 in the heart (Figure 12A), diaphragm (Figure 12B), gastrocnemius (Figure 12C), or tibialis anterior (Figure 12D), respectively, following administration of conjugates containing anti-TfR1 Fab conjugated to the indicated oligonucleotides. All ASOs were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3.

[0045] [Figure 13] Figures 13A-13D show that in a chronic experimental setting, a conjugate containing a control anti-TfR1 Fab conjugated to ASO1 reduced human mutant DMPK expression in various muscle tissues of a mouse model expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat. Figure 13A shows that the ASO1-conjugate knocked down human mutant DMPK in the heart by 9% 2 weeks after injection and 15% 4 weeks after injection. Figure 13B shows that the ASO1-conjugate knocked down human mutant DMPK in the diaphragm by 19% 2 weeks after injection and 34% 4 weeks after injection. Figure 13C shows that the ASO1-conjugate knocked down human mutant DMPK in the gastrocnemius muscle by 7% 2 weeks after injection and 17% 4 weeks after injection. FIG. 13D shows that the ASO1-conjugate knocked down human mutant DMPK in tibialis anterior muscle by 6% 2 weeks after injection and 0% 4 weeks after injection.

[0046] [Figure 14A-B] Figures 14A-14D show that a conjugate containing a control anti-TfR1 Fab conjugated to ASO1 reduced mouse Dmpk expression in the same mouse model as in Figures 13A-13D. Figure 14A shows that the ASO1-conjugate knocked down mouse Dmpk in the heart by 8% two weeks after injection and by 13% four weeks after injection. Figure 14B shows that the ASO1-conjugate knocked down mouse Dmpk in the diaphragm by 14% two weeks after injection and by 33% four weeks after injection. [Fig. 14C-D]Figure 14C shows that ASO1-conjugate knocked down mouse Dmpk in gastrocnemius muscle by 0% at 2 weeks post-injection and by 6% at 4 weeks post-injection. Figure 14D shows that ASO1-conjugate did not knock down mouse Dmpk in tibialis anterior muscle at 2 weeks post-injection and at 4 weeks post-injection.

[0047] [Figure 15] Figures 15A-15D show the amount of ASO1 in the heart (Figure 15A), diaphragm (Figure 15B), gastrocnemius muscle (Figure 15C), or tibialis anterior muscle (Figure 15D) following administration of a conjugate containing a control anti-TfR1 Fab conjugated to ASO1.

[0048] [Figure 16] Figures 16A-16D show the activity of a conjugate containing a control anti-TfR1 Fab conjugated to ASO1 in a different experimental design in the same mouse model as in Figures 13A-13D. The conjugate was administered at a different dose and frequency compared to that in Figures 13A-13D. Figure 16A shows that the ASO1-conjugate knocked down human mutant DMPK in the heart by 5% 5 weeks after injection. Figure 16B shows that the ASO1-conjugate knocked down human mutant DMPK in the diaphragm by 35% 5 weeks after injection. Figure 16C shows that the ASO1-conjugate did not appear to knock down human mutant DMPK in the gastrocnemius muscle 5 weeks after injection. Figure 16D shows that the ASO1-conjugate did not appear to knock down human mutant DMPK in the tibialis anterior muscle 5 weeks after injection.

[0049] [Figure 17A-B] Figures 17A-17D show that a conjugate containing a control anti-TfR1 Fab conjugated to ASO1 reduced mouse Dmpk expression in the same mouse model as in Figures 13A-13D. Figure 17A shows that the ASO1-conjugate knocked down mouse Dmpk in the heart by 13% 5 weeks after injection. Figure 17B shows that the ASO1-conjugate knocked down mouse Dmpk in the diaphragm by 41% 5 weeks after injection. [Fig. 17C-D] Figure 17C shows that the ASO1-conjugate knocked down mouse Dmpk by 5% in the gastrocnemius muscle 5 weeks after injection, and Figure 17D shows that the ASO1-conjugate knocked down mouse Dmpk by 10% in the tibialis anterior muscle 5 weeks after injection.

[0050] [Figure 18] Figures 18A-18D show the amount of ASO1 in the heart (Figure 18A), diaphragm (Figure 18B), gastrocnemius muscle (Figure 18C), or tibialis anterior muscle (Figure 18D) following administration of a conjugate containing a control anti-TfR1 Fab conjugated to ASO1.

[0051] [Figure 19] Figures 19A-19D show that conjugates containing anti-TfR1 Fab conjugated to ASO9 reduced human mutant DMPK expression in various muscle tissues of a mouse model expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat. ASO9 was conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Figure 19A shows that the ASO9-conjugate knocked down human mutant DMPK in the heart by 50% two weeks after injection. Figure 19B shows that the ASO9-conjugate knocked down human mutant DMPK in the diaphragm by 58% two weeks after injection. Figure 19C shows that the ASO9-conjugate knocked down human mutant DMPK in the tibialis anterior muscle by 30% two weeks after injection. Figure 19D shows that the ASO9-conjugate knocked down human mutant DMPK in the gastrocnemius muscle by 35% two weeks after injection.

[0052] [Figure 20A-B]Figures 20A-20D show that a conjugate containing anti-TfR1 Fab conjugated to ASO9 reduced mouse Dmpk expression in the same mouse model as in Figures 19A-19D. ASO9 was conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Figure 20A shows that the ASO9-conjugate knocked down mouse Dmpk in the heart by 48% two weeks after injection. Figure 20B shows that the ASO9-conjugate knocked down mouse Dmpk in the diaphragm by 68% two weeks after injection. [Figure 20C-D] Figure 20C shows that the ASO9-conjugate knocked down mouse Dmpk by 45% in the gastrocnemius muscle two weeks after injection, and Figure 20D shows that the ASO9-conjugate knocked down mouse Dmpk by 20% in the tibialis anterior muscle two weeks after injection.

[0053] [Figure 21] Figures 21A-21D show the amount of ASO9 following administration of a conjugate containing an anti-TfR1 Fab conjugated to ASO9 in the heart (Figure 21A), diaphragm (Figure 21B), gastrocnemius (Figure 21C), or tibialis anterior (Figure 21D), respectively. ASO9 was conjugated to anti-TfR1 Fab 3M12-VH4 / VK3.

[0054] [Figure 22] Figures 22A-22D show that conjugates containing a control anti-TfR1 Fab conjugated to ASO1 reduced DMPK expression in various muscle tissues of the non-human primate animal Cynomolgus macaque (cyno). Figure 22A shows that the ASO1-conjugate knocked down DMPK in the heart by 10% 7 weeks after injection. Figure 22B shows that the ASO1-conjugate did not appear to knock down DMPK in the diaphragm 7 weeks after injection. Figure 22C shows that the ASO1-conjugate knocked down DMPK in the gastrocnemius muscle 29% 7 weeks after injection. Figure 22D shows that the ASO1-conjugate knocked down DMPK in the tibialis anterior muscle 31% 7 weeks after injection.

[0055] [Diagram 23] Figures 23A-23D show the amount of ASO1 in the cyno heart (Figure 23A), diaphragm (Figure 23B), gastrocnemius muscle (Figure 23C), or tibialis anterior muscle (Figure 23D) two weeks after administration of a conjugate containing a control anti-TfR1 Fab conjugated to ASO1.

[0056] [Figure 24] Figures 24A-24D show that conjugates containing anti-TfR1 Fab conjugated to ASO10 reduced human mutant DMPK expression in various muscle tissues of a mouse model expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat. ASO10 was conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Figure 24A shows that ASO10-conjugates knocked down human mutant DMPK in the heart 28 days after injection at all doses tested. Figure 24B shows that ASO10-conjugates knocked down human mutant DMPK in the diaphragm 28 days after injection at all doses tested. Figure 24C shows that ASO10-conjugates knocked down human mutant DMPK in the gastrocnemius muscle 28 days after injection at all doses tested. FIG. 24D shows that the ASO10-conjugate knocked down human mutant DMPK in the tibialis anterior muscle 28 days after injection at all doses tested.

[0057] [Figure 25A-C]Figures 25A-25H show that ASO10 was delivered to the nucleus in mice injected with a conjugate containing anti-TfR1 Fab 3M12-VH4 / VK3 conjugated to ASO10 at a dose equivalent to 10 mg / kg of ASO10, and that the ASO10-conjugate reduced the accumulation of mutant human DMPK mRNA trapped in the nucleus. The ASO10-conjugate was tested in a mouse model expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat. Figure 25A shows that mutant human DMPK was trapped in the nucleus of muscle cells by subcellular fractionation of gastrocnemius muscle from mice injected with vehicle control. Figure 25B shows that Malat1 was used as a nuclear RNA marker. Figure 25C shows that Birc5 was used as a cytoplasmic RNA marker. [Fig. 25D-E] Figure 25D shows that Gapdh was used as a cytoplasmic RNA marker, and Figure 25E shows that the nuclear protein marker, Histone H3, is only present in the nuclear fraction. [Fig. 25F-G] Figure 25F shows that the cytoplasmic protein marker GAPDH is only present in the cytoplasmic fraction, and Figure 25G shows that ASO10 reduced mutant human DMPK in whole tissue extracts. [Fig. 25H] Figure 25H shows that ASO10 reduced mutant human DMPK in the nuclear fraction of gastrocnemius muscle cells.

[0058] [Figure 26A-B] Figures 26A-26H show that conjugates containing anti-TfR1 Fab conjugated to ASO10 or ASO26 reduced wild-type DMPK in Cynomolgus macaques (cyno). ASO10 or ASO26 was conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Figures 26A-26D show that ASO10 was present in the heart, diaphragm, gastrocnemius, and tibialis anterior in a dose-dependent manner. Figures 26E-26H show that ASO26 was present in the heart, diaphragm, gastrocnemius, and tibialis anterior in a dose-dependent manner. [Fig. 26C-H] Figures 26A-26H show that conjugates containing anti-TfR1 Fab conjugated to ASO10 or ASO26 reduced wild-type DMPK in Cynomolgus macaques (cyno). ASO10 or ASO26 was conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Figures 26A-26D show that ASO10 was present in the heart, diaphragm, gastrocnemius, and tibialis anterior in a dose-dependent manner. Figures 26E-26H show that ASO26 was present in the heart, diaphragm, gastrocnemius, and tibialis anterior in a dose-dependent manner.

[0059] [Figure 27A-B] Figures 27A-27D show that conjugates containing anti-TfR1 Fab conjugated to ASO10 were active in both heart and skeletal muscle of non-human primates, and conjugates containing anti-TfR1 Fab conjugated to ASO26 were active in skeletal muscle. ASO10 or ASO26 were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Figure 27A shows that the ASO10-conjugate was active in the heart, and the ASO26-conjugate appeared to have no activity in the heart. Figure 27B shows that both the ASO10-conjugate and the ASO26-conjugate were active in the diaphragm. [Figure 27C-D] Figure 27C shows that both the ASO10-conjugate and the ASO26-conjugate were active in the gastrocnemius muscle, and Figure 27D shows that both the ASO10-conjugate and the ASO26-conjugate were active in the tibialis anterior muscle.

[0060] [Fig. 28A-B]Figures 28A-28D show the DMPK knockdown activity of conjugates containing anti-TfR1 Fab 3M12-VH4 / VK3 conjugated to ASO10 or ASO26 in the heart, diaphragm, gastrocnemius, and tibialis anterior. ASO10-conjugates or ASO26-conjugates were administered at a dose equivalent to 10 mg / kg of ASO10 or ASO26 to mice expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat. Figure 28A shows that the ASO10-conjugate was active in the heart, and the ASO26-conjugate appeared to have no activity in the heart. Figure 28B shows that both the ASO10-conjugate and the ASO26-conjugate were active in the diaphragm. [Fig. 28C-D] Figure 28C shows that both the ASO10-conjugate and the ASO26-conjugate were active in the gastrocnemius muscle, and Figure 28D shows that both the ASO10-conjugate and the ASO26-conjugate were active in the tibialis anterior muscle.

[0061] [Figure 29] Figures 29A-29D show the human DMPK RNA knockdown ability of a conjugate containing anti-TfR1 Fab 3M12-VH4 / VK3 conjugated to ASO10 in the heart (Figure 29A), diaphragm (Figure 29B), tibialis anterior (Figure 29C), and gastrocnemius (Figure 29D) muscles of mice expressing both human TfR1 and two copies of a mutant human DMPK transgene harboring an expanded CTG repeat.

[0062] [Diagram 30]Figures 30A-30B show reduced DMPK foci in the nuclei of cardiac muscle fibers in mice expressing both human TfR1 and two copies of a mutant human DMPK transgene harboring an expanded CTG repeat and treated with anti-TfR1 Fab 3M12-VH4 / VK3 conjugated to ASO10. Figure 30A shows a representative image of a sample that underwent situ hybridization staining of DMPK foci and fluorescent staining of myofibrils (insert panel). In the microscopic image shown in Figure 30A, the bright circles indicate cell nuclei and the bright spots within the nuclei indicate DMPK foci. Figure 30B shows quantification of DMPK foci.

[0063] [Diagram 31] Figure 31 shows the splicing correction activity of a conjugate containing anti-TfR1 Fab 3M12-VH4 / VK3 conjugated to ASO10 in the hearts of mice expressing both human TfR1 and two copies of a mutant human DMPK transgene harboring an expanded CTG repeat (hTfR1 / DMSXL mice). Composite splicing indices based on splicing of Ldb3 exon 11, Mbnl2 exon 6, and Nfix exon 7 are shown for control mice treated with vehicle control ("hTfR1 - PBS"), hTfR1 / DMSXL mice treated with vehicle control ("hTfR1 / DMSXL - PBS"), and hTfR1 / DMSXL mice treated with anti-TfR1 Fab-ASO10 conjugate ("hTfR1 / DMSXL - conjugate").

[0064] [Diagram 32]Figure 32 shows the splicing correction activity of a conjugate containing anti-TfR1 Fab 3M12-VH4 / VK3 conjugated to ASO10 in the diaphragm of mice expressing both human TfR1 and two copies of a mutant human DMPK transgene containing an expanded CTG repeat (hTfR1 / DMSXL mice). A composite splicing index based on the splicing of Bin1 exon 11, Insr exon 11, Ldb3 exon 11, and Nfix exon 7 is shown for control mice treated with vehicle control ("hTfR1 - PBS"), hTfR1 / DMSXL mice treated with vehicle control ("hTfR1 / DMSXL - PBS"), and hTfR1 / DMSXL mice treated with anti-TfR1 Fab-ASO10 conjugate ("hTfR1 / DMSXL - conjugate").

[0065] [Diagram 33] Figure 33 shows the splicing correction activity of a conjugate containing anti-TfR1 Fab 3M12-VH4 / VK3 conjugated to ASO10 in the tibialis anterior muscle of mice expressing both human TfR1 and two copies of a mutant human DMPK transgene containing an expanded CTG repeat (hTfR1 / DMSXL mice). A composite splicing index based on the splicing of Bin1 exon 11, Ldb3 exon 11, Mbnl2 exon 6, and Nfix exon 7 is shown for control mice treated with vehicle control ("hTfR1 - PBS"), hTfR1 / DMSXL mice treated with vehicle control ("hTfR1 / DMSXL - PBS"), and hTfR1 / DMSXL mice treated with anti-TfR1 Fab-ASO10 conjugate ("hTfR1 / DMSXL - conjugate").

[0066] [Diagram 34]Figure 34 shows the splicing correction activity of a conjugate containing anti-TfR1 Fab 3M12-VH4 / VK3 conjugated to ASO10 in the gastrocnemius muscle of mice expressing both human TfR1 and two copies of a mutant human DMPK transgene harboring an expanded CTG repeat (hTfR1 / DMSXL mice). A composite splicing index based on the splicing of Mbnl2 exon 6, Nfix exon 7, and Ttn exon 313 is shown for control mice treated with vehicle control ("hTfR1 - PBS"), hTfR1 / DMSXL mice treated with vehicle control ("hTfR1 / DMSXL - PBS"), and hTfR1 / DMSXL mice treated with anti-TfR1 Fab-ASO10 conjugate ("hTfR1 / DMSXL - conjugate").

[0067] [Diagram 35] Figure 35 shows DMPK knockdown in DM1 patient myotubes and wild-type non-human primate (NHP) myotubes following incubation with a conjugate containing anti-TfR1 Fab 3M12-VH4 / Vk3 covalently linked to ASO10. Results are shown normalized to expression in DM1 patient myotubes or NHP myotubes treated with vehicle alone. Data are shown as mean + standard deviation for n=4 replicates per condition. Statistics were calculated by one-way ANOVA (*, P<0.05; **, P<0.01). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] Detailed Description Some aspects of the disclosure provide oligonucleotides designed to target DMPK RNA. In some embodiments, the disclosure provides oligonucleotides complementary to DMPK RNA useful for reducing the level of toxic DMPK with disease-associated repeat expansion, e.g., in subjects with or suspected of having myotonic dystrophy. In some embodiments, the oligonucleotides are designed to direct RNAe H-mediated degradation of the target DMPK RNA. In some embodiments, the oligonucleotides are designed to direct RNAe H-mediated degradation of the target DMPK RNA present in the nucleus of a cell (e.g., a muscle cell, e.g., a myotube). In some embodiments, the oligonucleotides are designed to direct RNAe H-mediated degradation of the target DMPK RNA present in the nucleus of a cell (e.g., a cell of the nervous system (e.g., a central nervous system (CNS) cell)). In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum-stability properties. In some embodiments, the oligonucleotides are designed to have desired binding affinity properties. In some embodiments, the oligonucleotides are designed to have a desired toxicity profile. In some embodiments, the oligonucleotides are designed to have low complement activation and / or cytokine induction properties.

[0069] In some aspects, the present disclosure provides a complex comprising a muscle targeting agent covalently linked to a DMPK targeting oligonucleotide described herein for effective delivery of the oligonucleotide to muscle cells. In some embodiments, the complex targets a DMPK allele comprising an expanded disease-associated repeat and is provided for treating a subject with DM1. In some embodiments, the complex provided herein may comprise an oligonucleotide that inhibits expression of a DMPK allele comprising an expanded disease-associated repeat. As another example, the complex may comprise an oligonucleotide that interferes with binding of disease-associated DMPK mRNA to Muscleblind-like proteins (e.g., MBNL1, 2, and / or (e.g., and) 3), thereby reducing the toxic effects of the disease-associated DMPK allele.

[0070] Further aspects of the disclosure, including descriptions of defined terms, are provided below.

[0071] I. Definition Administering: As used herein, the terms "administering" or "administration" mean providing a conjugate to a subject in a physiologically and / or pharmacologically useful manner (e.g., treating a disease in a subject).

[0072] about: As used herein, the term "approximately" or "about" when applied to one or more values ​​of interest refers to a value similar to a specified reference value. In some embodiments, the term "approximately" or "about" refers to a broad range of values ​​that fall within plus or minus (greater or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the specified reference value, unless otherwise stated or clear from the context (except when such number exceeds 100% of a feasible value).

[0073] 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 bispecific antibody. In some embodiments, the antibody comprises a framework having 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, the 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, the antibody comprises a constant region, for example an Fc region. Immunoglobulin constant region refers to a heavy or light chain constant region. Human IgG heavy and light chain constant region amino acid sequences and their functional variations are known. With respect to the heavy chain, the heavy chain of the antibody described herein in some embodiments can be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein can comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In specific embodiments, the antibody described herein comprises a human gamma 1 CH1 domain, CH2 domain, and / or (for example and) a CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any sequence known in the art.Non-limiting examples of human constant region sequences are described in the art, see, for example, U.S. Patent No. 5,693,780 and Kabat EA et al. (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, for example, via glycosylation, phosphorylation, sumoylation, and / or (for example, 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 (for example, and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, 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 together by peptide bonds 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 non-covalent 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).

[0074] CDR: As used herein, the term "CDR" refers to the complementarity determining region in an antibody variable sequence. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are usually involved in antigen binding. The VH and VL regions can be further subdivided into hypervariable regions, also known as "complementarity determining regions" ("CDRs"), interspersed with more conserved regions known as "framework regions" ("FRs"). Each VH and VL is typically composed of three CDRs and four FRs, arranged from 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.For example, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242; IMGT (registered trademark), the international ImMunoGeneTics information system (registered trademark) http: / / www.imgt.org, Lefranc, M.-P. et al., Nucleic Acids. Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[Epub],5:45-60(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,33:D593-597(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,37:D1006-1012(2009);Lefranc,M.-P.et al.,Nucleic Acids Res.,43:D413-422(2015);Chothia et al.,(1989)Nature 342:877;Chothia,C.et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDRs may refer to CDRs defined by any method known in the art.Two antibodies having the same CDRs means that the two antibodies have the same amino acid sequence of their CDRs, as determined by the same method, e.g., the IMGT definitions.

[0075] There are three CDRs in each of the heavy and light chain variable regions, which are designated CDR1, CDR2, and CDR3 for each variable region. The term "CDR set" as used herein refers to a group of three CDRs occurring in a single variable region capable of binding to an antigen. The exact boundaries of these CDRs are defined differently according to various systems. The system described by Kabat (Kabat et al., Sequence of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides 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 and heavy chain regions, respectively. These regions are sometimes referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Other boundaries 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 specific residues or groups of residues, or even entire CDRs, do 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. CDR definition [Table 5] 1 IMGT®, the international ImMunoGeneTics information system®, imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999) 2 Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 3 Chothia et al., J. Mol. Biol. 196:901-917 (1987))

[0076] CDR-grafted antibodies: The term "CDR-grafted antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species but in which the sequence of one or more of the CDR regions of its VH and / or VL have been replaced with CDR sequences from another species, such as an antibody having murine heavy and light chain variable regions but in which one or more of the murine CDRs (e.g., CDR3) have been replaced with human CDR sequences.

[0077] Chimeric antibodies: The term "chimeric antibody" refers to an antibody that contains heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to a human constant region.

[0078] Complementary: As used herein, the term "complementary" refers to the capacity for exact pairing between two nucleosides or two pairs of nucleosides. In particular, complementary is a term that characterizes the degree of hydrogen bond pairing that results in binding between two nucleosides or two pairs of nucleosides. For example, if the base of an oligonucleotide at a position can hydrogen bond with the base of a target nucleic acid (e.g., mRNA) at the corresponding position, then the bases are considered to be complementary to each other at that position. Base pairing may include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, an adenosine-type base (A) is complementary to a thymidine-type base (T) or a uracil-type base (U), a cytosine-type base (C) is complementary to a guanosine-type base (G), and a universal base such as 3-nitropyrrole or 5-nitroindole can hybridize with and is considered complementary to any A, C, U, or T. Inosine (I) is also considered in the art to be a universal base and is considered complementary to any A, C, U, or T.

[0079] Conservative Amino Acid Substitutions: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for modifying polypeptide sequences known to those skilled in the art, for example, references that summarize such methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FMAusubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative amino acid substitutions include those made to amino acids in the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0080] Covalently linked: As used herein, the term "covalently linked" refers to the characteristic of two or more molecules being linked together through at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond (e.g., a disulfide bond or a disulfide bridge) that acts as an intermolecular linker. However, in some embodiments, two or more molecules can be covalently linked together through a molecule that acts as a linker that connects two or more molecules together through multiple covalent bonds. In some embodiments, the linker can be a cleavable linker. However, in some embodiments, the linker can be a non-cleavable linker.

