Administration of muscle-targeted complexes for treatment of ankylosing muscular dystrophy

Abstract

Some aspects of the present disclosure relate to methods of reducing the expression or activity of DMPK (e.g., reducing the level of mutant or wild-type DMPK RNA, or the activity of a DMPK gene product) and / or methods of treating ankylosing muscular dystrophy (e.g., DM1) in a subject. In some embodiments, the methods comprise administering to the subject a composition comprising a complex (e.g., a muscle targeting complex) comprising an oligonucleotide (e.g., a DMPK targeting oligonucleotide) covalently linked to an antibody (e.g., an anti-TfR1 antibody).

Description

[0001] Related Applications

[0002] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Serial No. 63 / 368,691, filed on July 18, 2022, entitled “DOSING OF MUSCLE Targeting Complexes For TREATING MYOTONIC DYSTROPHY,” and U.S. Provisional Application Serial No. 63 / 331,733, filed on April 15, 2022, entitled “DOSING OF MUSCLE Targeting Complexes For TREATING MYOTONIC DYSTROPHY,” the entire contents of each of which are incorporated herein by reference. Technical Field

[0003] The present application relates to a targeting complex for delivering an effective amount of an oligonucleotide molecular payload to a cell and its use, in particular to the use for treating a disease.

[0004] References to Electronic Sequence Listings

[0005] The contents of the electronic sequence listing (D082470077WO00-SEQ-COB.xml; size: 56,306 bytes; and creation date: April 7, 2023) are incorporated herein by reference in their entirety. Background Art

[0006] Myotonic dystrophy (DM) is a dominant inherited genetic disease characterized by myotonia, muscle loss or degeneration, weakened muscle function, insulin resistance, arrhythmia, smooth muscle dysfunction and nervous system abnormalities. DM is the most common form of adult-onset muscular dystrophy, with a worldwide incidence of about 1 in 8000 people worldwide. Two types of the disease have been described, i.e., myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is the more common form of the disease, which is caused by repeated expansion of CTG trinucleotide repeats in the 3' non-coding region of DMPK on chromosome 19; DM2 is caused by repeated expansion of CCTG tetranucleotide repeats in the first intron of ZNF9 on chromosome 3. In DM1 patients, repetitive expansion of CTG trinucleotide repeats, which can include greater than about 50 to about 3,000 or more total repeats, results in the production of toxic RNA repeats that are capable of forming hairpin structures that bind with high affinity to essential intracellular proteins, such as muscleblind-like protein, leading to protein sequestration and the loss-of-function phenotype characteristic of the disease. There are currently no effective therapeutic agents available for DM1, other than supportive care and treatment to address disease symptoms. Summary of the invention

[0007] According to some aspects, the present disclosure provides methods (e.g., methods of delivering oligonucleotides to a subject, methods of administering a complex to a subject, methods of reducing the expression or activity of DMPK in a subject, and / or methods of treating myotonic dystrophy (e.g., DM1) in a subject), comprising administering to the subject a composition comprising an effective amount of a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to one or more oligonucleotides.

[0008] According to some aspects, the methods provided herein for reducing DMPK expression in a subject include administering to the subject a composition comprising an effective amount of a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to one or more oligonucleotides, wherein the effective amount provides the subject with 5 mg to 110 mg of the anti-TfR1 antibody / kg of the subject in the complex, wherein the antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a heavy chain complementary determining region 3 (CDR-H4) comprising a sequence as shown in SEQ ID NO: 6, 8 or 13. The light chain complementary determining region 3 (CDR-L3) of the sequence shown in NO: 6 or 16, wherein the oligonucleotide in the complex comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

[0009] In some embodiments, the oligonucleotide in the complex comprises a 5'-XYZ-3' configuration, wherein X and Z are flanking regions comprising one or more modified nucleosides, and Y is a spacer region comprising one or more 2'-deoxyribonucleosides.

[0010] In some embodiments, each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Containing a group of formula (Ia):

[0011]

[0012] Where R 3comprising an oligonucleotide comprising a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21) and comprising a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage;

[0013] Where R 2 comprising an anti-TfR1 antibody; and

[0014] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A. In some embodiments, the average value of n1 of the complexes in the composition is 0.5 to 5.

[0015] In some embodiments, each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of which:

[0016] Each R 1 Containing a group of formula (Ib):

[0017]

[0018] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage, and R 1 The oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0019] Where R 2 comprising an anti-TfR1 antibody; and

[0020] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A. In some embodiments, the average value of n1 of the complexes in the composition is 0.5 to 5.

[0021] In some embodiments, each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of which:

[0022] Each R 1 Containing a group of formula (Ic):

[0023]

[0024] Where R 2 comprising an anti-TfR1 antibody; and

[0025] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A. In some embodiments, the average value of n1 of the complexes in the composition is 0.5 to 5.

[0026] In some embodiments, each complex comprises the structure of Formula (Id):

[0027]

[0028] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage, and R 1 The oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0029] Where R 2 comprising an anti-TfR1 antibody; and

[0030] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A. In some embodiments, the average value of n1 of the complexes in the composition is 0.5 to 5.

[0031] In some embodiments, the anti-TfR1 antibody is a Fab fragment.

[0032] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0033] In some embodiments, administration occurs one or more times.

[0034] In some embodiments, the effective amount per administration provides the subject with 10 to 110 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0035] In some embodiments, the effective amount per administration provides to the subject 5 to 90 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0036] In some embodiments, the effective amount per administration provides the subject with 10 to 20 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0037] In some embodiments, the effective amount per administration provides the subject with 13 mg anti-TfR1 antibody / kg of subject in the complex.

[0038] In some embodiments, the effective amount per administration provides the subject with 18 mg to 36 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0039] In some embodiments, the effective amount per administration provides the subject with 25 mg anti-TfR1 antibody / kg of subject in the complex.

[0040] In some embodiments, the effective amount per administration provides the subject with 36 mg to 72 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0041] In some embodiments, the effective amount per administration provides the subject with 50 mg anti-TfR1 antibody / kg of the subject in the complex.

[0042] In some embodiments, the effective amount per administration provides the subject with 55 mg to 110 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0043] In some embodiments, the effective amount per administration provides the subject with 75 mg of anti-TfR1 antibody / kg of subject in the complex.

[0044] In some embodiments, the effective amount per administration provides the subject with 6 mg to 12 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0045] In some embodiments, the effective amount per administration provides the subject with 8 mg anti-TfR1 antibody / kg of the subject in the complex.

[0046] In some embodiments, the effective amount per administration provides the subject with 11 mg to 22 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0047] In some embodiments, the effective amount per administration provides the subject with 15 mg anti-TfR1 antibody / kg of the subject in the complex.

[0048] In some embodiments, the effective amount per administration provides the subject with 22 mg to 44 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0049] In some embodiments, the effective amount per administration provides the subject with 30 mg anti-TfR1 antibody / kg of the subject in the complex.

[0050] In some embodiments, the effective amount per administration provides the subject with 44 mg to 88 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0051] In some embodiments, the effective amount per administration provides the subject with 60 mg anti-TfR1 antibody / kg of the subject in the complex.

[0052] In some embodiments, the effective amount provides the subject with 20 mg to 43 mg anti-TfRl antibody / kg subject in the complex per administration. In some embodiments, the effective amount provides the subject with 29 mg anti-TfRl antibody / kg subject in the complex per administration.

[0053] In some embodiments, the effective amount per administration provides the subject with 26 mg to 53 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0054] In some embodiments, the effective amount per administration provides the subject with 37 mg anti-TfR1 antibody / kg subject in the complex.

[0055] In some embodiments, during the administration period, the composition is administered once every 4 weeks, once every 8 weeks, or once every 12 weeks. In some embodiments, the administration period is less than 10 years. In some embodiments, the administration period is the rest of the subject's life.

[0056] In some embodiments, the composition is administered once every 4 weeks during a first administration period, and then administered once every 8 weeks during a second administration period. In some embodiments, the first administration period is 8 to 16 weeks, and / or the second administration period is 16 weeks to the rest of the subject's life.

[0057] In some embodiments, the composition is in the form of an aqueous solution and further comprises tris(hydroxymethyl)aminomethane and sucrose. In some embodiments, tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 25 mM, sucrose is present in the aqueous solution at a concentration of 10 w / v%, and the pH of the aqueous solution is 7.5. In some embodiments, the complex is present in the composition at a concentration of 10 mg / mL to 50 mg / mL.

[0058] In some embodiments, administration reduces DMPK expression in the muscle cells of the subject. In some embodiments, reducing DMPK expression comprises reducing the amount of DMPK RNA in the muscle cells. In some embodiments, the amount of DMPK RNA in the nucleus of the muscle cells is reduced.

[0059] In some embodiments, reducing DMPK expression in a muscle cell comprises reducing the amount of DMPK protein in the muscle cell.

[0060] In some embodiments, the subject is a human.

[0061] In some embodiments, the complex is administered systemically. In some embodiments, the complex is administered intravenously. In some embodiments, the complex is administered by infusion.

[0062] In some embodiments, the composition further comprises one or more anti-TfR1 antibodies that are not covalently linked to the oligonucleotide. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figures 1A to 1D Shown are the effects of administration of a conjugate containing an anti-TfR1 Fab covalently linked to an ASO on the heart ( Figure 1A ), diaphragm( Figure 1B ), gastrocnemius( Figure 1C ) or tibialis anterior ( Figure 1D ) in the presence of DMPK targeting oligonucleotide (ASO).

[0064] Figures 2A to 2D The conjugate containing anti-TfR1 Fab conjugated to DMPK targeting oligonucleotide (ASO) was shown in the heart of mice ( Figure 2A ), diaphragm( Figure 2B ), tibialis anterior ( Figure 2C ) and gastrocnemius ( Figure 2D) mice expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats.

[0065] Figures 3A to 3B Shown are 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 containing expanded CTG repeats and treated with an anti-TfR1 Fab conjugated to a DMPK targeting oligonucleotide (ASO). Figure 3A Representative images of samples after in situ hybridization staining for DMPK foci and fluorescent staining of myofibers are shown (inset images). Figure 3A In the microscopy image shown in , the light-colored circles show the cell nuclei, and the bright spots within the nuclei show DMPK foci. Figure 3B Quantification of DMPK foci is shown.

[0066] Figure 4 The splicing correction activity of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) in the heart of mice (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats is shown. 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-ASO conjugates ("hTfR1 / DMSXL-conjugates").

[0067] Figure 5 The splicing correction activity of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) in the diaphragm of mice (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats is shown. Composite splicing indices based on splicing of Bin1 exon 11, Insr exon 11, Ldb3 exon 11, 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-ASO conjugates ("hTfR1 / DMSXL-conjugates").

[0068] Figure 6 The splicing correction activity of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) in the tibialis anterior muscle of mice (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing an expanded CTG repeat is shown. Composite splicing indices based on splicing of Bin1 exon 11, 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 an anti-TfR1 Fab-ASO conjugate ("hTfR1 / DMSXL-conjugate").

[0069] Figure 7 The splicing correction activity of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) in gastrocnemius muscle of mice (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CTG repeats is shown. Composite splicing indices based on splicing of Mbnl2 exon 6, Nfix exon 7, and Ttn exon 313 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-ASO conjugates ("hTfR1 / DMSXL-conjugates").

[0070] Figure 8 DMPK knockdown in DM1 patient myotubes and wild-type non-human primate (NHP) myotubes is shown, which is produced by incubation with a conjugate containing an anti-TfR1Fab covalently linked to a DMPK targeting oligonucleotide (ASO). The results shown are normalized for expression in DM1 patient myotubes or NHP myotubes treated with vehicle alone. The data show the mean + standard deviation of n=4 repetitions under each condition. Data were calculated by one-way ANOVA (*, P<0.05, **, P<0.01).

[0071] Figures 9A to 9D Figure 3 shows the heart of mice 12 weeks after administration of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting ASO ( Fig.9A ), diaphragm( Fig. 9B ), tibialis anterior ( Fig. 9C ) or gastrocnemius ( Fig.9D ) in the DMPK expression. DMPK expression is relative to that in vehicle-treated animals. Data are shown as mean ± standard deviation of n = 5 to 12 replicates per tissue. Statistics were calculated by ANOVA followed by uncorrected Fisher's least significant difference test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0072] Figures 10A to 10D Figures 2 and 3 show the heart of mice after administration of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting ASO ( Fig. 10A ), diaphragm( Fig. 10B ), tibialis anterior ( Fig. 10C ) or gastrocnemius ( Fig. 10D ) in tissue DMPK expression. The conjugate was administered with one 10 mg / kg ASO equivalent dose ("10"), two 5 mg / kg ASO equivalent doses, one week apart ("2×5 weekly"), or two 5 mg / kg ASO equivalent doses, two weeks apart ("2×5, every 2 weeks"). Tissue DMPK levels were measured four weeks after the last dose was administered and are shown relative to vehicle-treated controls ("vehicle"). Data are shown as mean + standard deviation of n=3 to 6 replicates per tissue. Statistics were calculated by one-way ANOVA (*, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001).

[0073] Figures 11A to 11D Figures 2 and 3 show the heart of mice after administration of different doses of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting ASO ( Fig.11A ), diaphragm( Fig. 11B ), tibialis anterior ( Fig. 11C ) or gastrocnemius ( Fig.11D ) in the DMPK expression. The conjugate was administered with a 5 mg / kg ASO equivalent dose ("5"), a 10 mg / kg ASO equivalent dose ("10"), or a 20 mg / kg ASO equivalent dose ("20"), and tissue DMPK levels were measured after four weeks. Data are shown as mean + standard deviation and are shown relative to vehicle-treated controls ("Vehicle"). Statistical data were calculated by one-way ANOVA and Dunnett's post hoc analysis (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

[0074] Figures 12A to 12D Figures 2 and 3 show the heart of mice after administration of different doses of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting ASO ( Fig. 12A ), diaphragm( Fig. 12B ), tibialis anterior ( Fig. 12C ) or gastrocnemius ( Fig.12D ) in the DMPK expression. The conjugate was administered as two 5 mg / kg ASO equivalent doses, administered one week apart ("2×5") or two 10 mg / kg ASO equivalent doses, administered one week apart ("2×10"). Tissue DMPK levels were measured four weeks after the first dose and are shown relative to vehicle-treated controls ("Vehicle"). Data are shown as mean + standard deviation of n=5 to 6 replicates per tissue. Statistics were calculated by Brown-Forsythe and Welch ANOVA test and Dunnett T3 multiple comparison test (*, P<0.05; **, P<0.01; ****, P<0.0001).

[0075] Figures 13A to 13D Figures 2 and 3 show the heart of mice after administration of different doses of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting ASO ( Fig.13A ), diaphragm( Fig. 13B ), tibialis anterior ( Fig. 13C ) or gastrocnemius ( Fig.13D ) in the DMPK expression. The conjugate was administered at four 5 mg / kg ASO equivalent doses ("4×5") or four 10 mg / kg ASO equivalent doses ("4×10"), each dose was administered four weeks apart (on days 0, 28, 56 and 84, respectively). 4 weeks after the last dose (16 weeks after the first dose), tissue DMPK levels were measured and shown relative to vehicle-treated controls ("vehicle"). Data are shown as mean + standard deviation (n = 6 mice / group). Statistical data were calculated by one-way ANOVA, followed by an uncorrected Fisher's least significant difference (Least Significant Difference, LSD) test (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

[0076] Figures 14A to 14B The results of the study on the gastrocnemius muscle of non-human primates after administration of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting ASO at a dose of 10 mg / kg ASO are shown. Fig.14A ) and tibialis anterior ( Fig. 14B ) DMPK expression in . Tissue DMPK levels were measured 4, 8 and 12 weeks after administration of the conjugate and are shown relative to vehicle-treated controls. Data are expressed as mean ± standard deviation. Statistical data were calculated by unpaired t-test (*, P < 0.05).

[0077] Figures 15A to 15D Figure 2 shows the cardiac function of non-human primates after administration of 5 mg / kg (“5”) or 10 mg / kg (“10”) ASO equivalents of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting ASO. Fig.15A ), diaphragm( Fig. 15B ), tibialis anterior ( Fig. 15C ) or gastrocnemius ( Fig.15D ) in the non-human primate model. After a single 5 mg / kg ASO equivalent dose or a single 10 mg / kg ASO equivalent dose was administered to the non-human primate model, tissue DMPK levels were measured after four weeks and are shown relative to the vehicle-treated control ("vehicle"). Data are shown as mean + standard deviation. Statistical data were calculated by one-way ANOVA (*, P < 0.05; **, P < 0.01).

[0078] Figures 16A to 16G Figure 2 shows the cardiac function of non-human primates after administration of two 5 mg / kg ASO equivalent doses ("2×5") or two 10 mg / kg ASO equivalent doses ("2×10") of a conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting ASO, respectively. Fig.16A ), diaphragm( Fig. 16B ), tibialis anterior ( Fig. 16C ), gastrocnemius( Fig.16D )、masseter muscle( Fig.16E ),esophagus( Fig.16F ) or duodenum ( Figure 16G ) in the non-human primate model. After two 5 mg / kg ASO equivalent doses or two 10 mg / kg ASO equivalent doses were administered to the non-human primate model, the tissue DMPK levels were measured four weeks after the last dose every four weeks and were shown relative to the vehicle-treated control ("vehicle"). The data are shown as mean + standard deviation. Statistical data were calculated by one-way ANOVA, followed by an uncorrected Fisher's least significant difference (LSD) test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). DETAILED DESCRIPTION

[0079] According to some aspects, the disclosure provides methods for reducing the expression or activity of DMPK in an object (e.g., reducing the level of mutant or wild-type DMPK RNA) and / or methods for treating myotonic dystrophy (e.g., DM1). In some embodiments, the methods described herein include administering to an object a composition comprising an effective amount of a muscle-targeted complex, each complex comprising an anti-transferrin receptor I (TfR1) antibody covalently linked to one or more oligonucleotides, wherein the effective amount provides 0.5 mg to 20 mg (e.g., about 1 mg to about 12 mg, about 1.8 mg, about 3.4 mg, about 6.8 mg, or about 10.2 mg) of oligonucleotides / kg object in the complex to the object. In some embodiments, the effective amount provides about 4 mg or about 5 mg of oligonucleotides / kg object in the complex to the object. In some embodiments, the effective amount provides 2 mg to 220 mg (e.g., about 5 mg to about 110 mg, about 13 mg, about 25 mg, about 50 mg, or about 75 mg) of anti-TfR1 antibody (e.g., Fab) / kg of the subject in the complex to the subject. In some embodiments, the effective amount provides about 29 mg or about 37 mg of anti-TfR1 antibody (e.g., Fab) / kg of the subject in the complex to the subject. In some embodiments, administration is once every two weeks to once every 12 weeks (e.g., once every two weeks, once every four weeks, once every 8 weeks, or once every 12 weeks). In some embodiments, the subject has a DMPK allele associated with DM1 (e.g., wherein the DMPK allele comprises a DM1 disease-associated repeat expansion).

[0080] Additional aspects of the disclosure are provided below, including a description of defined terms.

[0081] definition

[0082] Administering: As used herein, the term "administering" or variations thereof means providing a complex to a subject (eg, to treat a condition in a subject) in a physiologically and / or (eg, and) pharmacologically usable manner.

[0083] Approximately: The term "approximately" or "about" as used herein, when applied to one or more target values, refers to a value similar to the stated reference value. In certain embodiments, the term "approximately" or "approximately" refers to a range of values ​​that fall within (greater than or less than) 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction of the stated reference value, unless otherwise stated or otherwise apparent from the context (unless such a number exceeds 100% of the possible value).

[0084] Antibody: The term "antibody" as used herein refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one antigenic determinant (e.g., a paratope that specifically binds to an antigen). In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humanized antibody. However, in some embodiments, the antibody is a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a Fv fragment, or a 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 double antibody. In some embodiments, the antibody comprises a framework with a human germline sequence. In another embodiment, the antibody comprises a heavy chain constant domain selected from IgG, IgG1, IgG2, IgG2A, IgG2B, IgG2C, IgG3, IgG4, IgA1, IgA2, IgD, IgM, and IgE constant domains. In some embodiments, the antibody comprises a heavy (H) chain variable region (referred to herein as VH) and / or (e.g., and) a light (L) chain variable region (referred to herein as VL). In some embodiments, the antibody comprises a constant domain, such as an Fc region. An immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of human IgG heavy and light chain constant domains and their functional variations are known. With regard to the heavy chain, in some embodiments, the heavy chain of the antibodies described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibodies described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In a specific embodiment, the antibodies described herein comprise human γ1CH1, CH2, and / or (e.g., and) CH3 domains. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, such as any known in the art. Some non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) supra. In some embodiments, the VH domain comprises an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identity with any variable chain constant region provided herein. In some embodiments, the antibody is modified, for example, by glycosylation, phosphorylation, SUMOylation and / or (e.g., and) methylation. In some embodiments, the antibody is a glycosylated antibody conjugated to one or more sugar or carbohydrate molecules.In some embodiments, one or more sugar or carbohydrate molecules are conjugated to the antibody by N-glycosylation, O-glycosylation, C-glycosylation, glycosylphosphatidylinositolization (GPI anchor attachment) and / or (e.g., and) phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides or polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched polysaccharides. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units or phospholipid units. In some embodiments, the antibody is a construct comprising a polypeptide comprising one or more antigen-binding fragments of the present disclosure connected to a linker polypeptide or an immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues connected by a peptide bond and is used to connect one or more antigen-binding moieties. Some 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). In addition, an antibody may be part of a larger immunoadhesion molecule formed by covalent or non-covalent association of an antibody or antibody portion with one or more other proteins or peptides. Some examples of such immunoadhesion molecules include the use of a streptavidin core region to prepare tetrameric scFv molecules (Kipriyanov, SM, et al. (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine ​​residues, a marker peptide, and a C-terminal polyhistidine tag to prepare bivalent and biotinylated scFv molecules (Kipriyanov, SM, et al. (1994) Mol. Immunol. 31:1047-1058).

[0085] CDR: The term "CDR" as used herein refers to the complementarity determining region within an antibody variable sequence. A typical antibody molecule comprises a heavy chain variable region (VH) and a light chain variable region (VL), which are generally involved in antigen binding. The VH region and the VL region can be further subdivided into hypervariable regions, also known as "complementarity determining regions" ("complementarity determining regions, CDR"), interspersed with more conservative regions called "framework regions" ("framework regions, FR"). Each VH and VL is generally composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Methods known in the art can be used, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition and / or (e.g., and) contact definition (all of which are well known in the art) to accurately identify the scope of the framework region and CDR. See, for example,

[0086] Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIHPublication No. 91-3242; the international ImMunoGeneTics information 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, MP. 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. 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. al., (1989) Nature 342: 877; Chothia, C. et al. (1987) J. Mol. Biol. 196: 901-917, A1-lazikani et al. al (1997) J. Molec. Biol. 273: 927-948; and Almagro, J. Mol. Recognit. 17: 132-143 (2004).

[0087] See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. CDRs used herein may refer to CDRs defined by any method known in the art. Two antibodies having the same CDRs means that the amino acid sequences of the CDRs of the two antibodies are identical, as determined by the same method (e.g., IMGT definition).

[0088] There are three CDRs in each variable region of the heavy and light chains, referred to as CDR1, CDR2 and CDR3 for each variable region. The term "CDR set" as used herein refers to a group of three CDRs that are present in a single variable region and are capable of binding to an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides an unambiguous residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Subportions of the CDRs may be designated L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate the light chain and heavy chain regions, respectively. These regions may be referred to as Chothia CDRs, which have boundaries that overlap with the Kabat CDRs. Padlan (FASEB J. 9: 133-139 (1995)) and MacCallum (J Mol Biol 262 (5): 732-45 (1996)) have described other boundaries that define CDRs that overlap with the Kabat CDRs. Other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with the Kabat CDRs. CDR overlap, although they can be shortened or lengthened based on predictions or based on experimental findings that specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding. The methods used herein can utilize CDRs defined according to any one of these systems. Some examples of CDR definition systems are provided in Table 1.

[0089] Table 1. CDR definitions

[0090] <![CDATA[IMGT 1 ]]> <![CDATA[Kabat 2 ]]> <![CDATA[Chothia 3 ]]> CDR-H1 27-38 31-35 26-32 CDR-H2 56-65 50-65 53-55 CDR-H3 105-116 / 117 95-102 96-101 CDR-L1 27-38 24-34 26-32 CDR-L2 56-65 50-56 50-52 CDR-L3 105-116 / 117 89-97 91-96

[0091] 1 the international ImMunoGeneTics information imgt.orh, Leffanc, M.-P. et al., Nucleic Acids Res., 27: 209-212 (1999)

[0092] 2Kabat et al. (1991) Sequences of Proteins or Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242

[0093] 3 Chothia et al., J. Mol. Biol. 196: 901-917 (1987))

[0094] Complementary: The term "complementary" as used herein refers to the ability to accurately pair between two nucleotides or two groups of nucleotides. In particular, complementarity is a term that characterizes the degree of binding between two nucleotides or two groups of nucleotides caused by hydrogen bond pairing. For example, if the base at one position of an oligonucleotide can hydrogen bond with the base at the corresponding position of a target nucleic acid (e.g., mRNA), the bases are considered to be complementary to each other at this 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, adenosine bases (A) are complementary to thymidine bases (T) or uracil bases (U), cytosine bases (C) are complementary to guanosine bases (G), and universal bases such as 3-nitropyrrole or 5-nitroindole can hybridize with any A, C, U or T and are considered to be complementary. Inosine (I) is also considered in the art to be a universal base, and is considered complementary to any of A, C, U, or T.

[0095] Covalently linked: As used herein, the term "covalently linked" refers to the feature of two or more molecules being linked together by at least one covalent bond. In some embodiments, two molecules can be covalently linked together by a single bond, such as a disulfide bond or a disulfide bridge, that acts as a linker between the molecules. However, in some embodiments, two or more molecules can be covalently linked together by a molecule that acts as a linker that links the two or more molecules together by 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.

[0096] Disease-associated repeats: The term "disease-associated repeats" as used herein refers to a repetitive nucleotide sequence at a certain genomic position, wherein the number of units of the repetitive nucleotide sequence is associated with a genetic disease and / or (e.g., and) directly or indirectly contributes to or causes a genetic disease. The length of each repeat unit of the disease-associated repeat can be 2, 3, 4, 5 or more nucleotides. 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 CAG repeats, CTG repeats, CUG repeats, CGG repeats, CCTG repeats, or any complementary nucleotides thereof. In some embodiments, the disease-associated repeat is in the non-coding portion of the gene. However, in some embodiments, the disease-associated repeat is in the coding region of the gene. In some embodiments, the disease-associated repeat is amplified from a normal state to a length that directly or indirectly contributes to or causes a genetic disease. In some embodiments, the disease-associated repeat is in RNA (e.g., RNA transcript). In some embodiments, the disease-associated repeat is in DNA (e.g., chromosome, plasmid). In some embodiments, the disease-associated repeats are expanded in the chromosomes of germ cells. In some embodiments, the disease-associated repeats are expanded in the chromosomes of somatic cells. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the congenital onset of the disease. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the onset of the disease in childhood. In some embodiments, the disease-associated repeats are expanded to the number of repeat units associated with the onset of the disease in adults. In DM1, the DMPK gene comprises a disease-associated repeat of the CTG unit.

[0097] DMPK: The term "DMPK" as used herein refers to a gene encoding myotonic protein kinase (also known as myotonic dystrophy protein kinase or dystrophic myotonic protein kinase) which is a serine / threonine protein kinase. Substrates of this enzyme may include myogenin, the beta subunit of the L-type calcium channel, and phospholemman. In some embodiments, DMPK may be a human gene (Gene ID: 1760), a non-human primate gene (e.g., Gene ID: 456139, Gene ID: 715328, Gene ID: 102125829), or a rodent gene (e.g., Gene ID: 13400). In humans, 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 protein isoforms have been characterized (e.g., as annotated with the following GenBank RefSeq accession numbers: NM_001081563.2, NM_004409.4, NM_001081560.2, NM_001081562.2, NM_001288764.1, NM_001288765.1, and NM_001288766.1).

[0098] DMPK allele: The term "DMPK allele" as used herein refers to any 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 myotonic protein kinase that retains its normal and typical functions. In some embodiments, the DMPK allele may comprise one or more disease-associated repeat expansions. In some embodiments, a normal subject has two DMPK alleles comprising 5 to 37 repeat units. In some embodiments, the number of CTG repeat units in subjects with DM1 is from about 50 to about 3,000 or more, and the higher the number of repeats, the higher the severity of the disease. In some embodiments, a mildly affected DM1 subject has at least one DMPK allele having 50 to 150 repeat units. In some embodiments, a subject with typical DM1 has at least one DMPK allele having 100 to 1,000 or more repeat units. In some embodiments, a subject with congenital onset DM1 may have at least one DMPK allele comprising more than 2,000 repeat units.

[0099] Framework: The term "framework" or "framework sequence" used herein refers to the remaining sequence of the variable region minus CDR. Since the exact definition of CDR sequences can be determined by different systems, the meaning of framework sequences has different interpretations accordingly. 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 distinguish the framework on the light chain and the heavy chain into four subregions (FR1, FR2, FR3 and FR4) on each chain, wherein CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. In the case where a specific subregion is not designated as FR1, FR2, FR3 or FR4, the framework region mentioned by others represents the FR of the combination in the variable region of a single naturally occurring immunoglobulin chain. The FR used herein represents one of the four subregions, and a plurality of FRs represent two or more of the four subregions constituting the framework region. Human heavy chain and light chain receptor sequences are known in the art. In one embodiment, acceptor sequences known in the art can be used in the antibodies disclosed herein.

[0100] Human antibody: The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR, and particularly in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) have been grafted onto human framework sequences.

[0101] Humanized antibody: The term "humanized antibody" refers to an antibody comprising heavy and light chain variable region sequences from non-human species (e.g., mouse) but wherein at least a portion of the VH and / or (e.g., and) VL sequences have been altered to be more "human-like" (i.e., more similar to human germline variable sequences). One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences to replace the corresponding non-human CDR sequences. In one embodiment, humanized anti-transferrin receptor antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining mouse anti-transferrin receptor monoclonal antibodies using conventional hybridoma technology and then humanizing using in vitro genetic engineering, such as those disclosed in PCT Publication No. WO2005 / 123126A2 of Kasaian et al.

[0102] Kabat numbering: The terms "Kabat numbering," "Kabat definition," and "Kabat notation" are used interchangeably herein. These terms, recognized in the art, refer to a system for numbering amino acid residues in the variable regions of the heavy and light chains of an antibody, or antigen-binding portion thereof, that are more variable (i.e., hypervariable) than other amino acid residues (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 2001-0306). No.91-3242). For the heavy chain variable region, the hypervariable region of CDR1 is from amino acids 31 to 35, the hypervariable region of CDR2 is from amino acids 50 to 65, and the hypervariable region of CDR3 is from amino acids 95 to 102. For the light chain variable region, the hypervariable region of CDR1 is from amino acids 24 to 34, the hypervariable region of CDR2 is from amino acids 50 to 56, and the hypervariable region of CDR3 is from amino acids 89 to 97.

[0103] Myotonic dystrophy (DM): The term "myotonic dystrophy (DM)" as used herein refers to a genetic disease caused by mutations in the DMPK gene or the CNBP (ZNF9) gene, characterized by muscle loss, muscle weakness, and muscle function. Two types of the disease have been described, myotonic dystrophy type 1 (DM1) and myotonic dystrophy type 2 (DM2). DM1 is associated with an expansion of CTG trinucleotide repeats in the 3' noncoding region of DMPK. DM2 is associated with an expansion of CCTG tetranucleotide repeats in the first intron of ZNF9. In both DM1 and DM2, nucleotide expansion leads to toxic RNA repeats that are able to form hairpin structures that bind to key intracellular proteins (e.g., muscleblind-like proteins) with high affinity. Myotonic dystrophy, the genetic basis of the disease, and associated symptoms are described in the art (see, e.g., Thornton, CA, "Myotonic Dystrophy" Neurol Clin. (2014), 32(3):705-719.; and Konieczny et al. "Myotonic dystrophy: candidate small molecule therapeutics" Drug Discovery Today (2017), 22:11). In some embodiments, the subject is born with a DM1 variant known as congenital myotonic dystrophy. Symptoms of congenital myotonic dystrophy are present from birth and include weakness of all muscles, breathing problems, club feet, developmental delays, and intellectual disability. DM1 is associated with Online Mendelian Inheritance in Man (OMIM) Entry #160900. DM2 is associated with OMIM Entry #602668.

[0104] Oligonucleotide: The term "oligonucleotide" as used herein refers to an oligonucleotide compound up to 200 nucleotides in length. Some examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), microRNA, spacer polymers, mixed polymers, phosphorodiamidate morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., Cas9 guide RNA), etc. Oligonucleotides can be single-stranded or double-stranded. In some embodiments, the oligonucleotide may include one or more modified nucleosides (e.g., 2'-O-methyl sugar modifications, purine or pyrimidine modifications). In some embodiments, the oligonucleotide may include one or more modified internucleoside linkages. In some embodiments, the oligonucleotide may include one or more phosphorothioate linkages, which may be Rp or Sp stereochemical conformations.

[0105] Complementary region: The term "complementary region" as used herein refers to a nucleotide sequence, such as an oligonucleotide, that is fully complementary to a homologous nucleotide sequence, such as a target nucleic acid, such that the two nucleotide sequences can anneal to each other under physiological conditions (e.g., in a cell). In some embodiments, the complementary region is completely complementary to the homologous nucleotide sequence of the target nucleic acid. However, in some embodiments, the complementary region is partially complementary to the homologous nucleotide sequence of the target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementary). In some embodiments, the complementary region comprises 1, 2, 3 or 4 mismatches compared to the homologous nucleotide sequence of the target nucleic acid.

[0106] Specific binding: As used herein, the term "specific binding" refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from a suitable control in a binding assay or other binding context. With respect to antibodies, the term "specific binding" refers to the ability of an antibody to bind to a specific antigen with a degree of affinity or avidity that enables the antibody to be used to distinguish the specific antigen from other antigens, e.g., to a degree that allows preferential targeting of certain cells (e.g., muscle cells) by binding to an antigen as described herein. In some embodiments, if the K of an antibody binding to a target is D 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 -13 M or less, the antibody specifically binds to the target. In some embodiments, the antibody specifically binds to the transferrin receptor (eg, an epitope of the apical domain of the transferrin receptor).

[0107] Subject: The term "subject" as used herein refers to a mammal. In some embodiments, the subject is a non-human primate or a rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, such as a human patient suffering from or suspected of having a disease. In some embodiments, the subject is a human patient suffering from or suspected of having a disease caused by a disease-associated repeat expansion (e.g., a disease-associated repeat expansion in a DMPK allele).

[0108] Transferrin receptor: The term "transferrin receptor" (also referred to as TFRC, CD71, p90, TFR or TFR1) as used herein refers to a cell surface receptor that binds transferrin to promote internalization of iron uptake by endocytosis. In some embodiments, the transferrin receptor may be of human origin (NCBI Gene ID 7037), non-human primate origin (e.g., NCBI Gene ID 711568 or NCBI Gene ID 102136007), or rodent origin (e.g., NCBI Gene ID 22042). In addition, a variety of human transcript variants encoding different isoforms of the receptor have been characterized (e.g., as annotated with the following GenBank RefSeq accession numbers: NP_001121620.1, NP_003225.2, NP_001300894.1, and NP_001300895.1).

[0109] 2'-modified nucleosides: As used herein, the terms "2'-modified nucleosides" and "2'-modified ribonucleosides" are used interchangeably and refer to nucleosides having a modified sugar moiety at the 2' position. In some embodiments, the 2'-modified nucleoside is a 2'-4' bicyclic nucleoside, wherein the 2' and 4' positions of the sugar are bridged (e.g., bridged 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., wherein the 2' position of the sugar moiety is substituted. Some non-limiting examples of 2'-modified nucleosides include: 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamido (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 nucleotides and oligonucleotides comprising 2'-modified nucleosides having increased affinity for a target sequence relative to an unmodified oligonucleotide. Some examples of the structures of 2'-modified nucleosides are provided below:

[0110]

[0111] These examples are shown with phosphate groups, but any internucleoside linkages are contemplated to be between 2'-modified nucleosides.

[0112] Ranges: All ranges provided in this disclosure are inclusive of the endpoints.

[0113] Compound

[0114] Provided herein are complexes comprising targeting agents (e.g., antibodies) covalently linked to oligonucleotides. In some embodiments, complexes comprise muscle targeting antibodies covalently linked to one or more oligonucleotides. In some embodiments, oligonucleotides are antisense oligonucleotides (e.g., reducing the level of mutant or wild-type DMPK RNA, or the activity of DMPK gene products) targeting DMPK RNA to reduce the expression or activity of DMPK.

[0115] The complexes described herein generally comprise a linker that covalently links an antibody described herein (eg, an anti-TfR1 antibody) to an oligonucleotide (eg, an oligonucleotide comprising a 5'-XYZ-3' configuration). The linker comprises at least one covalent bond.

[0116] In some embodiments, the complex described herein comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 independently comprises a compound comprising an oligonucleotide (eg, an oligonucleotide comprising a 5'-XYZ-3' configuration), and R 2 comprising an antibody (eg, an anti-TfR1 antibody), and wherein in each complex, n1 is independently an integer (eg, 1 or greater) representing the number of R 1 In some embodiments, each R 1 In some embodiments, each R 1 In some embodiments, R 2 The antibody (e.g., anti-TfR1 antibody) comprises a heavy chain comprising a heavy chain variable region (VH) and a heavy chain constant region and a light chain comprising a light chain variable region (VL) and a light chain constant region. In some embodiments, each R in the complex 1 Independently with R 2 The different amino acid residues (e.g., lysine or cysteine) of the polypeptide are covalently linked.

[0117] In some embodiments, in each complex, n1 is independently an integer (e.g., 1 or greater). In some embodiments, the antibody comprises a sequence as shown in Table 2. For example, in some embodiments, the antibody comprises a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprises a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv. In some embodiments, the antibody is a Fab fragment.

[0118] In some embodiments, the value of n1 of each or any complex (e.g., any complex of any of the compositions or formulations disclosed herein) is an integer up to the number of amino acid residues (e.g., the number of lysine residues) desired or targeted for conjugation in the antibody. In some embodiments, in each complex, the value of n1 is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27. In some embodiments, in each complex, the value of n1 is independently selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26. In some embodiments, in each complex, the value of n1 is independently 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3. In some embodiments, the average value of n1 of the complexes in the composition is 1 to 5 (e.g., 1 to 5, 1 to 4, 1 to 3, 3 to 5, or 1 to 2). In some embodiments, the compositions described herein comprise a complex comprising formula (I): [R 1 ] n1 -R 2 wherein n1 is 0. In some embodiments, the average value of n1 of the complexes in the composition is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2). In some embodiments, in each complex type, n1 is independently an integer of 1 or greater, which represents the number of R 1 The invention relates to a method for preparing an aqueous solution of the present invention and wherein different composite types of the composition are characterized by having different n1 values ​​(e.g., n1 values ​​of 1 to 27, 1 to 26, 1 to 25, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3).

[0119] In some embodiments, compositions comprising a plurality of different complexes are provided (e.g., formulations comprising tris(hydroxymethyl)aminomethane and / or sucrose, as described herein). In some embodiments, a plurality of different complexes comprise a common targeting agent (e.g., an antibody) and a common oligonucleotide (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration, such as a DMPK targeting oligonucleotide). In such embodiments, different complex types are characterized by having different numbers of oligonucleotides covalently attached to the antibody. For example, in some embodiments, compositions comprising a plurality of complexes are provided, the complexes comprising formula (I): [R 1 ] n1 -R 2 The structure of each R 1 independently comprises a compound comprising an oligonucleotide (e.g., a DMPK targeting oligonucleotide), and R 2 comprising an antibody (eg, an anti-TfR1 antibody), and wherein n1 is a 1 In some embodiments, n1 is an integer representing the number of instances of the composition, and different complexes of the composition can have different n1 values ​​(e.g., n1 values ​​are 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, in the complexes in the composition, n1 is independently an integer. In some embodiments, the average value of n1 of the complexes in the composition is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2). In some embodiments, the compositions described herein comprise complexes wherein n1 is 0.

[0120] In some embodiments, the compositions described herein include antibodies that are not conjugated to oligonucleotides (e.g., in trace amounts) and antibodies that are conjugated to one or more oligonucleotides. In some embodiments, the antibodies that are not conjugated to oligonucleotides may be referred to as formula (I): 1 ] n1 -R 2 A compound of the structure wherein n1 is 0. Thus, in some embodiments, the composition for administration to a subject in the methods described herein comprises formula (I): [R 1 ] n1 -R 2A compound (e.g., a complex) of the structure wherein each R 1 independently comprising a group containing an oligonucleotide, R 2 comprising an antibody, and n1 is independently an integer of 0 or greater, which reflects the number of R 1 In some embodiments, the composition wherein n1 is 1 or greater has formula (I): [R 1 ] n1 -R 2 Compared to all compounds of the structure, the fraction of compounds of the structure in which n1 is 0 in the composition is less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05% or less than 0.01%. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0121] In some embodiments, R 1 Each instance of is covalently linked to a different amino acid residue of the antibody. 1 The covalently linked amino acid comprises an ε-amino group (e.g., lysine, arginine). However, in some embodiments, 1 The covalently linked amino acid is cysteine. 1 Directly covalently linked to an amino acid residue of the antibody. However, in some embodiments, R 1 Indirect covalent attachment to an amino acid residue of the antibody, for example, covalent attachment to a glycosylation site on an amino acid. 1 It is not covalently linked to amino acid residues present in the CDR regions of the antibody.

[0122] In some embodiments, a complex provided herein (eg, in a composition or formulation described herein) comprises formula (I): [R 1 ] n1 -R 2 The structure of R 1 Each instance of independently comprises a group of formula (Ia):

[0123]

[0124] Where R 3 comprising an oligonucleotide, such as an oligonucleotide comprising a 5'-XYZ-3' configuration; and R 1 At the connection point A and R 2 In some embodiments, R 2 The invention comprises an antibody comprising a sequence as shown in Table 2. For example, in some embodiments, R 2 The invention comprises an antibody comprising a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprising a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 The invention comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2In some embodiments, R3 comprises an oligonucleotide comprising the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). In some embodiments, R 3 An oligonucleotide is included, which includes the structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MoE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents thiophosphate internucleoside linkage.

[0125] In some embodiments, the complexes provided herein (eg, in a composition or formulation described herein) comprise formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Containing a group of formula (Ib):

[0126]

[0127] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-O-methoxyethyl (MoE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21), wherein n1 is an integer (e.g., 1 or greater) representing the number of R in each complex. 1 The number of instances, and each R 1 At the connection point A and R 2 In some embodiments, R 2 The invention comprises an antibody comprising a sequence as shown in Table 2. For example, in some embodiments, R 2The invention comprises an antibody comprising a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprising a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 The invention comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 Contains an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv.