[0081] Cross-reacting: As used herein, and in the context of targeting agents (e.g., antibodies), the term "cross-reacting" refers to the property of an agent that can specifically bind to more than one antigen of the same type or class (e.g., multiple homologues, paralogues, or orthologs of antigens) with similar affinity or avidity. For example, in some embodiments, an antibody that cross-reacts to a similar type or class of human and non-human primate antigens (e.g., human transferrin receptor and non-human primate transferrin receptor) can bind to a human antigen and a non-human primate antigen with similar affinity or avidity. In some embodiments, the antibody cross-reacts to a similar type or class of human antigen and rodent antigen. In some embodiments, the antibody cross-reacts to a similar type or class of rodent antigen and non-human primate antigen. In some embodiments, the antibody cross-reacts to a similar type or class of human antigen, non-human primate antigen, and rodent antigen.

[0082] Disease-associated repeats: As used herein, the term "disease-associated repeat" refers to a repeated nucleotide sequence at a genomic location where several units of the repeated nucleotide sequence correlate with and / or (for example, and) directly or indirectly contribute to or cause a genetic disease, such as DM1. Each repeat unit of the disease-associated repeat may be 2, 3, 4, 5 or more nucleotides in length. For example, in some embodiments, the disease-associated repeat is a dinucleotide repeat. In some embodiments, the disease-associated repeat is a trinucleotide repeat. In some embodiments, the disease-associated repeat is a tetranucleotide repeat. In some embodiments, the disease-associated repeat is a pentanucleotide repeat. In some embodiments, the disease-associated repeat comprises a CAG repeat, a CTG repeat, a CUG repeat, a CGG repeat, a CCTG repeat, or a nucleotide complement of any thereof. In some embodiments, the disease-associated repeat is in a non-coding portion of a gene. However, in some embodiments, the disease-associated repeat is in a coding region of a gene. In some embodiments, the disease-associated repeat is expanded from a normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, the disease-associated repeats are in RNA (e.g., RNA transcripts). In some embodiments, the disease-associated repeats are in DNA (e.g., chromosomes, plasmids). In some embodiments, the disease-associated repeats are expanded in chromosomes of germline cells. In some embodiments, the disease-associated repeats are expanded in chromosomes of somatic cells. In some embodiments, the disease-associated repeats are expanded into multiple repeat units associated with congenital onset. In some embodiments, the disease-associated repeats are expanded into multiple repeat units associated with childhood onset of the disease. In some embodiments, the disease-associated repeats are expanded into multiple repeat units associated with adult onset of the disease. In DM1, a trinucleotide repeat region of CTG units in the 3' untranslated region (3'-UTR) of DMPK is associated with the disease. Whereas normal DMPK alleles contain about 5 to about 37 CTG repeat units, in DM1 patients, the length of the CTG repeat region is significantly increased, up to hundreds or thousands of trinucleotide repeats.

[0083] DMPK: As used herein, the term "DMPK" refers to a gene encoding myotonin protein kinase (also known as myotonic dystrophy protein kinase or dystrophia myotonica protein kinase), a serine / threonine protein kinase. Substrates of this enzyme can include myogenin, beta subunit of L-type calcium channel, and phospholemman. In some embodiments, DMPK can be a human (Gene ID: 1760), non-human primate (e.g., Gene ID: 456139, Gene ID: 715328), or rodent gene (e.g., Gene ID: 13400). In humans, a CTG repeat expansion in the 3' non-coding untranslated region of DMPK is associated with myotonic dystrophy type I (DM1). In addition, multiple human transcript variants encoding different isoforms of the receptor (e.g., those annotated with GenBank RefSeq accession numbers: NM_001081563.2, NM_004409.4, NM_001081560.2, NM_001081562.2, NM_001288764.1, NM_001288765.1, and NM_006128) have been characterized.

[0084] DMPK alleles: As used herein, the term "DMPK allele" refers to any one of the alternative forms (e.g., wild-type or mutant forms) of the DMPK gene. In some embodiments, the DMPK allele may encode a wild-type myotonin protein kinase that retains its normal and typical function. In some embodiments, the DMPK allele may contain one or more disease-associated repeat expansions. In some embodiments, a normal subject has two DMPK alleles that contain repeat units ranging from 5 to 37. In some embodiments, the number of CTG repeat units in a subject with DM1 ranges from about 50 to about 3,000 or more, with the higher the number of repeats, the greater the severity of the disease. In some embodiments, a mild DM1 subject has at least one DMPK allele with repeat units ranging from 50 to 150. In some embodiments, a subject with classical DM1 has at least one DMPK allele with repeat units ranging from 100 to 1,000 or more. In some embodiments, a subject with congenital onset DM1 may have at least one DMPK allele that contains more than 2,000 repeat units.

[0085] Framework: As used herein, the term "framework" or "framework sequence" refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of the CDR sequence can be determined by various systems, the meaning of the framework sequence is subject to correspondingly different interpretations. The six CDRs (CDR-L1, CDR-L2, and CDR-L3 of the light chain, and CDR-H1, CDR-H2, and CDR-H3 of the heavy chain) also divide the framework region on the light chain and the heavy chain into four subregions (FR1, FR2, FR3, and FR4) on each chain, where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. The framework region that does not specify a specific subregion as FR1, FR2, FR3, or FR4, when referred to by others, represents the combined FR(s) in the variable region of a naturally occurring single immunoglobulin chain. As used herein, FR refers to one of the four subregions, and FR refers to two or more of the four subregions that contain framework regions. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

[0086] Human antibodies: The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may include, for example, in the CDRs, particularly CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted onto human framework sequences.

[0087] Humanized antibodies: The term "humanized antibody" refers to an antibody that contains heavy and light chain variable region sequences from a non-human species (e.g., mouse), but 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 to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-TfR1 antibodies and antigen-binding portions are provided. Such antibodies may be produced by obtaining a murine anti-TfR1 monoclonal antibody 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.).

[0088] Internalizing cell surface receptors: As used herein, the term "internalizing cell surface receptor" refers to a cell surface receptor that is internalized by a cell upon an external stimulus (e.g., a ligand binding to the receptor). In some embodiments, the internalizing cell surface receptor is internalized by endocytosis. In some embodiments, the internalizing cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalizing cell surface receptor is internalized by a clathrin-independent pathway, such as phagocytosis, macropinocytosis, caveolae- and raft-mediated uptake, or clathrin-independent constitutive endocytosis. In some embodiments, the internalizing cell surface receptor comprises an intracellular domain, a transmembrane domain, and / or (e.g., and) an extracellular domain, which optionally further comprises a ligand-binding domain. In some embodiments, the cell surface receptor becomes internalized by a cell after ligand binding. In some embodiments, the ligand may be a muscle-targeting agent or a muscle-targeting antibody. In some embodiments, the internalizing cell surface receptor is a transferrin receptor.

[0089] Isolated antibodies: An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (for example, an isolated antibody that specifically binds to the transferrin receptor is substantially free of antibodies that specifically bind to antigens other than the transferrin receptor). However, an isolated antibody that specifically binds to the transferrin receptor complex may have cross-reactivity to other antigens, such as transferrin receptor molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or (for example and) chemicals.

[0090] Kabat numbering: The terms "Kabat numbering", "Kabat definition", and "Kabat labeling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system for numbering amino acid residues that are more variable (i.e., more variable) than other amino acid residues in the heavy and light chain variable regions of an antibody or antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). In the heavy chain variable region, the hypervariable region spans amino acid positions 31-35 for CDR1, amino acid positions 50-65 for CDR2, and amino acid positions 95-102 for CDR3. In the light chain variable region, the hypervariable region spans amino acid positions 24-34 for CDR1, amino acid positions 50-56 for CDR2, and amino acid positions 89-97 for CDR3.

[0091] Molecular payload: As used herein, the term "molecular payload" refers to a molecule or species that functions to modulate a biological outcome. In some embodiments, the molecular payload is linked to or otherwise associated with a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, a protein, a peptide, a nucleic acid, or an oligonucleotide. In some embodiments, the molecular payload functions to modulate the transcription of a DNA sequence, to modulate the expression of a protein, or to modulate the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide that includes a strand having a region of complementarity to a target gene.

[0092] Muscle-targeting agents: As used herein, the term "muscle targeting agent" refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on a muscle cell may be a membrane protein, such as an integral or peripheral membrane protein. Typically, a muscle targeting agent specifically binds to an antigen on a muscle cell that facilitates internalization of the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalizing cell surface receptor on muscle and is capable of being internalized into the muscle cell through receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In some embodiments, the muscle targeting agent is linked to a molecular payload.

[0093] Muscle targeting antibodies: As used herein, the term "muscle-targeting antibody" refers to a muscle targeting agent that is an antibody that specifically binds to an antigen found in or on a muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an antigen on a muscle cell that facilitates internalization of the muscle-targeting antibody (and any attached molecular payload) into the muscle cell. In some embodiments, the muscle-targeting antibody specifically binds to an internalizing cell surface receptor present on a muscle cell. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0094] Myotonic Dystrophy (DM): As used herein, the term "myotonic dystrophy (DM)" refers to a genetic disease caused by mutations in the DMPK or CNBP (ZNF9) genes that is characterized by muscle loss, muscle weakness, and muscle function. Two types of the disease have been reported: myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is associated with an expansion of a CTG trinucleotide repeat in the 3' noncoding region of DMPK. DM2 is associated with an expansion of a CCTG tetranucleotide repeat in the first intron of ZNF9. In both DM1 and DM2, the nucleotide expansion leads to toxic RNA repeats that can form hairpin structures that bind with high affinity to important intracellular proteins, such as muscleblind-like proteins. Myotonic dystrophy, the genetic basis of the disease, and associated symptoms have been reported in the art (see, e.g., Thornton, CA, "Myotonic Dystrophy" Neurol Clin. (2014), 32(3): 705-719.; and Konieczny et al. "Myotonic dystrophy: candidate small molecule therapeutics" Drug Discovery Today (2017), 22:11). In some embodiments, the subject is born with a variation of DM1 called congenital myotonic dystrophy. Symptoms of congenital myotonic dystrophy are present from birth and include weakness of all muscles, breathing problems, clubfoot, developmental delay, and intellectual disability. DM1 is associated with Online Mendelian Inheritance in Man (OMIM) entry #160900. DM2 is associated with OMIM entry #602668.

[0095] Oligonucleotides: As used herein, the term "oligonucleotide" refers to an oligomeric nucleic acid compound up to 200 nucleotides in length. Examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, gapmer, mixmer, phosphorodiamidate morpholino, peptide nucleic acid, aptamer, guide nucleic acid (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 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 that may be in Rp or Sp stereochemical configuration.

[0096] Recombinant antibodies: The term "recombinant human antibody," as used herein, refers to any human antibody that is prepared, expressed, created, or isolated by recombinant means, e.g., antibodies expressed using a recombinant expression vector transfected into a host cell (as described in more detail in this disclosure), antibodies isolated from a recombinant combinatorial human antibody library (Hoogenboom HR, (1997) TIB Tech. 15:62-70; Azzazy H., and Highsmith WE, (2002) Clin. Biochem. 35:425-445; Gavilondo JV, and Larrick JW (2002) BioTechniques 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), antibodies isolated from a human immunoglobulin gene transgenic animal (e.g., a mouse) (see, e.g., Taylor, LD, et al., J. Immunology 29:128-145; Hoogenboom H., and Chames P. (2000) Immunology Today 21:371-378), or antibodies isolated from a human immunoglobulin gene transgenic animal (e.g., a mouse) (see, e.g., Taylor, LD, et al., J. Immunology 29:128-145; Hoogenboom ... al. (1992) Nucl. Acids Res. 20:6287-6295; Kellermann SA., and Green LL (2002) Current Opinion in Biotechnology 13:593-597; Little M. et al (2000) Immunology Today 21:364-370), or any other means involving splicing of human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when human Ig sequence transgenic animals are used, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, although derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur within the germline repertoire of human antibodies in vivo.One aspect of the disclosure provides fully human antibodies capable of binding to the human transferrin receptor that can be generated using techniques well known in the art, such as, but not limited to, techniques using human Ig phage libraries (e.g., those disclosed in PCT Publication No. WO 2005 / 007699 A2 to Jermutus et al.).

[0097] Regions of complementarity: As used herein, the term "region of complementarity" refers to a nucleotide sequence (e.g., a nucleotide sequence of an oligonucleotide) that is sufficiently complementary to a cognate nucleotide sequence (e.g., a nucleotide sequence of a target nucleic acid) such that the two nucleotide sequences are capable of annealing to each other under physiological conditions (e.g., in a cell). In some embodiments, the region of complementarity is fully complementary to the cognate nucleotide sequence of the target nucleic acid. However, in some embodiments, the region of complementarity is 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.

[0098] Specific binding to: As used herein, the term "specifically binds" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that can be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context. With respect to an antibody, the term "specifically binds" refers to the ability of an antibody to bind to a particular antigen, as compared to an appropriate reference antigen, or an antigen that can be used to distinguish the particular antigen from other antigens, with a degree of affinity or avidity (e.g., a degree that allows preferential targeting to certain cells (e.g., muscle cells) through binding to the antigen, as described herein). In some embodiments, the antibody binds to a target with at least about 10% affinity. -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 than this 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.

[0099] subject: As used herein, the term "subject" refers to a mammal. In some embodiments, the subject is a non-human primate animal or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, e.g., a human patient, having or suspected of having a disease caused by a disease-associated repeat expansion, e.g., a DMPK allele.

[0100] Transferrin Receptor: As used herein, the term "transferrin receptor" (also known as TFRC, CD71, p90, or TFR1) refers to an internalizing cell surface receptor that binds to transferrin to facilitate iron uptake by endocytosis. In some embodiments, the transferrin receptor may originate from humans (NCBI Gene ID 7037), non-human primates (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodents (e.g., NCBI Gene ID 22042). In addition, multiple human transcript variants encoding different isoforms of the receptor have been characterized (e.g., as annotated with GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0101] 2'-Modified Nucleosides: As used herein, the terms "2'-modified nucleoside" and "2'-modified ribonucleoside" are used interchangeably and refer to a nucleoside having a sugar moiety modified at the 2' position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, in which the 2' and 4' positions of the sugar are bridged (e.g., by a methylene, ethylene, or (S)-constrained ethyl bridge). In some embodiments, the 2'-modified nucleoside is a non-bicyclic 2'-modified nucleoside, e.g., in which the 2' position of the sugar moiety is substituted. Non-limiting examples of 2'-modified nucleosides include the following: 2'deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamide (2'-O-NMA), locked nucleic acid (LNA, methylene bridged nucleic acid), ethylene bridged nucleic acid (ENA), and (S)-constrained ethyl bridged nucleic acid (cEt). In some embodiments, the 2'-modified nucleosides described herein are high affinity modified nucleosides, and oligonucleotides comprising the 2'-modified nucleosides have increased affinity for target sequences compared to unmodified oligonucleotides. Examples of 2'-modified nucleosides structures are provided below: [ka] The examples are shown with a phosphate group, however, any internucleoside linkage is contemplated between 2'-modified nucleosides.

[0102] II. Complex Further provided herein is a conjugate comprising a targeting agent, for example an antibody, covalently linked to a molecular payload. In some embodiments, the conjugate comprises a muscle-targeting antibody covalently linked to an oligonucleotide. The conjugate may comprise an antibody that specifically binds to a single antigen site, or an antibody that binds to at least two antigen sites, which may be present on the same antigen or on different antigens.

[0103] The complex may be used to modulate the activity or function of at least one gene, protein, and / or (for example, and) nucleic acid. In some embodiments, the molecular payload present in the complex is responsible for the modulation of a gene, protein, and / or (for example, and) nucleic acid. The molecular payload may be a small molecule, a protein, a nucleic acid, an oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and / or (for example, and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets disease-associated repeats in muscle cells. In some embodiments, the molecular payload is an oligonucleotide that targets disease-associated repeats in CNS cells. In some embodiments, the molecular payload is an oligonucleotide that does not target disease-associated repeats. In some embodiments, the molecular payload is an oligonucleotide that targets a coding or non-coding region of a DMPK transcript (for example, pre-mRNA or mRNA), such as a 3'-untranslated region, an intron region, or an exon region in a cell (for example, a muscle cell or a CNS cell).

[0104] In some embodiments, the conjugate comprises a muscle targeting agent (e.g., an anti-TfR1 antibody) covalently linked to a molecular payload (e.g., an antisense oligonucleotide targeting DMPK, such as a nucleic acid comprising a disease-associated repeat (e.g., a DMPK allele)).

[0105] A. Muscle-targeting Agents Some aspects of the present disclosure provide muscle targeting agents, e.g., muscle targeting agents for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents are capable of binding to muscle cells and delivering the associated molecular payload to muscle cells, e.g., via specific binding to an antigen on the muscle cell. In some embodiments, the molecular payload is bound (e.g., covalently bound) to the muscle targeting agent and is internalized into the muscle cell, e.g., via endocytosis, when the muscle targeting agent binds to the antigen on the muscle cell. It should be understood that various types of muscle targeting agents may be used according to the present disclosure, and that any muscle target (e.g., muscle surface protein) may be targeted by any type of muscle targeting agent described herein. For example, the muscle targeting agent may include or consist of a small molecule, a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide). Exemplary muscle-targeting agents are described in further detail herein; however, it should be understood that the exemplary muscle-targeting agents provided herein are not intended to be limiting.

[0106] Some aspects of the disclosure provide muscle targeting agents that specifically bind to antigens on muscles, such as skeletal, smooth, or cardiac muscles. In some embodiments, any of the muscle targeting agents provided herein bind (e.g., specifically bind) to antigens on skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells.

[0107] Through interaction with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, molecules that are substrates for muscle uptake transporters are useful for delivering molecular payloads into muscle tissue. Binding to muscle surface recognition elements, followed by endocytosis, can allow even macromolecules, such as antibodies, to enter muscle cells. As another example, molecular payloads conjugated to transferrin or anti-TfR1 antibodies can be taken up by muscle cells via binding to the transferrin receptor and then endocytosed, e.g., via clathrin-mediated endocytosis.

[0108] The use of muscle targeting agents can be useful to concentrate molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells compared to other cell types in a subject. In some embodiments, muscle targeting agents concentrate bound molecular payloads in muscle cells (e.g., skeletal muscle cells, smooth muscle cells, or cardiomyocytes) at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold greater than the amount in non-muscle cells (e.g., liver cells, nerve cells, blood cells, or adipocytes). In some embodiments, the toxicity in a subject of a molecular payload when conjugated to a muscle targeting agent is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95% when delivered to a subject.

[0109] In some embodiments, a muscle recognition element (e.g., a muscle cell antigen) may be required to achieve muscle selectivity. As one example, the muscle targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, the muscle targeting agent may be an antibody that enters muscle cells via transporter-mediated endocytosis. As another example, the muscle targeting agent may be a ligand that binds to a cell surface receptor on muscle cells. It should be understood that while transporter-based approaches provide a direct pathway for cell entry, receptor-based targeting may also involve stimulated endocytosis to reach the desired site of action.

[0110] i. Muscle targeting antibody In some embodiments, the muscle targeting agent is an antibody. In general, the high specificity of antibodies to their target antigens provides the potential to selectively target muscle cells (e.g., skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiomyocytes). This specificity may also limit off-target toxicity. Examples of antibodies capable of targeting surface antigens of muscle cells have been reported and are within the scope of this disclosure. For example, antibodies that target the surface of muscle cells are described in Arahata K., et al. "Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide" Nature 1988;333:861-3; Song KS, et al. "Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins" J Biol Chem 1996;271:15160-5; and Weisbart RH et al., "Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb" Mol Immunol. 2003 Mar,39(13):78309; the entire contents of each of which are incorporated herein by reference.

[0111] a. Anti-transferrin receptor (TfR) antibody Some aspects of the present disclosure are based on the recognition that agents that bind to the transferrin receptor, e.g., anti-transferrin receptor antibodies, can target muscle cells. The transferrin receptor is an internalizing cell surface receptor that transports transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to the transferrin receptor. Accordingly, aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to the transferrin receptor. In some embodiments, the binding proteins that bind to the transferrin receptor are internalized into muscle cells along with any molecular payloads attached. As used herein, antibodies that bind to the transferrin receptor may be interchangeably referred to as transferrin receptor antibodies, anti-transferrin receptor antibodies, or anti-TfR1 antibodies. Antibodies that bind, e.g., specifically bind, to the transferrin receptor may be internalized into cells upon binding to the transferrin receptor, e.g., through receptor-mediated endocytosis.

[0112] It should be understood that anti-TfR1 antibodies may be produced, synthesized, and / or (for example and) derivatized using several known methodologies, for example library design using phage display. Exemplary methodologies have been characterized in the art and are incorporated by reference (Diez, P. et al. "High-throughput phage-display screening in array format", Enzyme and microbial technology, 2015, 79, 34-41.; Christoph MH and Stanley, JR "Antibody Phage Display: Technique and Applications" J Invest Dermatol. 2014, 134:2.; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies." 1985, Springer). In other embodiments, anti-TfR1 antibodies have been previously characterized or disclosed.Antibodies that specifically bind to the transferrin receptor are known in the art (see, e.g., U.S. Pat. No. 4,364,934, filed Dec. 4, 1979, "Monoclonal antibody to a human early thymocyte antigen and methods for preparing same"; U.S. Pat. No. 8,409,573, filed Jun. 14, 2006, "Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells"; U.S. Pat. No. 9,708,406, filed May 20, 2014, "Anti-transferrin receptor antibodies and methods of use"; U.S. Pat. No. 9,611,323, filed Dec. 19, 2014, "Low affinity blood brain barrier receptor antibodies and uses therefor"; WO 2015 / 098989, filed Dec. 24, 2014, "Novel anti-Transferrin receptor antibody that passes through blood-brain barrier"; Schneider C. et al. "Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9.”J Biol Chem.1982,257:14,8516-8522.;Lee et al.“Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse”2000,J Pharmacol. Exp. Ther., 292:1048-1052).

[0113] In some embodiments, the anti-TfR1 antibodies described herein bind to the transferrin receptor with high specificity and affinity. In some embodiments, the anti-TfR1 antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor or epitope that becomes exposed to the antibody. In some embodiments, the anti-TfR1 antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR1 antibodies provided herein bind to the human transferrin receptor. In some embodiments, the anti-TfR1 antibodies described herein bind to an amino acid segment of the human or non-human primate transferrin receptor provided in SEQ ID NOs: 105-108. In some embodiments, the anti-TfR1 antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor as represented in SEQ ID NO: 105 that is not in the apical domain of the transferrin receptor.

[0114] In some embodiments, the anti-TfR1 antibodies described herein (e.g., anti-TfR clone 8 in Table 2 below) bind to an epitope in TfR1, where the epitope includes residues at amino acids 214-241 and / or amino acids 354-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope including residues at amino acids 214-241 and amino acids 354-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope including one or more of residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1 as represented in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope comprising residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1 as set forth in SEQ ID NO:105.

[0115] In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope in TfR1, where the epitope includes residues at amino acids 258-291 and / or amino acids 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope including residues at amino acids 258-291 and amino acids 358-381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope including one or more of residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as represented in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and its variants in Table 2 below) bind to an epitope including residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 as represented by SEQ ID NO:105.

[0116] An example of a human transferrin receptor amino acid sequence, which corresponds to the NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens), is as follows: (Sequence number 105).

[0117] An example of a primate non-human animal transferrin receptor amino acid sequence, which corresponds to the NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Macaca mulatta), is as follows: (Sequence number 106).

[0118] An example of a primate non-human animal transferrin receptor amino acid sequence that corresponds to the NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows: (Sequence number 107).

[0119] An example of a mouse transferrin receptor amino acid sequence, which corresponds to the NCBI sequence NP_001344227.1 (transferrin receptor protein 1, mus musculus), is as follows: MMDQARSAFSNLFGGEPLSYTRFSLARQVDGDNSHVEMKLAADEEENADNNMKASVRKPKRFNGRLCFAAIALVIFFLIGFMSGYLGYCKRVEQKEECVKLAETEETDKSETMETEDVPTSSRLYWADLKTLLSEKLNSIEFADTIKQLSQNTYTPREAGSQKDESLAYYIENQFHEFKFSKVWRDEHYVKIQVKSSIGQNMVTIVQSNGNLDPVESPEGYVAFSKPTEVSGKLVHANFGTKKDFEELSYSVNGSLVIVRAGEITFAEKVANAQSFNAIGVLIYMDKNKFPVVEADLALFGHAHLGTGDPYTPGFPSFNHTQFPPSQSSGLPNIPVQTISRAAAEKLFGKMEGSCPARWNIDSSCKLELSQNQNVKLIVKNVLKERRILNIFGVIKGYEEPDRYVVVGAQRDALGAGVAAKSSVGTGLLLKLAQVFSDMISKDGFRPSRSIIFASWTAGDFGAVGATEWLEGYLSSLHLKAFTYINLDKVVLGTSNFKVSASPLLYTLMGKIMQDVKHPVDGKSLYRDSNWISKVEKLSFDNAAYPFLAYSGIPAVSFCFCEDADYPYLGTRLDTYEALTQKVPQLNQMVRTAAEVAGQLIIKLTHDVELNLDYEMYNSKLLSFMKDLNQFKTDIRDMGLSLQWLYSARGDYFRATSRLTTDFHNAEKTNRFVMREINDRIMKVEYHFLSPYVSPRESPFRHIFWGSGSHTLSALVENLKLRQKNITAFNETLFRNQLALATWTIQGVANALSGDIWNIDNEF(SEQ ID NO: 108).

[0120] In some embodiments, the anti-TfR1 antibodies bind to the following amino acid segment of the receptor: FVKIQVKDSAQNSVIIVDKNGRLVYLVENPGGYVAYSKAATVTGKLVHANFGTKKDFEDLYTPVNGSIVIVRAGKITFAEKVANAESLNAIGVLIYMDQTKFPIVNAELSFFGHAHLGTGDPYTPGFPSFNHTQFPPSRSSGLPNIPVQTISRAAAEKLFGNMEGDCPSDWKTDSTCRMVTSESKNVKLTVSNVLKE (SEQ ID NO: 109) and do not inhibit the binding interaction between the transferrin receptor and transferrin and / or (by way of example and) human hemochromatosis protein (also known as HFE). In some embodiments, the anti-TfR1 antibodies described herein do not bind to the epitope of SEQ ID NO: 109.

[0121] Suitable methodologies may be used to obtain and / or (for example and) produce antibodies, antibody fragments, or antigen-binding agents, for example through the use of recombinant DNA protocols. In some embodiments, antibodies may also be produced through the generation of hybridomas (see, for example, Kohler, G and Milstein, C. "Continuous cultures of fused cells secreting antibody of predefined specificity" Nature, 1975, 256:495-497). An antigen of interest may be used as an immunogen of any type or entity, for example, recombinant or naturally occurring type or entity. Hybridomas are screened using standard methods, for example, ELISA screening, to find at least one hybridoma that produces an antibody that targets a specific antigen. Antibodies may also be produced through screening of protein expression libraries (for example, phage display libraries) that express antibodies. Phage display library design may also be used in some embodiments (see, e.g., U.S. Patent No. 5,223,409, filed 3 / 1 / 1991, "Directed evolution of novel binding proteins"; WO 1992 / 18619, filed 4 / 10 / 1992, "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, filed 5 / 1 / 1991, "Recombinant library screening methods"; WO 1992 / 20791, filed 5 / 15 / 1992, "Methods for producing members of specific binding pairs"; and WO 1992 / 15679, filed 2 / 28 / 1992, "Improved epitope displaying phage"). In some embodiments, the antigen of interest may be used to immunize a non-human animal, e.g., a rodent animal or a goat.In some embodiments, once the antibody is obtained from the non-human animal, it may then be optionally modified using a number of methodologies, for example using recombinant DNA techniques. Additional examples of antibody production and methodologies are also known in the art (see, for example, Harlow et al. "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988).

[0122] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, optionally deficient in an enzyme (e.g., glycosyltransferase) in the N- or O-glycosylation pathway. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0123] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VL domain and / or (for example and) a VH domain of any one of the anti-TfR1 antibodies selected from any one of Tables 2-7, and comprises a constant region comprising the amino acid sequence of the constant region of an immunoglobulin molecule of an IgG, IgE, IgM, IgD, IgA, or IgY, of any class (for example, IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or of any subclass (for example, IgG2a and IgG2b). Non-limiting examples of human constant regions are described in the art. See, for example, Kabat EA et al., (1991) supra.

[0124] In some embodiments, agents that bind to the transferrin receptor, e.g., anti-TfR1 antibodies, can target muscle cells and / or (e.g., and) mediate transport of agents across the blood-brain barrier (e.g., to CNS cells). The transferrin receptor is an internalizing cell surface receptor that transports transferrin across the cell membrane and participates in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to the transferrin receptor. Antibodies that bind, e.g., specifically bind, to the transferrin receptor may be internalized into cells upon binding to the transferrin receptor, e.g., through receptor-mediated endocytosis.

[0125] In some embodiments, provided herein are humanized antibodies that bind to the transferrin receptor with high specificity and affinity. In some embodiments, the anti-TfR1 antibodies described herein specifically bind to any extracellular epitope of the transferrin receptor, or an epitope that becomes exposed to the antibody. In some embodiments, the anti-TfR1 antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR1 antibodies provided herein bind to the human transferrin receptor. In some embodiments, the anti-TfR1 antibodies described herein bind to an amino acid segment of the human or non-human primate transferrin receptor, such as those provided in SEQ ID NOs: 105-108. In some embodiments, the anti-TfR1 antibodies described herein bind to an amino acid segment corresponding to amino acids 90-96 of the human transferrin receptor as set forth in SEQ ID NO: 105, which is not in the apical domain of the transferrin receptor. In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 but not to TfR2.

[0126] In some embodiments, the anti-TfR1 antibody is at least about 10 -4 M, 10 -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 (e.g., human or non-human primate TfR1) with a binding affinity (e.g., as indicated by a Kd) of 10 M or less. In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 with a Kd in the sub-nanomolar range. In some embodiments, the anti-TfR1 antibodies described herein selectively bind to transferrin receptor 1 (TfR1) but not to transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR1 antibodies described herein bind to human TfR1 and cynomolgus TfR1 (e.g., as indicated by a Kd) of 10 M or less. -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or smaller Kd) does not bind to mouse TfR1. The affinity and binding kinetics of the anti-TfR antibodies can be tested using any suitable method, including but not limited to biosensor technology (e.g., OCTET or BIACORE). In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit transferrin binding to TfR1. In some embodiments, the binding of any one of the anti-TfR1 antibodies described herein does not compete with or inhibit HFE-beta2-microglobulin binding to TfR1.

[0127] Non-limiting examples of anti-TfR1 antibodies are provided in Table 2. Table 2. Examples of anti-TfR1 antibodies [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4]

[0128] In some embodiments, an anti-TfR1 antibody of the disclosure is a variant of any one of the anti-TfR1 antibodies provided in Table 2. In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 in any one of the anti-TfR1 antibodies provided in Table 2, and comprises a heavy chain variable region and / or (by way of example and) a light chain variable region.

[0129] Exemplary amino acid sequences of anti-TfR1 antibodies described herein are provided in Table 3. Table 3. Variable regions of anti-TfR1 antibodies [Table 7-1] [Table 7-2] [Table 7-3] [Table 7-4]

[0130] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising a CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework regions when compared to the respective VHs provided in Table 3. Alternatively or additionally (e.g., in addition), an anti-TfR1 antibody of the present disclosure comprises a VL comprising a CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework regions when compared to the respective VLs provided in Table 3.

[0131] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VH comprising CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identical amino acid sequence in the framework region compared to the respective VH provided in Table 3. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibody of the present disclosure comprises a VL comprising CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identical amino acid sequence in the framework region compared to the respective VL provided in Table 3.

[0132] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:69 and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0133] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:71 and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0134] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:72 and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0135] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73 and a VL comprising the amino acid sequence of SEQ ID NO:74.

[0136] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73 and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0137] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:74.

[0138] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0139] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77 and a VL comprising the amino acid sequence of SEQ ID NO:78.

[0140] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:79 and a VL comprising the amino acid sequence of SEQ ID NO:80.

[0141] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77 and a VL comprising the amino acid sequence of SEQ ID NO:80.

[0142] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:154 and a VL comprising the amino acid sequence of SEQ ID NO:155.

[0143] In some embodiments, the anti-TfR1 antibodies described herein are full-length IgGs that may include heavy and light chain constant regions from a human antibody. In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, such as human, mouse, rat, or rabbit. In one specific example, the heavy chain constant region is from a human IgG (gamma heavy chain), such as IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 81).

[0144] In some embodiments, the heavy chain of any of the anti-TfR1 antibodies described herein comprises a mutant human IgG1 constant region. For example, the introduction of LALA mutations on the CH2 domain of human IgG1 (a mutant derived from mAb b12 mutated to replace the lower hinge residues Leu234 Leu235 with Ala234 and Ala235) is known to reduce Fcγ receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The mutant human IgG1 constant region is provided below (mutations in bold and underlined): [ka] (Sequence number 82).

[0145] In some embodiments, the light chain constant region of any of the anti-TfR1 antibodies described herein can be any light chain constant region known in the art. In some examples, it is a kappa light chain or a lambda light chain. In some embodiments, the light chain constant region is a kappa light chain, the sequence of which is provided below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 83).

[0146] Heavy and light chain constant regions of other antibodies are well known in the art and are provided, for example, in the IMGT database (imgt.org) or at www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0147] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region that contains no more than 25 amino acid 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) compared to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region as represented by SEQ ID NO: 81. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof, and a heavy chain constant region as set forth in SEQ ID NO:82.

[0148] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any variants thereof listed in Table 3, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any variants thereof listed in Table 3, and a light chain constant region that contains no more than 25 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) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any variants thereof listed in Table 3, and a light chain constant region as represented by SEQ ID NO: 83.

[0149] Examples of IgG heavy and light chain amino acid sequences of the described anti-TfR1 antibodies are provided in Table 4 below. Table 4. Heavy and light chain sequences of example anti-TfR1 IgGs [Table 8-1] [Table 8-2] [Table 8-3] [Table 8-4]

[0150] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain that contains no more than 25 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) compared to a heavy chain as represented by any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a light chain that contains no more than 25 amino acid variations (for example, 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) compared to a light chain as represented by any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0151] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or additionally (e.g., in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. Alternatively or additionally (by way of example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0152] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:84 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0153] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:86 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0154] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:87 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0155] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0156] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0157] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0158] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0159] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0160] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:94 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0161] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0162] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:156 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0163] In some embodiments, the anti-TfR1 antibody is a Fab fragment, Fab' fragment, or F(ab')2 fragment of an intact antibody (full-length antibody). Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared via routine methods (e.g., recombinantly or by digesting the heavy chain constant region of a full-length IgG using an enzyme such as papain). For example, F(ab')2 fragments can be generated by pepsin or papain digestion of an antibody molecule, and Fab fragments can be produced by reducing disulfide bridges of F(ab')2 fragments. In some embodiments, the heavy chain constant region on the Fab fragment of the anti-TfR1 antibodies described herein is as follows: It contains the amino acid sequence of ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHT (SEQ ID NO: 96).

[0164] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region that contains no more than 25 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) compared to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs or any variants thereof listed in Table 3, and a heavy chain constant region as represented by SEQ ID NO: 96.

[0165] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any variants thereof listed in Table 3, and a light chain constant region that is at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any variants thereof listed in Table 3, and a light chain constant region that contains no more than 25 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) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs or any variants thereof listed in Table 3, and a light chain constant region as represented by SEQ ID NO: 83.

[0166] Examples of Fab heavy and light chain amino acid sequences of the described anti-TfR1 antibodies are provided in Table 5 below. Table 5. Heavy and light chain sequences of example anti-TfR1 Fabs [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5]

[0167] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain that contains no more than 25 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) compared to a heavy chain as represented in any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a light chain that contains no more than 25 amino acid variations (for example, 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) compared to a light chain as represented by any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0168] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively or additionally (e.g., in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98%, or 99%) identical to any one of SEQ ID NOs: 97-103, 158, and 159. Alternatively or additionally (by way of example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95, and 157.

[0169] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:97 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0170] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:98 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0171] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0172] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0173] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0174] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0175] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0176] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0177] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:103 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0178] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0179] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:158 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0180] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:159 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0181] Other known anti-TfR1 antibodies Any other suitable anti-TfR1 antibody known in the art can be used as a muscle targeting agent in the conjugates disclosed herein. Examples of known anti-TfR1 antibodies (including related references and binding epitopes) are listed in Table 6. In some embodiments, the anti-TfR1 antibody comprises the complementarity determining region (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3) of any of the anti-TfR1 antibodies provided herein, for example, the anti-TfR1 antibodies listed in Table 6.

[0182] Table 6 - List of anti-TfR1 antibody clones with relevant references and binding epitope information. [Table 10-1] [Table 10-2] [Table 10-3]

[0183] In some embodiments, an anti-TfR1 antibody of the disclosure includes one or more of the CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, an anti-TfR1 antibody includes CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, an anti-TfR1 antibody includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-TfR1 antibodies selected from Table 6.

[0184] In some embodiments, the anti-TfR1 antibodies of the disclosure include any antibody that includes a heavy chain variable domain and / or (by way of example and) a light chain variable domain of any anti-TfR1 antibody, such as any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the anti-TfR1 antibodies of the disclosure include any antibody that includes a heavy chain variable and light chain variable pair of any anti-TfR1 antibody, such as any one of the anti-TfR1 antibodies selected from Table 6.

[0185] Aspects of the present disclosure provide anti-TfR1 antibodies having heavy chain variable (VH) and / or (for example and) light chain variable (VL) domain amino acid sequences homologous to any of those described herein. In some embodiments, the anti-TfR1 antibodies comprise a heavy chain variable sequence or a light chain variable sequence that is at least 75% (for example, 80%, 85%, 90%, 95%, 98%, or 99%) identical to the heavy chain variable sequence and / or any light chain variable sequence of any anti-transferrin receptor antibody, such as any one of the anti-TfR1 antibodies selected from Table 6. In some embodiments, the homologous heavy chain variable and / or (for example and) light chain variable amino acid sequence does not vary in any of the CDR sequences provided herein. For example, in some embodiments, a degree of sequence variation (for example, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) may occur within the heavy chain variable and / or (for example and) light chain variable sequence that excludes any of the CDR sequences provided herein. In some embodiments, any of the anti-TfR1 antibodies provided herein comprise heavy chain and light chain variable sequences that comprise framework sequences that are at least 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the framework sequences of any anti-TfR1 antibody, such as any one of the anti-TfR1 antibodies selected from Table 6.

[0186] An example of a transferrin receptor antibody that may be used in accordance with the present disclosure is described in International Application Publication WO 2016 / 081643, which is incorporated herein by reference. The amino acid sequence of this antibody is provided in Table 7. Table 7. Heavy and light chain CDRs of examples of known anti-TfR1 antibodies [Table 11-1] [Table 11-2]

[0187] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-H1, CDR-H2, and CDR-H3 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a CDR-L1, CDR-L2, and CDR-L3 that are the same as the CDR-L1, CDR-L2, and CDR-L3 shown in Table 7.

[0188] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 that contains no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) when compared to a CDR-L3 as shown in Table 7. In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 that contains 1 amino acid variation when compared to a CDR-L3 as shown in Table 7. In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system). In some embodiments, an anti-TfR1 antibody of the disclosure comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 that are the same as the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7, and comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) according to the Kabat and Chothia definition system) or QHFAGTPL (SEQ ID NO: 127) according to the Contact definition system).

[0189] In some embodiments, anti-TfR1 antibodies of the disclosure comprise heavy chain CDRs that, together, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs shown in Table 7. Alternatively or additionally (e.g., in addition), anti-TfR1 antibodies of the disclosure comprise light chain CDRs that, together, are at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identical to the light chain CDRs shown in Table 7.

[0190] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 124. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 125.

[0191] In some embodiments, an anti-TfR1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 128. Alternatively or additionally (for example, in addition), an anti-TfR1 antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 129.

[0192] In some embodiments, the anti-TfR1 antibodies of the present disclosure include a VH that contains 25 amino acid variations or less (e.g., 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations or less) when compared to the VH as set forth in SEQ ID NO: 128. Alternatively or in addition (e.g., in addition), the anti-TfR1 antibodies of the present disclosure include a VL that contains 15 amino acid variations or less (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations or less) when compared to the VL as set forth in SEQ ID NO: 129.

[0193] In some embodiments, the heavy chain of any of the anti-TfR1 antibodies as described herein may comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may belong to any suitable origin, e.g., human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is from human IgG (gamma heavy chain), e.g., IgG1, IgG2, or IgG4. An example of a human IgG1 constant region is given below: ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (sequence number 81).

[0194] In some embodiments, the light chain of any of the anti-TfR1 antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. In some embodiments, the CL is a kappa light chain, the sequence of which is given below: RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (sequence number 83).

[0195] In some embodiments, the anti-TfR1 antibodies described herein are chimeric antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 132. Alternatively or additionally (for example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0196] In some embodiments, the anti-TfR1 antibodies described herein are fully human antibodies comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 134. Alternatively or additionally (for example, in addition), the anti-TfR1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0197] In some embodiments, the anti-TfR1 antibody is an antigen-binding fragment (Fab) of an intact antibody (full-length antibody). In some embodiments, the anti-TfR1 Fab described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 136. Alternatively or additionally (for example, in addition), the anti-TfR1 Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-TfR1 Fab described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 137. Alternatively or additionally (for example, in addition), the anti-TfR Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0198] The anti-TfR1 antibodies described herein can be in any antibody form, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (such as Fab, Fab', F(ab')2, Fv, etc.), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the anti-TfR1 antibodies described herein are scFv. In some embodiments, the anti-TfR1 antibodies described herein are scFv-Fab (e.g., scFv fused with a portion of a constant region). In some embodiments, the anti-TfR1 antibodies described herein are scFv fused with a constant region (e.g., human IgG1 constant region as represented by SEQ ID NO:81).

[0199] In some embodiments, conservative mutations can be introduced into an antibody sequence (e.g., CDR or framework sequences) at positions where the residues are unlikely to be involved in interactions with the target antigen (e.g., transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) of an anti-TfR1 antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or (e.g., and) antigen-dependent cellular cytotoxicity to cells.

[0200] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased) as described, e.g., in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0201] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0202] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. For example, see International Publication Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that will alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0203] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-TfR1 antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (e.g., and) in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al. (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, termed "YTE mutant", has been shown to exhibit a 4-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.

[0204] In some embodiments, one, two or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the anti-TfR1 antibody. The effector ligand with altered affinity to itself can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce binding of circulating antibodies to Fc receptors, thereby increasing tumor localization. See, for example, U.S. Pat. Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0205] In some embodiments, one or more amino acids in the constant region of the anti-TfR1 antibody described herein may be replaced with a different amino acid residue, such that the antibody may have altered C1q binding and / or (for example and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are altered, thereby altering the antibody's ability to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or (for example and) to increase the affinity of the antibody to Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0206] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein may be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be appreciated by one of skill in the art, any variant, CDR-grafted, chimeric, humanized, or composite antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and will retain specific binding ability to the transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it was derived.

[0207] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibodies. For example, to avoid potential complications due to Fab-arm exchange that is known to occur in native IgG4 mAbs, the antibodies provided herein may contain a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include a stabilizing "Adair" mutation.

[0208] In some embodiments, the antibody is modified (e.g., modified via glycosylation, phosphorylation, sumoylation, and / or (e.g., and) methylation). In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules. In some embodiments, the one or more sugar or carbohydrate molecules are conjugated to the antibody via N-glycosylation, O-glycosylation, C-glycosylation, glypiation (GPI anchor attachment), and / or (e.g., and) phosphoglycosylation. In some embodiments, the one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, the one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, the one or more sugar or carbohydrate molecules include mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, there are about 1-10, about 1-5, about 5-10, about 1-4, about 1-3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell (optionally deficient in an enzyme (e.g., glycosyltransferase) in the N- or O-glycosylation pathway). In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, published May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof."

[0209] In some embodiments, any one of the anti-TfR1 antibodies described herein may include a signal peptide (e.g., an N-terminal signal peptide) on the heavy and / or (e.g., and) light chain sequences. In some embodiments, the anti-TfR1 antibodies described herein include any one of the VH and VL sequences, any one of the IgG heavy and light chain sequences, or any one of the F(ab') heavy and light chain sequences described herein, and further include a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide includes the amino acid sequence of MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0210] In some embodiments, the antibody provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also referred to as pyroglutamic acid formation (pyroGlu), may occur in antibodies at N-terminal glutamic acid (Glu) and / or glutamine (Gln) residues during production. Therefore, it should be understood that an antibody identified as having a sequence that includes an N-terminal glutamic acid or glutamine residue encompasses an antibody that has undergone pyroglutamic acid formation as a result of post-translational modification. In some embodiments, pyroglutamic acid formation occurs in heavy chain sequences. In some embodiments, pyroglutamic acid formation occurs in light chain sequences.

[0211] b. Other muscle-targeting antibodies In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin Iib, or CD63. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a myogenic precursor protein. Exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin alpha7, integrin alpha7beta1, MYF-5, MyoD, myogenin, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a skeletal muscle protein. Exemplary skeletal muscle proteins include, without limitation, alpha-sarcoglycan, beta-sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha 7, integrin alpha 7 beta 1, integrin beta 1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, the muscle-targeting antibody is an antibody that specifically binds to a smooth muscle protein. Exemplary smooth muscle proteins include, without limitation, alpha-smooth muscle actin, VE-cadherin, caldesmon / CALD1, calponin 1, desmin, histamine H2 R, motilin R / GPR38, Transgelin / TAGLN, and vimentin, however, it should be understood that antibodies to additional targets are within the scope of the present disclosure and that the exemplary list of targets provided herein is not intended to be limiting.

[0212] c. Antibody Features / Modifications In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., a CDR or framework sequence) at a position where the residue is unlikely to be involved in interactions with the target antigen (e.g., the transferrin receptor) as determined, for example, based on a crystal structure. In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region) of a muscle-targeting antibody described herein to alter one or more functional properties of the antibody, such as serum half-life, complement binding, Fc receptor binding, and / or (e.g., and) antigen-dependent cellular cytotoxicity to cells.

[0213] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region can be varied (e.g., increased or decreased) as described, e.g., in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain can be altered, e.g., to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody, or to facilitate linker conjugation.

[0214] In some embodiments, one or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of a muscle-targeting antibody described herein (e.g., in the CH2 domain (residues 231-340 of human IgG1), and / or (e.g., and) in the CH3 domain (residues 341-447 of human IgG1), and / or (e.g., and) in the hinge region, numbered according to the Kabat numbering system (e.g., EU index of Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor (e.g., an activated Fc receptor) on the surface of an effector cell. Mutations in the Fc region of an antibody that increase or decrease the affinity of the antibody for an Fc receptor, and techniques for introducing such mutations into an Fc receptor or fragment thereof, are known to those of skill in the art. Examples of mutations in the Fc receptor of an antibody that can be made to alter the affinity of the antibody to the Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Patent No. 6,737,056, and International Publications Nos. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0215] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., increase or decrease) the half-life of the antibody in vivo. For example, see International Publications WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745 for mutations that may alter (e.g., increase or decrease) the half-life of the antibody in vivo.