[0128] In some embodiments, the complexes provided herein (eg, in a composition or formulation described herein) comprise formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Containing a group of formula (Ic):

[0129]

[0130] Where R1 At the connection point A and R 2 In some embodiments, R 2 The invention comprises an antibody comprising a sequence as shown in Table 2. For example, in some embodiments, R 2 The invention comprises an antibody comprising a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 Contains an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv.

[0131] In some embodiments, a complex provided herein (eg, in a composition or formulation described herein) comprises the structure of Formula (Id):

[0132]

[0133] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-O-methoxyethyl (MoE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); wherein R 2 The invention comprises an antibody comprising a sequence as shown in Table 2; wherein n1 is an integer (e.g., 1 or greater) representing the number of instances of the group enclosed by square brackets, wherein each instance of the group enclosed by square brackets is covalently linked to a different amino acid residue of the antibody, optionally wherein each different amino acid residue is lysine. In some embodiments, R 2 The invention comprises an antibody comprising a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprising a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention also comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R2 The invention also includes an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 Contains an antibody that is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv.

[0134] In some embodiments, the complex described herein comprises a structure of Formula (A):

[0135]

[0136] (A), wherein y is 0 to 15 (e.g., 3) and z is 0 to 15 (e.g., 4). In some embodiments, the amide adjacent to the antibody (e.g., anti-TfR1 antibody) shown in structure (A) is produced by a reaction with an amine (e.g., lysine ε amine) of the antibody. In some embodiments, the complex described herein comprises an anti-TfR1 antibody (e.g., anti-TfR1 Fab) covalently linked to the 5' end of an oligonucleotide (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration) via a lysine of the antibody. In some embodiments, the antibody comprises a sequence as shown in Table 2. For example, in some embodiments, the antibody comprises a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprises a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv.

[0137] Antibody

[0138] In some embodiments, the complex described herein comprises an antibody that binds to human transferrin receptor 1 (TfR1). An exemplary human TfR1 amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, homo sapiens) is as follows:

[0139]

[0140]

[0141] Table 2 provides some examples of sequences of anti-TfR1 antibodies that can be used in the complexes provided herein.

[0142] Table 2. Some examples of anti-TfR1 antibody sequences

[0143]

[0144]

[0145] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO:1 (according to the IMGT definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO:2 (according to the IMGT definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO:3 (according to the IMGT definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO:4 (according to the IMGT definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO:5 (according to the IMGT definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO:6 (according to the IMGT definition system).

[0146] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO:7 (according to the Kabat definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO:8 (according to the Kabat definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO:9 (according to the Kabat definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO:10 (according to the Kabat definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO:11 (according to the Kabat definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO:6 (according to the Kabat definition system).

[0147] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain complementary determining region 1 (CDR-H1) of SEQ ID NO:12 (according to the Chothia definition system), a heavy chain complementary determining region 2 (CDR-H2) of SEQ ID NO:13 (according to the Chothia definition system), a heavy chain complementary determining region 3 (CDR-H3) of SEQ ID NO:14 (according to the Chothia definition system), a light chain complementary determining region 1 (CDR-L1) of SEQ ID NO:15 (according to the Chothia definition system), a light chain complementary determining region 2 (CDR-L2) of SEQ ID NO:5 (according to the Chothia definition system), and a light chain complementary determining region 3 (CDR-L3) of SEQ ID NO:16 (according to the Chothia definition system).

[0148] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain variable region (VH), which comprises no more than 25 amino acid variations in the framework region compared to the VH comprising the amino acid sequence of SEQ ID NO: 17 (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). As an alternative or supplement (e.g., supplement), the anti-TfR1 antibody of the present disclosure comprises a light chain variable region (VL), which comprises no more than 25 amino acid variations in the framework region compared to the VL comprising the amino acid sequence of SEQ ID NO: 18 (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).

[0149] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VH comprising an amino acid sequence in the framework region that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to a VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or additionally (e.g., supplementing), in some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VL comprising an amino acid sequence in the framework region that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to a VL comprising the amino acid sequence of SEQ ID NO: 18.

[0150] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 17. Alternatively or additionally (eg, supplemented), in some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO:18.

[0151] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the amino acid sequence of SEQ ID NO: 19. As an alternative or supplement (e.g., supplement), the anti-TfR1 antibody of the present disclosure comprises a light chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-TfR1 antibody of the present disclosure is a Fab comprising a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity with the amino acid sequence of SEQ ID NO: 19. Alternatively or additionally (e.g., supplementally), the anti-TfR1 antibody of the present disclosure is a Fab comprising a light chain comprising an amino acid sequence that is at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identical to the amino acid sequence of SEQ ID NO:20.

[0152] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. As an alternative or in addition (e.g., supplement), the anti-TfR1 antibody of the present disclosure comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the anti-TfR1 antibody of the present disclosure is a Fab comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. As an alternative or in addition (e.g., supplement), the anti-TfR1 antibody of the present disclosure is a Fab comprising a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0153] In some embodiments, the anti-TfR1 antibodies provided herein may have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also known as pyroglutamate (pyro-Glu) formation, may occur at the N-terminal glutamic acid (Glu) and / or glutamine (Gln) residues of the antibody during production. Therefore, it should be understood that antibodies designated as having sequences comprising N-terminal glutamic acid or glutamine residues encompass antibodies that have undergone pyroglutamate formation caused by post-translational modifications. In some embodiments, pyroglutamate formation occurs in the heavy chain sequence. In some embodiments, pyroglutamate formation occurs in the light chain sequence.

[0154] Oligonucleotides

[0155] In some embodiments, the oligonucleotide in the complex described herein is a single-stranded oligonucleotide. In some embodiments, the oligonucleotide can be used for targeting DMPK (e.g., for reducing the expression or activity of DMPK RNA, such as the level of mutant or wild-type DMPK RNA). In some embodiments, the oligonucleotide can be used for targeting DMPK RNA (e.g., for reducing the expression or activity of DMPK RNA, such as the level of mutant or wild-type DMPK RNA). In some embodiments, the oligonucleotide comprises a complementary region to DMPK RNA. In some embodiments, the oligonucleotide can be used to reduce the level of toxic DMPK with disease-related repeat expansion, such as in a subject suffering from or suspected of suffering from myotonic dystrophy. In some embodiments, the oligonucleotides are designed to direct RNase H-mediated degradation of a target DMPK RNA located in the nucleus of a cell, e.g., a muscle cell (e.g., a myotube) or a nervous system cell (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 desired toxicity characteristics. In some embodiments, the oligonucleotides are designed to have low complement activation and / or cytokine induction properties.

[0156] In some embodiments, the DMPK targeting oligonucleotides described herein are designed to cause RNase H-mediated degradation of DMPK mRNA. It should be understood that, in some embodiments, one form of oligonucleotide (e.g., antisense oligonucleotide) can be appropriately adapted to another form (e.g., siRNA oligonucleotide) by incorporating a functional sequence (e.g., antisense strand sequence) from one form into another form.

[0157] Some examples of oligonucleotides that can be used to target DMPK are provided in the following: U.S. Patent Application Publication 20100016215A1, which was published on January 1, 2010, and is entitled Compound And Method For Treating Myotonic Dystrophy; U.S. Patent Application Publication 20130237585A1, which was published on July 19, 2010, Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression; U.S. Patent Application Publication 20150064181A1, which was published on March 5, 2015, and is entitled "Antisense Conjugates For Decreasing Expression Of Dmpk"; U.S. Patent Application Publication 20150238627A1, which was published on August 27, 2015, and is entitled "Peptide-Linked Morpholino Antisense Oligonucleotides For Treatment Of Myotonic Dystrophy”; and U.S. Patent Application Publication No. 20160304877A1, published on October 20, 2016, entitled “Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (Dmpk) Expression”, the contents of each of which are incorporated herein in their entirety.

[0158] In some embodiments, the oligonucleotide may comprise a complementary region to the sequence shown below, which is an exemplary human DMPK gene sequence (Gene ID 1760; NM_001081560.2):

[0159]

[0160]

[0161]

[0162] In some embodiments, an oligonucleotide can comprise a complementary region to a sequence as shown below, which is an exemplary mouse DMPK gene sequence (Gene ID 13400; NM_001190490.1).

[0163]

[0164]

[0165] In some embodiments, the oligonucleotides may comprise complementary regions to DMPK gene sequences of multiple species (eg, selected from human, mouse, and a non-human species such as cynomolgus monkey).

[0166] In some embodiments, the oligonucleotide may comprise a complementary region to a mutant form of DMPK, such as a mutant form as reported in: Botta A. et al. "The CTG repeat expansion size correlates with the splicing defects observed in muscles from myotonic dystrophy type 1 patients." J Med Genet. 2008 Oct; 45(10): 639-46.; and Machuca-Tzili L. et al. "Clinical and molecular aspects of the myotonic dystrophies: a review." Muscle Nerve. 2005 Jul; 32(1): 1-18.; the contents of each of which are incorporated herein in their entirety.

[0167] In some embodiments, the oligonucleotides provided herein are antisense oligonucleotides targeting DMPK. In some embodiments, the oligonucleotides targeting DMPK are any antisense oligonucleotides targeting DMPK, as described in U.S. Patent Application Publication US20160304877A1, which was disclosed on October 20, 2016, entitled "Compounds And Methods For Modulation Of Dystrophia Myotonica-Protein Kinase (DMPK) Expression," which is incorporated herein by reference). In some embodiments, the DMPK targeting oligonucleotide targets the DMPK gene sequence (SEQ ID NO: 24) as shown in Genbank Accession No. NM_001081560.2 or the DMPK gene sequence (SEQ ID NO: 26) as shown in Genbank Accession No. NG_009784.1.

[0168] In some embodiments, the DMPK targeting oligonucleotides provided herein comprise a nucleotide sequence comprising a complementary region to a target region, wherein the target region is at least 8 consecutive nucleotides (e.g., at least 8, at least 9, at least 10, at least 12, at least 14, at least 16, at least 18, at least 20, or more consecutive nucleotides) of SEQ ID NO: 24.

[0169] In some embodiments, the DMPK targeting oligonucleotides provided herein are 10 to 35 (e.g., 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 35, 20 to 30, 13 to 18, 14 to 17, 15 to 18, 20 to 30, 15 to 17, 27 to 30, 25 to 35, or 30 to 35) nucleotides in length. In some embodiments, the DMPK targeting oligonucleotides provided herein are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides in length, optionally 15 to 30 or 16 nucleotides in length. In some embodiments, a DMPK targeting oligonucleotide provided herein is 16 nucleotides in length.

[0170] In some embodiments, the DMPK targeting oligonucleotides provided herein comprise a complementary region of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more) consecutive nucleotides of DMPK RNA.

[0171] In some embodiments, the DMPK targeting oligonucleotide provided herein comprises a complementary region of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more) consecutive nucleotides of the DMPK sequence shown in SEQ ID NO: 24 or 25.

[0172] In some embodiments, the DMPK targeting oligonucleotides provided herein comprise a complementary region of at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, or 16) consecutive nucleotides of the target sequence shown in SEQ ID NO: 22 (TGACTGGTGGGCGCTG). In some embodiments, the oligonucleotides useful for targeting DMPK comprise at least 8 (e.g., at least 8, 9, 10, 11, 12, 13, 14, 15, or 16) consecutive nucleotides of the sequence shown in SEQ ID NO: 21 (CAGCGCCCACCAGUCA). In some embodiments, the oligonucleotides useful for targeting DMPK comprise the nucleotide sequence of SEQ ID NO: 21.

[0173] In some embodiments, the DMPK targeting oligonucleotide comprises a 5'-XYZ-3' configuration. An oligonucleotide comprising a 5'-XYZ-3' configuration may refer to such a chimeric antisense compound: wherein a spacer region having a plurality of nucleosides supporting RNase H cleavage is located between the flanking regions having one or more nucleosides, wherein the nucleosides comprising the spacer region are chemically different from one or more nucleosides comprising the flanking region. In some embodiments, the oligonucleotides described herein (e.g., the DMPK targeting oligonucleotides described herein) comprise a 5'-XYZ-3' configuration, wherein X and Z are flanking regions around the spacer region Y. In some embodiments, the spacer region Y comprises one or more 2'-deoxyribonucleosides. In some embodiments, each nucleoside in the spacer region Y is a 2'-deoxyribonucleoside, and the flanking region X or the flanking region Z do not comprise any 2'-deoxyribonucleosides.

[0174] In some embodiments, the spacer Y comprises a continuous segment of 6 or more 2'-deoxyribonucleosides, which can recruit RNases, such as RNase H. In some embodiments, the oligonucleotide binds to the target nucleic acid, recruits RNases at this point, and can then cut the target nucleic acid. In some embodiments, each of the flanking regions X and Z comprises one or more modified nucleosides. In some embodiments, each of the flanking regions X and Z comprises one or more high-affinity modified nucleosides, such as one to six high-affinity modified nucleosides. Some 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 length of the flanking regions X and Z can be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides. The flanking regions X and Z can have similar lengths or different lengths. In some embodiments, spacer Y may comprise a nucleotide sequence of 5 to 20 nucleotides, 5 to 15 nucleotides, 5 to 12 nucleotides, or 6 to 10 nucleotides in length.

[0175] In some embodiments, spacer Y comprises one or more unmodified internucleoside linkages. In some embodiments, one or both flanking regions X and Z each independently comprise a phosphorothioate internucleoside linkage (e.g., a phosphorothioate internucleoside linkage or other linkage) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, spacer Y and both flanking regions X and Z each independently comprise a modified internucleoside linkage (e.g., a phosphorothioate internucleoside linkage or other linkage) between at least two, at least three, at least four, at least five or more nucleotides.

[0176] In some embodiments, the length of the spacer Y in the spacer polymer is 5 to 20 nucleosides. For example, the length of the spacer Y can be 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15 or 15 to 20 nucleosides. In some embodiments, the length of the spacer Y is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 nucleosides. In some embodiments, each nucleoside in the spacer Y is a 2'-deoxyribonucleoside. In some embodiments, all nucleosides in the spacer Y are 2'-deoxyribonucleosides. In some embodiments, one or more nucleosides in the spacer Y are modified nucleosides (for example, 2' modified nucleosides, such as those described herein). In some embodiments, one or more cytosines in the spacer Y are optionally 5-methyl-cytosines. In some embodiments, each cytosine in the spacer Y is 5-methyl-cytosine.

[0177] In some embodiments, the flanking region X of the oligonucleotide (X in a 5'-XYZ-3' configuration) and the flanking region Z of the oligonucleotide (Z in a 5'-XYZ-3' configuration) are independently 1 to 20 nucleosides long. For example, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide 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 long. In some embodiments, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide are independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides long. In some embodiments, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide have the same length. In some embodiments, the flanking region X of the oligonucleotide and the flanking region Z of the oligonucleotide have different lengths. In some embodiments, the flanking region X of the oligonucleotide is longer than the flanking region Z of the oligonucleotide. In some embodiments, the flanking region X of the oligonucleotide is shorter than the flanking region Z of the oligonucleotide.

[0178] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide) comprises the following 5'-XYZ-3' configuration:

[0179] 5-10-5，4-12-4，3-14-3，2-16-2，1-18-1，3-10-3，2-10-2，1-10-1，2-8-2，4-6-4，3-6-3，2-6-2，4-7-4，3-7-3，2-7-2，4-8-4，3-8-3，2-8-2，1-8-1，2-9-2，1-9-1，2-10-2，1-10-1，1-12-1，1-16-1，2-15-1，1-15-2，1-14-3，3-14-1，2-14-2，1-13-4，4-13-1，2-13-3，3-13-2，1-12-5，5-12-1，2-12-4，4-12-2，3-12-3，1-11-6.6-11-1，2-11-5，5-11-2，3-11-4，4-11-3，1-17-1，2-16-1.1-16-2，1-15-3.3-15-1，2-15-2.1-14-4，4-14-1，2-14-3，3-14-2，1-13-5，5-13-1，2-13-4，4-13-2，3-13-3，1-12-6.6-12-1，2-12-5，5-12-2，3-12-4，4-12-3，1-11-7，7-11-1，2-11-6，6-11-2，3-11-5，5-11-3，4-11-4，1-18-1，1-17-2，2-17-1，1-16-3，1-16-3，2-16-2，1-15-4，4-15-1，2-15-3.3-15-2，1-14-5，5-14-1，2-14-4，4-14-2，3-14-3，1-13-6，6-13-1，2-13-5，5-13-2，3-13-4，4-13-3，1-12-7，7-12-1，2-12-6，6-12-2，3-12-5，5-12-3，1-11-8，8-11-1，2-11-7，7-11-2，3-11-6，6-11-3，4-11-5，5-11-4，1-18-1，1-17-2，2-17-1，1-16-3，3-16-1，2-16-2，1-15-4，4-15-1，2-15-3，3-15-2，1-14-5，2-14-4，4-14-2，3-14-3，1-13-6，6-13-1，2-13-5，5-13-2,3-13-4，4-13-3，1-12-7，7-12-1,2-12-6，6-12-2，3-12-5，5-12-3，1-11-8，8-11-1，2-11-7，7-11-2，3-11-6，6-11-3，4-11-5，5-11-4，1-19-1，1-18-2.2-18-1，1-17-3.3-17-1，2-17-2，1-16-4，4-16-1，2-16-3，3-16-2，1-15-5，2-15-4，4-15-2，3-15-3，1-14-6，6-14-1，2-14-5，5-14-2，3-14-4，4-14-3，1-13-7，7-13-1，2-13-6，6-13-2.3-13-5，5-13-3，4-13-4，1-12-8，8-12-1，2-12-7，7-12-2，3-12-6，6-12-3，4-12-5，5-12-4，2-11-8，8-11-2，3-11-7，7-11-3，4-11-6，6-11-4，5-11-5，1-20-1，1-19-2，2-19-1，1-18-3，3-18-1，2-18-2，1-17-4，4-17-1，2-17-3，3-17-2，1-16-5，2-16-4，4-16-2，3-16-3，1-15-6.6-15-1，2-15-5，5-15-2.3-15-4，4-15-3，1-14-7，7-14-1，2-14-6，6-14-2，3-14-5，5-14-3，4-14-4，1-13-8，8-13-1，2-13-7，7-13-2，3-13-6.6-13-3, 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 ,7-12-4,5-12-6,6-12-5,4-11-8,8-11-4,5-11-7,7-11-5,6-11-6,1-22-1,1-21-2,2-21-1,1-21-3,3-20-1,2-20-2,1-19-4,4-19-1,2-19-3,3-19-2,1-18-5,2-18-4,4-18-2,3-18-3,1-17-6,6-17-1,2-17-5,5-17-2,3-17-4,4-17-3,1-16-7,7-16-1,2-16-6,6-16-2,3-1 6-5, 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.

[0180] The numbers indicate the number of nucleosides in the X, Y and Z regions, respectively, of an oligonucleotide comprising a 5'-XYZ-3' configuration.

[0181] In some embodiments, one or more nucleosides in the flanking region X of the oligonucleotide (X in a 5'-XYZ-3' configuration) or the flanking region Z of the oligonucleotide (Z in a 5'-XYZ-3' configuration) are modified nucleosides (e.g., high affinity modified nucleosides). In some embodiments, the modified nucleosides (e.g., high affinity modified nucleosides) are 2'-modified nucleosides. In some embodiments, the 2'-modified nucleosides are 2'-4' bicyclic nucleosides or non-bicyclic 2'-modified nucleosides. In some embodiments, the high affinity modified nucleoside is a 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2'-modified nucleoside (e.g., 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethyloxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamido (2'-O-NMA)).

[0182] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein 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 (the 5'-most position is position 1) of X is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein 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 (the 5'-most position is position 1) of Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 2 to 7 (e.g., 2, 3, 4, 5, 6, or 7) nucleosides in length, and Y is 6 to 10 (e.g., 6, 7, 8, 9, or 10) nucleosides in length, wherein at least one but not all of the 1st, 2nd, 3rd, 4th, 5th, 6th, or 7th positions (the 5'-most position is the 1st position) in X 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 (the 5'-most position is position 1) of X and Z are non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), wherein the remaining nucleosides in both X and Z are 2'-4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside.

[0183] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide) is 10 to 20 nucleosides (e.g., 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleosides) in length, comprises a complementary region to at least 8 consecutive nucleosides (e.g., at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16 consecutive nucleosides) of SEQ ID NO: 22 (TGACTGGTGGGCGCTG), and comprises a 5'-XYZ-3' configuration, wherein X comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one nucleoside in X is a 2'-modified nucleoside (e.g., 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2'-MOE modified nucleoside, 2 '-O-Me modified nucleosides, LNA, cEt or ENA); Y comprises 6 to 10 (e.g., 6, 7, 8, 9 or 10) linked 2'-deoxyribonucleosides, wherein each cytosine in Y is optionally and independently 5-methyl-cytosine; and Z comprises 3 to 5 (e.g., 3, 4 or 5) linked nucleosides, wherein at least one nucleoside in Z is a 2'-modified nucleoside (e.g., 2'-MOE modified nucleosides, 2'-O-Me modified nucleosides, LNA, cEt or ENA).

[0184] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises at least 8 consecutive nucleosides (e.g., at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or 16 consecutive nucleosides) of a nucleotide sequence of SEQ ID NO: 21 (CAGCGCCCACCAGUCA), and comprises a 5'-XYZ-3' configuration, wherein X comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one nucleoside in X is a 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside). In some embodiments, each thymine base (T) of the nucleotide sequence of the antisense oligonucleotide can be independently and optionally replaced by a uracil base (U), and each U can be independently and optionally replaced by a T.

[0185] In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises the nucleotide sequence of SEQ ID NO: 21, and comprises a 5'-XYZ-3' configuration, wherein X comprises 3 to 5 (e.g., 3, 4, or 5) linked nucleosides, wherein at least one nucleoside 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, wherein 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, wherein at least one nucleoside 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) of the nucleotide sequence of the antisense oligonucleotide can be independently and optionally replaced by a uracil base (U), and each U can be independently and optionally replaced by a T.

[0186] In some embodiments, X comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside), and / or (e.g., and) Z comprises at least one 2'-4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) and at least one non-bicyclic 2'-modified nucleoside (e.g., a 2'-MOE modified nucleoside or a 2'-O-Me modified nucleoside).

[0187] 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.

[0188] In some embodiments, the nucleosides of oligonucleotides are linked together by phosphorothioate internucleoside linkages, phosphodiester internucleoside linkages or their combinations. In some embodiments, oligonucleotides only comprise phosphorothioate internucleoside linkages connecting each nucleoside (i.e., oligonucleotides comprise complete phosphorothioate backbones). In some embodiments, oligonucleotides comprise at least one phosphorothioate internucleoside linkage. In some embodiments, oligonucleotides comprise a mixture of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages. In some embodiments, oligonucleotides only comprise phosphorothioate internucleoside linkages connecting every pair of 2'-deoxyribonucleosides, and a mixture of phosphorothioate internucleoside linkages and phosphodiester internucleoside linkages connecting the remaining nucleosides.

[0189] In some embodiments, the oligonucleotide comprises a 5'-XYZ-3' configuration of LLEE-(D)8-EELL, wherein "E" is a 2'-MOE modified ribonucleoside; "L" is LNA; "D" is a 2'-deoxyribonucleoside; and "10" or "8" is the number of 2'-deoxyribonucleosides in Y, and wherein the oligonucleotide comprises phosphorothioate internucleoside linkages, phosphodiester internucleoside linkages, or a combination thereof.

[0190] In some embodiments, each cytidine (e.g., 2'-modified cytidine) in X and / or Z of the oligonucleotide is optionally and independently 5-methyl-cytidine, and / or each uridine (e.g., 2'-modified uridine) in X and / or Z is optionally and independently 5-methyl-uridine.

[0191] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide described herein) comprises a 5'-XYZ-3' configuration and comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). In some embodiments, an oligonucleotide described herein (e.g., a DMPK targeting oligonucleotide described herein) comprises a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-O-methoxyethyl (MOE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage.

[0192] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide described herein) comprises the structure of Formula (Ie):

[0193]

[0194] In some embodiments, an oligonucleotide described herein (eg, a DMPK targeting oligonucleotide described herein) can be in the form of a salt, for example, a sodium salt, a potassium salt, or a magnesium salt.

[0195] In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of an oligonucleotide is optionally conjugated to an amine group 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, there is a phosphodiester linkage between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of an oligonucleotide described herein is covalently linked to a spacer, and the spacer 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 -,-NRAC(=O)O-,-NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-,-S(O)2NR A , -NR A S(O)2-, or a combination thereof; each R A is independently hydrogen or substituted or unsubstituted alkyl. In certain embodiments, the spacer is substituted or unsubstituted alkylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-or-C(=O)N(R A )2, or a combination thereof.

[0196] In some embodiments, the 5' or 3' nucleoside of the oligonucleotide is conjugated to a compound of the formula -NH2-(CH2)n-, wherein n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11 or 12. In some embodiments, in the formula -NH2-(CH2) n - There is a phosphodiester linkage between the compound and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the compound of 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.

[0197] In some embodiments, the oligonucleotide is conjugated to a targeting agent, such as a muscle targeting agent (eg, an anti-TfR1 antibody), such as via an amine group of a lysine of the targeting agent.

[0198] In some embodiments, it is understood that methylation of the nucleobase uracil at the C5 position forms thymine. Therefore, in some embodiments, a nucleotide or nucleoside having a C5 methylated uracil (or 5-methyl-uracil) can be equivalently identified as a thymine nucleotide or nucleoside.

[0199] In some embodiments, any one or more thymine bases (T) in any oligonucleotide provided herein may independently and optionally be a uracil base (U), and / or any one or more U in the oligonucleotide provided herein (e.g., the oligonucleotide shown in SEQ ID NO: 21) may independently and optionally be T.

[0200] Composition

[0201] In some embodiments, the composition comprising the complex (i.e., multiple complexes) is prepared in a manner suitable for the methods described herein. In some embodiments, the composition comprising the muscle-targeted complex is delivered to the object in such a manner that degradation is minimized, delivery is promoted and / or (e.g., and) uptake is provided or other beneficial properties are provided for the complex in the composition. Therefore, in some embodiments, the composition comprising the complex (e.g., a plurality of complexes comprising oligonucleotides covalently linked to Fab) comprises tris (hydroxymethyl) aminomethane and / or sucrose. In some embodiments, in an aqueous solution, the composition comprising the muscle-targeted complex (e.g., a complex comprising oligonucleotides covalently linked to Fab) comprises tris (hydroxymethyl) aminomethane and / or sucrose. In some embodiments, the composition comprising multiple complexes, tris (hydroxymethyl) aminomethane and sucrose can be lyophilized (e.g., for storage). In some embodiments, the lyophilized composition can be reconstituted (e.g., with water) to be applied to the object. In some embodiments, the composition comprising multiple complexes, tris (hydroxymethyl) aminomethane and sucrose can be frozen (e.g., for storage). In some embodiments, the frozen composition can be thawed prior to administration to a subject, e.g., to produce an aqueous solution. The composition can be suitably prepared (e.g., in an aqueous solution, in a frozen composition, or in a lyophilized composition) so that when it is administered to a subject, whether administered to the immediate environment of the target cells or systemically, a sufficient amount of the complex enters the target muscle cells.

[0202] In some embodiments, the composition for administering to the subject in the methods described herein (e.g., in aqueous solution) comprises a complex (i.e., multiple complexes), each of which comprises an oligonucleotide covalently linked to an antibody (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration). In some embodiments, the composition for administering to the subject in the methods described herein (e.g., in aqueous solution) comprises a complex, wherein each complex comprises an oligonucleotide covalently linked to an anti-TfR1 antibody (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration), optionally wherein the antibody of such a complex comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as shown in Table 2, and in addition, in some embodiments, wherein the composition further comprises tris(hydroxymethyl)aminomethane and sucrose. In some embodiments, the antibody is an anti-TfR1Fab.

[0203] In some embodiments, the composition for administration to a subject in the methods described herein (e.g., in aqueous solution) comprises a complex (i.e., a plurality of complexes) comprising Formula (I): [R 1 ] n1 -R 2 The structure of each R 1 independently comprises a compound containing an oligonucleotide (eg, an oligonucleotide containing a 5'-XYZ-3' configuration) and R 2 Covalently linked, where R 2 comprising an antibody (eg, an anti-TfR1 antibody), and wherein in each complex, n1 is independently an integer of 1 or greater, which represents the number of R 1 The number of instances of .

[0204] In some embodiments, the value of n1 for each complex in the composition is independently and optionally from 1 up to antibody (R 2) is an integer of the number of amino acid residues (e.g., the number of lysine residues) desired or targeted for conjugation in the composition. In some embodiments, the value of n1 for each complex in the composition is independently and optionally selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27. In some embodiments, the value of n1 for each complex in the composition is independently and optionally selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, and 26. In some embodiments, the value of n1 for each complex in the composition is independently and optionally selected from an integer of 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3. In some embodiments, the average value of n1 of the complexes in the composition is 1-2, 1-3, 1-5, 1-10, 1-26, or 1-27.

[0205] In some embodiments, the composition for administration to a subject in the methods described herein comprises an unconjugated antibody (e.g., in trace amounts) and an antibody conjugated to one or more oligonucleotides. In some embodiments, the unconjugated antibody may be referred to as comprising formula (I): 1 ] n1 -R 2 A compound of the structure wherein n1 is 0. Therefore, in some embodiments, the composition for administration to a subject in the methods described herein comprises a compound of formula (I): [R 1 ] n1 -R 2 A compound (e.g., a complex) of the structure wherein each R 1 independently comprising a group containing an oligonucleotide, R 2 The antibody comprises an antibody and n1 is independently an integer of 0 or greater, which reflects the number of R 1 In some embodiments, all compounds having formula (I) in which n1 is 1 or greater: 1 ] n1 -R 2Compared with the compound having the structure, the fraction of the compound containing the structure in which n1 is 0 in the composition is less than 10%, less than 5%, less than 1%, less than 0.5%, less than 0.1%, less than 0.05% or less than 0.01%. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0206] In some embodiments, R 1 Each instance of is conjugated to a different amino acid residue of the antibody. In some embodiments, each different amino acid comprises an ε-amino group (e.g., lysine, arginine). However, in some embodiments, 1 Each different amino acid that is covalently linked is cysteine. In some embodiments, R 1 Directly covalently linked to an amino acid residue of the antibody. However, in some embodiments, R 1 Indirect covalent attachment to an amino acid of the antibody, for example, covalent attachment to a glycosylation site on an amino acid. In some embodiments, a composition is provided wherein R 1 The complex covalently linked to the amino acid residues present in the CDR region of the antibody is present only in trace amounts, or in undetectable amounts, or not present at all. In some embodiments, a composition is provided in which R is not detectable in the composition using standard detection techniques. 1 A complex covalently linked to amino acid residues present in the CDR regions of an antibody.

[0207] In some embodiments, a composition for administration to a subject in the methods described herein (eg, in aqueous solution) comprises a complex comprising Formula (I): [R 1 ] n1 -R 2 The structure of the present invention, wherein the complex R 1 Each example of independently comprises a group of formula (Ia):

[0208]

[0209] Where R3 comprising an oligonucleotide, such as an oligonucleotide comprising a 5'-XYZ-3' configuration; and R 1 At the connection point A and R 2 In some embodiments, R 2 The invention comprises an antibody comprising a sequence as shown in Table 2. For example, in some embodiments, R 2 The invention comprises an antibody comprising a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprising a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 The invention comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody which is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, R 3In some embodiments, R3 comprises an oligonucleotide comprising a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21). In some embodiments, R3 comprises an oligonucleotide comprising a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, the composition for administration to a subject in the methods described herein (e.g., in an aqueous solution) further comprises a complex in which n1 is 0.

[0210] In some embodiments, a composition for administration to a subject in the methods described herein (eg, in aqueous solution) comprises a complex comprising Formula (I): [R 1 ] n1 -R 2 The structure of the present invention, wherein the complex R 1 Each instance of comprises a group of formula (Ib):

[0211]

[0212] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-O-methoxyethyl (MoE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21), wherein n1 is an integer (e.g., 1 or greater) representing the number of R in each complex. 1 The number of instances, and each R 1 At the connection point A and R 2 In some embodiments, R 2 The invention comprises an antibody comprising a sequence as shown in Table 2. For example, in some embodiments, R 2The invention comprises an antibody comprising a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention also comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody which is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, R 2 The invention relates to an antibody comprising a Fab fragment. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, the composition for administration to a subject in the methods described herein (e.g., in an aqueous solution) further comprises a complex in which n1 is 0.

[0213] In some embodiments, a composition for administration to a subject in the methods described herein (eg, in aqueous solution) comprises a complex comprising Formula (I): [R 1 ] n1 -R 2 The structure of the present invention, wherein the complex R 1 Each instance of comprises a group of formula (Ic):

[0214]

[0215] Where R 1 At the connection point A and R 2 In some embodiments, R 2 The invention comprises an antibody comprising a sequence as shown in Table 2. For example, in some embodiments, R 2 The invention comprises an antibody comprising a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention also comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2The invention also includes an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody which is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, R 2 The invention relates to an antibody comprising a Fab fragment. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, the composition for administration to a subject in the methods described herein (e.g., in an aqueous solution) further comprises a complex in which n1 is 0.

[0216] In some embodiments, a composition for administration to a subject in the methods described herein (eg, in aqueous solution) comprises a complex comprising the structure of Formula (Id):

[0217]

[0218] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-O-methoxyethyl (MoE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents thiophosphate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); wherein R2 comprises an antibody comprising a sequence as shown in Table 2; wherein n1 is an integer (e.g., 1 or greater) representing the number of instances of the group enclosed by square brackets, wherein each instance of the group enclosed by square brackets is covalently attached to a different amino acid residue of the antibody, optionally wherein each different amino acid residue is lysine. In some embodiments, R 2The invention comprises an antibody comprising a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprising a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, R 2 The invention comprises an antibody comprising a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, R 2 The invention comprises an antibody comprising a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, R 2 The invention also includes an antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, R 2 The invention comprises an antibody which is a Fab fragment, a full-length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv or a Fv. In some embodiments, R 2 The invention relates to an antibody comprising a Fab fragment. In some embodiments, in each complex, n1 is independently an integer (e.g., an integer from 1 to 27, 1 to 26, 1 to 10, 1 to 5, or 1 to 3). In some embodiments, the composition for administration to a subject in the methods described herein (e.g., in an aqueous solution) further comprises a complex in which n1 is 0.

[0219] In some embodiments, the composition for administration to a subject in the methods described herein (eg, in aqueous solution) comprises a structure of Formula (A):

[0220]

[0221] (A), wherein y is 0 to 15 (e.g., 3), and z is 0 to 15 (e.g., 4). In some embodiments, the amide adjacent to the antibody (e.g., anti-TfR1 antibody) shown in structure (A) is produced by a reaction with an amine (e.g., lysine epsilon amine) of the antibody. In some embodiments, the complex described herein comprises an anti-TfR1 antibody (e.g., anti-TfR1 Fab) covalently linked to an oligonucleotide (e.g., an oligonucleotide comprising a 5'-XYZ-3' configuration) via a lysine of the antibody. In some embodiments, the antibody comprises a sequence as shown in Table 2. For example, in some embodiments, the antibody comprises a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, and a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14; and / or comprises a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16. In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 17, and / or comprises a light chain variable region (VL) comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17, and / or comprises a VL comprising the amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 19, and / or comprises a light chain comprising an amino acid sequence at least 85% (e.g., at least 95%) identical to SEQ ID NO: 20. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and / or comprises a light chain comprising the amino acid sequence of SEQ ID NO: 20. In some embodiments, the antibody is a Fab fragment, a full length IgG, a Fab' fragment, a F(ab')2 fragment, a scFv, or a Fv. In some embodiments, the antibody is a Fab fragment.

[0222] In some embodiments, the composition is formulated to be compatible with its intended route of administration. Some examples of routes of administration include parenteral administration, such as intravenous, intradermal, subcutaneous administration. Typically, the route of administration is intravenous or subcutaneous.

[0223] Methods of use / treatment / administration

[0224] As described herein, a complex comprising an anti-TfR1 antibody (e.g., Fab) covalently linked to a molecular payload (e.g., a DMPK targeting oligonucleotide) is effective in treating a subject with myotonic dystrophy (e.g., DM1). In some embodiments, the complex comprises a molecular payload that is an oligonucleotide, such as an oligonucleotide that promotes expression or activity reduction of DMPK in a subject (e.g., a subject with DM1) (e.g., a mutant or wild-type DMPK RNA level reduction).

[0225] In some embodiments, the subject can be a human subject, a non-human primate subject (e.g., cynomolgus monkey), a rodent subject, or any suitable mammalian subject. In some embodiments, the subject is a human. In some embodiments, the subject is a neonate or young pediatric human subject (e.g., less than 4 years old, less than 3 years old, less than 2 years old, less than 1 year old, about 11 months old, about 10 months old, about 9 months old, about 8 months old, about 7 months old, about 6 months old, about 5 months old, about 4 months old, about 3 months old, about 2 months old, or about 1 month old human subject). In some embodiments, the subject is a human subject of 2 to 60 years old (e.g., 2 to 60 years old, 2 to 50 years old, 2 to 40 years old, 2 to 30 years old, 2 to 20 years old, 2 to 10 years old). In some embodiments, the subject is a human subject between 5 and 30 years old (e.g., 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, or 30 years old). In some embodiments, the subject is a human subject between 18 and 50 years old (e.g., 18 to 50, 18 to 45, 18 to 40, 18 to 35, 18 to 30, 18 to 25, 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 years old). In some embodiments, the subject is a human subject 60 years old or older (e.g., about 60 years old, about 65 years old, about 70 years old, about 75 years old, or about 80 years old). In some embodiments, the subject is a human subject between 5 and 12 years old (e.g., 5, 6, 7, 8, 9, 10, 11, or 12 years old).In some embodiments, the subject is between 4 and 16 years old (e.g., 4 to 16, 5 to 16, 6 to 16, 7 to 16, 8 to 16, 9 to 16, 10 to 16, 11 to 16, 12 to 16, 13 to 16, 14 to 16, 15 to 16, 4 to 15, 5 to 15, 6 to 15, 7 to 15, 8 to 15, 9 to 15, 10 to 15, 11 to 15, 12 to 15, 13 to 15, 14 to 15, 4 to 14, 5 to 14, 6 to 14, 7 to 14, 8 to 14, 9 to 14, 10 to 14, 11 to 14, 12 to 14, 13 to 14, 4 to 13, 5 to 13, 6 to 13, 7 to 13 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 4 to 7, 5 to 7, 6 to 7, 4 to 6, 5 to 6, or 4 to 5 years old). In some embodiments, the subject is a human subject of about 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 years old.

[0226] In some embodiments, the subject may have myotonic dystrophy, such as DM1. In some embodiments, the subject has a DMPK allele, which may optionally include a disease-associated repeat, such as a CTG trinucleotide repeat expansion. In some embodiments, the subject may have a DMPK allele with an expanded disease-associated repeat comprising about 2 to 10 repeat units, about 2 to 50 repeat units, about 2 to 100 repeat units, about 50 to 1,000 repeat units, about 50 to 500 repeat units, about 50 to 250 repeat units, about 50 to 100 repeat units, about 500 to 10,000 repeat units, about 500 to 5,000 repeat units, about 500 to 2,500 repeat units, about 500 to 1,000 repeat units, or about 1,000 to 10,000 repeat units. In some embodiments, the subject suffers from symptoms of DM1, such as muscle atrophy, muscle loss, excessive daytime sleepiness, or cognitive delay. In some embodiments, the subject does not suffer from symptoms of DM1. In some embodiments, the subject suffers from congenital myotonic dystrophy. In some embodiments, the subject is ambulatory. In some embodiments, the subject is non-ambulatory.

[0227] One aspect of the present disclosure includes methods involving administering to a subject an effective amount of a composition (e.g., an aqueous solution) comprising a complex as described herein. In some embodiments, an effective amount of a composition (e.g., an aqueous solution) comprising a complex may be administered to a subject in need of treatment, the complex comprising an anti-TfR1 antibody (e.g., Fab) described herein covalently linked to an oligonucleotide (e.g., a DMPK targeting oligonucleotide) as described herein. In some embodiments, the composition (e.g., an aqueous solution) is administered systemically. 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, administration may be performed by intravenous, intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intraarticular, intrasynovial or intrathecal routes. In some embodiments, a composition (e.g., an aqueous solution) comprising a complex as described herein is administered by infusion (e.g., intravenous infusion).

[0228] In some embodiments, the composition comprising a plurality of complexes described herein can be in solid form, aqueous form, or liquid form. In some embodiments, the aqueous or liquid form can be atomized or lyophilized. In some embodiments, the lyophilized form can be reconstituted with an aqueous solution or liquid solution (e.g., before administration, such as by intravenous infusion).

[0229] In some embodiments, methods and / or uses for treating an object having a DMPK allele associated with myotonic dystrophy (e.g., DM1) are provided, comprising administering to the object a composition comprising one or more of the compounds described herein with an effective amount of the compound. In some embodiments, methods and / or uses for reducing the expression or activity of DMPK in an object (e.g., reducing the level of mutant or wild-type DMPK RNA, or the activity of a DMPK gene product) are provided, the method comprising contacting a cell with a composition comprising a plurality of compounds described herein with an effective amount of the compound. In some embodiments, the method comprises administering a lyophilized form (e.g., lyophilized powder) of a composition comprising a plurality of compounds described herein, comprising reconstructing the lyophilized form of the composition in an aqueous solution, and administering the aqueous solution to an object in need thereof. For example, in some embodiments, a lyophilized form of a composition comprising one or more compounds is transported and / or stored in a lyophilized form, reconstituted in an aqueous solution at an administration site (e.g., a health care provider site), and administered by injection or intravenous (e.g., by infusion) in a reconstituted form (e.g., as an aqueous solution). In some embodiments, the subject has a DMPK allele, which may optionally comprise a disease-associated repeat, such as a CTG trinucleotide repeat expansion.

[0230] In some embodiments, the composition is administered by site-specific or local delivery techniques. Some examples of these techniques include implantable reservoir sources of the complex, local delivery catheters, site-specific carriers, direct injection, or direct application.