[0216] In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to decrease the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant region or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or (e.g., and) in the third constant (CH3) domain (residues 341-447 of human IgG1) numbered according to the EU index of Kabat (Kabat EA et al. (1991) supra). In some embodiments, the IgG1 constant region of the antibody described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG, referred to as a "YTE mutant", has been shown to exhibit a 4-fold increased half-life compared to the wild-type version of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant region comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389, and 428-436, numbered according to the EU index as in Kabat.

[0217] In some embodiments, one or more amino acid substitutions are introduced into the IgG constant region Fc region to alter the effector function(s) of the anti-transferrin receptor antibody. The effector ligand with altered affinity to itself can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation of the constant region domain (through point mutation or other means) can reduce binding of circulating antibodies to Fc receptors, thereby increasing tumor localization. See, for example, U.S. Pat. Nos. 5,585,097 and 8,591,886 for a description of mutations that delete or inactivate constant regions, thereby increasing tumor localization. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites in the Fc region, which may reduce binding to Fc receptors (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0218] In some embodiments, one or more amino acid residues in the constant region of the muscle-targeting antibody described herein may be replaced with a different amino acid residue, such that the antibody may have altered C1q binding and / or (by way of example and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Patent No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibody described herein are altered, thereby altering the antibody's ability to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibody described herein is modified to increase the antibody's ability to mediate antibody-dependent cellular cytotoxicity (ADCC) to cells and / or (by way of example and) to increase the affinity of the antibody to Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0219] In some embodiments, the heavy and / or (by way of example) light chain variable domain(s) sequence(s) of the antibodies provided herein may be used to generate, for example, CDR-grafted, chimeric, humanized, or composite human antibodies, or antigen-binding fragments, as described elsewhere herein. As will be appreciated by one of skill in the art, any variant, CDR-grafted, chimeric, humanized, or composite antibody derived from any of the antibodies provided herein may be useful in the compositions and methods described herein, and will retain specific binding ability to the transferrin receptor, such that the variant, CDR-grafted, chimeric, humanized, or composite antibody may have at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to the transferrin receptor compared to the original antibody from which it was derived.

[0220] In some embodiments, the antibodies provided herein contain mutations that confer desired properties to the antibody. For example, to avoid potential complications due to Fab-arm exchange that is known to occur in native IgG4 mAbs, the antibodies provided herein may contain a stabilizing "Adair" mutation (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody", Mol Immunol 30, 105-108; 1993), in which serine 228 (EU numbering; residue 241 Kabat numbering) is converted to a proline resulting in an IgG1-like hinge sequence. Consequently, any of the antibodies may include a stabilizing "Adair" mutation.

[0221] As provided herein, the antibody of the present disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be attached at its C-terminus to a light chain constant region like Cκ or Cλ. Similarly, the VH domain or a portion thereof may be attached to all or a portion of a heavy chain like IgA, IgD, IgE, IgG, and IgM, and any subclass of isotype. The antibody may include a suitable constant region (see, for example, Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Thus, the antibody within the scope of the present disclosure may include VH and VL domains, or antigen-binding portions thereof, combined with any suitable constant region.

[0222] ii. Muscle-targeting peptides Some aspects of the disclosure provide muscle-targeting peptides as muscle-targeting agents. Short peptide sequences (e.g., peptide sequences between 5 and 20 amino acids in length) that bind to specific cell types have been described. For example, cell-targeting peptides can be found in Vines e.,et al.,A. “Cell-penetrating and cell-targeting peptides in drug delivery” Biochim Biophys Acta 2008,1786:126-38; Jarver P.,et al., “In vivo biodistribution and efficacy of peptide mediated delivery” Trends Pharmacol Sci 2010;31:528-35; Samoylova TI,et al., “Elucidation of muscle-binding peptides by phage display screening” Muscle Nerve 1999;22:460-6; ​​U.S. Patent No. 6,329,501, issued December 11, 2001, entitled “METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE”; and Samoylov AM,et al., “Recognition of cell-specific binding of phage display derived peptides using an acoustic wave sensor." Biomol Eng 2002;18:269-72; the entire contents of each of which are incorporated herein by reference. By designing peptides to interact with specific cell surface antigens (e.g., receptors), selectivity for a desired tissue, e.g., muscle, can be obtained. Skeletal muscle targeting has been explored, and a wide range of molecular payloads can be delivered. These approaches, without many of the practical disadvantages of large antibodies or viral particles, may have high selectivity for muscle tissue. Consequently, in some embodiments, the muscle targeting agent is a muscle-targeting peptide of 4 to 50 amino acids in length.In some embodiments, the muscle-targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Muscle-targeting peptides can be generated using any of several methods, such as phage display.

[0223] In some embodiments, the muscle targeting peptide may bind to an internalizing cell surface receptor (e.g., transferrin receptor) that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells. In some embodiments, the muscle targeting peptide may target (e.g., bind to) the transferrin receptor. In some embodiments, the transferrin receptor targeting peptide may include a segment of a naturally occurring ligand, e.g., transferrin. In some embodiments, the transferrin receptor targeting peptide is as described in U.S. Patent No. 6,743,893, 11 / 30 / 2000 application, "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR." In some embodiments, the transferrin receptor targeting peptide is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells." BMC Cancer. 2011 Aug 18;11:359. In some embodiments, the transferrin receptor targeting peptide is as described in U.S. Patent No. 8,399,653, filed 5 / 20 / 2011, entitled "TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY."

[0224] As mentioned above, examples of muscle-targeting peptides have been reported. For example, muscle-specific peptides have been identified using phage display libraries that display surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 205) binds to C2C12 mouse myotubes in vitro and to mouse muscle tissue in vivo. As a result, in some embodiments, the muscle-targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 205). This peptide exhibits improved specificity for binding to myocardial and skeletal muscle tissues after intravenous injection into mice, with reduced binding to the liver, kidney, and brain. Additional muscle-specific peptides have been identified using phage display. For example, in the context of treatment for DMD, a 12-amino acid peptide has been identified by a phage display library for muscle targeting. See Yoshida D., et al., "Targeting of salicylate to skin and muscle following topical injections in rats." Int J Pharm 2002;231:177-84, the entire contents of which are hereby incorporated by reference, wherein a 12 amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO:206) was identified, and this muscle-targeting peptide showed improved binding to C2C12 cells compared to the ASSLNIA (SEQ ID NO:205) peptide.

[0225] Any additional method for identifying peptides selective for muscle (e.g., skeletal muscle) over other cell types includes in vitro selection, as described in Ghosh D., et al., "Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting" J Virol 2005;79:13667-72; the entire contents of which are incorporated herein by reference. Non-specific cell binders were selected by pre-incubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After undergoing rounds of selection, the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 207) emerged most frequently. Consequently, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 140).

[0226] The muscle targeting agent may be an amino acid-containing molecule or peptide. The muscle targeting peptide may correspond to a sequence of a protein that preferentially binds to a protein receptor found on muscle cells. In some embodiments, the muscle targeting peptide is highly hydrophobic in nature (e.g., valine) so that the peptide may preferentially target muscle cells. In some embodiments, the muscle targeting peptide has not been previously characterized or disclosed. These peptides may be conceived, produced, synthesized, and / or (for example, and) derivatized using any of several methodologies, for example, phage-displayed peptide libraries, one-bead one-compound peptide libraries, or positional scanning synthetic peptide combinatorial libraries. Exemplary methodologies are characterized in the art and are incorporated by reference (Gray, BP and Brown, KC "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides" Chem Rev. 2014, 114:2, 1020-1081.; Samoylova, TI and Smith, BF "Elucidation of muscle-binding peptides by phage display screening." Muscle Nerve, 1999, 22:4.460-6).In some embodiments, muscle-targeting peptides have been previously disclosed (see, e.g., Writer MJ et al. "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display." J. Drug Targeting. 2004;12:185; Cai, D. "BDNF-mediated enhancement of inflammation and injury in the aging heart." Physiol Genomics. 2006,24:3,191-7.; Zhang, L. "Molecular profiling of heart endothelial cells." Circulation, 2005,112:11,1601-11.; McGuire, MJ et al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004,342:1,171-82). Exemplary muscle-targeting peptides include the following groups of amino acid sequences: CQAQGQLVC (SEQ ID NO: 208), CSERSMNFC (SEQ ID NO: 209), CPKTRRVPC (SEQ ID NO: 210), WLSEAGPVVTVRALRGTGSW (SEQ ID NO: 211), ASSLNIA (SEQ ID NO: 205), CMQHSMRVC (SEQ ID NO: 212), and DDTRHWG (SEQ ID NO: 213). In some embodiments, muscle-targeting peptides may include about 2-25 amino acids, about 2-20 amino acids, about 2-15 amino acids, about 2-10 amino acids, or about 2-5 amino acids. Muscle-targeting peptides may include naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring amino acids, or modified amino acids.Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the muscle-targeting peptides can be linear; in other embodiments, the muscle-targeting peptides can be cyclic (e.g., bicyclic) (see, e.g., Silvana, MGet al. Mol. Therapy, 2018, 26:1, 132-147).

[0227] iii. Muscle-targeted receptor ligands The muscle targeting agent may be a ligand, e.g., a ligand that binds to a receptor protein. The muscle targeting ligand may be a protein, e.g., transferrin, that binds to an internalized cell surface receptor expressed by muscle cells. Accordingly, in some embodiments, the muscle targeting agent is transferrin or a derivative thereof that binds to the transferrin receptor. The muscle targeting ligand may alternatively be a small molecule, e.g., a lipophilic small molecule that preferentially targets muscle cells over other cell types. Exemplary lipophilic small molecules that may target muscle cells include compounds that include cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linoleic acid, linoleic acid, myristic acid, sterol, dihydrotestosterone, testosterone derivatives, glycerin, alkyl chains, trityl groups, and alkoxy acids.

[0228] iv. Muscle-targeting aptamers The muscle targeting agent may be an aptamer, e.g., an RNA aptamer, that preferentially targets muscle cells over other cell types. In some embodiments, the muscle targeting aptamer has not been previously characterized or disclosed. These aptamers may be conceived, produced, synthesized, and / or (e.g., and) derivatized using any of several methodologies, e.g., Systematic Evolution of Ligands by Exponential Enrichment. Exemplary methodologies have been characterized in the art and are incorporated by reference (Yan, AC and Levy, M. "Aptamers and aptamer targeted delivery" RNA biology, 2009, 6:3, 316-20.; Germer, K. et al. "RNA aptamers and their therapeutic and diagnostic applications." Int. J. Biochem. Mol. Biol. 2013; 4:27-40). In some embodiments, muscle-targeting aptamers have been previously disclosed (see, e.g., Phillippou, S. et al. "Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers." Mol Ther Nucleic Acids. 2018, 10:199-214.; Thiel, W H et al. "Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation." Mol Ther. 2016, 24:4, 779-87). Exemplary muscle-targeting aptamers include the A01B RNA aptamer and RNA Apt 14. In some embodiments, the aptamer is a nucleic acid-based aptamer, an oligonucleotide aptamer, or a peptide aptamer. In some embodiments, the aptamer may be about 5-15 kDa, about 5-10 kDa, about 10-15 kDa, about 1-5 Da, about 1-3 kDa, or smaller.

[0229] v. Other muscle-targeting agents One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use a substrate of a muscle transporter protein, such as a transporter protein expressed on the sarcolemma. In some embodiments, the muscle targeting agent is a substrate of an influx transporter specific to muscle tissue. In some embodiments, the influx transporter is specific to skeletal muscle tissue. The two major classes of transporters expressed on the sarcolemma of skeletal muscle are (1) the adenosine triphosphate (ATP)-binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue, and (2) the solute carrier (SLC) superfamily, which can facilitate the influx of substrates into skeletal muscle. In some embodiments, the muscle targeting agent is a substrate that binds to the ABC or SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a naturally occurring substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-naturally occurring substrate, such as a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0230] In some embodiments, the muscle targeting agent is any muscle targeting agent described herein that targets the SLC superfamily of transporters (e.g., antibodies, nucleic acids, small molecules, peptides, aptamers, lipids, sugar moieties). In some embodiments, the muscle targeting agent is a substrate of the SLC superfamily of transporters. SLC transporters are either equilibria or use the proton or sodium ion gradient created across the membrane to drive transport of the substrate. Exemplary SLC transporters with high expression in skeletal muscle include, without limitation, the SATT transporter (ASCT1; SLC1A4), the GLUT4 transporter (SLC2A4), the GLUT7 transporter (GLUT7; SLC2A7), the ATRC2 transporter (CAT-2; SLC7A2), the LAT3 transporter (KIAA0245; SLC7A6), the PHT1 transporter (PTR4; SLC15A4), the OATP-J transporter (OATP5A1; SLC21A15), the OCT3 transporter (EMT; SLC22A3), the OCTN2 transporter (FLJ46769; SLC22A5), the ENT transporters (ENT1; SLC29A1 and ENT2; SLC29A2), the PAT2 transporter (SLC36A2), and the SAT2 transporter (KIAA1382; SLC38A2). These transporters may facilitate the entry of substrates into skeletal muscle, providing opportunities for muscle targeting.

[0231] In some embodiments, the muscle targeting agent is a substrate for equilibrative nucleoside transporter 2 (ENT2) transporter. Compared to other transporters, ENT2 has one of the highest mRNA expression in skeletal muscle. Human ENT2 (hENT2) is expressed in most body organs, such as brain, heart, placenta, thymus, pancreas, prostate, and kidney, but is particularly abundant in skeletal muscle. Human ENT2 facilitates the uptake of its substrates according to their concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. hENT2 transporter has low affinity for all nucleosides except inosine (adenosine, guanosine, uridine, thymidine, and cytidine). Consequently, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3'-dideoxyinosine, and clofarabine. In some embodiments, any of the muscle targeting agents provided herein are associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular payload.

[0232] In some embodiments, the muscle targeting agent is a substrate for the organic cation / carnitine transporter (OCTN2), a sodium ion-dependent high affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, mildronate, acetylcarnitine, or any derivative thereof that binds to OCTN2. In some embodiments, carnitine, mildronate, acetylcarnitine, or a derivative thereof is covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0233] A muscle targeting agent may be a protein, which is a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, hemojuvelin may be full-length or a fragment or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant may be a soluble fragment, may lack the N-terminal signaling and / or (by way of example and) may lack the C-terminal anchoring domain. In some embodiments, the hemojuvelin may be annotated as GenBank RefSeq Accession No. NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It is to be understood that the hemojuvelin may be of human, non-human primate, or rodent origin.

[0234] B. Molecular Payload Some aspects of the disclosure provide oligonucleotides designed to target a molecular payload, e.g., DMPK RNA, to modulate DMPK expression or activity. In some embodiments, modulating DMPK expression or activity includes reducing the level of DMPK RNA and / or (e.g., and) protein. In some embodiments, the DMPK RNA is disease-associated, e.g., has a disease-associated repeat expansion or is encoded from an allele that has a disease-associated repeat expansion. In some embodiments, the DMPK RNA contains a CUG repeat expansion or the allele from which it is encoded contains a CTG repeat expansion. In some embodiments, the disclosure provides oligonucleotides complementary to DMPK RNA that are useful for reducing the level of toxic DMPK with a disease-associated repeat expansion, e.g., in subjects having or suspected of having myotonic dystrophy. In some embodiments, the oligonucleotides are designed to target RNAe H-mediated degradation of the target DMPK RNA. In some embodiments, the oligonucleotides are designed to target RNAe H-mediated degradation of the target DMPK RNA present in the nucleus of a cell (e.g., a muscle cell, e.g., a myotube). In some embodiments, the oligonucleotides are designed to target RNAe H-mediated degradation of a target DMPK RNA present in the nucleus of a cell (e.g., a CNS cell (e.g., a neuron)). In some embodiments, the oligonucleotides are designed to have desired bioavailability and / or serum-stability properties. In some embodiments, the oligonucleotides are designed to have desired binding affinity properties. In some embodiments, the oligonucleotides are designed to have a desired toxicity profile. In some embodiments, the oligonucleotides are designed to have low complement activation and / or cytokine induction properties.

[0235] In some embodiments, the oligonucleotide is linked or otherwise attached to a muscle targeting agent described herein. In some embodiments, such oligonucleotides can target DMPK in muscle cells, for example, via specific binding to a DMPK sequence in muscle cells followed by delivery to the muscle cells by the attached muscle targeting agent. It should be understood that various types of muscle targeting agents may be used in accordance with the present disclosure. In some embodiments, the oligonucleotide comprises a region of complementarity to a DMPK allele that contains a disease-associated repeat expansion. Exemplary oligonucleotides that target DMPK RNA are described in further detail herein, however, it should be understood that the exemplary molecular payloads provided herein are not intended to be limiting.

[0236] i. Oligonucleotides In some embodiments, the DMPK-targeting oligonucleotides described herein are designed to trigger RNase H-mediated degradation of DMPK mRNA. It should be understood that in some embodiments, oligonucleotides of one format (e.g., antisense oligonucleotides) may be suitably adapted to another format (e.g., siRNA oligonucleotides) by incorporating functional sequences from one format (e.g., antisense strand sequences) into the other format.

[0237] Examples of oligonucleotides useful for targeting DMPK are described in U.S. Patent Application Publication No. 20100016215A1, published on January 1, 2010, entitled Compound And Method For Treating Myotonic Dystrophy; U.S. Patent Application Publication No. 20130237585A1, published on July 19, 2010, entitled Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; U.S. Patent Application Publication No. 20150064181A1, published on March 5, 2015, entitled "Antisense Conjugates For Decreasing Expression Of Dmpk; U.S. Patent Application Publication No. 20150238627A1, published on August 27, 2015, entitled "Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy; and U.S. Patent Application Publication No. 20150238627A1, published on August 27, 2015, entitled "Compounds And Methods For Treating Myotonic Dystrophy." and U.S. Patent Application Publication No. 20160304877A1, published on October 20, 2016, entitled "Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression," the entire contents of each of which are incorporated herein by reference.

[0238] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence depicted below, which is an example of the human DMPK gene sequence (Gene ID 1760; NM_001081560.2):

[0239] In some embodiments, the oligonucleotide may have a region of complementarity to the sequence depicted below, which is an example of the mouse DMPK gene sequence (Gene ID 13400; NM_001190490.1):

[0240] In some embodiments, the oligonucleotide may have a region of complementarity to a mutant form of DMPK, such as those reported in Botta A. et al. "The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients" J Med Genet. 2008 Oct; 45(10): 639-46.; and Machuca-Tzili L. et al. "Clinical and molecular aspects of the myotonic dystrophies: a review" Muscle Nerve. 2005 Jul; 32(1): 1-18. (the entire contents of each of which are incorporated herein).

[0241] In some embodiments, the oligonucleotide provided herein is an antisense oligonucleotide targeting DMPK. In some embodiments, the oligonucleotide targeting is any one of the antisense oligonucleotides (e.g., Gapmer) targeting DMPK described in US Patent Application Publication US20160304877A1, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression", published on October 20, 2016, which is incorporated herein by reference. In some embodiments, the DMPK targeting oligonucleotide targets the region of the DMPK gene sequence represented by Genbank Accession No. NM_001081560.2 (SEQ ID NO: 130) or represented by Genbank Accession No. NG_009784.1.

[0242] In some embodiments, the DMPK targeting oligonucleotide comprises a nucleotide sequence that includes a region complementary to a target region that is at least 10 consecutive nucleotides in SEQ ID NO:130 (e.g., at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, or more consecutive nucleotides).

[0243] In some embodiments, the DMPK-targeting oligonucleotide comprises a gapmer motif. "Gapmer" refers to a chimeric antisense compound in which an internal region having a number of nucleotides that support RNase H cleavage is located between external regions having one or more nucleotides, where the nucleotides that comprise the internal region are chemically distinct from the nucleotides or nucleotides that comprise the external region. The internal region may be referred to as a "gap segment" and the external region may be referred to as a "wing segment". In some embodiments, the DMPK-targeting oligonucleotide comprises one or more modified nucleotides and / or (for example and) one or more modified internucleoside linkages. In some embodiments, the internucleoside linkages are phosphorothioate linkages. In some embodiments, the oligonucleotide comprises a complete phosphorothioate backbone. In some embodiments, the oligonucleotide is a DNA gapmer with a cET end (for example, 3-10-3; cET-DNA-cET). In some embodiments, the DMPK targeted oligonucleotide comprises one or more 6'-(S)-CH3 bicyclic nucleotides, one or more β-D-2'-deoxyribonucleotides, and / or (by way of example and) one or more 5 methyl-cytosine nucleotides.

[0244] Oligonucleotide size / sequence Oligonucleotides may be of a variety of different lengths, depending, for example, on the format. In some embodiments, the oligonucleotides are 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 nucleotides in length or longer. In some embodiments, the oligonucleotides are 8-50 nucleotides in length, 8-40 nucleotides in length, 8-30 nucleotides in length, 10-15 nucleotides in length, 10-20 nucleotides in length, 15-25 nucleotides in length, 21-23 nucleotides in length, etc. In some embodiments, the oligonucleotides are 15-20 nucleotides in length or 20-25 nucleotides in length.

[0245] In some embodiments, for purposes of the present disclosure, a complementary nucleic acid sequence of an oligonucleotide is specifically hybridizable to or specific for a target nucleic acid when the binding of said sequence to a target molecule (e.g., an mRNA) interferes with the normal function of the target (e.g., an mRNA) causing loss of activity (e.g., inhibition of translation) or loss of expression (e.g., degradation of the target mRNA) and when there is a sufficient degree of complementarity to avoid non-specific binding of said sequence to non-target sequences under conditions where avoidance of non-specific binding is desired, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatments and in the case of in vitro assays, where the assay is performed under suitable conditions of stringency. Thus, in some embodiments, the oligonucleotide may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the consecutive nucleotides of the target nucleic acid. In some embodiments, the complementary nucleotide sequence does not need to be 100% complementary to the sequence of the target that is specifically hybridizable to or specific to the target nucleic acid. In some embodiments, the oligonucleotide comprises one or more mismatched nucleobases compared to the target nucleic acid. In some embodiments, the activity of the target is reduced by such mismatch, while the activity of the non-target is reduced by a greater amount (i.e., the selectivity for the target nucleic acid is increased and the off-target effect is reduced). In some embodiments, the target nucleic acid is a pre-mRNA molecule or an mRNA molecule.

[0246] In some embodiments, the oligonucleotide comprises a region of complementarity to a target nucleic acid that is in the range of 8-15, 8-30, 8-40, or 10-50, or 5-50, or 5-40 nucleotides in length. In some embodiments, the oligonucleotide comprises a region of complementarity to a nucleic acid that is in the range of 15-20 or 20-25 nucleotides in length. In some embodiments, the region of complementarity of the oligonucleotide to a target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the region of complementarity is complementary to at least 8 consecutive nucleotides of the target nucleic acid.In some embodiments, the oligonucleotide may contain 1, 2 or 3 base mismatches compared to the consecutive nucleotides of a portion of the target nucleic acid.In some embodiments, the oligonucleotide may have up to 3 mismatches over 15 bases or up to 2 mismatches over 10 bases.

[0247] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides of a sequence comprising any one of SEQ ID NOs: 173-192 and 196-201. In some embodiments, the oligonucleotide comprises a sequence comprising any one of SEQ ID NOs: 173-192 and 196-201. In some embodiments, the oligonucleotide comprises a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity with at least 12 or at least 15 consecutive nucleotides of any one of SEQ ID NOs: 173-192 and 196-201.

[0248] In some embodiments, the oligonucleotide comprises a region of complementarity to a nucleotide sequence represented by any one of SEQ ID NOs: 160-172 and 193-195. In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides (e.g., consecutive nucleotides) that are complementary to a nucleotide sequence represented by any one of SEQ ID NOs: 160-172 and 193-195. In some embodiments, the oligonucleotide comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%; 99%, or 100% complementary to at least 12 or at least 15 consecutive nucleotides of any one of SEQ ID NOs: 160-172 and 193-195.

[0249] In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to the target sequence of any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 8). In some embodiments, such target sequence is 100% complementary to the oligonucleotides listed in Table 8 or Table 17.

[0250] It should be understood that in some embodiments, methylation at the C5 position of the nucleobase uracil 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.

[0251] In some embodiments, one or more of the thymine bases (T) of any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 8) can independently and optionally be a uracil base (U), and / or any one or more of the U can independently and optionally be a T.

[0252] b. Oligonucleotide Modification: The oligonucleotides described herein may be modified, including, for example, modified sugar moieties, modified internucleoside linkages, modified nucleotides, and / or combinations thereof.In addition, in some embodiments, the oligonucleotides may exhibit one or more of the following properties: do not mediate alternative splicing; are not immunostimulatory; are nuclease resistant; have improved cellular uptake compared to unmodified oligonucleotides; are not toxic to cells or mammals; have improved endosomal exit into cells; minimize TLR stimulation; or avoid pattern recognition receptors.Any of the modified chemistries or formats of the oligonucleotides described herein may be combined with each other.For example, 1, 2, 3, 4, 5, or more different types of modifications may be included in the same oligonucleotide.

[0253] In some embodiments, certain nucleotide modifications may be used that make the oligonucleotides that incorporate the modifications more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides remain intact longer than unmodified oligonucleotides.Specific examples of modified oligonucleotides include those that include modified backbones, such as phosphorothioates, phosphotriesters, methylphosphonates, short alkyl or cycloalkyl intersugar linkages, or modified internucleoside linkages, such as short heteroatom or heterocyclic intersugar linkages.As a result, the oligonucleotides of the present disclosure can be stabilized against nucleic acid degradation by modifications, such as the incorporation of nucleotide modifications.

[0254] In some embodiments, the oligonucleotide may be an oligonucleotide up to 50 nucleotides or up to 100 nucleotides in length, with 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30, 2-40, 2-45, or more nucleotides of the oligonucleotide being modified nucleotides. The oligonucleotide may be an oligonucleotide 8-30 nucleotides in length, with 2-10, 2-15, 2-16, 2-17, 2-18, 2-19, 2-20, 2-25, 2-30 nucleotides of the oligonucleotide being modified nucleotides. The oligonucleotide may be an oligonucleotide 8-15 nucleotides in length, with 2-4, 2-5, 2-6, 2-7, 2-8, 2-9, 2-10, 2-11, 2-12, 2-13, 2-14 nucleotides of the oligonucleotide being modified nucleotides. Optionally, the oligonucleotide may be modified at every nucleotide except 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Oligonucleotide modifications are further described herein.

[0255] C modified nucleosides In some embodiments, the oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of the sugar. In some embodiments, the oligonucleotide comprises at least one 2' modified nucleoside. In some embodiments, all of the nucleosides on the oligonucleotide are 2' modified nucleosides.

[0256] Modified nucleotides include, for example, 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-O-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-O-N-methylacetamido (2'-O-NMA) modified nucleosides.

[0257] In some embodiments, the oligonucleotides described herein include one or more 2'-4' bicyclic nucleosides, in which the ribose ring in the nucleoside includes a bridging moiety that connects two atoms in the ring (e.g., connecting the 2'-O atom to the 4'-C atom via a methylene (LNA) bridge, an ethylene (ENA) bridge, or an (S)-constrained ethyl (cEt) bridge). Examples of LNAs are described in International Patent Application Publication WO / 2008 / 043753, entitled "RNA Antagonist Compounds For The Modulation Of PCSK9," published on April 17, 2008, the contents of which are incorporated herein by reference in their entirety. Other modifications that may be used in the oligonucleotides disclosed herein include ethylene-bridged nucleic acids (ENAs). ENAs include, but are not limited to, 2'-O,4'-C-ethylene-bridged nucleic acids. Examples of ENAs are provided in International Patent Publication No. WO 2005 / 042777, published May 12, 2005, entitled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006; and Horie et al., Nucleic Acids Symp. Ser(Oxf), 49:171-172, 2005, the disclosures of which are incorporated herein by reference in their entireties. Examples of cEt are provided in US Patents 7,101,993; 7,399,845 and 7,569,686, each of which is incorporated herein by reference in its entirety.

[0258] In some embodiments, the oligonucleotide comprises a modified nucleoside disclosed in one of the following U.S. patents or patent application publications: U.S. Patent 7,399,845, issued July 15, 2008, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,741,457, issued June 22, 2010, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 8,022,193, issued September 20, 2011, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,569,686, issued August 4, 2009, entitled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. Patent 7,335,765, issued February 26, 2008, entitled "Novel Nucleoside And Oligonucleotide Analogs"; No. 7,314,923, issued Jan. 1, 2008, entitled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. Patent No. 7,816,333, issued Oct. 19, 2010, entitled "Oligonucleotide Analogues And Methods Utilizing The Same" and U.S. Publication No. 2011 / 0009471, now U.S. Patent No. 8,957,201, issued Feb. 17, 2015, entitled "Oligonucleotide Analogues And Methods Utilizing The Same", the entire contents of each of which are incorporated herein by reference for all purposes.

[0259] In some embodiments, an oligonucleotide comprises at least one modified nucleoside that results in an increase in the Tm of the oligonucleotide in the range of 1° C., 2° C., 3° ​​C., 4° C., or 5° C., compared to an oligonucleotide that does not have at least one modified nucleoside. An oligonucleotide may have multiple modified nucleosides that, in total, result in an increase in the Tm of the oligonucleotide in the range of 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., or more, compared to an oligonucleotide that does not have the modified nucleoside.

[0260] An oligonucleotide may contain a mix of different kinds of nucleosides. For example, an oligonucleotide may contain a mix of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. An oligonucleotide may contain a mix of deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. An oligonucleotide may contain a mix of 2'-fluoro modified nucleosides and 2'-O-methyl modified nucleosides. An oligonucleotide may contain a mix of bridged nucleosides and 2'-fluoro or 2'-O-methyl modified nucleosides. An oligonucleotide may contain a mix of non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). The oligonucleotide may comprise a mix of 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. The oligonucleotide may comprise a mix of 2'-4' bicyclic nucleosides and 2'MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. The oligonucleotide may comprise a mix of non-bicyclic 2' modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0261] Oligonucleotides may contain different types of alternating nucleosides. For example, oligonucleotides may contain alternating 2'-deoxyribonucleosides or ribonucleosides and 2'-fluoro modified nucleosides. Oligonucleotides may contain alternating deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. Oligonucleotides may contain alternating 2'-fluoro modified nucleosides and 2'-O-Me modified nucleosides. Oligonucleotides may contain alternating bridged nucleosides and 2'-fluoro or 2'-O-methyl modified nucleosides. Oligonucleotides may contain alternating non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). The oligonucleotides may contain alternating 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluoro, or 2'-O-Me modified nucleosides. The oligonucleotides may contain alternating non-bicyclic 2' modified nucleosides (e.g., 2'-MOE, 2'-fluoro, or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

[0262] In some embodiments, the oligonucleotides described herein contain 5'-vinylphosphonate modifications, one or more abasic residues, and / or one or more inverted abasic residues.

[0263] d. Internucleoside linkage / backbone In some embodiments, the oligonucleotide may contain phosphorothioate or other modified internucleoside linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between at least two nucleosides. In some embodiments, the oligonucleotide comprises phosphorothioate internucleoside linkages between all nucleosides. For example, in some embodiments, the oligonucleotide comprises modified internucleoside linkages at the first, second, and / or (for example, and) third internucleoside linkages at the 5'-end or 3'-end of the nucleotide sequence.

[0264] Phosphorus-containing linkages which may be used include, but are not limited to, normal 3'-5' linkages, 2'-5' linked analogs of these, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3' alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates, as well as those of reverse polarity (where adjacent pairs of nucleoside units are 3'-5' to 5'-3' or 6'-7' to 8'-9'). 2'-5' to 5'-2') and the like; U.S. Patents Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,39 See Nos. 9,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0265] In some embodiments, oligonucleotides may have heteroatom backbones such as methylene (methylimino) or MMI backbones; amide backbones (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28:366-374); morpholino backbones (see Summerton and Weller, U.S. Pat. No. 5,034,506); or peptide nucleic acid (PNA) backbones (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone and the nucleotides are attached directly or indirectly to aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

[0266] e. Stereospecific oligonucleotides In some embodiments, the phosphorus atom between oligonucleotides of oligonucleotides is chiral, and the properties of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atom. In some embodiments, suitable methods may be used to synthesize P-chiral oligonucleotide analogs in a stereocontrolled manner (e.g., as described in Oka N, Wada T, Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms.Chem Soc Rev.2011 Dec;40(12):5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided that include nucleoside units linked together by either substantially all Sp phosphorothioate intersugar linkages or substantially all Rp phosphorothioate intersugar linkages. In some embodiments, such phosphorothioate oligonucleotides with substantially chiral pure intersugar linkages are prepared by enzymatic synthesis or chemical synthesis, for example, as described in U.S. Patent 5,587,261, issued December 12, 1996, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the chiral controlled oligonucleotide provides a selective cleavage pattern of the target nucleic acid.For example, in some embodiments, the chiral controlled oligonucleotide provides a single cleavage site within the complementary sequence of the nucleic acid, as described, for example, in U.S. Patent Application Publication No. 20170037399 A1, entitled "CHIRAL DESIGN," published on February 2, 2017 (the contents of which are incorporated herein by reference in their entirety).

[0267] h.Gapmer In some embodiments, the oligonucleotides described herein are gapmers. Gapmer oligonucleotides generally have the formula 5'-XYZ-3' with X and Z as flanking regions surrounding gap region Y. In some embodiments, flanking region X of formula 5'-XYZ-3' is also referred to as X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, flanking region Z of formula 5'-XYZ-3' is also referred to as Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, gap region Y of formula 5'-XYZ-3' is also referred to as Y region, Y segment, or gap segment Y. In some embodiments, each nucleoside of gap region Y is a 2'-deoxyribonucleoside, and neither 5' wing region X nor 3' wing region Z contains any 2'-deoxyribonucleosides. In some embodiments, the gapmer oligonucleotide comprises a region of complementarity to at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleosides) of a target sequence provided in Table 8 (e.g., any one of SEQ ID NOs: 160-172) and / or at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleosides) of a nucleotide sequence of an antisense sequence, gapmer sequence, or ASO structure provided in Table 8 (e.g., any one of SEQ ID NOs: 173-192), wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by a T.

[0268] In some embodiments, the Y region is a stretch of nucleotides, e.g., a region of six or more DNA nucleotides, that can recruit an RNAse, such as RNAse H. In some embodiments, the gapmer binds to the target nucleic acid, at which point an RNAse can be recruited and then cleave the target nucleic acid. In some embodiments, the Y region is flanked on both 5' and 3' by regions X and Z that contain high affinity modified nucleosides, e.g., 1 to 6 high affinity modified nucleosides. Examples of high affinity modified nucleosides include, but are not limited to, 2' modified nucleosides (e.g., 2'-MOE, 2'O-Me, 2'-F) or 2'-4' bicyclic nucleosides (e.g., LNA, cEt, ENA). In some embodiments, the flanking sequences X and Z can be 1-20 nucleotides, 1-8 nucleotides, or 1-5 nucleotides in length. The flanking sequences X and Z can be of similar or dissimilar lengths. In some embodiments, the gap segment Y can be a nucleotide sequence between 5 and 20 nucleotides, between 5 and 15 nucleotides, between 5 and 12 nucleotides, or between 6 and 10 nucleotides in length.

[0269] In some embodiments, the gap region of the gapmer oligonucleotide may contain, in addition to DNA nucleotides, modified nucleotides known to be permissive for efficient RNase H action, such as C4'-substituted nucleotides, acyclic nucleotides, and arabino-type nucleotides. In some embodiments, the gap region contains one or more unmodified internucleoside linkages. In some embodiments, one or both flanking regions each independently contain one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least 2, at least 3, at least 4, at least 5, or more nucleotides. In some embodiments, the gap region and the two flanking regions each independently contain modified internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least 2, at least 3, at least 4, at least 5, or more nucleotides.

[0270] Gapmers may be produced using any suitable method. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gapmers include U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,6 No. 23,065; No. 5,652,355; No. 5,652,356; No. 5,700,922; No. 5,898,031; No. 7,015,315; No. 7,1 No. 01,993; No. 7,399,845; No. 7.432,250; No. 7,569,686; No. 7,683,036; No. 7,750,131; No. 8,5 No. 80,756; No. 9,045,754; No. 9,428.534; No. 9,695,418; No. 10,017,764; No. 10,260,069; No. 9.428,534; No. 8,580,756; U.S. Patent Publication Nos. US20050074801, US20090221685; US200902869 69, US20100197762, and US20110112170; PCT Publication Nos. W02004069991; W02005023825; W02008049085 and W02009090182; and European Patent No. EP 2,149,605, each of which is incorporated herein by reference in its entirety.

[0271] In some embodiments, gapmers are 10-40 nucleosides in length. For example, gapmers can be 10-40, 10-35, 10-30, 10-25, 10-20, 10-15, 15-40, 15-35, 15-30, 15-25, 15-20, 20-40, 20-35, 20-30, 20-25, 25-40, 25-35, 25-30, 30-40, 30-35, or 35-40 nucleosides in length. In some embodiments, the gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleosides in length.

[0272] In some embodiments, the gap region Y on the gapmer is 5-20 nucleosides in length. For example, the gap region Y can be 5-20, 5-15, 5-10, 10-20, 10-15, or 15-20 nucleosides in length. In some embodiments, the gap region Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, each nucleoside in the gap region Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides in the gap region Y are 2'-deoxyribonucleosides. In some embodiments, one or more of the nucleosides in the gap region Y are modified nucleosides (e.g., 2'-modified nucleosides, such as those described herein). In some embodiments, one or more cytosines in gap region Y are optionally 5-methylcytosines. In some embodiments, each cytosine in gap region Y is a 5-methylcytosine.

[0273] In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are independently 1 to 20 nucleosides in length. For example, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') can independently be 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, 2 to 5, 2 to 7, 3 to 5, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleosides in length. In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides in length. In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are the same length. In some embodiments, the 5' wing region of the gapmer (X of the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z of the formula 5'-XYZ-3') are different lengths. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is longer than the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is shorter than the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula).

[0274] In some embodiments, the gapmers are 5-10-5, 4-12-4, 3-14-3, 2-16-2, 1-18-1, 3-10-3, 2-10-2, 1-10-1, 2-8-2, 4-6-4, 3-6-3, 2-6-2, 4-7-4, 3-7-3, 2-7-2, 4-8-4, 3-8-3, 2-8-2, 1-8-1, 2-9-2, 1-9-1, 2-10-2, 1-10-1, 1-12-1, 1-16-1, 2-15-1, 1-15-2, 1-14-3, 3-14-1, 2-14-2, 1-13-4, 4-13-1, 2-13-3, 3-13-2, 1-12- 5, 5-12-1, 2-12-4, 4-12-2, 3-12-3, 1-11-6, 6-11-1, 2-11-5, 5-11-2, 3-11-4, 4-11-3, 1-17-1, 2-16-1, 1-16-2, 1-15-3, 3-15-1, 2-15-2, 1-14-4, 4-14 -1, 2-14-3, 3-14-2, 1-13-5, 5-13-1, 2-13-4, 4-13-2, 3-13-3, 1-12-6, 6-12-1, 2-12-5, 5-12-2, 3-12-4, 4-12-3, 1-11-7, 7-11-1, 2-11-6, 6-11-2, 3-1 1-5, 5-11-3, 4-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 1-16-3, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 5-14-1, 2-14-4, 4-14-2, 3-14-3, 1- 13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4 -11-5, 5-11-4, 1-18-1, 1-17-2, 2-17-1, 1-16-3, 3-16-1, 2-16-2, 1-15-4, 4-15-1, 2-15-3, 3-15-2, 1-14-5, 2-14-4, 4-14-2, 3-14-3, 1-13-6, 6-13-1, 2-13-5, 5-13-2, 3-13-4, 4-13-3, 1-12-7, 7-12-1, 2-12-6, 6-12-2, 3-12-5, 5-12-3, 1-11-8, 8-11-1, 2-11-7, 7-11-2, 3-11-6, 6-11-3, 4-11-5, 5-11-4,1-19-1、1-18-2、2-18-1、1-17-3、3-17-1、2-17-2、1-16-4、4-16-1、2-16-3、3-16-2、1-15-5、2-15-4、4-15-2、3-15-3、1-14-6、6-14-1、2-14-5、5-14-2、3-14-4、4-14-3、1-13-7、7-13-1、2-13-6、6-13-2、3-13-5、5-13-3、4-13-4、1-12-8、8-12-1、2-12-7、7-12-2、3-12-6、6-12-3、4-12-5、5-12-4、2-11-8、8-11-2、3-11-7、7-11-3、4-11-6、6-11-4、5-11-5、1-20-1、1-19-2、2-19-1、1-18-3、3-18-1、2-18-2、1-17-4、4-17-1、2-17-3、3-17-2、1-16-5、2-16-4、4-16-2、3-16-3、1-15-6、6-15-1、2-15-5、5-15-2、3-15-4、4-15-3、1-14-7、7-14-1、2-14-6、6-14-2、3-14-5、5-14-3、4-14-4、1-13-8、8-13-1、2-13-7、7-13-2、3-13-6、6-13-3、4-13-5、5-13-4、2-12-8、8-12-2、3-12-7、7-12-3、4-12-6、6-12-4、5-12-5、3-11-8、8-11-3、4-11-7、7-11-4、5-11-6、6-11-5、1-21-1、1-20-2、2-20-1、1-20-3、3-19-1、2-19-2、1-18-4、4-18-1、2-18-3、3-18-2、1-17-5、2-17-4、4-17-2、3-17-3、1-16-6、6-16-1、2-16-5、5-16-2、3-16-4、4-16-3、1-15-7、7-15-1、2-15-6、6-15-2、3-15-5、5-15-3、4-15-4、1-14-8、8-14-1、2-14-7、7-14-2、3-14-6、6-14-3、4-14-5、5-14-4、2-13-8、8-13-2、3-13-7、7-13-3、4-13-6、6-13-4、5-13-5、1-12-10、10-12-1、2-12-9、9-12-2、3-12-8、8-12-3、4-12-7、7-12-4、5-12-6、6-12-5、4-11-8, 8-11-4, 5-11-7, 7-11-5, 6-11-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1 -15-8, 8-15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6 5'-XYZ-3'. The numbers indicate the number of nucleosides in the X, Y, and Z regions of the 5'-XYZ-3' gapmer.

[0275] In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) or the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are modified nucleosides (e.g., high affinity modified nucleosides). In some embodiments, the modified nucleoside (e.g., high affinity modified nucleoside) is a 2'-modified nucleoside. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside or a non-bicyclic 2' modified nucleoside. In some embodiments, the high affinity modified nucleoside is a 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2'-modified nucleoside (e.g., 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-ODMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamide (2'-O-NMA)).

[0276] In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) are high affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is a high affinity modified nucleoside. In some embodiments, one or more nucleosides in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are high affinity modified nucleosides. In some embodiments, each nucleoside in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a high affinity modified nucleoside. In some embodiments, one or more nucleosides in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) are high affinity modified nucleosides and one or more nucleosides in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) are high affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) is a high affinity modified nucleoside and each nucleoside in the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) is a high affinity modified nucleoside.

[0277] In some embodiments, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) comprises the same high affinity nucleosides as the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula). For example, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) can comprise one or more non-bicyclic 2' modified nucleosides (e.g., 2'-MOE or 2'-O-Me). In another example, the 5' wing region of the gapmer (X of the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z of the 5'-XYZ-3' formula) can comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z in the formula 5'-XY-Z-3') is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me). In some embodiments, each nucleoside in the 5' wing region of the gapmer (X in the formula 5'-XYZ-3') and the 3' wing region of the gapmer (Z in the formula 5'-XYZ-3') is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt).

[0278] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1 to 7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, each nucleoside in X and Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), and each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, each nucleoside in X and Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside in Y is a 2'-deoxyribonucleoside. In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) comprises a different high affinity nucleoside as the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) may contain one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) may contain one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0279] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where each nucleoside in X is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), each nucleoside in Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside in Y is a 2'-deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 1-7 (e.g., 1, 2, 3, 4, 5, 6, or 7) nucleosides in length and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, where each nucleoside in X is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), each nucleoside in Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), and each nucleoside in Y is a 2'-deoxyribonucleoside.

[0280] In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) comprises one or more non-bicyclic 2' modified nucleosides (e.g., 2'-MOE or 2'-OMe) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) comprises one or more non-bicyclic 2' modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) contain one or more non-bicyclic 2' modified nucleosides (e.g., 2'-MOE or 2'-OMe) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0281] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 of X (the most 5' position is position 1) is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside of Y is a 2' deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 of Z (the most 5' position is position 1) is a non-bicyclic 2' modified nucleoside (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside of Y is a 2' deoxyribonucleoside.In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, where X and Z are independently 2-7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6-10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, and at least one, but not all (e.g., 1, 2, 3, 4, 5, 6, or 7) of positions 1, 2, 3, 4, 5, 6, or 7 of X and positions 1, 2, 3, 4, 5, or 6 of Z are 5' to 7'. At least one of positions 1, 2, 3, 4, 5, 6, or 7, but not all of positions (e.g., positions 1, 2, 3, 4, 5, or 6) 1, 2, 3, 4, 5, 6, or 7 of X are non-bicyclic 2' modified nucleosides (e.g., 2'-MOE or 2'-O-Me), the remainder of the nucleosides of both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and each nucleoside of Y is a 2' deoxyribonucleoside.