[0231] In some aspects, provided herein is a method for reducing the expression or activity of DMPK in a subject (e.g., reducing the level of mutant or wild-type DMPK RNA, or the activity of a DMPK gene product). In some aspects, provided herein is a method for treating myotonic dystrophy (e.g., DM1) in a subject. In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of complexes, each complex comprising an anti-transferrin receptor 1 (TfR1) antibody (e.g., Fab) covalently linked to one or more oligonucleotides, wherein the antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16, wherein the oligonucleotide comprises CAGCGCCCACCAGUCA (SEQ ID NO: 21),

[0232] Optionally wherein the oligonucleotide comprises the structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage;

[0233] Also optionally, the antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and also optionally, the antibody (e.g., Fab) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0234] In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of a complex, wherein each complex comprises formula (I): [R 1 ]n1-R 2 The structure of the present invention, wherein the complex R 1 Each instance of comprises a group of formula (Ia):

[0235]

[0236] Where R 3 An oligonucleotide comprising a nucleobase sequence comprising CAGCGCCCACCAGUCA (SEQ ID NO: 21), optionally wherein R 3 an oligonucleotide comprising a structure comprising +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage;

[0237] Where R 2Comprising an anti-TfR1 antibody (e.g., Fab), comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16, optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 18. A heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20;

[0238] Each R 1 At the connection point A and R 2 covalently linked, optionally wherein each R 1 At the connection point A and R 2 covalently linking different amino acid residues of an antibody (e.g., Fab) of the invention, optionally wherein each of the different amino acid residues is lysine; and

[0239] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R 1 , optionally wherein the average value of n1 of the complexes in the composition is 1 to 5. In some embodiments, the composition for administration to a subject in the methods described herein (e.g., in aqueous solution) further comprises a complex comprising formula (I): [R 1 ] n1 -R 2, wherein n1 is 0. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0240] In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of a complex, wherein each complex comprises formula (I): [R 1 ]n1-R 2 The structure of the present invention, wherein the complex R 1 Each instance of comprises a group of formula (Ib):

[0241]

[0242] wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-O-methoxyethyl (MoE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0243] Where R 2The invention comprises an anti-TfR1 antibody (e.g., Fab) comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16, optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 19, and optionally wherein the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 21, A heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20;

[0244] Each R 1 At the connection point A and R 2 covalently linked, optionally wherein each R 1 At the connection point A and R 2 covalently linking different amino acid residues of an antibody (e.g., Fab) of the invention, optionally wherein each of the different amino acid residues is lysine; and

[0245] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R 1 , optionally wherein the average value of n1 of the complexes in the composition is 1 to 5. In some embodiments, the composition for administration to a subject in the methods described herein (e.g., in aqueous solution) further comprises a complex comprising formula (I): [R 1 ] n1 -R 2, wherein n1 is 0. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0246] In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of a complex, wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of each R in the complex 1 Containing a group of formula (Ic):

[0247]

[0248] Where R 2 The invention comprises an anti-TfR1 antibody (e.g., Fab) comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16, optionally wherein the antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VH comprising the amino acid sequence of SEQ ID NO: 18. NO: 18, and optionally wherein the antibody (e.g., Fab) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20;

[0249] Each R 1 At the connection point A and R 2 covalently linked, optionally wherein each R 1At the connection point A and R 2 covalently linking different amino acid residues of an antibody (e.g., Fab) of the invention, optionally wherein each of the different amino acid residues is lysine; and

[0250] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R 1 , optionally wherein the average value of n1 of the complexes in the composition is 1 to 5. In some embodiments, the composition for administration to a subject in the methods described herein (e.g., in aqueous solution) further comprises a complex comprising formula (I): [R 1 ] n1 -R 2 , wherein n1 is 0. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0251] In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of a muscle-targeted complex, wherein each complex comprises a group of formula (Id):

[0252]

[0253] wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-O-methoxyethyl (MoE) modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0254] Where R 2The invention comprises an anti-TfR1 antibody (e.g., Fab) comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16, optionally wherein the antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VH comprising the amino acid sequence of SEQ ID NO: 18. NO: 18, and optionally wherein the antibody (e.g., Fab) comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; and

[0255] Wherein in each complex, n1 is independently an integer of 1 or greater, optionally wherein the average value of n1 of the complex in the composition is 1 to 5, optionally wherein the anti-TfR1 antibody (e.g., Fab) is covalently linked through different amino acid residues of the antibody (e.g., Fab), optionally wherein each different amino acid residue is lysine. In some embodiments, the composition for administration to a subject in the methods described herein (e.g., in an aqueous solution) further comprises a complex wherein n1 is 0. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0256] In some embodiments, in any method described herein, the composition is in an aqueous solution and further comprises tris(hydroxymethyl)aminomethane and sucrose, wherein the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 5mM to 50mM, and the sucrose is present in the aqueous solution at a concentration of 5w / v% to 15w / v%. In some embodiments, in any method described herein, the composition is in an aqueous solution and further comprises tris(hydroxymethyl)aminomethane and sucrose, wherein the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 25mM or about 25mM, the sucrose is present in the aqueous solution at a concentration of 10w / v% or about 10w / v%, and the pH of the composition is 7.5 or about 7.5. In some embodiments, the complex is present in the composition at a concentration of 10 mg / mL to 50 mg / mL (e.g., 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL).

[0257] In some embodiments, the methods described herein include administering to a subject a composition comprising an effective amount of a complex comprising Formula (I) as described herein: [R 1 ] n1 -R 2 ) (e.g., comprising a group of Formula (Ia), (Ib), (Ic) or (Id)). In some embodiments, in any of the methods described herein, the effective amount of the complex provides a certain amount of anti-TfR1 antibody (e.g., Fab) / kg of the subject in the complex to the subject, and the amount of the oligonucleotide in the complex provided to the subject / kg of the subject can be derived using the following equation (Equation A):

[0258]

[0259] wherein "MW" means molecular weight (g / mol), and "average value of n1" means the average value of n1 of the complexes in the composition.

[0260] In some embodiments, the methods described herein include administering to a subject a composition comprising an effective amount of a complex comprising Formula (I) as described herein: [R 1 ] n1 -R 2 In some embodiments, in any of the methods described herein, an effective amount of the complex provides a certain amount of oligonucleotide in the complex / kg subject to the subject, and the amount of oligonucleotide in the complex provided to the subject / kg subject can be derived using the following equation (Equation B):

[0261]

[0262] wherein "MW" means molecular weight (g / mol), and "average value of n1" means the average value of n1 of the complexes in the composition.

[0263] It should be understood that the average value of n1 does not have to be an integer and can be a decimal. In some embodiments, the present disclosure contemplates a variation in the average value of n1 of up to 30% (e.g., ± up to 30%, ± up to 25%, ± up to 20%, ± up to 15%, ± up to 10%, ± up to 5%, ± up to 3%, or ± up to 1%). In some embodiments, the average value of n1 of the complexes in the composition is 0.5 to 5 (e.g., 0.5 to 5, 0.5 to 4.5, 0.5 to 4, 0.5 to 3.5, 0.5 to 3, 0.5 to 2.5, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 1 to 5, 1 to 4.5, 1 to 4, 1 to 3.5, 1 to 3, 1 to 2.5, 1 to 2, 1 to 1.5, 1.5 to 5, 1.5 to 4. 5, 1.5 to 4, 1.5 to 3.5, 1.5 to 3, 1.5 to 2.5, 1.5 to 2, 2 to 5, 2 to 4.5, 2 to 4, 2 to 3.5, 2 to 3, 2 to 2.5, 2.5 to 5, 2.5 to 4.5, 2.5 to 4, 2.5 to 3.5, 2.5 to 3, 3 to 5, 3 to 4.5, 3 to 4, 3 to 3.5, 3.5 to 5, 3.5 to 4.5, 3.5 to 4, 4 to 5, 4 to 4.5 4.5 to 5, 0.8 to 1.5, 0.9 to 1.4, or 1 to 1.3), which has a variation of up to 30% (e.g., ± up to 30%, ± up to 25%, ± up to 20%, ± up to 15%, ± up to 10%, ± up to 5%, ± up to 3%, or ± up to 1%). In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of a complex comprising formula (I) as described herein: [R 1 ] n1 -R 2The composition comprises a structure (e.g., comprising a group of Formula (Ia), (Ib), (Ic) or (Id)), wherein the average value of n1 of the complexes in the composition is 0.5 to 5 (e.g., 0.5 to 5, 0.5 to 4.5, 0.5 to 4, 0.5 to 3.5, 0.5 to 3, 0.5 to 2.5, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 1 to 5, 1 to 4.5, 1 to 4, 1 to 3.5, 1 to 3, 1 to 2.5, 1 to 2, 1 to 1. 5, 1.5 to 5, 1.5 to 4.5, 1.5 to 4, 1.5 to 3.5, 1.5 to 3, 1.5 to 2.5, 1.5 to 2, 2 to 5, 2 to 4.5, 2 to 4, 2 to 3.5, 2 to 3, 2 to 2.5, 2.5 to 5, 2.5 to 4.5, 2.5 to 4, 2.5 to 3.5, 2.5 to 3, 3 to 5, 3 to 4.5, 3 to 4, 3 to 3.5, 3.5 to 5, 3.5 to 4.5, 3.5 to 4, 4 to 5, 4 to 4.5 4.5 to 5, 0.8 to 1.5, 0.9 to 1.4, or 1 to 1.3), which has a variation of up to 30% (e.g., ± up to 30%, ± up to 25%, ± up to 20%, ± up to 15%, ± up to 10%, ± up to 5%, ± up to 3%, or ± up to 1%). In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of a complex comprising formula (I) as described herein: [R 1 ] n1 -R 2 In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of a complex comprising formula (I) as described herein: [R 1 ] n1 -R 2 structures (e.g., comprising a group of Formula (Ia), (Ib), (Ic) or (Id)), wherein the average value of n1 of the complexes in the composition is about 1.1 or 1.15, which has a variation of up to 30% (e.g., ± up to 30%, ± up to 25%, ± up to 20%, ± up to 15%, ± up to 10%, ± up to 5%, ± up to 3%, or ± up to 1%).

[0264] It should be understood that any amount of anti-TfR1 antibody (e.g., Fab) provided herein can be expressed as the amount of oligonucleotide (e.g., DMPK targeting oligonucleotide) according to equation A. Therefore, it should be understood that the description herein involving providing any amount of anti-TfR1 antibody (e.g., Fab) can be understood as providing the corresponding amount of oligonucleotide (e.g., DMPK targeting oligonucleotide) according to equation A. Conversely, any amount of oligonucleotide (e.g., DMPK targeting oligonucleotide) provided herein can be expressed as the amount of anti-TfR1 antibody (e.g., Fab) according to equation B. Therefore, it should be understood that the description herein about providing any amount of oligonucleotide (e.g., DMPK targeting oligonucleotide) can be understood as providing the corresponding amount of anti-TfR1 antibody (e.g., Fab) according to equation B.

[0265] In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of a complex comprising Formula (I) as described herein: [R 1 ] n1 -R 2 structure (e.g., comprising a group of Formula (Ia), (Ib), (Ic) or (Id)), wherein an effective amount of the complex provides a certain amount of anti-TfR1 antibody (e.g., Fab) / kg of the subject in the complex to the subject, and wherein the amount of the oligonucleotide provided to the subject / kg of the subject is derived using Equation A provided herein based on the average value of n1 of the complexes in the composition and the amount of anti-TfR1 antibody (e.g., Fab) / kg of the subject in the complex provided to the subject. In some embodiments, the amount of the oligonucleotide differs from the amount derived from Equation A by up to 30% (e.g., ±up to 30%, ±up to 25%, ±up to 20%, ±up to 15%, ±up to 10%, ±up to 5%, ±up to 3%, or ±up to 1%).

[0266] In some embodiments, the methods provided herein include administering to a subject a composition comprising an effective amount of a complex comprising Formula (I) as described herein: [R 1 ] n1 -R 2The structure of , wherein the composition comprises an amount of oligonucleotides in the complexes / kg subject, and wherein the amount of anti-TfR1 antibody (e.g., Fab) provided to the subject / kg subject is derived based on the average value of n1 of the complexes in the composition and the amount of oligonucleotides in the complexes provided to the subject / kg subject using Equation B provided herein. In some embodiments, the amount of anti-TfR1 antibody (e.g., Fab) differs from the amount derived from Equation B by at most 45% (e.g., ± at most 45%, ± at most 40%, ± at most 35%, ± at most 30%, ± at most 25%, ± at most 20%, ± at most 15%, ± at most 10%, ± at most 5%, ± at most 3%, or ± at most 1%).

[0267] In some embodiments, for the purpose of determining the molecular weight of an oligonucleotide, an oligonucleotide is represented by the following structure:

[0268]

[0269] This corresponds to a molecular weight of 5,667 g / mol.

[0270] In some embodiments, the molecular weight of the anti-TfR1 Fab (eg, an anti-TfR1 Fab comprising the heavy chain and light chain provided in Table 2) in the complex used in the methods described herein is 47,986 g / mol.

[0271] Thus, in some embodiments, the methods described herein include administering to a subject a composition comprising an effective amount of a complex comprising Formula (I) as described herein: [R 1 ] n1 -R 2 (e.g., comprising a group of Formula (Ia), (Ib), (Ic) or (Id)), wherein an effective amount of the complex provides to the subject an amount of anti-TfR1 Fab / kg subject and an amount of oligonucleotide / kg subject in the complex, wherein the amount of the oligonucleotide is:

[0272]

[0273] Provided that the average value of n1 for the complexes in the composition is 1.15. In some embodiments, the amount of the oligonucleotide differs by at most 30% (e.g., ± at most 30%, ± at most 25%, ± at most 20%, ± at most 15%, ± at most 10%, ± at most 5%, ± at most 3%, or ± at most 1%) from the amount deduced from Equation A.

[0274] In some embodiments, the methods described herein include administering to a subject a composition comprising an effective amount of a complex comprising Formula (I) as described herein: [R 1 ]n1 -R 2 (e.g., comprising a group of Formula (Ia), (Ib), (Ic) or (Id)), wherein an effective amount of the complex provides to the subject an amount of anti-TfR1 Fab / kg subject and an amount of oligonucleotide / kg subject in the complex, wherein the amount of the oligonucleotide is:

[0275]

[0276] Provided that the average value of n1 for the complexes in the composition is 1.15. In some embodiments, the amount of the oligonucleotide differs by at most 30% (e.g., ± at most 30%, ± at most 25%, ± at most 20%, ± at most 15%, ± at most 10%, ± at most 5%, ± at most 3%, or ± at most 1%) from the amount deduced from Equation A.

[0277] In some embodiments, the methods described herein include administering to a subject a composition comprising an effective amount of a complex comprising Formula (I) as described herein: [R 1 ] n1 -R 2 , wherein an effective amount of the complex provides to the subject an amount of oligonucleotide / kg of the subject and an amount of anti-TfR1 Fab / kg of the subject in the complex, wherein the amount of anti-TfR1 Fab is:

[0278]

[0279] Provided that the average value of n1 of the complexes in the composition is 1.15. In some embodiments, the amount of anti-TfR1 antibody (e.g., Fab) differs from the amount derived from Equation B by at most 45% (e.g., ± at most 45%, ± at most 40%, ± at most 35%, ± at most 30%, ± at most 25%, ± at most 20%, ± at most 15%, ± at most 10%, ± at most 5%, ± at most 3%, or ± at most 1%).

[0280] In some embodiments, the methods described herein include administering to a subject a composition comprising an effective amount of a complex comprising Formula (I) as described herein: [R 1 ] n1 -R 2 , wherein an effective amount of the complex provides to the subject a certain amount of oligonucleotide / kg of the subject and a certain amount of anti-TfR1 Fab / kg of the subject in the complex, wherein the amount of anti-TfR1 Fab is:

[0281]

[0282] Provided that the average value of n1 of the complexes in the composition is 1.15. In some embodiments, the amount of anti-TfR1 antibody (e.g., Fab) differs from the amount derived from Equation B by up to 45% (e.g., ± up to 45%, ± up to 40%, ± up to 35%, ± up to 30%, ± up to 25%, ± up to 20%, ± up to 15%, ± up to 10%, ± up to 5%, ± up to 3%, or ± up to 1%). In some embodiments, administration occurs once or more. In some embodiments, a single dose of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, multiple doses of a composition comprising an effective amount of a complex described herein (e.g., a complex comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising formula (I): [R 1 ] n1 -R 2 [R] ]) to a subject. For example, in some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)) once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, or once every 16 weeks. 1 ] n1 -R 2 The invention also provides a composition comprising a complex of a structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id).

[0283] In some embodiments, the composition is administered to the subject during the administration period in any of the methods described herein. In some embodiments, the administration period is 1 to 24 (e.g., 1 to 24, 2 to 24, 3 to 24, 4 to 24, 5 to 24, 6 to 24, 7 to 24, 8 to 24, 9 to 24, 10 to 24, 11 to 24, 12 to 24, 13 to 24, 14 to 24, 15 to 24, 16 to 24, 17 to 24, 18 to 24, 1 to 18, 1 to 12, 1 to 8, 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, 2 to 18, 2 to 12, 2 to 8, 2 to 6, 2 to 4, 4 to 18, 4 to 12, 4 to 8, 4 to 6, 6 to 18, 6 to 12, 6 to 8, 8 to 18, 8 to 12, or 12 to 18) months. In some embodiments, the administration period is less than 1 month, less than 2 months, less than 3 months, less than 4 months, less than 5 months, less than 6 months, less than 7 months, less than 8 months, less than 9 months, less than 10 months, less than 11 months, less than 12 months, less than 13 months, less than 14 months, less than 15 months, less than 16 months, less than 17 months, or less than 18 months. In some embodiments, the administration period is 1 to 20 (e.g., 1 to 20, 2 to 20, 3 to 20, 4 to 20, 5 to 20, 6 to 20, 7 to 20, 8 to 20, 9 to 20, 10 to 20, 11 to 20, 12 to 20, 13 to 20, 14 to 20, 15 to 20, 16 to 20, 17 to 20, 18 to 20, 19 to 20, 2 to 18, 2 to 12, 2 to 10, 2 to 5, 4 to 18, 4 to 12, 4 to 8, 6 to 18, 6 to 12, or 6 to 8) years. In some embodiments, the administration period is less than 1 year, less than 2 years, less than 3 years, less than 4 years, less than 5 years, less than 6 years, less than 7 years, less than 8 years, less than 9 years, less than 10 years, less than 11 years, less than 12 years, less than 13 years, less than 14 years, less than 15 years, less than 16 years, less than 17 years, less than 18 years, less than 19 years, or less than 20 years. In some embodiments, the administration period is at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, at least 10 years, at least 11 years, at least 12 years, at least 13 years, at least 14 years, at least 15 years, at least 16 years, at least 17 years, at least 18 years, at least 19 years, or at least 20 years. In some embodiments, the administration period is 1 to 50 years (e.g., 1 to 50 years, 2 to 50 years, 3 to 50 years, 4 to 50 years, 5 to 50 years, 6 to 50 years, 7 to 50 years, 8 to 50 years, 9 to 50 years, 10 to 50 years, 20 to 50 years, 30 to 50 years, 40 to 50 years, 10 to 20 years, 10 to 30 years, 10 to 40 years, 20 to 30 years, 20 to 40 years, or 30 to 40 years). In some embodiments, the administration period is the remainder of the subject's life.

[0284] In some embodiments, the methods described herein comprise administering to a subject an effective amount of a complex described herein (e.g., a drug comprising Formula (I): [R] once a week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, or once every 16 weeks for the rest of the subject's life. 1 ] n1 -R 2 In some embodiments, the methods described herein comprise administering to the subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)) once every 4 weeks for the rest of the subject's life. 1 ] n1 -R 2 In some embodiments, the methods described herein comprise administering to the subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)) once every 8 weeks for the rest of the subject's life. 1 ] n1 -R 2 In some embodiments, the methods described herein comprise administering to the subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)) once every 12 weeks for the rest of the subject's life. 1 ] n1 -R 2 The invention also provides a composition comprising a complex of a structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id).

[0285] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising Formula (I): [R] once a week to once every four weeks (e.g., once a week, once every 2 weeks, once every 3 weeks, or once every 4 weeks) during a first administration period. 1 ] n1 -R 2The composition of the invention is a composition comprising a complex of the structure described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic) or (Id)), and then during the second administration period, once every 4 weeks to once every 16 weeks (e.g., once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, or once every 16 weeks) to the subject. 1 ] n1 -R 2 In some embodiments, the first administration period is 2 to 24 weeks (e.g., 2 to 24, 2 to 20, 2 to 26, 2 to 12, 2 to 8, 2 to 4, 4 to 24, 4 to 20, 4 to 16, 4 to 12, 4 to 8, 8 to 24, 8 to 20, 8 to 16, 8 to 12, 12 to 24, 12 to 20, 12 to 16, 16 to 24, 16 to 20, or 20 to 24 weeks). In some embodiments, the first administration period is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 weeks. In some embodiments, the second administration period is 4 weeks to the rest of the subject's life (e.g., 4, 8, 12, 16, 18, 20, 22, 24, 28, 32, 36, 40, 44, 48, 52, 56, 60, 64, 68, 72, 76, 80, 84, 88, 92, 96 weeks or longer to the rest of the subject's life). In some embodiments, the first administration period is 8 weeks and the second administration period is 16 weeks. In some embodiments, the first administration period is 8 weeks and the second administration period is the rest of the subject's life.

[0286] In some embodiments, each administration during the first administration period provides the subject with the same amount of the complex (e.g., comprising Formula (I): [R 1 ] n1 -R 2 In some embodiments, each administration during the first administration period provides the subject with a different amount of the complex (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)) than each administration during the second administration period. 1 ] n1 -R 2 A complex of structure, for example a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id)).

[0287] In some embodiments, the methods described herein comprise administering to a subject once every 4 weeks during a first administration period of 8 weeks, 12 weeks, or 16 weeks a composition comprising an effective amount of a complex described herein (e.g., a composition comprising Formula (I): [R 1 ] n1 -R 2 The subject is administered a composition comprising an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)) once every 8 weeks during a second administration period of 16 weeks, 24 weeks, 32 weeks, 40 weeks, 48 ​​weeks, 56 weeks, 64 weeks, 72 weeks, 80 weeks, 88 weeks, 96 weeks, 104 weeks, 112 weeks, 120 weeks, 128 weeks, 136 weeks, 144 weeks, 152 weeks, 160 weeks or longer, or the rest of the subject's life. 1 ] n1 -R 2 The composition is a complex of structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic) or (Id), wherein each administration during the first administration period provides the subject with the same amount of the complex (e.g., comprising Formula (I): [R 1 ] n1 -R 2 A complex of structure, for example a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id)).