[0282] Non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and a mix of 2'-4' bicyclic nucleosides (e.g., LNA or cEt) in the 5'-wing region of a gapmer (X in the 5'-X-Y-Z-3' formula) and / or in the 3'-wing region of a gapmer (Z in the 5'-X-Y-Z-3' formula) Non-limiting examples of gapmer arrangements having are: BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAA; KKK-(D)n-KKKAA; LLL-(D)n-LLLAA; BBB-(D)n-BBBEE; KKK-(D)n-KKKEE; LLL-(D)n-LLLEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BBB-(D)n-BBBAAA; KKK-(D)n-KKKAAA; LLL-(D)n-LLLAAA; BBB-(D)n-BBBEEE; KKK-(D)n-KKKEEE; LLL-(D)n-LLLEEE; BABA-(D)n-ABAB; KAKA-(D)n-AKAK; LALA-(D)n-ALAL; BEBE-(D)n-EBEB; KEKE-(D)n-EKEK; LELE-(D)n-ELEL; BABA-(D)n-ABAB; KAKA-(D)n-AKAK; LALA-(D)n-ALAL; BEBE-(D)n-EBEB; KEKE-(D)n-EKEK; LELE-(D)n-ELEL; ABAB-(D)n-ABAB; AKAK-(D)n-AKAK; ALAL-(D)n-ALAL; EBEB-(D)n-EBEB; EKEK-(D)n-EKEK; ELEL-(D)n-ELEL; ABAB-(D)n-ABAB; AKAK-(D)n-AKAK; ALAL-(D)n-ALAL; EBEB-(D)n-EBEB; EKEK-(D)n-EKEK; ELEL-(D)n-ELEL; AABB-(D)n-BBAA; BBAA-(D)n-AABB; AAKK-(D)n-KKAA; AALL-(D)n-LLAA; EEBB-(D)n-BBEE; EEKK-(D)n-KKEE;EELL-(D)n-LLEE;AABB-(D)n-BBAA;AAKK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;BBB-(D)n-BBA;KKK-(BBA; D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE; E;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;ABBB-(D)n-BBBA;ACC-KK-KKA; ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK-(D)n-KKKE;ELLL-(D)n-LLLE;ABBB-(D)n-BBBA;AKKK-(D)n-KKKA;ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK -(D)n-CCKE;ELLL-(D)n-LLLE;ABBB-(D)n-BBBAA;ACC-(D)n-CCKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;ECC-(D)n-CCKEE;ELLL-(D)n-LLLEE;ACC-(D)n-LLLAA; BBB-(D)n-BBBAA;ACC-(D)n-CCKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;CCK-(D)n-CCKEE;ELLL-(D)n-LLLEE;AABBB-(D)n-BBB;AACCC-(D)n-CCKK ;AALLL-(D)n-LLL;EEBBB-(D)n-BBB;EEKKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBB;AACKK-(D)n-KKK;AALLL-(D)n-LLL; KKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBBA;AACKK-(D)n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKK-(D)n-KKKE;EELLL-(D)n-LLLE;AABBB-(D)n-BBBA;AAKKK-(D)n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE;ABBAABB-(D)n-BB;AKKAA ALLAALL-( D)n-LL;EBBEEBB-(D)n-BB;EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALLL-(D)n-LLL;EBBEBB-(D)n-B BB;EKKEKK-(D)n-KKK;ELLELL-(D)n-LLL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALL-(D)n-LLL;EBBEBB-(D)n-BBB;EKKEKK-(D)n-KKK;ELL ELL-(D)n-LLL;EEEK-(D)n-EEEEEEEE;EEK-(D)n-EEEEEEEEEE;EK-(D)n-EEEEEEEEEE;EK-(D)n-EEEKK;K-(D)n-EEEKEKE;K-(D)n-EEEKEKEE;K- (D)n-EEKEK;EK-(D)n-EEEEKEKE;EK-(D)n-EEEKEK;EEK-(D)n-KEEKE;EK-(D)n-EEKEK;EK-(D)n-KEEK;EEK-(D)n-EEEEKEK;EK-(D)n-KEEEKEE;E K-(D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK; "A" represents a 2' modified nucleoside; "B" represents a 2'-4' bicyclic nucleoside; "K" represents a constrained ethyl nucleoside (cEt); "L" represents an LNA nucleoside; "E" represents a 2'-MOE modified ribonucleoside; "D" represents a 2' deoxyribonucleoside; "n" represents the length of the gap segment (Y in the 5'-XYZ-3' configuration) and is an integer between 1 and 20;

[0283] In some embodiments, any one of the gapmers described herein comprises one or more modified nucleoside linkages (e.g., phosphorothioate linkages) in each of the X, Y, and Z regions. In some embodiments, each internucleoside linkage in any one of the gapmers described herein is a phosphorothioate linkage. In some embodiments, each of the X, Y, and Z regions independently comprises a mix of phosphorothioate and phosphodiester linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate linkage, 5' wing region X comprises a mix of phosphorothioate and phosphodiester linkages, and 3' wing region Z comprises a mix of phosphorothioate and phosphodiester linkages.

[0284] Non-limiting examples of DMPK targeting oligonucleotides are provided in Table 8. Table 8. Examples of DMPK-targeting oligonucleotides (ASOs) [Table 12-1] [Table 12-2] [Table 12-3] [Table 12-4] [Table 12-5] [Table 12-6]

[0285] In some embodiments, the DMPK-targeting oligonucleotides described herein are 15-20 nucleosides in length (e.g., 15, 16, 17, 18, 19, or 20 nucleosides), contain a region of complementarity to at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 consecutive nucleosides) of any one of SEQ ID NOs: 160-172 and 193-195, and contain a 5'-XYZ-3' configuration, where X contains 3-5 (e.g., 3, 4, or 5) linked nucleosides, where at least one of the nucleosides in X is a 5'-XYZ-3' arrangement. another is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA); Y comprises 6 to 10 (e.g., 6, 7, 8, 9, or 10) linked 2'-deoxyribonucleosides, where each cytosine in Y is optionally and independently a 5-methyl-cytosine; and Z comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, where at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA).

[0286] In some embodiments, the antisense oligonucleotide comprises at least 15 contiguous nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or 20 contiguous nucleosides) of any one of the nucleotide sequences of SEQ ID NOs: 174, 177, 179-182, and 184-192, and comprises a 5'-XYZ-3' configuration, where X comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, and where at least one of the nucleosides in X is a 2'-modified nucleoside (e.g., 2'-M Y is a 2'-OE modified nucleoside, a 2'-O-Me modified nucleoside, an LNA, cEt, or an ENA); Y comprises 6 to 10 (e.g., 6, 7, 8, 9, or 10) linked 2'-deoxyribonucleosides, where each cytosine in Y is optionally and independently a 5-methyl-cytosine; and Z comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, where at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, an LNA, cEt, or an ENA). In some embodiments, each thymine base (T) in the nucleotide sequence of the antisense oligonucleotide may be independently and optionally replaced with a uracil base (U), and each U may be independently and optionally replaced with a T.

[0287] In some embodiments, the antisense oligonucleotide comprises a nucleotide sequence of any one of SEQ ID NOs: 174, 177, 179-182, and 184-192, and comprises a 5'-XYZ-3' configuration, where X comprises 3-5 (e.g., 3, 4, or 5) linked nucleosides, and where at least one of the nucleosides in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). Y comprises 6 to 10 (e.g., 6, 7, 8, 9, or 10) linked 2'-deoxyribonucleosides, where each cytosine in Y is optionally and independently a 5-methyl-cytosine; and Z comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, where at least one of the nucleosides in Z is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside, a 2'-O-Me modified nucleoside, LNA, cEt, or ENA). In some embodiments, each thymine base (T) in the nucleotide sequence of the antisense oligonucleotide may be independently and optionally replaced with a uracil base (U), and each U may be independently and optionally replaced with a T.

[0288] In some embodiments, each nucleoside in X is a 2'-modified nucleoside and / or (for example and) each nucleoside in Z is a 2'-modified nucleoside. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside (for example, LNA, cEt, or ENA) or a non-bicyclic 2'-modified nucleoside (for example, a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside).

[0289] In some embodiments, each nucleoside in X is a non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside) and / or (e.g., and) each nucleoside in Z is a non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside). In some embodiments, each nucleoside in X is a 2'-4' bicyclic nucleoside (e.g., an LNA, cEt, or ENA) and / or (e.g., and) each nucleoside in Z is a 2'-4' bicyclic nucleoside (e.g., an LNA, cEt, or ENA).

[0290] In some embodiments, X comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE or 2'-O-Me modified nucleoside), and / or (e.g., and) Z comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE or 2'-O-Me modified nucleoside).

[0291] In some embodiments, the DMPK targeted oligonucleotide comprises one or more phosphorothioate internucleoside linkages. In some embodiments, each internucleoside linkage in the DMPK targeted oligonucleotide is a phosphorothioate internucleoside linkage. In some embodiments, the DMPK targeted oligonucleotide comprises one or more phosphodiester internucleoside linkages, optionally wherein the phosphodiester internucleoside linkages are in X and / or Z. In some embodiments, the DMPK targeted oligonucleotide comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages. In some embodiments, the DMPK targeted oligonucleotide comprises 1 phosphodiester internucleoside linkage (PO), 2 PO, 3 PO, 4 PO, 5 PO, 6 PO, 7 PO, 8 PO, 9 PO, 10 PO, 11 PO, 12 PO, 13 PO, 14 PO, 15 PO, 16 PO, 17 PO, 18 PO, 19 PO, 20 PO, 21 PO, 22 PO, 23 PO, 24 PO, 25 PO, 26 PO, 27 PO, 28 PO, or 29 PO, and the remaining internucleoside linkages are phosphorothioate internucleoside linkages (PS). For example, a 20 nucleotide DMPK-targeting oligonucleotide may contain 1 PO and 18 PS, 2 PO and 17 PS, 3 PO and 16 PS, 4 PO and 15 PS, 5 PO and 14 PS, 6 PO and 13 PS, 7 PO and 12 PS, 8 PO and 11 PS, 9 PO and 10 PS, 10 PO and 9 PS, 11 PO and 8 PS, 12 PO and 7 PS, 13 PO and 6 PS, 14 PO and 5 PS, 15 PO and 4 PS, 16 PO and 3 PS, 17 PO and 2 PS, or 18 PO and 1 PS.In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate internucleoside linkage, X comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages, and Z comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate internucleoside linkage, each internucleoside linkage in X is a phosphorothioate internucleoside linkage, and Z comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages. In some embodiments, each internucleoside linkage in gap region Y is a phosphorothioate internucleoside linkage, X comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages, and each internucleoside linkage in Z is a phosphorothioate internucleoside linkage. For example, a DMPK-targeted oligonucleotide may contain wing regions X and Z with mixed phosphodiester / phosphorothioate backbones and gap region Y with an all phosphorothioate backbone, or it may contain one wing region (i.e., X or Z) with a mixed phosphodiester / phosphorothioate backbone, the other wing region with an all phosphorothioate backbone, and gap region Y with an all phosphorothioate backbone. In some embodiments, gap region Y comprises one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages, and wing regions X and Y each independently either have an all phosphorothioate backbone or comprise one or more phosphorothioate internucleoside linkages and one or more phosphodiester internucleoside linkages. For example, a DMPK-targeted oligonucleotide may comprise wing regions X and Z with a mixed phosphodiester / phosphorothioate backbone and gap region Y with a mixed phosphodiester / phosphorothioate backbone.

[0292] In some embodiments, there is provided an antisense oligonucleotide having the following formula: (L) X1 (E) X2 (L) X3 (D) X4 (L) X5 (E) X6 (L) X7 : wherein each (L) is a 2'-4' bicyclic nucleoside; wherein each (E) is a non-bicyclic 2'-modified nucleoside; wherein each (D) is a 2'-deoxyribonucleoside; where X1 is independently an integer from 0 to 5 representing the corresponding number of L; where X2 is independently an integer from 0 to 5 representing the corresponding number of E; where X3 is independently an integer from 0 to 5 representing the corresponding number of L; where X4 is independently an integer from 5 to 12 representing the number of D; where X5 is independently an integer from 0 to 5 representing the corresponding number of L; where X6 is independently an integer from 0 to 5 representing the corresponding number of E; where X7 is independently an integer from 0 to 5 representing the corresponding number of L; and wherein at least one of X1, X2, and X3 is in the range of 1 to 5, and at least one of X5, X6, and X7 is in the range of 1 to 5.

[0293] In some embodiments, X1, X3, X5, and X7 are each 0, and X2 and X6 are independently 1, 2, 3, 4, or 5.

[0294] In some embodiments, X1, X2, X5, and X6 are each 0, and X3 and X7 are independently 1, 2, 3, 4, or 5.

[0295] In some embodiments, X3 and X5 are each 0, and X1, X2, X6 and X7 are independently 1, 2, 3, 4, or 5.

[0296] In some embodiments, X1 and X7 are each 0, and X2, X3, X5 and X6 are independently 1, 2, 3, 4, or 5.

[0297] In some embodiments, X4 is 5, 6, 7, 8, 9, or 10.

[0298] In some embodiments, the 2'-4' bicyclic nucleoside is selected from LNA, cEt, and ENA nucleosides. In some embodiments, the non-bicyclic 2'-modified nucleoside is a 2'-MOE modified nucleoside or a 2'-OMe modified nucleoside.

[0299] In some embodiments, the nucleosides of the oligonucleotide are linked together by phosphorothioate internucleoside linkages, phosphodiester internucleoside linkages, or a combination thereof. In some embodiments, the oligonucleotide contains only phosphorothioate internucleoside linkages linking each nucleoside. In some embodiments, the oligonucleotide contains at least one phosphorothioate internucleoside linkage. In some embodiments, the oligonucleotide contains a mix of phosphorothioate and phosphodiester internucleoside linkages. In some embodiments, the oligonucleotide contains only phosphorothioate internucleoside linkages linking each pair of 2'-deoxyribonucleosides and a mix of phosphorothioate and phosphodiester internucleoside linkages linking the remaining nucleosides.

[0300] In some embodiments, the oligonucleotide has the following 5'-XYZ-3' configuration: [Table 13] where "E" is a 2'-MOE modified ribonucleoside; "L" is LNA; "D" is a 2'-deoxyribonucleoside; and "10" or "8" is the number of 2'-deoxyribonucleosides in Y, and where the oligonucleotide comprises phosphorothioate internucleoside linkages, phosphodiester internucleoside linkages, or combinations thereof.

[0301] In some embodiments, in any one of the DMPK-targeting oligonucleotides described herein, each cytidine in X and / or Z (e.g., a 2'-modified cytidine) is optionally and independently a 5-methyl-cytidine, and / or each uridine in X and / or Z (e.g., a 2'-modified uridine) is optionally and independently a 5-methyl-uridine.

[0302] In some embodiments, the DMPK targeting oligonucleotide is selected from ASO 1-29 listed in Table 8. In some embodiments, any one of the DMPK targeting oligonucleotides can be in the form of a salt, e.g., as a sodium, potassium, magnesium salt.

[0303] In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8) is conjugated to an amine group, optionally via a spacer. In some embodiments, the spacer comprises an aliphatic moiety. In some embodiments, the spacer comprises a polyethylene glycol moiety. In some embodiments, a phosphodiester linkage is present between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8) is conjugated to a spacer, which is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -, -NR A C(=O)-, -NR A C(=O)R A -, -C(=O)R A -, -NR A C(=O)O-, -NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-, -S(O)NR A -, -NR A S(O)2-, or a combination thereof; each R A are independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, the spacer is a substituted or unsubstituted alkylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted heteroarylene, -O-, -N(R A )-, or -C(=O)N(R A )2, or a combination of these.

[0304] In some embodiments, the 5' or 3' nucleoside of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 8) has the formula -NH2-(CH2) n -, where n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11, or 12. In some embodiments, the phosphodiester linkage is conjugated to a compound of the formula NH2-(CH2) n - is present between the compound represented by the formula NH2-(CH2)6- and the 5' or 3' nucleoside. In some embodiments, the compound represented by the formula NH2-(CH2)6- is conjugated to the oligonucleotide via a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide.

[0305] In some embodiments, the oligonucleotide is conjugated, e.g., via an amine group, to a targeting agent, e.g., a muscle targeting agent such as an anti-TfR1 antibody.

[0306] C. Linker The conjugates described herein generally include a linker that connects any one of the anti-TfR1 antibodies described herein to a molecular payload. The linker includes at least one covalent bond. In some embodiments, the linker may be a single bond, such as a disulfide bond or a disulfide bridge, that connects the anti-TfR1 antibody to the molecular payload. However, in some embodiments, the linker may connect any one of the anti-TfR1 antibodies described herein to the molecular payload through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker. The linker is generally stable in vitro and in vivo, and may be stable in a certain cellular environment. In addition, the linker generally does not negatively affect the functional properties of either the anti-TfR1 antibody or the molecular payload. Examples and methods of linker synthesis are known in the art (see, for example, Kline, T. et al. "Methods to Make Homogenous Antibody Drug Conjugates." Pharmaceutical Research, 2015, 32:11, 3480-3493.; Jain, N. et al. "Current ADC Linker Chemistry" Pharm Res. 2015, 32:11, 3526-3540.; McCombs, JR and Owen, SC "Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry" AAPS J. 2015, 17:2, 339-351.).

[0307] The precursor of the linker will typically contain two different reactive species that can be attached to both the anti-TfR1 antibody and the molecular payload. In some embodiments, the two different reactive species may be nucleophiles and / or (for example and) electrophiles. In some embodiments, the linker is connected to the anti-TfR1 antibody via conjugation to a lysine or cysteine ​​residue of the anti-TfR1 antibody. In some embodiments, the linker is connected to the cysteine ​​residue of the anti-TfR1 antibody via a maleimide-containing linker, where optionally, the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some embodiments, the linker is connected to the cysteine ​​residue of the anti-TfR1 antibody or a thiol-functionalized molecular payload via a 3-arylpropionitrile functional group. In some embodiments, the linker is connected to the lysine residue of the anti-TfR1 antibody. In some embodiments, the linker is connected to the anti-TfR1 antibody and / or the molecular payload (by way of example and not limitation) via an amide bond, a carbamate bond, a hydrazide, a triazole, a thioether, or a disulfide bond.

[0308] i. Cleavable Linker The cleavable linker may be a protease-sensitive linker, a pH-sensitive linker, or a glutathione-sensitive linker. These linkers are generally only cleavable intracellularly and are preferably stable in the extracellular environment, e.g., outside of muscle cells or CNS cells.

[0309] Protease-sensitive linkers are cleavable by protease enzyme activity. These linkers typically comprise peptide sequences and may be 2-10 amino acids, about 2-5 amino acids, about 5-10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length. In some embodiments, the peptide sequence may comprise naturally occurring amino acids, such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the protease-sensitive linker comprises a valine-citrulline or alanine-citrulline sequence. In some embodiments, the protease-sensitive linker may be cleaved by lysosomal proteases, such as cathepsin B, and / or endosomal proteases.

[0310] A pH-sensitive linker is a covalent linkage that is easily degraded in a high or low pH environment. In some embodiments, the pH-sensitive linker may be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH-sensitive linker is cleaved in an endosome or lysosome.

[0311] In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by disulfide exchange reaction with glutathione species inside the cell. In some embodiments, the disulfide moiety further comprises at least one amino acid, for example a cysteine ​​residue.

[0312] In some embodiments, the linker is a Val-cit linker (e.g., as described in US Pat. No. 6,214,345, incorporated herein by reference). In some embodiments, the val-cit linker prior to conjugation has the following structure: [ka] Yes.

[0313] In some embodiments, the val-cit linker after conjugation has the following structure: [ka] Yes.

[0314] In some embodiments, the Val-cit linker is attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation). In some embodiments, the val-cit linker attached to a highly reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) prior to click chemistry conjugation has the following structure: [ka] where n is any number from 0 to 10. In some embodiments, n is 3.

[0315] In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) is conjugated (e.g., via a different chemical moiety) to a molecular payload (e.g., an oligonucleotide). In some embodiments, the val-cit linker attached to a reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) and conjugated to a molecular payload (e.g., an oligonucleotide) has the following structure (prior to click chemistry conjugation): [ka] where n is any number from 0 to 10. In some embodiments, n is 3.

[0316] In some embodiments, after conjugation to a molecular payload (e.g., an oligonucleotide), the val-cit linker has the following structure: [ka] where n is any number from 0 to 10, and where m is any number from 0 to 10. In some embodiments, n is 3 and m is 4.

[0317] ii. Non-cleavable linker In some embodiments, a non-cleavable linker may be used. In general, a non-cleavable linker cannot be easily degraded in a cellular or physiological environment. In some embodiments, a non-cleavable linker comprises an optionally substituted alkyl group, where the substitution may include halogen, hydroxyl group, oxygen species, and other common substitutions. In some embodiments, a linker may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one unnatural amino acid, a truncated glycan, a sugar or sugars that cannot be enzymatically degraded, an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, a biotin, a biphenyl, a repeating unit of polyethylene glycol or an equivalent compound, an acid ester, an acid amide, a sulfamide, and / or (for example, and) an alkoxy-amine linker. In some embodiments, sortase-mediated ligation will be utilized to link an anti-TfR1 antibody containing an LPXT sequence to a molecular payload containing a (G)n sequence (see, e.g., Proft T. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1):1-10).

[0318] In some embodiments, the linker may comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; an imino, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), such as polyethylene oxide or polypropylene oxide.

[0319] iii. Linker conjugation In some embodiments, the linker is connected to the anti-TfR1 antibody and / or (for example and) the molecular payload via a phosphate, thioether, ether, carbon-carbon, carbamate, or amide bond. In some embodiments, the linker is connected to the oligonucleotide through a phosphate or phosphorothioate group, for example, a phosphate at the end of the oligonucleotide backbone. In some embodiments, the linker is connected to the anti-TfR1 antibody through a lysine or cysteine ​​residue present on the anti-TfR1 antibody.

[0320] In some embodiments, the linker is connected to the anti-TfR antibody and / or (for example and) molecular payload by a cycloaddition reaction between an azide and an alkyne forming a triazole, where the azide and alkyne may be located on the anti-TfR antibody, the molecular payload, or the linker. In some embodiments, the alkyne may be a cyclic alkyne, for example, a cyclooctyne. In some embodiments, the alkyne may be a bicyclononyne (also known as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctane is as described in International Patent Application Publication WO2011136645, entitled "Fused Cyclooctyne Compounds And Their Use In Metal-free Click Reactions," published on November 3, 2011. In some embodiments, the azide may be a sugar or carbohydrate molecule containing an azide. In some embodiments, the azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the azide-containing sugar or carbohydrate molecule is as described in International Patent Application Publication WO2016170186, published on October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase."In some embodiments, the cycloaddition reaction between an azide and an alkyne to form a triazole (wherein the azide or alkyne may be located on the anti-TfR1 antibody, the molecular payload, or the linker) is as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled "Modified antibody, antibody-conjugate and process for the preparation thereof"; or International Patent Application Publication WO2016170186, published on October 27, 2016, entitled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From A β(1,4)-N-Acetylgalactosaminyltransferase."

[0321] In some embodiments, the linker further comprises a spacer, e.g., a polyethylene glycol spacer or an acyl / carbamoyl sulfamide spacer, e.g., a HydraSpace™ spacer. In some embodiments, the spacer is as described in Verkade, JMM et al., “A Polar Sulfamide Spacer Significantly Enhances the Manufacturing Capacity, Stability, and Therapeutic Index of Antibody-Drug Conjugates”, Antibodies, 2018, 7, 12.

[0322] In some embodiments, the linker is connected to the anti-TfR1 antibody and / or (for example and) the molecular payload by a Diels-Alder reaction between a dienophile and a diene / hetero-diene, where the dienophile and the diene / hetero-diene may be located on the anti-TfR1 antibody, the molecular payload, or the linker. In some embodiments, the linker is connected to the anti-TfR1 antibody and / or (for example and) the molecular payload by other pericyclic reactions, for example, ene reactions. In some embodiments, the linker is connected to the anti-TfR1 antibody and / or (for example and) the molecular payload by an amide, thioamide, or sulfonamide coupling reaction. In some embodiments, the linker is connected to the anti-TfR1 antibody and / or (for example and) the molecular payload by a condensation reaction forming an oxime group, a hydrazone group, or a semicarbazide group present between the linker and the anti-TfR1 antibody and / or (for example and) the molecular payload.

[0323] In some embodiments, the linker is connected to the anti-TfR1 antibody and / or (for example and) the molecular payload by a conjugate addition reaction between a nucleophile (for example, an amine group or a hydroxyl group) and an electrophile (for example, a carboxylic acid, carbonate, or aldehyde). In some embodiments, the nucleophile may be present on the linker and the electrophile may be present on the anti-TfR1 antibody or the molecular payload prior to the reaction between the linker and the anti-TfR1 antibody or the molecular payload. In some embodiments, the electrophile may be present on the linker and the nucleophile may be present on the anti-TfR1 antibody or the molecular payload prior to the reaction between the linker and the anti-TfR1 antibody or the molecular payload. In some embodiments, the electrophile may be an azide, pentafluorophenyl, silicon center, carbonyl, carboxylic acid, anhydride, isocyanate, thioisocyanate, succinimidyl ester, sulfosuccinimidyl ester, maleimide, alkyl halide, alkyl pseudohalide, epoxide, episulfide, aziridine, aryl, activated phosphorus center, and / or (by way of example and) activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxyl group, an amino group, an alkylamino group, an anilide group, or a thiol group.

[0324] In some embodiments, the val-cit linker attached to the highly reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) has the following structure: [ka] where m is any number from 0 to 10. In some embodiments, m is 4.

[0325] In some embodiments, the val-cit linker attached to the highly reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) has the following structure: [ka] (G) where m is any number from 0 to 10. In some embodiments, m is 4. It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (G) results from reaction of the anti-TfR1 antibody with an amine, such as the lysine epsilon amine.

[0326] In some embodiments, the val-cit linker attached to a highly reactive chemical moiety (e.g., SPAAC for click chemistry conjugation) and conjugated to an anti-TfR1 antibody has the following structure: [ka] (F) where n is any number from 0 to 10, and where m is any number from 0 to 10. In some embodiments, n is 3, and / or (for example and) m is 4. In some embodiments, the oligonucleotide is covalently linked to a compound comprising a structure represented by formula (F), thereby forming a conjugate comprising a structure represented by formula (D). It should be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (F) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0327] In some embodiments, the val-cit linker linking the anti-TfR1 antibody and the molecular payload has the following structure: [ka] where n is any number from 0 to 10, and m is any number from 0 to 10. In some embodiments, n is 3, and / or (for example, and) m is 4. In some embodiments, n is 3, and / or (for example, and) m is 4. In some embodiments, X is NH (for example, NH from the amine group of lysine), S (for example, S from the thiol group of cysteine), O (O from the hydroxyl group of serine, threonine, or tyrosine).

[0328] In some embodiments, the conjugates described herein have the following structure: [ka] where n is any number from 0 to 10, and where m is any number from 0 to 10. In some embodiments, n is 3 and m is 4.

[0329] In Structural Formulas (A), (B), (C), and (D), L, in some embodiments, is a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(RA)-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NRA-, -NRAC(=O)-, -NRAC(=O)RA-, -C(=O)RA-, -NRAC(=O)O-, -NRAC(=O)N(RA)-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(RA)-, -S(O)2NRA-, -NRAS(O)2-, or a combination thereof, wherein each R A is independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, L is [ka] where L2 is [ka] where a labels a site that is directly linked to the carbamate moiety of formulas (A), (B), (C), and (D); and b labels a site that is covalently linked (directly or via an additional chemical moiety) to the oligonucleotide.

[0330] In some embodiments, L1 is one of the following: [ka] where a labels a site that is directly linked to the carbamate moiety of formulas (A), (B), (C), and (D); and b labels a site that is covalently linked (directly or via an additional chemical moiety) to the oligonucleotide.

[0331] In some embodiments, L1 is [ka] It is.

[0332] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide. In some embodiments, the linkage of L1 to the 5' phosphate of the oligonucleotide forms a phosphodiester bond between L1 and the oligonucleotide.

[0333] In some embodiments, L1 is optional (eg, does not have to be present).

[0334] In some embodiments, any one of the conjugates described herein has the following structure: [ka] wherein n is 0 to 15 (for example, 3) and m is 0 to 15 (for example, 4).

[0335] C. Examples of Antibody-Molecular Payload Conjugates Further provided herein are non-limiting examples of conjugates comprising any one of the anti-TfR1 antibodies described herein covalently linked to any one of the molecular payloads described herein (e.g., oligonucleotides). In some embodiments, the anti-TfR1 antibody (e.g., any one of the anti-TfR1 antibodies provided in Tables 2-7) is covalently linked to the molecular payload (e.g., an oligonucleotide such as the oligonucleotide provided in Table 8) via a linker. Any of the linkers described herein may be used. In some embodiments, when the molecular payload is an oligonucleotide, the linker is linked to the 5' end, 3' end, or internally of the oligonucleotide. In some embodiments, the linker is linked to the anti-TfR1 antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the anti-TfR1 antibody). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an anti-TfR1 antibody described herein) via an amine group (e.g., via a lysine in the antibody). In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide targeting a gene listed in Table 8).

[0336] An example structure of a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload via a Val-cit linker is provided below: [ka] Here, the linker is linked to the antibody via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody). In some embodiments, the molecular payload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8).

[0337] Another example of the structure of a conjugate comprising an anti-TfR1 antibody covalently linked to a molecular payload via a Val-cit linker is provided below: [ka] wherein n is a number between 0 and 10, wherein m is a number between 0 and 10, wherein the linker is linked to the antibody via an amine group (e.g., on a lysine residue) and / or (e.g., and) the linker is linked to the oligonucleotide (e.g., at the 5' end, the 3' end, or internally). In some embodiments, the linker is linked to the antibody via lysine, the linker is linked to the oligonucleotide at the 5' end, n is 3, and m is 4. In some embodiments, the molecular payload is an oligonucleotide comprising a sense strand and an antisense strand, and the linker is linked to the sense strand or the antisense strand at the 5' end or the 3' end. In some embodiments, the molecular payload is a DMPK targeting oligonucleotide (e.g., a DMPK targeting oligonucleotide listed in Table 8). In some embodiments, L1 is [ka] It is to be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (D) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0338] It should be understood that antibodies can be linked to molecular payloads with various stoichiometries, a property sometimes referred to as drug-antibody ratio (DAR), where the "drug" is the molecular payload. In some embodiments, one molecular payload is linked to the antibody (DAR=1). In some embodiments, two molecular payloads are linked to the antibody (DAR=2). In some embodiments, three molecular payloads are linked to the antibody (DAR=3). In some embodiments, four molecular payloads are linked to the antibody (DAR=4). In some embodiments, a mixture of different conjugates, each having a different DAR, is provided. In some embodiments, the average DAR of the conjugates in such a mixture may range from 1-3, 1-4, 1-5 or more. The DAR may be increased by conjugating the molecular payloads to various sites on the antibody and / or (by way of example and) by conjugating multimers to one or more sites on the antibody. For example, a DAR of 2 may be achieved by conjugating a single molecular payload to two different sites on an antibody, or by conjugating a dimeric molecular payload to a single site on an antibody.

[0339] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody described herein (e.g., an antibody provided in Tables 2-7) covalently linked to a molecular payload. In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody described herein (e.g., an antibody provided in Tables 2-7) covalently linked to a molecular payload via a linker (e.g., a Val-cit linker). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an anti-TfR1 antibody described herein) via a thiol-reactive linkage (e.g., via a cysteine ​​in the antibody). In some embodiments, the linker (e.g., a Val-cit linker) is linked to the antibody (e.g., an anti-TfR1 antibody described herein) via an amine group (e.g., via a lysine in the antibody).

[0340] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of any one of the antibodies listed in Table 2. In some embodiments, the molecular payload is a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8).

[0341] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 69, SEQ ID NO: 71, or SEQ ID NO: 72, and a VL comprising the amino acid sequence of SEQ ID NO: 70. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0342] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 74. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0343] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 76, and a VL comprising the amino acid sequence of SEQ ID NO: 75. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0344] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77, and a VL comprising the amino acid sequence of SEQ ID NO: 78. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0345] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 77 or SEQ ID NO: 79, and a VL comprising the amino acid sequence of SEQ ID NO: 80. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0346] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 154, and a VL comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0347] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 84, SEQ ID NO: 86, or SEQ ID NO: 87, and a VL comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0348] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91, and a VL comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0349] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 88 or SEQ ID NO: 91, and a VL comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0350] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 92 or SEQ ID NO: 94, and a VL comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0351] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 92, and a VL comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0352] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 156, and a VL comprising the amino acid sequence of SEQ ID NO: 157. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0353] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 97, SEQ ID NO: 98, or SEQ ID NO: 99, and a VL comprising the amino acid sequence of SEQ ID NO: 85. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0354] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101, and a VL comprising the amino acid sequence of SEQ ID NO: 89. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0355] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 100 or SEQ ID NO: 101, and a VL comprising the amino acid sequence of SEQ ID NO: 90. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0356] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 102, and a VL comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0357] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 102 or SEQ ID NO: 103, and a VL comprising the amino acid sequence of SEQ ID NO: 95. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0358] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked to a molecular payload, wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 158 or SEQ ID NO: 159, and a VL comprising the amino acid sequence of SEQ ID NO: 157. In some embodiments, the molecular payload is a DMPK-targeted oligonucleotide (e.g., a DMPK-targeted oligonucleotide listed in Table 8).

[0359] In any of the example conjugates described herein, in some embodiments, the anti-TfR1 antibody has the following structure: [ka] where n is 3, m is 4, X is NH (e.g., NH from the amine group of lysine), and L1 is [ka] It is.

[0360] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked via a lysine in the anti-TfR1 antibody to the 5' end of a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8), where the anti-TfR1 antibody comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of any one of the antibodies listed in Table 2, and where the conjugate has the following structure: [ka] where n is 3 and m is 4, and where L1 is [ka] It is to be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (D) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0361] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked via a lysine in the anti-TfR1 antibody to the 5' end of a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8), where the anti-TfR1 antibody comprises a VH and VL of any one of the antibodies listed in Table 3, and where the conjugate has the following structure: [ka] where n is 3 and m is 4, and where L1 is [ka] It is to be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (D) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0362] In some embodiments, the conjugates described herein comprise an anti-TfR1 antibody covalently linked via a lysine in the anti-TfR1 antibody to the 5' end of a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8), where the anti-TfR1 antibody comprises the heavy and light chains of any one of the antibodies listed in Table 4, and where the conjugate has the following structure: [ka] where n is 3 and m is 4, and where L1 is [ka] It is to be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (D) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0363] In some embodiments, the conjugates described herein comprise an anti-TfR1 Fab covalently linked to the 5' end of a DMPK-targeting oligonucleotide (e.g., a DMPK-targeting oligonucleotide listed in Table 8) via a lysine in the anti-TfR1 Fab, where the anti-TfR1 Fab comprises the heavy and light chains of any one of the antibodies listed in Table 5, and where the conjugate has the following structure: [ka] where n is 3 and m is 4, and where L1 is [ka] It is to be understood that the amide shown adjacent to the anti-TfR1 antibody in formula (D) results from reaction with an amine of the anti-TfR1 antibody, such as the lysine epsilon amine.

[0364] In some embodiments, L1 is linked to the 5' phosphate of the oligonucleotide.

[0365] In some embodiments, L1 is optional (eg, does not have to be present).

[0366] III. Preparations The conjugates provided herein may be formulated in any suitable manner. In general, the conjugates provided herein are formulated in a manner suitable for pharmaceutical use. For example, the conjugates may be delivered to a subject using a formulation that minimizes degradation, facilitates delivery and / or (for example and) uptake, or provides another beneficial property to the conjugate in the formulation. In some embodiments, provided herein is a composition comprising the conjugate and a pharma- ceutically acceptable carrier. Such compositions may be suitably formulated so that a sufficient amount of the conjugate can enter the target muscle cell when administered either into the environment surrounding the target cell of the subject or into the whole body of the subject. Such compositions may be suitably formulated so that a sufficient amount of the conjugate can enter the target CNS cell when administered either into the environment in which the target cell of the subject is located or into the whole body of the subject. In some embodiments, the conjugates are formulated in a buffer solution, such as phosphate buffered saline solution, in a liposome, in a micellar structure, and in a capsid.

[0367] It should be understood that in some embodiments, a composition may individually include one or more components of the conjugates provided herein (e.g., a muscle targeting agent, a linker, a molecular payload, or a precursor molecule of any one of these).

[0368] In some embodiments, the complex is formulated in water or an aqueous solution (e.g., pH-adjusted water). In some embodiments, the complex is formulated in a basic buffered aqueous solution (e.g., PBS). In some embodiments, the formulation as disclosed herein includes an excipient. In some embodiments, the excipient confers improved stability, improved absorption, improved solubility, and / or (e.g., and) therapeutic enhancement of the active ingredient to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, Tris base, or sodium hydroxide) or a vehicle (e.g., a buffer solution, petrolatum, dimethyl sulfoxide, or mineral oil).

[0369] In some embodiments, the complex or a component thereof (e.g., an oligonucleotide or an antibody) is lyophilized to extend its shelf life and then brought into solution prior to use (e.g., administration to a subject).Accordingly, the excipient in a composition comprising the complex or a component thereof described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone), or a collapse temperature modifier (e.g., dextran, ficoll, or gelatin).

[0370] In some embodiments, a pharmaceutical composition is formulated to be compatible with its intended route of administration. Examples of routes of administration include parenteral administration, such as intravenous administration, intradermal administration, and subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous administration.

[0371] Pharmaceutical compositions suitable for use in injections include sterile aqueous solutions (wherein they are soluble in water) or dispersions, and sterile powders for extemporaneous preparation of sterile injection solutions or dispersions. Carriers can be, for example, solvents or dispersion media containing water, ethanol, polyols (for example, glycerol, propylene glycol, liquid polyethylene glycol, etc.), and suitable mixtures thereof. In some embodiments, the formulations include isotonic agents in the composition, for example, polyalcohols such as sugar, mannitol, sorbitol, and sodium chloride. Sterile injection solutions can be prepared by incorporating the required amount of the complex with one or a combination of the above-listed ingredients in the selected solvent, and then optionally filtering and sterilizing.

[0372] In some embodiments, the composition may contain at least about 0.1% of the complex or its components, although the percentage of active ingredient(s) may be between about 1% and about 80% or more by weight or volume of the total composition.Factors such as solubility, bioavailability, biological half-life, route of administration, shelf life of the product, and other pharmacological considerations will be taken into account by those skilled in the art who prepare such pharmaceutical formulations.Therefore, different dosages and treatment schedules may be desired.

[0373] IV. Method of Use / Treatment A complex comprising a muscle targeting agent covalently linked to a molecular payload as described herein is effective for treating myotonic dystrophy. In some embodiments, the complex is effective for treating myotonic dystrophy type 1 (DM1). In some embodiments, DM1 is associated with an expansion of a CTG / CUG trinucleotide repeat in the 3' non-coding region of DMPK. In some embodiments, the nucleotide expansion leads to a toxic RNA repeat that can form a hairpin structure that binds with high affinity to important intracellular proteins, such as muscleblind-like proteins.

[0374] In some embodiments, the subject may be a human subject, a non-human primate animal subject, a rodent animal subject, or any suitable mammalian animal subject. In some embodiments, the subject may have myotonic dystrophy. In some embodiments, the subject may have a DMPK allele that may optionally contain disease-associated repeats. In some embodiments, the subject may have a DMPK allele that has expanded disease-associated repeats, including about 2-10 repeat units, about 2-50 repeat units, about 2-100 repeat units, about 50-1,000 repeat units, about 50-500 repeat units, about 50-250 repeat units, about 50-100 repeat units, about 500-10,000 repeat units, about 500-5,000 repeat units, about 500-2,500 repeat units, about 500-1,000 repeat units, or about 1,000-10,000 repeat units. In some embodiments, the subject suffers from symptoms of DM1, such as muscle atrophy or muscle loss. In some embodiments, the subject does not suffer from symptoms of DM1. In some embodiments, the subject has congenital myotonic dystrophy.

[0375] Aspects of the present disclosure include methods involving administering to a subject an effective amount of a complex as described herein. In some embodiments, an effective amount of a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently linked to a molecular payload may be administered to a subject in need of treatment. In some embodiments, a pharmaceutical composition comprising a complex as described herein may be administered by a suitable route, 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 a 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.

[0376] Compositions for intravenous administration may contain a variety of carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). Water-soluble antibodies for intravenous injection may be administered by drip infusion, whereby a pharmaceutical formulation containing the antibody and a pharmaceutically acceptable excipient is infused. Pharmaceutically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. Intramuscular preparations, for example, a sterile formulation of a suitable soluble salt form of the antibody, may be administered dissolved in a pharmaceutical excipient such as water for injection, 0.9% saline, or 5% glucose solution.

[0377] In some embodiments, pharmaceutical compositions comprising a conjugate comprising a muscle-targeting agent covalently linked to a molecular payload are administered via site-specific or localized delivery techniques, examples of which include an implanted depot source of the conjugate, a localized delivery catheter, a site-specific carrier, direct injection, or direct application.

[0378] In some embodiments, the pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload is administered at an effective concentration that provides a therapeutic effect to the subject. The effective amount will vary depending on the severity of the disease, the specific characteristics of the subject being treated, such as age, physical condition, health, or weight, the duration of treatment, the nature of any concomitant treatments, the route of administration, and related factors, as will be recognized by those skilled in the art. These related factors are known to those skilled in the art and can be addressed with a little routine experimentation. In some embodiments, the effective concentration is the maximum dose that is considered safe for the patient. In some embodiments, the effective concentration will be the lowest concentration feasible that provides maximum efficacy.

[0379] 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 the treatment.

[0380] The efficacy of treatment can be assessed by any suitable method.In some embodiments, the efficacy of treatment can be assessed by evaluation of symptoms related to DM1, for example muscle atrophy or muscle weakness, through subject's self-reported outcome measurement, for example mobility, self-care, normal activity, pain / discomfort and anxiety / depression, or by quality of life indicators, for example life span.

[0381] In some embodiments, a pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload described herein is administered to a subject at an effective concentration sufficient to inhibit target gene activity or expression by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% as compared to a control (e.g., a baseline level of gene expression prior to treatment).

[0382] In some embodiments, a single dose or administration to a subject of a pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload as described herein is sufficient to inhibit target gene activity or expression for at least 1-5 days, 1-10 days, 5-15 days, 10-20 days, 15-30 days, 20-40 days, 25-50 days, or longer. In some embodiments, a single dose or administration to a subject of a pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload as described herein is sufficient to inhibit target gene activity or expression for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, or 24 weeks. In some embodiments, a single dose or administration to a subject of a pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload as described herein is sufficient to inhibit activity or expression of a target gene for at least 1-5, 1-10, 2-5, 2-10, 4-8, 4-12, 5-10, 5-12, 5-15, 8-12, 8-15, 10-12, 10-15, 10-20, 12-15, 12-20, 15-20, or 15-25 weeks. In some embodiments, a single dose or administration to a subject of a pharmaceutical composition comprising a conjugate comprising a muscle targeting agent covalently linked to a molecular payload as described herein is sufficient to inhibit activity or expression of a target gene for at least 1, 2, 3, 4, 5, or 6 months.

[0383] In some embodiments, the pharmaceutical composition may include more than one conjugate comprising a muscle targeting agent covalently linked to a molecular payload. In some embodiments, the pharmaceutical composition may further include any other suitable therapeutic agent for the treatment of a subject, for example, a human subject with DM1. In some embodiments, the other therapeutic agent may enhance or complement the efficacy of the conjugates described herein. In some embodiments, the other therapeutic agent may function to treat a different condition or disease than the conjugates described herein.

[0384] example Example 1. In vitro activity of conjugates containing anti-TfR1 Fab conjugated to DMPK-targeted oligonucleotides (ASOs) In vitro experiments were performed to determine the activity of DMPK-targeting oligonucleotides (ASOs) listed in Table 8 in reducing DMPK mRNA expression in rhabdomyosarcoma (RD) and 32F cells, and correcting the BIN1 exon 11 splicing defect in DM1-32F primary cells (32F cells; Cook MyoSite, Pittsburg, PA), which express a mutant DMPK mRNA containing 380 CTG repeats (Figure 1A). All ASOs were conjugated to anti-TfR1 Fab (3M12-VH4 / VK3).

[0385] RD cells were expanded and seeded into 96-well plates at a density of 20,000 cells / well. Cells were allowed to recover overnight at 37C. The next day, medium was changed and cells were treated with conjugates equivalent to 500 nM ASO and incubated for 72 hours. After 72 hours, total RNA was extracted using the PureLink Pro 96 RNA extraction kit and cDNA was generated using the qScript cDNA synthesis kit. cDNA was used to assess knockdown of total DMPK using Taqman PCR. Data were normalized to PPIB expression and 2 -ΔΔCt The method was used to determine DMPK knockdown compared to vehicle only control. Data are plotted as mean values ​​with standard deviations.

[0386] DM1 32F primary cells were thawed and allowed to recover, then seeded in 96-well plates at a density of 50,000 cells / well in growth medium and allowed to recover overnight. The following day, growth medium was replaced with low serum differentiation medium and cells were treated with conjugates equivalent to 100 nM ASO. Cells were incubated for 10 days, then total RNA was harvested using the Qiagen MiRNeasy extraction kit and cDNA was synthesized using the qScript cDNA synthesis kit.

[0387] cDNA was used to assess knockdown of total DMPK using Taqman PCR. Data were normalized to PPIB expression and expressed as 2 -ΔΔCt The method was used to determine DMPK knockdown compared to vehicle-only control. Data are presented as the mean % DMPK knockdown with standard deviation (Table 9). In addition, modification of DM1-mediated aberrant splicing was assessed using multiplexed Taqman qPCR to evaluate aberrantly spliced ​​versus normal transcripts. These data are presented as the mean ratio of aberrantly spliced ​​to normal with standard deviation (Table 9). A ratio of 1 means no change in aberrant splicing when compared to vehicle control treated with DM1 patient myotubes. A ratio greater than 1 means more transcripts have wild-type splicing patterns. A ratio less than 1 means more transcripts have DM1-mediated splicing patterns. Table 9. DMPK knockdown and correction of BIN1 exon 11 splicing defect [Table 14-1] [Table 14-2] [Table 14-3]

[0388] Example 2. In vitro activity of conjugates containing anti-TfR1 Fab conjugated to DMPK-targeting ASO in patient-derived cells In vitro experiments were performed to determine the activity of the DMPK-targeting oligonucleotide ASO1 in reducing DMPK mRNA expression, correcting the BIN1 exon 11 splicing defect, and reducing nuclear foci (measured as the ratio of nuclear foci area to nuclear area) in DM1-32F primary cells (32F cells; Cook MyoSite, Pittsburg, PA) expressing mutant DMPK mRNA containing 380 CTG repeats (Figure 1A) and in DM1-CL5 immortalized cells (CL5 cells) expressing mutant DMPK mRNA containing 2600 CTG repeats (Figure 1B). We first confirmed that ASO1 could reduce mutant DMPK mRNA in DM1-32F and DM1-CL5 cells, but did not affect DMPK mRNA levels in healthy cells according to RT-PCR (Figures 1A-1B).

[0389] Conjugates containing a control anti-TfR1 Fab conjugated to ASO1 or ASO 32 were tested for their ability to reduce DMPK mRNA expression, correct the BIN1 exon 11 splicing defect, and reduce nuclear foci (measured as the ratio of nuclear foci area to nuclear area) in 32F cells. 32F cells were cultured at 156,000 cells / cm as described (Arandel et al., Disease Models & Mechanisms 2017 10: 487-497, incorporated herein by reference). 2 Cells were seeded at a density of 100x and allowed to recover for 24 hours, transferred to differentiation medium to induce myotube formation, and subsequently exposed to ASO32- and ASO1-conjugates at a payload concentration of 500 nM. In parallel, cultures exposed to the vehicle PBS served as negative controls. Cells were harvested after 10 days of culture.

[0390] For gene expression analysis, cells were harvested by Qiazol for total RNA extraction with Qiagen miRNAeasy kit. Purified RNA was reverse transcribed and levels of DMPK, PPIB, BIN1 transcripts, and exon 11-containing BIN1 mRNA isoforms were determined by qRT-PCR with specific TaqMan assays (ThermoFisher). Log fold changes in DMPK expression were calculated as 2. -ΔΔCT PPIB was used as the reference gene, and vehicle-exposed cells were used as the control group. The Log fold change in the levels of the BIN1 isoform containing exon 11 was calculated according to the 2 -ΔΔCT The calculation was performed according to the method described above. BIN1 was used as the reference gene, and cells exposed to the vehicle were used as the control group.