[0288] In some embodiments, the methods described herein comprise administering to a subject once every 4 weeks during a first administration period of 8 weeks, 12 weeks, or 16 weeks a composition comprising an effective amount of a complex described herein (e.g., a composition comprising Formula (I): [R 1 ] n1 -R 2 The subject is then administered a composition comprising an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)) during a second administration period of 16 weeks, 24 weeks, 32 weeks, 40 weeks, 48 ​​weeks, 56 weeks, 64 weeks, 72 weeks, 80 weeks, 88 weeks, 96 weeks, 104 weeks, 112 weeks, 120 weeks, 128 weeks, 136 weeks, 144 weeks, 152 weeks, 160 weeks or longer, or the rest of the subject's life. 1 ] n1 -R 2 A composition of the invention comprising a complex of structure (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic) or (Id)), wherein each administration during a first administration period provides a different amount of the complex (e.g., a complex comprising Formula (I): [R 1] n1 -R 2 A complex of structure, for example a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id)).

[0289] In some embodiments, in any of the methods described herein, additional administrations can be given to the subject during any administration period.

[0290] In some embodiments, in any of the methods described herein, an effective amount of a complex (eg, comprising formula (I): [R 1 ] n1 -R 2A complex of structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)) is provided to a subject in an amount of 2 to 220 mg (e.g., 2 to 220 mg, 5 to 220 mg, 5 to 210 mg, 5 to 200 mg, 5 to 190 mg, 5 to 180 mg, 5 to 170 mg, 5 to 160 mg, 5 to 150 mg, 5 to 140 mg, 5 to 130 mg, 5 to 120 mg, 5 to 110 mg, 5 to 100 mg, 5 to 90 mg, 5 to 80 mg, 5 to 75 mg, 5 to 70 mg, 5 to 60 mg, 5 to to 50mg, 5mg to 40mg, 5mg to 30mg, 5mg to 20mg, 5mg to 10mg, 8mg to 220mg, 8mg to 210mg, 8mg to 200mg, 8mg to 190mg, 8mg to 180mg, 8mg to 170mg, 8mg to 160mg, 8mg to 150mg, 8mg to 140mg, 8mg to 130mg, 8mg to 120mg, 8mg to 110mg, 8mg to 100mg, 8mg to 90mg, 8mg to 80mg, 8mg to 75mg, 8mg to 70mg, 8mg to 60mg, 8mg to 50mg, 8mg to 40mg, 8mg to 35mg, 8mg to 3 0mg, 8mg to 25mg, 8mg to 20mg, 8mg to 15mg, 8mg to 13mg, 10mg to 150mg, 10mg to 140mg, 10mg to 130mg, 10mg to 120mg, 10mg to 110mg, 10mg to 100mg, 10mg to 90mg, 10mg to 80mg, 10mg to 70mg, 10mg to 60mg, 10mg to 50mg, 10mg to 40mg, 10mg to 30mg, 10mg to 20mg, 13mg to 150mg, 13mg to 140mg, 13mg to 130mg, 13mg to 120mg, 13mg to 110mg, 13mg to 100mg 13mg to 90mg, 13mg to 80mg, 13mg to 75mg, 13mg to 70mg, 13mg to 60mg, 13mg to 50mg, 13mg to 40mg, 13mg to 35mg, 13mg to 30mg, 13mg to 25mg, 13mg to 20mg, 13mg to 15mg, 15mg to 150mg, 15mg to 140mg, 15mg to 130mg, 15mg to 120mg, 15mg to 110mg, 15mg to 100mg, 15mg to 90mg, 15mg to 80mg, 15mg to 75mg, 15mg to 70mg, 15mg to 60mg, 15mg to 50mg,15mg to 40mg, 15mg to 35mg, 15mg to 30mg, 15mg to 25mg, 15mg to 20mg, 20mg to 100mg, 20mg to 90mg, 20mg to 80mg, 20mg to 75mg, 20mg to 70mg, 20mg to 60mg, 20mg to 50mg, 20mg to 40mg, 20mg to 30mg, 20mg to 25mg, 25mg to 90mg, 25mg to 80mg, 25mg to 75mg, 25mg to 70mg, 25mg to 60mg, 25mg to 50mg, 25mg to 40mg, 25mg to 35mg, 25mg to 30mg, 30mg to 100mg, 30mg to 90mg, 3 0mg to 80mg, 30mg to 75mg, 30mg to 70mg, 30mg to 60mg, 30mg to 50mg, 30mg to 40mg, 40mg to 100mg, 40mg to 90mg, 40mg to 80mg, 40mg to 75mg, 40mg to 70mg, 40mg to 60mg, 40mg to 50mg, 50mg to 100mg, 50mg to 90mg, 50mg to 80mg, 50mg to 75mg, 50mg to 70mg, 50mg to 60mg, 60mg to 100mg, 60mg to 90mg, 60mg to 80mg, 60mg to 75mg, or 60mg to 70mg) of anti-TfR1 antibody (e.g., Fab) / kg of subject. In some embodiments, in any of the methods described herein, an effective amount of a complex (e.g., comprising Formula (I): [R, 1 ] n1 -R 2In some embodiments, the present invention provides about 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 11 mg, 12 mg, 13 mg, 14 mg, 15 mg, 16 mg, 17 mg, 18 mg, 19 mg, 20 mg, 21 mg, 22 mg, 23 mg, 24 mg, 25 mg, 26 mg, 27 mg, 28 mg, 29 mg, 30 mg, 31 mg, 32 mg, 33 mg, 34 mg, 35 mg of the complex. mg, 36mg, 37mg, 38mg, 39mg, 40mg, 41mg, 42mg, 43mg, 44mg, 45mg, 46mg, 47mg, 48mg, 49mg, 50mg, 51mg, 52mg, 53mg, 54mg, 55mg, 56mg, 57mg, 58mg, 59mg, 60mg, 61mg, 62mg, 63mg, 64mg, 65mg, 66mg, 67mg, 68mg, 69mg, 70mg, 71mg, 72mg, 73mg, 74m g, 75mg, 76mg, 77mg, 78mg, 79mg, 80mg, 81mg, 82mg, 83mg, 84mg, 85mg, 86mg, 87mg, 88mg, 89mg, 90mg, 91mg, 92mg, 93mg, 9 4mg, 95mg, 96mg, 97mg, 98mg, 99mg, 100mg, 101mg, 102mg, 103mg, 104mg, 105mg, 106mg, 107mg, 108mg, 109mg, 110mg, 111 or 200 mg, 205 mg, 210 mg, 215 mg, or 220 mg of anti-TfRl antibody (e.g., Fab) / kg of subject. In some embodiments, with respect to any of the foregoing amounts of anti-TfR1 antibodies (e.g., Fab), the values ​​may vary by up to ±45% (e.g., ±up to 45%, ±up to 40%, ±up to 35%, ±up to 30%, ±up to 25%, ±up to 20%, ±up to 15%, ±up to 10%, ±up to 5%, ±up to 3%, or ±up to 1%).

[0291] In some embodiments, in any of the methods described herein, an effective amount of a complex (eg, comprising formula (I): [R 1 ]n1 -R 2 A complex of structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)) is provided to a subject with 0.5 mg to 20 mg (e.g., 0.5 mg to 20 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 9 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 6 mg, 1 mg to 5 mg, 1 mg to 4 mg, 1 mg to 3 mg, 1 mg to 2 mg, 2 mg to 20 mg, 2 mg to 15 mg, 2 mg to 12 mg, 2 mg to 10 mg, 2 mg to 9 mg) of the complex. g, 2mg to 8mg, 2mg to 7mg, 2mg to 6mg, 2mg to 5mg, 2mg to 4mg, 2mg to 3mg, 3mg to 20mg, 3mg to 15mg, 3mg to 12mg, 3mg to 10mg, 3mg to 9mg, 3mg to 8mg, 3mg to 7mg, 3mg to 6mg, 3mg to 5mg, 3mg to 4mg, 3.5mg to 20mg, 3.5mg to 15mg, 3.5mg to 12mg, 3.5mg to 10mg, 3.5mg to 9mg, 3.5mg to 8mg, 3.5mg to 7mg, 3.5mg to 6mg mg, 3.5mg to 5mg, 3.5mg to 4mg, 4mg to 20mg, 4mg to 15mg, 4mg to 12mg, 4mg to 10mg, 4mg to 9mg, 4mg to 8mg, 4mg to 7mg, 4mg to 6mg, 4mg to 5mg, 5mg to 20mg, 5mg to 15mg, 5mg to 12mg, 5mg to 10mg, 5mg to 9mg, 5mg to 8mg, 5mg to 7mg, 5mg to 6mg, 6mg to 20mg, 6mg to 15mg, 6mg to 12mg, 6mg to 10mg, 6mg to 9mg, 6mg to 12 mg, 12 mg to 20 mg, 12 mg to 15 mg, 12 mg to 20 mg, 12 mg to 15 mg, or 15 mg to 20 mg) oligonucleotide / kg subject. In some embodiments, in any of the methods described herein, an effective amount of a complex (e.g., comprising formula (I): [R 1 ] n1 -R 2A complex of structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic) or (Id)) provides about 0.5 mg, 1 mg, 1.1 mg, 1.5 mg, 1.8 mg, 2 mg, 2.5 mg, 3 mg, 3.4 mg, 3.5 mg, 4 mg, 4.1 mg, 4.5 mg, 5 mg, 5.5 mg, 6 mg, 6.5 mg, 6.8 mg, 7 mg, 7.5 mg, 8 mg, 8. 10mg, 10.2mg, 10.5mg, 11mg, 11.5mg, 12mg, 12.5mg, 13mg, 13.5mg, 14mg, 14.5mg, 15mg, 15.5mg, 16mg, 16.5mg, 17mg, 17.5mg, 18mg, 18.5mg, 19mg, 19.5mg, or 20mg oligonucleotide / kg subject. In some embodiments, for any of the foregoing amounts of oligonucleotide, the values ​​may vary by up to ±30% (e.g., ±up to 30%, ±up to 25%, ±up to 20%, ±up to 15%, ±up to 10%, ±up to 5%, ±up to 3%, or ±up to 1%).

[0292] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2A composition comprising a complex of structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic) or (Id), wherein the effective amount (e.g., the effective amount per administration) provides to the subject 10 mg to 110 mg (e.g., 10 mg to 110 mg, 10 mg to 100 mg, 10 mg to 90 mg, 10 mg to 80 mg, 10 mg to 70 mg, 10 mg to 60 mg, 10 mg to 50 mg, 10 mg to 40 mg, 10mg to 30mg, 10mg to 20mg, 20mg to 110mg, 20mg to 100mg, 20mg to 90mg, 20mg to 80mg, 20mg to 70mg, 20mg to 60mg, 20mg to 50mg, 20mg to 40mg, 20mg to 30mg, 30mg to 110mg, 30mg to 100mg, 30mg to 90mg, 30mg to 80mg, 30mg to 70mg, 30mg to g to 60mg, 30mg to 50mg, 30mg to 40mg, 40mg to 110mg, 40mg to 100mg, 40mg to 90mg, 40mg to 80mg, 40mg to 70mg, 40mg to 60mg, 40mg to 50mg, 50mg to 110mg, 50mg to 100mg, 50mg to 90mg, 50mg to 80mg, 50mg to 70mg, 50mg to 60mg, 60mg to 1 10mg, 60mg to 100mg, 60mg to 90mg, 60mg to 80mg, 60mg to 70mg, 70mg to 110mg, 70mg to 100mg, 70mg to 90mg, 70mg to 80mg, 80mg to 110mg, 80mg to 100mg, 80mg to 90mg, 90mg to 110mg, 90mg to 100mg, 100mg to 110mg) anti-TfR1 antibody / kg subject. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the effective amount (e.g., the effective amount per administration) provides the subject with about 13 mg, about 25 mg, about 50 mg, or about 75 mg of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the effective amount (e.g., the effective amount per administration) provides the subject with about 29 mg or about 37 mg of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, with respect to any of the foregoing amounts of anti-TfR1 antibody (e.g., Fab), the value may vary by up to ±45% (e.g., ±up to 45%, ±up to 40%, ±up to 35%, ±up to 30%, ±up to 25%, ±up to 20%, ±up to 15%, ±up to 10%, ±up to 5%, ±up to 3%, or ±up to 1%). In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks), and then administered once every 8 weeks during a second administration period (e.g., 16 weeks to the rest of the subject's life).

[0293] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition of the invention comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id), wherein an effective amount (e.g., an effective amount per administration) provides the subject with 10 mg to 20 mg (e.g., 10 mg to 20 mg, 10.5 mg to 18 mg, 11 mg to 16 mg, 11.5 mg to 15 mg, 12 mg to 14 mg, or 12.5 mg to 13 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0294] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition of the invention comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 18 mg to 36 mg (e.g., 18 mg to 36 mg, 19 mg to 33 mg, 20 mg to 30 mg, 22 mg to 27 mg, or 23 mg to 26 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to the subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0295] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition of the invention comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id), wherein an effective amount (e.g., an effective amount per administration) provides the subject with 20 mg to 43 mg (e.g., 20 mg to 43 mg, 22 mg to 40 mg, 24 mg to 34 mg, 26 mg to 32 mg, or 28 mg to 30 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0296] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition of the invention comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 26 mg to 53 mg (e.g., 26 mg to 53 mg, 29 mg to 50 mg, 31 mg to 45 mg, 33 mg to 41 mg, or 35 mg to 40 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to the subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0297] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition of the invention comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id), wherein an effective amount (e.g., an effective amount per administration) provides the subject with 36 mg to 72 mg (e.g., 36 mg to 72 mg, 37 mg to 69 mg, 38 mg to 66 mg, 39 mg to 63 mg, 40 mg to 60 mg, 41 mg to 57 mg, 42 mg to 54 mg, or 45 mg to 51 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0298] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 55 mg to 110 mg (e.g., 55 mg to 110 mg, 58 mg to 100 mg, 61 mg to 95 mg, 64 mg to 90 mg, 67 mg to 85 mg, or 70 mg to 80 mg, or 73 mg to 76 mg) of anti-TfR1 antibody / kg of the subject. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0299] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2A composition comprising a complex of structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id), wherein an effective amount (e.g., an effective amount per administration) provides to a subject 5 mg to 90 mg (e.g., 5 mg to 90 mg, 5 mg to 80 mg, 5 mg to 70 mg, 5 mg to 60 mg, 5 mg to 50 mg, 5 mg to 40 mg, 5 mg to 30 mg, 5 mg to 20 mg, 5 mg to 10 mg, 10 mg to 90 mg, 10 mg to 80 mg, 10 mg to 70 mg, 10 mg to 60 mg, 10 mg to 50 mg, 10 mg to 40 mg, 10 mg to 30 mg, 10 mg to 20 mg, 20 mg to 90 mg, 20 mg to 80 mg, 20 mg to 70mg, 20mg to 60mg, 20mg to 50mg, 20mg to 40mg, 20mg to 30mg, 30mg to 90mg, 30mg to 80mg, 30mg to 70mg, 30mg to 60mg, 30mg to 50mg, 30mg to 40mg, 40mg to 90mg, 40mg to 80mg, 40mg to 70mg, 40mg to 60mg, 40mg to 50mg, 50mg to 90mg, 50mg to 80mg, 50mg to 70mg, 50mg to 60mg, 60mg to 90mg, 60mg to 80mg, 60mg to 70mg, 70mg to 90mg, 70mg to 80mg, or 80mg to 90mg) of anti-TfR1 antibody / kg of the subject. In some embodiments, the methods described herein include administering to the subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2A composition of the invention (e.g., a complex of a structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic) or (Id)), wherein an effective amount (e.g., an effective amount per administration) provides the subject with about 8 mg, about 15 mg, about 30 mg, or about 60 mg of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, with respect to any of the foregoing amounts of anti-TfR1 antibody (e.g., Fab), the value may vary by up to ±45% (e.g., ±up to 45%, ±up to 40%, ±up to 35%, ±up to 30%, ±up to 25%, ±up to 20%, ±up to 15%, ±up to 10%, ±up to 5%, ±up to 3%, or ±up to 1%). In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks), and then administered once every 8 weeks during a second administration period (e.g., 16 weeks to the rest of the subject's life).

[0300] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition of the invention comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 6 to 12 mg (e.g., 6 to 12 mg, 6.5 to 11 mg, 7 to 10 mg, 7.5 to 9 mg, or 8 to 8.5 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to the subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0301] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id), wherein an effective amount (e.g., an effective amount per administration) provides the subject with 11 mg to 22 mg (e.g., 11 mg to 22 mg, 11.5 mg to 20 mg, 12 mg to 19 mg, 12.5 mg to 18 mg, 13 mg to 17 mg, 14 mg to 16 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0302] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition of the invention comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id), wherein an effective amount (e.g., an effective amount per administration) provides the subject with 22 mg to 44 mg (e.g., 22 mg to 44 mg, 23 mg to 42 mg, 24 mg to 40 mg, 25 mg to 38 mg, 26 mg to 36 mg, 27 mg to 34 mg, 28 mg to 32 mg, or 29 mg to 31 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0303] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition of the invention comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic), or (Id), wherein an effective amount (e.g., an effective amount per administration) provides the subject with 44 to 88 mg (e.g., 44 to 88 mg, 46 to 84 mg, 48 to 80 mg, 50 to 76 mg, 52 to 72 mg, 54 to 68 mg, 56 to 64 mg, or 58 to 62 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0304] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 A composition comprising a complex of structure, such as a complex comprising a group of formula (Ia), (Ib), (Ic) or (Id), wherein an effective amount (e.g., an effective amount per administration) provides the subject with 36.8 mg to 58.9 mg (e.g., 36.8 mg to 58.9 mg, 36.8 mg to 51.5 mg, 36.8 mg to 44.2 mg, 44.2 mg to 58.9 mg, 44.2 mg to 51.5 mg, or 51.5 mg to 58.9 mg) of anti-TfR1 antibody / kg of the subject in the complex. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising formula (I): [R 1 ] n1 -R 2A composition of the invention (e.g., a complex of a structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic) or (Id)), wherein an effective amount (e.g., an effective amount per administration) provides the subject with about 36.8 mg of anti-TfR1 antibody / kg of subject in the complex. In some embodiments, with respect to any of the foregoing amounts of anti-TfR1 antibody, the value may vary by up to ±30% (e.g., ±up to 30%, ±up to 25%, ±up to 20%, ±up to 15%, ±up to 10%, ±up to 5%, ±up to 3%, or ±up to 1%). In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0305] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2A composition comprising a complex of structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic) or (Id), wherein the effective amount (e.g., the effective amount per administration) provides to the subject 1 mg to 14 mg (e.g., 1 mg to 14 mg, 1 mg to 13 mg, 1 mg to 12 mg, 1 mg to 11 mg, 1 mg to 10 mg, 1 mg to 9 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 6 mg, 1 mg to 5 mg, 1 mg to 4 mg, 1 mg to 3 mg, 1 mg to 2 mg, 2 mg to 14 mg, 2 mg to 13 mg, 2 mg to 12 mg, 2 mg to g to 11 mg, 2 mg to 10 mg, 2 mg to 9 mg, 2 mg to 8 mg, 2 mg to 7 mg, 2 mg to 6 mg, 2 mg to 5 mg, 2 mg to 4 mg, 2 mg to 3 mg, 3 mg to 14 mg, 3 mg to 13 mg, 3 mg to 12 mg, 3 mg to 11 mg, 3 mg to 10 mg, 3 mg to 9 mg, 3 mg to 8 mg, 3 mg to 7 mg, 3 mg to 6 mg, 3 mg to 5 mg, 3 mg to 4 mg, 4 mg to 14 mg, 4 mg to 13 mg, 4 mg to 12 mg, 4 mg to 11 mg, 4 mg to 10 mg, 4 mg to 9 mg, 4 mg to 8mg, 4mg to 7mg, 4mg to 6mg, 4mg to 5mg, 5mg to 14mg, 5mg to 13mg, 5mg to 12mg, 5mg to 11mg, 5mg to 10mg, 5mg to 9mg, 5mg to 8mg, 5mg to 7mg, 5mg to 6mg, 6mg to 14mg, 6mg to 13mg, 6mg to 12mg, 6mg to 11mg, 6mg to 10mg, 6mg to 9mg, 6mg to 8mg, 6mg to 7mg, 7mg to 14mg, 7mg to 13mg, 7mg to 12mg, 7mg to 11mg, 7mg to 10mg, 7mg to 9 mg, 7 mg to 8 mg, 8 mg to 14 mg, 8 mg to 13 mg, 8 mg to 12 mg, 8 mg to 11 mg, 8 mg to 10 mg, 8 mg to 9 mg, 9 mg to 14 mg, 9 mg to 13 mg, 9 mg to 12 mg, 9 mg to 11 mg, 9 mg to 10 mg, 10 mg to 14 mg, 10 mg to 13 mg, 10 mg to 12 mg, 10 mg to 11 mg, 11 mg to 14 mg, 11 mg to 13 mg, 11 mg to 12 mg, 12 mg to 14 mg, 12 mg to 13 mg, or 13 mg to 14 mg) of oligonucleotide / kg of subject. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising Formula (I): [R 1 ] n1 -R 2In some embodiments, the effective amount (e.g., the effective amount per administration) provides about 1.8 mg, about 3.4 mg, about 6.8 mg, or about 10.2 mg of the oligonucleotide in the complex per kg of the subject. In some embodiments, the effective amount (e.g., the effective amount per administration) provides about 4 mg or about 5 mg of the oligonucleotide in the complex per kg of the subject. In some embodiments, with respect to any of the foregoing amounts of the oligonucleotide, the values ​​may vary by up to ±30% (e.g., ±up to 30%, ±up to 25%, ±up to 20%, ±up to 15%, ±up to 10%, ±up to 5%, ±up to 3%, or ±up to 1%). In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks), and then administered once every 8 weeks during a second administration period (e.g., 16 weeks to the rest of the subject's life).

[0306] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 1.2 mg to 2.4 mg (e.g., 1.2 mg to 2.4 mg, 1.1 mg to 2.3 mg, 1.2 mg to 2.2 mg, 1.3 mg to 2.1 mg, 1.4 mg to 2 mg, 1.5 mg to 1.9 mg, or 1.6 mg to 1.85 mg) of oligonucleotide / kg of the subject. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0307] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject a composition comprising an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 2.2 mg to 4.5 mg (e.g., 2.2 mg to 4.5 mg, 2.4 mg to 4.4 mg, 2.5 mg to 4.3 mg, 2.6 mg to 4.2 mg, 2.7 mg to 4.1 mg, 2.8 mg to 4 mg, 2.9 mg to 3.9 mg, 3 mg to 3.8 mg, 3.1 mg to 3.7 mg, 3.2 mg to 3.6 mg, or 3.3 mg to 3.5 mg) of the oligonucleotide / kg of the subject in the complex. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0308] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 2.7 mg to 5.3 mg (e.g., 2.7 mg to 5.3 mg, 2.8 mg to 5.8 mg, 3 mg to 5.5 mg, 3.2 mg to 5 mg, or 3.8 mg to 4.2 mg) of the oligonucleotide / kg of the subject in the complex. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0309] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 3 to 7 mg (e.g., 3 to 7 mg, 3.5 to 6.6 mg, 4 to 6 mg, 4.2 to 5.8 mg, or 4.8 to 5.2 mg) of oligonucleotide / kg of the subject in the complex. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0310] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 4.5 mg to 9 mg (e.g., 4.5 mg to 9 mg, 4.7 mg to 8.8 mg, 4.9 mg to 8.6 mg, 5.1 mg to 8.4 mg, 5.3 mg to 8.2 mg, 5.5 mg to 8 mg, 5.7 mg to 7.8 mg, 5.9 mg to 7.6 mg, 6.1 mg to 7.4 mg, 6.3 mg to 7.2 mg, 6.5 mg to 7 mg, or 6.6 mg to 6.8 mg) of the oligonucleotide / kg of the subject. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0311] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 7 to 14 mg (e.g., 7 to 14 mg, 7.5 to 13.5 mg, 8 to 13 mg, 8.5 to 12.5 mg, 9 to 12 mg, 9.5 to 11.5 mg, or 10 to 11 mg) of oligonucleotide / kg of the subject in the complex. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0312] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2A composition comprising a complex of structure, such as a complex comprising a group of Formula (Ia), (Ib), (Ic) or (Id), wherein the effective amount (e.g., the effective amount per administration) provides to the subject 0.5 mg to 12 mg (e.g., 0.5 mg to 12 mg, 0.5 mg to 12 mg, 0.5 mg to 11 mg, 0.5 mg to 10 mg, 0.5 mg to 9 mg, 0.5 mg to 8 mg, 0.5 mg to 7 mg, 0.5 mg to 6 mg, 0.5 mg to 5 mg, 0.5 mg to 4 mg, 0.5 mg to 3 mg) of the complex. g, 0.5mg to 2mg, 0.5mg to 1mg, 1mg to 12mg, 1mg to 11mg, 1mg to 10mg, 1mg to 9mg, 1mg to 8mg, 1mg to 7mg, 1mg to 6mg, 1mg to 5mg, 1mg to 4mg, 1mg to 3mg, 1mg to 2mg, 2mg to 12mg, 2mg to 11mg, 2mg to 10mg, 2mg to 9mg, 2mg to 8mg, 2mg to 7mg, 2mg to 6mg, 2mg to 5mg, 2mg to 4mg, 2mg to 3mg, 3mg g to 12mg, 3mg to 11mg, 3mg to 10mg, 3mg to 9mg, 3mg to 8mg, 3mg to 7mg, 3mg to 6mg, 3mg to 5mg, 3mg to 4mg, 4mg to 12mg, 4mg to 11mg, 4mg to 10mg, 4mg to 9mg, 4mg to 8mg, 4mg to 7mg, 4mg to 6mg, 4mg to 5mg, 5mg to 12mg, 5mg to 11mg, 5mg to 10mg, 5mg to 9mg, 5mg to 8mg, 5mg to 7mg, 5mg to 6mg , 6 mg to 12 mg, 6 mg to 11 mg, 6 mg to 10 mg, 6 mg to 9 mg, 6 mg to 8 mg, 6 mg to 7 mg, 7 mg to 12 mg, 7 mg to 11 mg, 7 mg to 10 mg, 7 mg to 9 mg, 7 mg to 8 mg, 8 mg to 12 mg, 8 mg to 11 mg, 8 mg to 10 mg, 8 mg to 9 mg, 9 mg to 12 mg, 9 mg to 11 mg, 9 mg to 10 mg, 10 mg to 12 mg, 10 mg to 11 mg, or 11 mg to 12 mg) of oligonucleotide / kg of subject. In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., comprising Formula (I): [R 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0313] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject a composition comprising an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 0.7 mg to 1.5 mg (e.g., 0.7 mg to 1.5 mg, 0.8 mg to 1.4 mg, 0.9 mg to 1.3 mg, or 1 mg to 1.2 mg) of oligonucleotide / kg of the subject in the complex. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0314] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 1.4 mg to 2.7 mg (e.g., 1.4 mg to 2.7 mg, 1.5 mg to 2.6 mg, 1.6 mg to 2.5 mg, 1.7 mg to 2.4 mg, 1.8 mg to 2.3 mg, 1.9 mg to 2.2 mg, or 1.95 mg to 2.1 mg) of oligonucleotide / kg of the subject. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0315] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 2.7 mg to 5.4 mg (e.g., 2.7 mg to 5.4 mg, 2.9 mg to 5.2 mg, 3.1 mg to 5 mg, 3.3 mg to 4.8 mg, 3.5 mg to 4.6 mg, 3.7 mg to 4.3 mg, 3.9 mg to 4.2 mg, or 4 mg to 4.15 mg) of the oligonucleotide / kg of the subject. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0316] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 5 to 11 mg (e.g., 5 to 11 mg, 5.5 to 10.5 mg, 6 to 10 mg, 6.5 to 9.5 mg, 7 to 9 mg, 7.5 to 8.5 mg, or 8 to 8.3 mg) of the oligonucleotide / kg of the subject in the complex. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0317] It should be understood that once every 4 weeks is substantially similar to once a month, once every 8 weeks is substantially similar to once every two months, once every 12 weeks is substantially similar to once every three months, and once every 16 weeks is substantially similar to once every four months. Thus, in some embodiments, once every 4 weeks may mean once a month; once every 8 weeks may mean once every two months; once every 12 weeks may mean once every three months; and once every 16 weeks may mean once every four months. Similarly, in some embodiments, once every 4 weeks may mean 12 times a year; once every 8 weeks may mean 6 times a year; and once every 12 weeks may mean 4 times a year.

[0318] In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (eg, comprising formula (I): [R 1 ] n1 -R 2 In some embodiments, the methods described herein include administering to a subject an effective amount of a complex described herein (e.g., a complex comprising a group of Formula (Ia), (Ib), (Ic), or (Id)), wherein the effective amount (e.g., the effective amount per administration) provides the subject with 5 to 8 mg (e.g., 5 to 8 mg, 5 to 7 mg, 5 to 6 mg, 6 to 8 mg, 6 to 7 mg, or 7 to 8 mg) of oligonucleotide / kg of the subject in the complex. 1 ] n1 -R 2In some embodiments, the composition is administered once every 4 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 8 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 12 weeks (e.g., for a period of time, such as the rest of the subject's life). In some embodiments, the composition is administered once every 4 weeks during a first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks) and then once every 8 weeks during a second administration period (e.g., 16 weeks to the remainder of the subject's life).

[0319] In some embodiments, the methods described herein comprise intravenously administering (e.g., by intravenous infusion) an effective amount of a composition comprising a complex (e.g., in aqueous solution), wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Containing a group of formula (Ia):

[0320]

[0321] Where R 3 An oligonucleotide comprising a nucleobase sequence comprising CAGCGCCCACCAGUCA (SEQ ID NO: 21), optionally wherein R 3 an oligonucleotide comprising a structure comprising +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-MOE modified ribonucleoside, oC represents 5-methyl 2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage;

[0322] Where R 2The invention comprises an anti-TfR1 antibody (e.g., Fab) comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16, optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 19, and optionally wherein the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO: 21, a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20;

[0323] Each R 1 At the connection point A and R 2 covalently linked, optionally wherein each R 1 At the connection point A and R 2 covalently link different amino acid residues of an antibody (e.g., Fab) of the invention, optionally wherein each different amino acid residue is lysine; and

[0324] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R 1 , optionally wherein the average value of n1 of the complexes in the composition is 1 to 5; and

[0325] Wherein each effective amount of administration provides 5mg to 110mg (e.g., 8mg to 60mg or 13mg to 75mg) of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the object. In some embodiments, the effective amount of each administration provides about 13mg, about 25mg, about 50mg, or about 75mg of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the object. In some embodiments, the effective amount of each administration provides about 5mg, about 15mg, about 30mg, or about 60mg of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the object. In some embodiments, the composition is administered to the object once every four weeks or once every eight weeks. In some embodiments, the composition is administered to the object once every four weeks during the first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks), and then administered to the object once every eight weeks during the second administration period (e.g., 16 weeks to the rest of the life of the object). In some embodiments, the composition comprising a complex for administration to a subject in the methods described herein further comprises a complex comprising Formula (I): [R 1 ] n1 -R 2 , wherein n1 is 0. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0326] In some embodiments, the methods described herein comprise intravenously administering (e.g., by intravenous infusion) an effective amount of a composition comprising a complex (e.g., in aqueous solution), wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of each R 1 Containing a group of formula (Ib):

[0327]

[0328] wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-O-methoxyethyl (MoE) modified ribonucleoside, oC represents 5-methyl 2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0329] Where R 2 The invention comprises an anti-TfR1 antibody (e.g., Fab) comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16, optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 18. A heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20;

[0330] Each R 1 At the connection point A and R 2 covalently linked, optionally wherein each R 1 At the connection point A and R 2 covalently link different amino acid residues of an antibody (e.g., Fab) of the invention, optionally wherein each different amino acid residue is lysine; and

[0331] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R 1 number of instances of, optionally wherein the average value of n1 of the complexes in the composition is 1 to 5;

[0332] Wherein each effective amount of administration provides 5mg to 110mg (e.g., 8mg to 60mg or 13mg to 75mg) of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the subject. In some embodiments, the effective amount of each administration provides about 13mg, about 25mg, about 50mg, or about 75mg of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the subject. In some embodiments, the effective amount of each administration provides about 5mg, about 15mg, about 30mg, or about 60mg of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the subject. In some embodiments, the composition is administered to the subject once every four weeks or once every eight weeks. In some embodiments, the composition is administered to the subject once every four weeks during the first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks), and then administered to the subject once every eight weeks during the second administration period (e.g., 16 weeks to the rest of the life of the subject). In some embodiments, the composition comprising a complex for administration to a subject in the methods described herein further comprises a complex comprising Formula (I): [R 1 ] n1 -R 2 , wherein n1 is 0. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0333] In some embodiments, the methods described herein comprise intravenously administering (e.g., by intravenous infusion) an effective amount of a composition comprising a complex (e.g., in aqueous solution), wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of each R in the complex 1 Containing a group of formula (Ic):

[0334]

[0335] Where R 2Comprising an anti-TfR1 antibody (e.g., Fab), comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16, optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and optionally wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 18. A heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20;

[0336] Each R 1 At the connection point A and R 2 covalently linked, optionally wherein each R 1 At the connection point A and R 2 covalently link different amino acid residues of an antibody (e.g., Fab) of the invention, optionally wherein each different amino acid residue is lysine; and

[0337] Wherein in each complex, n1 is independently an integer of 1 or greater, which represents R 1 number of instances of, optionally wherein the average value of n1 of the complexes in the composition is 1 to 5;

[0338] Wherein each effective amount of administration provides 5mg to 110mg (e.g., 8mg to 60mg or 13mg to 75mg) of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the subject. In some embodiments, the effective amount of each administration provides about 13mg, about 25mg, about 50mg, or about 75mg of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the subject. In some embodiments, the effective amount of each administration provides about 5mg, about 15mg, about 30mg, or about 60mg of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the subject. In some embodiments, the composition is administered to the subject once every four weeks or once every eight weeks. In some embodiments, the composition is administered to the subject once every four weeks during the first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks), and then administered to the subject once every eight weeks during the second administration period (e.g., 16 weeks to the rest of the life of the subject). In some embodiments, the composition comprising a complex for administration to a subject in the methods described herein further comprises a complex comprising Formula (I): [R 1 ] n1 -R 2 , wherein n1 is 0. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0339] In some embodiments, the methods described herein comprise intravenously administering (e.g., by intravenous infusion) an effective amount of a composition comprising a complex (e.g., in aqueous solution), wherein each complex comprises a group of formula (Id):

[0340]

[0341] wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-O-methoxyethyl (MoE) modified ribonucleoside, oC represents 5-methyl 2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0342] Where R 2 The invention comprises an anti-TfR1 antibody (e.g., Fab), comprising: a heavy chain complementary determining region 1 (CDR-H1) comprising a sequence as shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising a sequence as shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising a sequence as shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising a sequence as shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising a sequence as shown in SEQ ID NO: 5 or 11, and a light chain complementary determining region 3 (CDR-L3) comprising a sequence as shown in SEQ ID NO: 6 or 16, optionally wherein the antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and optionally wherein the antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 17 and a VL comprising the amino acid sequence of SEQ ID NO: 18, and optionally wherein the antibody (e.g., Fab) comprises a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 19. NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; and

[0343] wherein in each complex, n1 is independently an integer of 1 or greater, optionally wherein the average value of n1 of the complexes in the composition is from 1 to 5;

[0344] Wherein each effective amount of administration provides 5mg to 110mg (e.g., 8mg to 60mg or 13mg to 75mg) of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the object. In some embodiments, the effective amount of each administration provides about 13mg, about 25mg, about 50mg, or about 75mg of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the object. In some embodiments, the effective amount of each administration provides about 5mg, about 15mg, about 30mg, or about 60mg of anti-TfR1 antibody (e.g., Fab) / kg object in the complex to the object. In some embodiments, the composition is administered to the object once every four weeks or once every eight weeks. In some embodiments, the composition is administered to the object once every four weeks during the first administration period (e.g., 8 weeks, 12 weeks, or 16 weeks), and then administered to the object once every eight weeks during the second administration period (e.g., 16 weeks to the rest of the life of the object). In some embodiments, the composition comprising a complex for administration to a subject in the methods described herein further comprises a complex comprising Formula (I): [R 1 ] n1 -R 2 , wherein n1 is 0. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is 0.5 to 5 (e.g., 0.5 to 5, 1 to 5, 1 to 4, 1 to 3, 3 to 5, 0.5 to 4, 0.5 to 3, 0.5 to 2, 0.5 to 1.5, 0.5 to 1, 0.7 to 1.5, 1 to 1.6, 1 to 1.5, 1 to 1.4, 1 to 1.3, 1 to 1.2, 1.1 to 1.5, 0.8 to 2, 0.8 to 1.5, 0.8 to 1.3, 0.8 to 1.2, 0.8 to 1.1, 0.9 to 3, 0.9 to 2, 0.9 to 1.8, 0.9 to 1.6, 0.9 to 1.5, 0.9 to 1.4, 0.9 to 1.3, or 0.9 to 1.2).

[0345] In some embodiments, the methods described herein include intravenously administering (e.g., by intravenous infusion) an effective amount of a composition comprising a complex (e.g., in an aqueous solution), wherein each complex comprises a group of formula (Ia), (Ib), (Ic) or (Id), wherein the effective amount for each administration provides the subject with 0.5 mg to 20 mg (e.g., 1 mg to 14 mg or 0.5 mg to 12 mg) of oligonucleotide / kg of the subject in the complex. In some embodiments, the effective amount for each administration provides the subject with about 1.8 mg, about 3.4 mg, about 6.8 mg, or about 10.2 mg of oligonucleotide / kg of the subject in the complex. In some embodiments, the effective amount for each administration provides the subject with about 1.1 mg, about 2 mg, about 4.1 mg, or about 8.2 mg of oligonucleotide / kg of the subject in the complex. In some embodiments, the composition is administered to the subject once every four weeks or once every eight weeks. In some embodiments, the composition comprising the complex (e.g., in an aqueous solution) for administration to the subject in the methods described herein further comprises a complex wherein n1 is 0. Thus, in some embodiments, the average value of n1 of the complexes in the compositions disclosed herein is from 0.5 to 5 (e.g., from 0.5 to 5, from 1 to 5, from 1 to 4, from 1 to 3, from 3 to 5, from 0.5 to 4, from 0.5 to 3, from 0.5 to 2, from 0.5 to 1.5, from 0.5 to 1, from 0.7 to 1.5, from 1 to 1.6, from 1 to 1.5, from 1 to 1.4, from 1 to 1.3, from 1 to 1.2, from 1.1 to 1.5, from 0.8 to 2, from 0.8 to 1.5, from 0.8 to 1.3, from 0.8 to 1.2, from 0.8 to 1.1, from 0.9 to 3, from 0.9 to 2, from 0.9 to 1.8, from 0.9 to 1.6, from 0.9 to 1.5, from 0.9 to 1.4, from 0.9 to 1.3, or from 0.9 to 1.2).

[0346] In some embodiments, administration of a composition comprising an effective amount of a complex according to any of the methods described herein reduces the expression or activity of DMPK in a subject (e.g., a subject suffering from DM1) (e.g., reduces the level of mutant or wild-type DMPK RNA, or reduces the activity of a DMPK gene product). In some embodiments, administration of a composition comprising an effective amount of a complex according to any of the methods described herein results in a decrease in the expression or activity of DMPK in a subject (e.g., the level of mutant or wild-type DMPK RNA, or the activity of a DMPK gene product) by at least 1% relative to a control (e.g., at least 1%, at least 2%, at least 3%, at least 4%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%). In some embodiments, the control is the DMPK expression or activity level in a healthy subject (e.g., a subject not suffering from myotonic dystrophy). In some embodiments, the control is the DMPK expression or activity level in a subject that does not have a DMPK RNA with a mutation. In some embodiments, the control is the DMPK expression or activity level in the subject before administering a composition comprising an effective amount of the complex to the subject.

[0347] In some embodiments, any of the methods described herein may further comprise the additional step of administering to a subject a composition comprising a complex described herein.

[0348] Example

[0349] Example 1. In vivo tissue distribution of a conjugate containing an anti-TfR1 Fab conjugated to a DMPK targeting oligonucleotide in the DM1 mouse model

[0350] A conjugate comprising an anti-TfR1 Fab conjugated to a DMPK targeting oligonucleotide (ASO) was tested in a mouse model expressing both human TfR1 and a human DMPK mutant containing expanded CUG repeats. The anti-TfR1 Fab used had a VH / VL sequence provided in Table 2. The Fab was covalently linked (conjugated by lysine) to a DMPK targeting oligonucleotide comprising a nucleobase sequence of SEQ ID NO: 21 via a linker comprising a valine-citrulline sequence. The conjugate comprises a structure of formula (Id):

[0351]

[0352] wherein +N represents an LNA (2'-4' methylene bridge) ribonucleoside, dN represents a 2'-deoxyribonucleoside, oN represents a 2'-O-methoxyethyl (MoE) modified ribonucleoside, oC represents 5-methyl 2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents a phosphorothioate internucleoside linkage, and wherein the oligonucleotide comprises a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21), and wherein R 2 comprising an anti-TfR1 antibody provided in Table 2, and wherein in each conjugate, n1 is independently an integer from 1 to 3.

[0353] The conjugate was administered intravenously to mice at a dose equivalent to 9.7 mg / kg ASO on days 0 and 7. Tissue exposure of ASO was tested by hybrid ELISA (Burki et al., Nucleic Acid Ther. 2015 Oct; 25(5): 275-84; which is incorporated herein by reference), and ASO levels in tissues were plotted. Figure 1A , 1B , 1C and 1D show the amount of ASO in the heart, diaphragm, gastrocnemius or tibialis anterior muscle two weeks after the first injection, respectively. These results indicate that conjugates comprising anti-TfR1 antibodies (e.g., anti-TfR1 Fabs having VH and VL sequences provided in Table 2) are capable of delivering oligonucleotides (e.g., DMPK targeting ASOs) to a variety of muscle tissues after intravenous administration.

[0354] Example 2. Sustained knockdown of toxic human DMPK in hTfR1 / DMSXL homozygous mice 4 weeks after repeated administration of anti-TfR1 Fab-ASO conjugates

[0355] The conjugates comprising anti-TfR1Fab covalently linked to DMPK targeting oligonucleotides (ASOs) as described in Example 1 (labeled in this example as "anti-TfR1Fab-ASO conjugates") were tested in a mouse model (hTfR1 / DMSXL mice) expressing both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CUG repeats. On days 0 and 7, vehicle controls (PBS) or 10 mg / kg ASO equivalent doses of anti-TfR1Fab-ASO conjugates were administered to mice. Mice were sacrificed on day 28 (4 weeks after the first dose of anti-TfR1Fab-ASO conjugates were administered) and tissues were collected. RNA was extracted and selected tissue samples were fixed, paraffin-embedded, and sectioned, followed by in situ hybridization. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) of RNA samples was performed to measure human DMPK and mouse Ppib (peptidylprolyl isomerase) as an internal control. DMPK expression in Figures 2A to 2D The data are shown as geometric mean + / - standard deviation (n=6 to 9). The significance was assessed by Student's t-test (****P<0.0001).