[0391] To measure the area of ​​mutant DMPK nuclear foci, cells were fixed with 4% formalin, permeabilized with 0.1% Triton X-100, and hybridized with a CAG peptide nucleic acid probe conjugated to a Cy5 fluorophore (PNA Bio) at 70°C. After multiple washes with hybridization buffer and 2x SSC solution, nuclei were counterstained with DAPI. Images were collected by confocal microscopy at 400x magnification, and the focus area was measured as the area of ​​Cy5 signal contained within the area of ​​DAPI signal. Data were expressed as the ratio of the nuclear focus area to the nuclear area.

[0392] The results show that a single dose of ASO32-conjugate or ASO1-conjugate resulted in reduced mutant DMPK expression (Figure 2A), corrected BIN1 exon 11 splicing defect (Figure 2B), and approximately 40% reduction in nuclear foci (Figures 2C-2D).

[0393] Similar experiments were also performed on CL5 cells, and the results show that a single dose of ASO32-conjugate or ASO1-conjugate led to reduced mutant DMPK expression (Figure 3A), corrected BIN1 exon 11 splicing defect (Figure 3B), and that the ASO32-conjugate reduced nuclear foci by approximately 30%, whereas the ASO1-conjugate reduced nuclear foci by approximately 3% (Figures 3C-3D).

[0394] In addition, conjugates containing anti-TfR1 Fab (3M12-VH4 / VK3) conjugated to other DMPK-targeting oligonucleotides, such as ASO32, ASO10, ASO8, ASO26, and ASO1, were also tested for their activity in reducing DMPK mRNA expression, correcting the BIN1 exon 11 splicing defect, and reducing nuclear foci in 32F cells (Figure 4A). Experiments were performed as described above.

[0395] The results show that a single dose of ASO32-, ASO10-, ASO8-, ASO26-, or ASO1-conjugate resulted in reduced mutant DMPK expression (Figure 4B), corrected BIN1 exon 11 splicing defect (Figure 4C), and approximately at least 20% reduced nuclear foci (Figures 4D-4E). Figure 4F shows that ASO32-, ASO10-, ASO8-, ASO26-, and ASO1-conjugates were able to dose-dependently reduce DMPK expression in 32F cells (cells were exposed to DMPK-targeting oligonucleotide-conjugates at ASO concentrations of 14 nM, 45 nM, and 150 nM). Figure 4G shows that ASO32-conjugate, ASO10-conjugate, ASO8-conjugate, ASO26-conjugate, and ASO1-conjugate were able to dose-dependently correct BIN1 exon 11 splicing defect in 32F cells (cells were exposed to DMPK-targeting oligonucleotide-conjugate at ASO concentrations of 14 nM, 45 nM, and 150 nM). Figure 4H shows that ASO32-conjugate, ASO10-conjugate, ASO8-conjugate, ASO26-conjugate, and ASO1-conjugate were able to dose-dependently reduce CUG foci in 32F cells (cells were exposed to DMPK-targeting oligonucleotide-conjugate at ASO concentrations of 14 nM, 45 nM, and 150 nM).

[0396] A similar experiment was also carried out in CL5 cells (Figure 5A). In this experiment, all DMPK-targeting oligonucleotides tested were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. The experiment was carried out as described above.

[0397] The results show that a single dose of ASO32-, ASO10-, ASO8-, ASO26-, or ASO1-conjugate resulted in reduced mutant DMPK expression (Figure 5B) and corrected BIN1 exon 11 splicing defects (Figure 5C). The ASO32-, ASO10-, and ASO8-conjugates reduced nuclear foci by approximately 30%, the ASO26-conjugate reduced nuclear foci by approximately 10%, and the ASO1-conjugate did not appear to reduce nuclear foci (Figures 5D-5E). Figure 5F shows that ASO32-conjugate, ASO10-conjugate, ASO8-conjugate, ASO26-conjugate, and ASO1-conjugate can reduce DMPK expression in CL5 cells in a dose-dependent manner (cells were exposed to DMPK-targeting oligonucleotide-conjugate at ASO concentrations of 14 nM, 45 nM, and 150 nM). Figure 5G shows that ASO32-conjugate, ASO10-conjugate, ASO8-conjugate, ASO26-conjugate, and ASO1-conjugate can correct BIN1 exon 11 splicing defect in CL5 cells in a dose-dependent manner (cells were exposed to DMPK-targeting oligonucleotide-conjugate at ASO concentrations of 14 nM, 45 nM, and 150 nM). Figure 5H shows that ASO32-, ASO10-, and ASO8-conjugates were able to reduce CUG foci in CL5 cells in a dose-dependent manner. The ASO26-conjugate reduced nuclear foci at the highest concentration, and the ASO1-conjugate did not appear to reduce nuclear foci (cells were exposed to DMPK-targeting oligonucleotide-conjugates at ASO concentrations of 14 nM, 45 nM, and 150 nM).

[0398] Moreover, ASO10-, ASO8-, and ASO26-conjugates were able to knock down DMPK expression in rhabdomyosarcoma (RD) cells in a dose-dependent manner, and ASO1-conjugate was able to knock down DMPK in non-human primate (NHP) cells in a dose-dependent manner (cells were exposed to ASO at 4 nM, 20 nM, 100 nM, or 500 nM) (Figure 6). All ASOs were conjugated to anti-TfR1 Fab 3M12 - VH4 / VK3.

[0399] Example 3. Chemical modification of DMPK-targeted oligonucleotides affects the efficacy of conjugates containing anti-TfR1 Fab conjugated to the oligonucleotide To test how different chemical modifications can affect the efficacy of DMPK-targeting oligonucleotides, the tool DMPK-targeting oligonucleotide ASO32 with different chemical modification patterns was tested for its activity in reducing DMPK expression. ASO30, ASO31, and ASO32 have the same nucleotide sequence but contain different modification patterns (see Table 8). All oligonucleotides were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3 prior to contacting rhabdomyosarcoma (RD) cells. The cells were contacted with ASO32-conjugate, ASO31-conjugate, or ASO30-conjugate at ASO concentrations of 4 nM, 20 nM, 100 nM, or 500 nM, and DMPK expression levels were evaluated to determine the oligonucleotide ability in knocking down DMPK expression. All tested oligonucleotide-conjugates were able to reduce DMPK expression in a dose-dependent manner. At an oligo concentration of 500 nM, the ASO32-conjugate was able to reduce DMPK expression by 88%, the ASO31-conjugate was able to reduce DMPK expression by 70%, and the ASO30-conjugate was able to reduce DMPK expression by 39% (Figure 7).

[0400] Similar experiments were performed on other DMPK-targeting oligonucleotides to show that length and different chemical modifications affect the efficacy of the oligonucleotides. In this experiment, conjugates containing anti-TfR1 Fab (3M12 - VH4 / VK3) conjugated to ASO32, ASO2, ASO8, ASO9, ASO10, ASO11, ASO20, and ASO26 were tested. ASO32, ASO2, ASO8, ASO9, ASO10, ASO11, ASO20, and ASO26 are gapmers with different modification patterns (see Table 8). Human RD cells were exposed to ASO32-conjugate, ASO10-conjugate, ASO8-conjugate, ASO9-conjugate, ASO11-conjugate, ASO20-conjugate, ASO26-conjugate, and ASO2-conjugate at ASO concentrations of 4 nM, 20 nM, 100 nM, or 500 nM, and DMPK expression levels were assessed to determine the ability of the oligonucleotides in knocking down DMPK expression. All oligonucleotide-conjugates tested were able to reduce DMPK expression in a dose-dependent manner. At an oligo concentration of 500 nM, the ASO10-conjugate was able to reduce DMPK expression by approximately 80%, the ASO8-conjugate was able to reduce DMPK expression by approximately 70%, the ASO9-conjugate was able to reduce DMPK expression by approximately 60% (Figure 8A), the ASO11-conjugate was able to reduce DMPK expression by approximately 40%, the ASO20-conjugate was able to reduce DMPK expression by approximately 40%, the ASO26-conjugate was able to reduce DMPK expression by approximately 30%, and the ASO2-conjugate was able to reduce DMPK expression by approximately 40% (Figure 8B).

[0401] We also performed hTfR1 ELISA experiments and confirmed that ASO10-conjugates (EC 50 11nM ASO equivalent), ASO8-conjugate (EC 50 29nM ASO equivalent), ASO26-conjugate (EC 50 1 nM ASO equivalent), and ASO1-conjugate (EC 50 EC 50 was measured.

[0402] Example 4. In vivo activity of conjugates containing anti-TfR1 Fab conjugated to DMPK-targeted oligonucleotides in a DM1 mouse model Conjugates containing anti-TfR1 Fab conjugated to various DMPK-targeting oligonucleotides were tested in a mouse model expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat. In the first experiment, ASO32 was conjugated to a control anti-TfR1 Fab, and the conjugate was administered to mice on days 0 and 7 by intravenous injection at a dose equivalent to 10 mg / kg ASO32. Mice were sacrificed on day 14, and human mutant DMPK expression was evaluated in various muscle tissues. The results show that the ASO32-conjugate reduced human mutant DMPK by 36% in the tibialis anterior muscle (Figure 9A), 46% in the diaphragm (Figure 9B), and 42% in the heart (Figure 9C).

[0403] Furthermore, a conjugate containing anti-TfR1 Fab 3M12-VH4 / VK3 conjugated to ASO32 was tested in a mouse model expressing human TfR1. The ASO32-conjugate reduced mouse wild-type dmpk by 79% in tibialis anterior muscle (Figure 9D), 76% in gastrocnemius muscle (Figure 9E), 70% in heart (Figure 9F), and 88% in diaphragm (Figure 9G). ASO32 distribution in tibialis anterior muscle, gastrocnemius muscle, heart, and diaphragm is shown in Figures 9H-9K. All tissues showed increased levels of ASO32 compared to vehicle control.

[0404] ASO10, ASO8, ASO26, and ASO1 were tested in the same mouse model described above expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat. ASO32 was included as a control and was conjugated to a control anti-TfR1 Fab. ASO10, ASO8, ASO26, and ASO1 were conjugated to anti-TfR1 Fab 3M12-VH4 / VK3. Mice were injected with oligonucleotides on days 0 and 7 and sacrificed on day 14. Experimental groups included: (i) vehicle control was injected into mice expressing human TfR1 (n=4); (ii) vehicle control was injected into mice expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat (n=10); (iii) ASO10-conjugate was injected into mice expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat at a dose equivalent to 2x9.7mg / kg ASO10 (n=6); (iv) ASO8-conjugate was injected into mice expressing both human TfR1 and a human DMPK mutant harboring an expanded CUG repeat at a dose equivalent to 2x9.2mg / kg ASO8 (n=6). (v) ASO26-conjugate was injected into mice expressing both human TfR1 and a human DMPK mutant containing an expanded CUG repeat at a dose equivalent to 2x12.3 mg / kg ASO26 (n=6); and (vi) ASO1-conjugate was injected into mice expressing both human TfR1 and a human DMPK mutant containing an expanded CUG repeat at a dose equivalent to 2x12.7 mg / kg ASO1 (n=6). After the mice were sacrificed, human mutant DMPK expression was evaluated in various muscle tissues. FIG. 10A shows that the ASO32-conjugate reduced human mutant DMPK in the heart by 42%, the ASO10-conjugate reduced human mutant DMPK in the heart by 60%, the ASO8-conjugate reduced human mutant DMPK in the heart by 67%, the ASO26-conjugate reduced human mutant DMPK in the heart by 49%; and the ASO1-conjugate reduced human mutant DMPK in the heart by 15%.FIG. 10B shows that the ASO32-conjugate reduced human mutant DMPK in the diaphragm by 46%, the ASO10-conjugate reduced human mutant DMPK in the diaphragm by 56%, the ASO8-conjugate reduced human mutant DMPK in the diaphragm by 58%, the ASO26-conjugate reduced human mutant DMPK in the diaphragm by 38%; and the ASO1-conjugate reduced human mutant DMPK in the diaphragm by 35%. FIG. 10C shows that the ASO32-conjugate reduced human mutant DMPK in gastrocnemius by 25%, the ASO10-conjugate reduced human mutant DMPK in gastrocnemius by 39%, the ASO8-conjugate reduced human mutant DMPK in gastrocnemius by 42%, the ASO26-conjugate reduced human mutant DMPK in gastrocnemius by 26%; and the ASO1-conjugate did not appear to reduce human mutant DMPK in gastrocnemius. Figure 10D shows that ASO32-conjugate reduced human mutant DMPK in tibialis anterior muscle by 36%, ASO10-conjugate reduced human mutant DMPK in tibialis anterior muscle by 54%, ASO8-conjugate reduced human mutant DMPK in tibialis anterior muscle by 51%, ASO26-conjugate reduc...

Claims

1. 1. A conjugate comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide, wherein the antibody is: (i) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 27, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 28, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 29, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 30, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32; (ii) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 33, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 34, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 35, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 36, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 32; or (iii) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 38, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 39, CDR-H3 comprising the amino acid sequence of SEQ ID NO: 40, CDR-L1 comprising the amino acid sequence of SEQ ID NO: 41, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 42; And wherein the oligonucleotide is 15-20 nucleotides in length and comprises a region of complementarity to at least 15 consecutive nucleosides of any one of SEQ ID NOs: 166 and comprises a 5'-XYZ-3' configuration; where X comprises a 3-5 linked nucleoside; wherein at least one of the nucleosides in X is a 2'-modified nucleoside; Y comprises a 6-10 linked 2'-deoxyribonucleoside; wherein each cytosine in Y is optionally and independently 5-methyl-cytosine; and The above conjugates, wherein Z comprises 3-5 linked nucleosides, wherein at least one of the nucleosides in Z is a 2'-modified nucleoside.

2. The conjugate of claim 1 , wherein the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 76 and a VL comprising the amino acid sequence of SEQ ID NO:

75.

3. The conjugate of claim 1 or 2, wherein the anti-TfR1 antibody is a Fab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO:

90.

4. The complex of any one of claims 1 to 3, wherein the region of complementarity is 16 nucleotides in length.

5. The complex of any one of claims 1 to 4, wherein the oligonucleotide is 16 nucleotides in length.

6. The conjugate of any one of claims 1 to 5, wherein the heavy chain of the antibody comprises an N-terminal pyroglutamic acid.

7. The complex according to any one of claims 1 to 6, wherein the oligonucleotide comprises the nucleotide sequence of SEQ ID NO:

179.

8. 8. The conjugate of any one of claims 1 to 7, wherein X comprises at least one 2'-4' bicyclic nucleoside and at least one non-bicyclic 2'-modified nucleoside and / or Z comprises at least one 2'-4' bicyclic nucleoside and at least one non-bicyclic 2'-modified nucleoside.

9. The conjugate of claim 8, wherein at least one non-bicyclic 2'-modified nucleoside is a 2'-MOE modified nucleoside and at least one 2'-4' bicyclic nucleoside is selected from LNA, cEt, and ENA.

10. 9. The conjugate of claim 8, wherein at least one non-bicyclic 2'-modified nucleoside is a 2'-MOE modified nucleoside and at least one 2'-4' bicyclic nucleoside is LNA.

11. The oligonucleotide is: LLEE (D) 8 EELL comprising the 5'-XYZ-3' arrangement, 11. The conjugate of any one of claims 1 to 10, wherein "E" is a 2'-MOE modified ribonucleoside; "L" is LNA; "D" is a 2'-deoxyribonucleoside; and "8" is the number of 2'-deoxyribonucleosides in Y.

12. The conjugate of claim 11 , wherein the oligonucleotide comprises one or more phosphorothioate internucleoside linkages.

13. 13. The conjugate of claim 12, wherein each internucleoside linkage in the oligonucleotide is a phosphorothioate internucleoside linkage.

14. The complex according to any one of claims 1 to 13, wherein the oligonucleotide is in the form of a salt.

15. 15. The complex of claim 14, wherein the salt is a sodium, potassium, or magnesium salt.

16. The oligonucleotide comprises: + C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (sequence number 179), + C*+A*+G*xdC*dG*dC*dC*dC*dC*dA*dC*dC*dA*dG*+U*+C*+A (SEQ ID NO:179), oC*oA*oG*xdC*dG*dC*dC*dC*dC*dA*dC*dC*dA*dG*oU*oC*oA (SEQ ID NO:179), oC*oC*oC*oA*oG*xdC*dG*dC*dC*dC*dA*dC*dC*dA*dG*oU*oC*oA*oC*oA (SEQ ID NO: 174), + C*+C*oA*oG*xdC*dG*dC*dC*dC*dC*dA*dC*dC*oA*oG*+U*+C (sequence number 180), + A*+G*oC*oG*dC*dC*dC*dA*dC*dC*dA*dG*oU*oC*+A*+C (SEQ ID NO:181), + C*+C*oC*oA*oG*xdC*dG*dC*dC*dC*dA*dC*dC*dA*dG*oU*oC*oA*+C*+A (Sequence number 174), oC*oCoC*oAoG*xdC*dG*dC*dC*dC*dC*dA*dC*dC*dA*dG*oUoC*oAoC*oA (SEQ ID NO:174), and oC*oCoCoAoG*xdC*dG*dC*dC*dC*dC*dA*dC*dC*dA*dG*oUoCoAoC*oA (SEQ ID NO:174), An oligonucleotide comprising an oligonucleotide selected from where "xdC" is 5-methyl-deoxycytidine; "dN" is a 2'-deoxyribonucleoside; "+N" is an LNA nucleoside; "oN" is a 2'-MOE modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "+C" is 5-methyl-2'-4'-bicyclic-cytidine (2'-4' methylene bridge); "oU" is is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl-2'-4'-bicyclic-uridine (2'-4' methylene bridge); "*" indicates a phosphorothioate internucleoside linkage; and the absence of an "*" between nucleosides indicates a phosphodiester internucleoside linkage.

17. The oligonucleotide comprises the structure + C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 179), 17. The conjugate of claim 16, wherein "dN" is a 2'-deoxyribonucleoside; "+N" is an LNA nucleoside; "oN" is a 2'-MOE modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "+C" is 5-methyl-2'-4'-bicyclic-cytidine (2'-4' methylene bridge); "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl-2'-4'-bicyclic-uridine (2'-4' methylene bridge); and "*" indicates a phosphorothioate internucleoside linkage.

18. The conjugate of any one of claims 1 to 17, wherein the antibody is covalently linked to the oligonucleotide via: (i) a cleavable linker; or (ii) A non-cleavable linker.

19. 20. The conjugate of claim 18, wherein the antibody is covalently linked to the oligonucleotide via a cleavable linker, and the cleavable linker comprises a valine-citrulline sequence.

20. 20. The conjugate of claim 18, wherein the antibody is covalently linked to the oligonucleotide via a non-cleavable linker, and the non-cleavable linker comprises an optionally substituted alkyl group.

21. The conjugate of any one of claims 18 to 20, wherein the oligonucleotide is covalently linked to the antibody via conjugation to a cysteine ​​residue of the antibody.

22. The conjugate of any one of claims 18 to 20, wherein the oligonucleotide is covalently linked to the antibody via conjugation to a lysine residue of the antibody.

23. The following structure: 【Chemistry 1】 Including, where n is 3 and m is 4, Here, L1 is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene, -O-, -N(RA)-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NRA-, -NRAC(=O)-, -NRAC(=O)RA-, -C(=O)RA-, -NRAC(=O)O-, -NRAC(=O)N(RA)-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(RA)-, -S(O) 2 NRA-, -NRAS(O) 2 -, or a combination thereof, Here, each R A The conjugate of any one of claims 1 to 19 and 22, wherein is independently hydrogen or substituted or unsubstituted alkyl.

24. L1: 【Chemistry 2】 24. The complex of claim 23, comprising:

25. A conjugate comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to the 5' end of an oligonucleotide targeted to DMPK RNA, wherein the complex has the following structure: 【Chemistry 3】 Including, where n is 3 and m is 4, Here, L1 【Chemistry 4】 Including, wherein the antibody is a Fab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 101 and a light chain comprising the amino acid sequence of SEQ ID NO: 90; And Here, the oligonucleotide + C*+A*oG*oC*dG*dC*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 179), wherein "dN" is a 2'-deoxyribonucleoside; "+N" is an LNA nucleoside; "oN" is a 2'-MOE modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "+C" is 5-methyl-2'-4'-bicyclic-cytidine (2'-4' methylene bridge); "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl-2'-4'-bicyclic-uridine (2'-4' methylene bridge); and "*" indicates a phosphorothioate internucleoside linkage, said conjugates.

26. The conjugate of claim 25 , wherein the heavy chain of the antibody comprises an N-terminal pyroglutamic acid.

27. 27. The complex of claim 25 or 26, wherein the oligonucleotide is in the form of a salt.

28. 28. The complex of claim 27, wherein the salt is a sodium, potassium, or magnesium salt.

29. 29. The conjugate of any one of claims 1 to 28 for use in a method of treating a disease or condition that can be ameliorated or prevented by reducing DMPK expression in a cell, said method comprising contacting said cell with said conjugate in an amount effective to promote internalization of an oligonucleotide into said cell.

30. 30. The complex of claim 29, wherein the cell comprises a DMPK allele that includes the disease-associated repeat.

31. 31. The complex of claim 29 or 30, wherein reducing DMPK expression comprises reducing RNA levels of DMPK.

32. 32. The complex for use according to claim 31, wherein the reduced RNA level is in the nucleus of the cell.

33. The complex for use according to any one of claims 29 to 32, wherein the cell is a muscle cell.

34. 30. The conjugate of any one of claims 1 to 28 for use in a method for treating myotonic dystrophy, said method comprising administering to a subject an effective amount of said conjugate.

35. 35. The conjugate for use according to claim 34, wherein the subject is a human.

36. The oligonucleotide comprises: 【Table 1】 and the oligonucleotides wherein "xdC" is 5-methyl-deoxycytidine; "dN" is a 2'-deoxyribonucleoside; "+N" is an LNA nucleoside; "oN" is a 2'-MOE modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "+C" is 5-methyl-2'-4'-bicyclic-cytidine (2'-4' methylene bridge); "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl-2'-4'-bicyclic-uridine (2'-4' methylene bridge); "*" indicates a phosphorothioate internucleoside linkage; and the absence of an "*" between nucleosides indicates a phosphodiester internucleoside linkage.

37. The oligonucleotide is: 【Table 2】 and where "xdC" is 5-methyl-deoxycytidine; "dN" is a 2'-deoxyribonucleoside; "+N" is an LNA nucleoside; "oN" is a 2'-MOE modified ribonucleoside; "oC" is 5-methyl-2'-MOE-cytidine; "+C" is 5-methyl-2'-4'-bicyclic-cytidine (2'-4' methylene bridge); "oU" is 5-methyl-2'-MOE-uridine; "+U" is 5-methyl-2'-4'-bicyclic-uridine (2'-4' methylene bridge); "*" indicates a phosphorothioate internucleoside linkage; and the absence of a "*" between nucleosides indicates a phosphodiester internucleoside linkage, and Here, the phosphodiester linkage is 5'-NH 2 -(CH 2 ) 6 The antisense oligonucleotide of claim 36, which is located between - and the antisense oligonucleotide.

38. 38. A composition comprising the oligonucleotide of claim 36 or 37 in the form of a salt, wherein the salt is a sodium, potassium, or magnesium salt;

39. 39. The composition of claim 38, wherein the salt is a sodium salt.

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