[0356] Figure 2A It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in the heart by 49% relative to PBS-treated mice. Figure 2B It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in diaphragm muscle by 40% relative to PBS-treated mice. Figure 2C It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in tibialis anterior muscle by 49% relative to PBS-treated mice. Figure 2D It was shown that anti-TfR1 Fab-ASO conjugate knocked down DMPK expression in gastrocnemius muscle by 44% relative to PBS-treated mice.

[0357] Figure 3A and 3B It was shown that anti-TfR1 Fab-ASO conjugate reduced DMPK foci within myofiber nuclei. Figure 3A Reduced DMPK foci were shown by in situ hybridization, and Figure 3B Quantification of DMPK foci in fluorescence microscopy images is shown, demonstrating that the conjugate reduced foci area by 49%. Data are presented as mean + / - standard deviation (n=7). Significance was assessed by t-test (*P<0.05).

[0358] These results demonstrate that administration of anti-TfR1 Fab-ASO conjugates results in robust, sustained knockdown of human toxic DMPK in cardiac and skeletal muscle.

[0359] Example 3. Correction of splicing defects in hTfR1 / DMSXL homozygous mice by anti-TfR1 Fab-ASO conjugates

[0360] A conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) as described in Example 1 (labeled in this example as "anti-TfR1 Fab-ASO conjugate") was tested in a mouse model ("hTfR1 / DMSXL") that expresses both human TfR1 and two copies of a mutant human DMPK transgene containing expanded CUG repeats. These mice are known to exhibit splicing defects consistent with those observed in patients with DM1 (Huguet, et al. (2012) PLOS Genetics 8(11): e1003043). On days 0 and 7, mice were administered vehicle control ("hTfR1 / DMSXL-PBS") or a 10 mg / kg ASO equivalent dose of anti-TfR1 Fab-ASO conjugate ("hTfR1 / DMSXL-conjugate"). Mice expressing only human TfR1 but not mutant human DMPK transgenes (hTfR1 mice) and mice treated with PBS ("hTfR1-PBS") were used as another control to determine the extent of the splicing phenotype in hTfR1 / DMSXL mice and to assess the extent of the effect of the conjugate on splicing. Mice were sacrificed on day 28 (four weeks after administration of the first dose of anti-TfR1 Fab-ASO conjugate), tissues were collected and RNA was extracted. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to measure exon inclusion in a set of RNAs known to be mis-spliced ​​during DM1 progression in humans and mice (Nakamori, et al. (2013) Ann. Neurol. 74(6):862-872; Huguet, et al. (2012) PLOS Genetics 8(11):e1003043). Exon inclusion was calculated as the normalized percent spliced ​​in (PSI) for each spliced ​​RNA marker and using RNA from heart ( Figure 4 ), diaphragm( Figure 5 ), tibialis anterior ( Figure 6 ) and gastrocnemius ( Figure 7) were used to calculate the composite splicing index using the normalized PSI values ​​of the splicing markers in . The composite splicing index was calculated as previously described (Tanner MK, et al. (2021) Nucleic Acids Res. 49: 2240-2254) and is shown as mean + / - standard deviation.

[0361] Figure 4 It is shown that anti-TfR1 Fab-ASO conjugate corrects splicing in hTfR1 / DMSXL mouse heart tissue, as shown in the composite splicing index data. The normalized PSI values ​​used to generate the composite splicing index data show correction of Mbnl2 exon 6 (E6) and Nfix E7 splicing in hTfR1 / DMSXL mouse heart tissue by treatment with anti-TfR1 Fab-ASO conjugate, but not correction of Ldb3E11 splicing. Figure 4 The composite splicing index data shown in are based on Ldb3E11, Mbnl2E6, and Nfix E7 splicing data; Bin1E11, Dtna E12, Insr E11, and Mbnl2E5 were not included because their normalized PSI values ​​in cardiac tissue were not altered in hTfR1 / DMSXL mice relative to hTfR1 mice under the experimental conditions tested.

[0362] Figure 5 Anti-TfR1 Fab-ASO conjugates are shown to correct splicing in hTfR1 / DMSXL mouse diaphragm tissue, as shown in the composite splicing index data. The normalized PSI values ​​used to generate the composite splicing index data show the correction of Bin1E11, Insr E11, Ldb3E11 and Nfix E7 splicing in hTfR1 / DMSXL mouse diaphragm tissue by treatment with anti-TfR1 Fab-ASO conjugates. Figure 5 The composite splicing index data shown in are based on Bin1E11, Insr E11, Ldb3E11, and Nfix E7 splicing data; Dtna E12, Mbnl2E5, Mbnl2E6, and Ttn E313 are not included because their normalized PSI values ​​in diaphragm tissue were not altered in hTfR1 / DMSXL mice relative to hTfR1 mice under the experimental conditions tested.

[0363] Figure 6It is shown that the anti-TfR1 Fab-ASO conjugate corrects splicing in hTfR1 / DMSXL mouse tibialis anterior muscle tissue, as shown in the composite splicing index data. The normalized PSI values ​​used to generate the composite splicing index data show correction of Bin1E11, Ldb3E11 and NfixE7 splicing in hTfR1 / DMSXL mouse tibialis anterior muscle tissue by treatment with anti-TfR1 Fab-ASO conjugate, but not correction of Mbnl2E6 splicing. Figure 6 The composite splicing index data shown in are based on Bin1E11, Ldb3E11, Mbnl2E6, and Nfix E7 splicing data; Dtna E12, Insr E11, Mbnl2E5, and Ttn E313 are not included because their normalized PSI values ​​in tibialis anterior muscle tissue were not altered in hTfR1 / DMSXL mice relative to hTfR1 mice under the experimental conditions tested.

[0364] Figure 7 It is shown that anti-TfR1 Fab-ASO conjugate corrects splicing in gastrocnemius muscle tissue of hTfR1 / DMSXL mice, as shown in the composite splicing index data. The normalized PSI values ​​used to generate the composite splicing index data show the correction of Mbnl2E6, Nfix E7 and TtnE313 splicing in gastrocnemius muscle tissue of hTfR1 / DMSXL mice by treatment with anti-TfR1 Fab-ASO conjugate. Figure 7 The composite splicing index data shown in are based on Mbnl2E6, Nfix E7, and Ttn E313 splicing data; Bin1E11, Dtna E12, Insr E11, Ldb3E11, and Mbnl2E5 are not included because their normalized PSI values ​​in gastrocnemius muscle tissue were not altered in hTfR1 / DMSXL mice relative to hTfR1 mice under the experimental conditions tested.

[0365] These results suggest that administration of anti-TfR1 Fab-ASO conjugate facilitates the correction of DM1 splicing defects in cardiac and skeletal muscles.

[0366] Example 4. DMPK knockdown in non-human primate and DM1 patient myotubes

[0367] The conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) as described in Example 1 (labeled in this example as "anti-TfR1 Fab-ASO conjugate") was tested in human DM1 patient myotubes (32F cells) and non-human primate (NHP) myotubes. The DM1 patient myotubes used expressed both wild-type DMPK mRNA and a mutant DMPK mRNA containing 380 CUG repeats. The NHP myotubes used expressed only wild-type DMPK.

[0368] DM1 patient cells or NHP cells were seeded into 96-well plates in growth medium at a density of 50,000 cells / well and allowed to recover overnight. The next day, the growth medium was changed to low serum differentiation medium and the cells were treated with conjugates at concentrations equivalent to 125nM, 250nM, or 500nM ASO. The cells were incubated for 10 days and then cDNA was synthesized using the Cells-to-Ct kit, with crude cell lysate as the source of total RNA.

[0369] The cDNA was used to assess total DMPK knockdown using Taqman PCR. Data were normalized to PPIB expression and expressed using 2-ΔΔ C The t method was used to determine DMPK knockdown compared to PBS-treated controls ("Vehicle"). Figure 8 Data shown in are presented as mean DMPK expression + standard deviation relative to species-matched vehicle controls (n=4 replicates per condition).

[0370] The results showed that when treated at physiologically relevant concentrations, anti-TfR1Fab-ASO conjugates achieved knockdown of DMPK expression in both normal NHP myotubes and DM1 patient myotubes, with a higher degree of knockdown of DMPK expression in DM1 patient cells (expressing both wild-type DMPK mRNA and DMPK mRNA containing 380 CUG repeats) compared to NHP cells (expressing only wild-type DMPK mRNA) ( Figure 8 ). At an ASO equivalent concentration of 125 nM, the conjugate achieved approximately 40% DMPK knockdown in NHP myotubes and approximately 65% ​​DMPK knockdown in DM1 patient myotubes relative to the vehicle-only control. At an ASO equivalent concentration of 250 nM, the conjugate achieved approximately 45% DMPK knockdown in NHP myotubes and approximately 80% DMPK knockdown in DM1 patient myotubes relative to the vehicle-only control. At an ASO equivalent concentration of 500 nM, the conjugate achieved approximately 60% DMPK knockdown in NHP myotubes and approximately 90% DMPK knockdown in DM1 patient myotubes relative to the vehicle-only control.

[0371] These results indicate that the conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide can achieve a greater degree of DMPK knockdown in human myotubes expressing wild-type DMPK mRNA and mutant DMPK mRNA (with expanded CUG repeats) relative to cynomolgus monkey myotubes expressing wild-type DMPK.

[0372] Example 5. DMPK knockdown in hTfR1 / DMSXL hemizygous mice 12 weeks after administration of anti-TfR1 Fab-ASO conjugate

[0373] The conjugates containing anti-TfR1Fab covalently linked to DMPK targeting oligonucleotides (ASO) as described in Example 1 were tested in the hTfR1 / DMSXL hemizygous mouse model. After intravenous administration to mice via the tail vein, mutant human DMPK expression was monitored in a variety of tissues for 12 weeks. The conjugates were administered to mice at 20 mg / kg ASO equivalent doses. Heart, diaphragm, gastrocnemius and tibialis anterior muscle tissues were collected. Mutant human DMPK was knocked down by quantitative PCR (qPCR) for DMPK mRNA expression. DMPK knockdown in treated mice was determined in 1 week (7 days), 2 weeks (14 days), 4 weeks (28 days), 8 weeks (56 days) and 12 weeks (84 days) after the administration of anti-TfR1Fab-ASO conjugates.

[0374] Fig.9A , Fig. 9B , Fig. 9C and Fig.9D Relative to control (PBS treated) mice, mutant human DMPK expression in the heart, diaphragm, tibialis anterior and gastrocnemius tissues of treated mice is shown respectively. The maximum knockdown of DMPK is usually reached 2 to 4 weeks after treatment, after which DMPK knockdown decreases. After administration of the conjugate, the expression of mutant human DMPK in the heart remains suppressed for up to 3 months. Data are expressed as mean + / - standard deviation (n = 5 to 12 replicates for each tissue).

[0375] These data indicate that the efficacy of the administered anti-TfR1 Fab-ASO conjugate persists for at least two weeks following treatment.

[0376] Example 6. DMPK knockdown in hTfR1 / DMSXL hemizygous mice following administration of anti-TfR1 Fab-ASO conjugates

[0377] The conjugates containing anti-TfR1Fab covalently linked to DMPK targeting oligonucleotides (ASOs) as described in Example 1 were further tested in the hTfR1 / DMSXL hemizygous mouse model. DMPK expression in different tissues was evaluated 4 weeks after treatment with the same dose of anti-TfR1Fab-ASO conjugates (administered in a single dose or two separate doses). Mice received a single dose of conjugate equivalent to 10 mg / kg ASO, or received two doses of conjugate (each dose equivalent to 5 mg / kg ASO). Mice receiving two 5 mg / kg doses received the doses one week apart or two weeks apart. DMPK knockdown and ASO levels were evaluated in multiple muscle tissues 4 weeks (28 days) after the last dose (10 mg / kg dose or the second dose in two 5 mg / kg doses).

[0378] Fig. 10A , 10B , 10C and 10D show the expression of DMPK in the heart, diaphragm, tibialis anterior and gastrocnemius tissues of treated mice, respectively, relative to control (vehicle treated) mice. Mice treated with all dosing regimens continued to knock down toxic human DMPK for up to 4 weeks. Dividing the 10 mg / kg dose into two separate 5 mg / kg doses improved the knockdown of DMPK in some tested muscle tissues. A single 10 mg / kg dose resulted in a 39% knockdown in the heart, a 50% knockdown in the diaphragm, a 38% knockdown in the tibialis anterior, and a 31% knockdown in the gastrocnemius; two 5 mg / kg doses administered one week apart resulted in a 51% knockdown in the heart, a 45% knockdown in the diaphragm, a 46% knockdown in the tibialis anterior, and a 50% knockdown in the gastrocnemius; and two 5 mg / kg doses administered two weeks apart resulted in a 55% knockdown in the heart, a 35% knockdown in the diaphragm, a 47% knockdown in the tibialis anterior, and a 40% knockdown in the gastrocnemius, as measured 28 days after the last dose of the complex was administered. Data are expressed as mean + standard deviation (n = 6 replicates for each tissue). Significance was determined by a one-way ANOVA test.

[0379] These results suggest that anti-TfR1 Fab-ASO conjugates can be administered to a subject in temporally spaced doses to enhance the efficacy of DMPK knockdown without increasing the total dose of ASO administered to the subject.

[0380] Example 7. DMPK knockdown in hTfR1 / DMSXL hemizygous mice following administration of anti-TfR1 Fab-ASO conjugates

[0381] The conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) as described in Example 1 was further tested in the hTfR1 / DMSXL hemizygous mouse model administered as a single dose or two doses one week apart. DMPK expression in various tissues of mice after treatment with the conjugate was assessed relative to expression in control (vehicle-treated) mouse tissues.

[0382] Fig.11A , 11B , 11C and 11D show the expression of mutant human DMPK in the heart, diaphragm, tibialis anterior and gastrocnemius tissues of mice treated with a 5 mg / kg ASO equivalent dose, a 10 mg / kg ASO equivalent dose or a 20 mg / kg ASO equivalent dose of the conjugate, respectively.DMPK was assessed to be low 28 days after the conjugate was administered. A dose-dependent trend of toxic human DMPK being low was observed, with approximately the same effect at the 10 mg / kg and 20 mg / kg doses tested at the 28th day time point. The 5 mg / kg dose resulted in a 30% knockdown in the heart, a 26% knockdown in the diaphragm, a 39% knockdown in the tibialis anterior, and a 19% knockdown in the gastrocnemius; the 10 mg / kg dose resulted in a 46% knockdown in the heart, a 51% knockdown in the diaphragm, a 46% knockdown in the tibialis anterior, and a 42% knockdown in the gastrocnemius; and the 20 mg / kg dose resulted in a 41% knockdown in the heart, a 47% knockdown in the diaphragm, a 53% knockdown in the tibialis anterior, and a 40% knockdown in the gastrocnemius, as measured 28 days after administration of the complex. Data are expressed as mean + standard deviation. Significance was determined by a one-way ANOVA test.

[0383] Fig. 12A , 12B, 12C and 12D respectively show the human mutant DMPK expression in the heart, diaphragm, tibialis anterior and gastrocnemius tissue of mice treated with the conjugate of two 5mg / kg ASO equivalent doses (10mg / kg in total) or two 10mg / kg ASO equivalent doses (20mg / kg in total). Two 5mg / kg ASO equivalent doses and two 10mg / kg ASO equivalent doses were administered at intervals of one week. DMPK was assessed to be knocked down 28 days after the first dose was administered to mice (i.e., 21 days after the second dose was administered). The dose-dependent effect of knocking down DMPK was observed in all assay tissues. Two 5 mg / kg doses of the complex resulted in 36% knockdown in the heart, 34% knockdown in the diaphragm, 36% knockdown in the gastrocnemius, and 38% knockdown in the tibialis anterior; two 10 mg / kg doses of the complex resulted in 51% knockdown in the heart, 78% knockdown in the diaphragm, 64% knockdown in the gastrocnemius, and 57% knockdown in the tibialis anterior, as measured 28 days after administration of the first dose of the complex. Data are expressed as mean + standard deviation (n = 5 to 6 replicates per tissue). Significance was determined by Brown-Forsythe and Welch ANOVA test with Dunnett T3 multiple comparison test.

[0384] Anti-TfR1 Fab-ASO conjugates were also tested in hTfR1 / DMSXL hemizygous mice after four monthly doses of the conjugate were administered to mice. The conjugates were administered as follows: four 5 mg / kg ASO-equivalent doses or four 10 mg / kg ASO-equivalent doses of the conjugate, with each administration spaced 4 weeks apart (on days 0, 28, 56, and 84, respectively). DMPK knockdown in multiple tissues was assessed 112 days (16 weeks) after the first dose was administered to mice. Fig.13A , 13B, 13C and 13D respectively show the expression of human mutant DMPK in the heart, diaphragm, tibialis anterior and gastrocnemius tissues of mice treated with conjugates. DMPK knockdown was observed in all tested tissues, and the knockdown of both the tibialis anterior and gastrocnemius of mice administered with four 10mg / kg ASO equivalent doses was statistically significantly improved relative to mice receiving four 5mg / kg doses. The complex of four 5mg / kg monthly doses resulted in 50% knockdown in the heart, 43% knockdown in the diaphragm, 45% knockdown in the gastrocnemius, and 47% knockdown in the tibialis anterior; and the complex of four 10mg / kg monthly doses resulted in 48% knockdown in the heart, 51% knockdown in the diaphragm, 64% knockdown in the gastrocnemius, and 59% knockdown in the tibialis anterior, as measured 112 days after the first dose of the complex was administered. Data are expressed as mean + standard deviation (n = 5 to 6 replicates for each tissue). Significance was determined by ANOVA test followed by uncorrected Fisher's least significant difference test.

[0385] These results show the effect of different doses of anti-TfR1 Fab-ASO conjugate on the expression of toxic human DMPK and suggest that the efficacy of the conjugate can be enhanced by splitting the dose of the conjugate.

[0386] Example 8. DMPK knockdown in non-human primates following administration of anti-TfR1 Fab-ASO conjugates

[0387] A conjugate comprising an anti-TfR1 Fab covalently linked to a DMPK targeting oligonucleotide (ASO) as described in Example 1 was tested in a non-human primate (NHP) model (cynomolgus monkey; Macaca fascicularis) at doses informed by the murine studies described in Examples 5 to 7. DMPK expression in various tissues was assessed following treatment with the conjugate as measured using a quantitative PCR assay (qPCR) for DMPK mRNA expression.

[0388] Fig.14A and 14B The DMPK expression in the gastrocnemius and tibialis anterior muscle tissues of NHP treated with the conjugate of about 10mg / kgASO equivalent dose measured at 4, 8 and 12 weeks after intravenous infusion of the conjugate is shown respectively. Expression is shown relative to the control animals of vehicle only. At each time point tested, the expression in the animals of the conjugate was reduced relative to the control (the relative expression in the gastrocnemius was 0.54, 0.47 and 0.61 respectively at 4, 8 and 12 weeks after administration; the relative expression in the tibialis anterior was 0.75, 0.48 and 0.72 respectively at 4, 8 and 12 weeks after administration). Data are expressed as mean + / - standard deviation. Significance was determined by unpaired t test.

[0389] Fig.15A , 15B , 15C and 15D show DMPK expression in heart, diaphragm, tibialis anterior and gastrocnemius tissues of NHP treated with 5mg / kg or 10mg / kg ASO equivalent doses of conjugates, respectively, relative to control (vehicle treated) animals. DMPK knockdown was assessed four weeks after the conjugate was administered. The dose-dependent effect of the conjugate on DMPK knockdown in cardiac muscle and skeletal muscle was observed. At the assay time, the 5mg / kg dose resulted in 21% knockdown in the heart, 43% knockdown in the diaphragm, 22% knockdown in the tibialis anterior and 25% knockdown in the gastrocnemius; and the 10mg / kg dose resulted in 38% knockdown in the heart, 60% knockdown in the diaphragm, 30% knockdown in the tibialis anterior and 35% knockdown in the gastrocnemius. These results are consistent with the dose effects previously observed in the hTfR1 / DMSXL mouse model. Data are expressed as mean + standard deviation. Significance was determined by one-way ANOVA test with Dunnett's post hoc analysis.

[0390] Fig.16A , 16B , 16C, 16D, 16E, 16F and 16G respectively show the DMPK expression in the heart, diaphragm, tibialis anterior, gastrocnemius, masseter, esophagus and duodenum tissues of NHP treated with two 5mg / kg or two 10mg / kg ASO equivalent doses of conjugates, wherein the two doses were administered at four weeks (28 days) apart.DMPK was assessed to be low after 8 weeks of administration of the first dose of the conjugate.Anti-TfR1 Fab-ASO conjugates were administered monthly resulting in continuous DMPK being knocked down in all tissues measured.Administering 10mg / kg ASO equivalent doses monthly resulted in the maximum and most significant knockdown of WT DMPK in tissues associated with DM1 pathology. The composite of two 5 mg / kg ASO equivalent doses resulted in 24% knockdown in the heart, 41% knockdown in the diaphragm, 56% knockdown in the gastrocnemius, 45% knockdown in the tibialis anterior, 39% knockdown in the masseter, 21% knockdown in the esophagus, and 28% knockdown in the duodenum; the composite of two 10 mg / kg ASO equivalent doses resulted in 34% knockdown in the heart, 64% knockdown in the diaphragm, 70% knockdown in the gastrocnemius, 65% knockdown in the tibialis anterior, 60% knockdown in the masseter, 49% knockdown in the esophagus, and 67% knockdown in the duodenum, as measured 8 weeks after the first dose was administered. Data are expressed as mean + standard deviation. Significance was determined by one-way ANOVA followed by an uncorrected Fisher's least significant difference (LSD) test.

[0391] These results demonstrate that the anti-TfR1 Fab-ASO conjugate is effective in the NHP model. These results also indicate that the efficacy of the conjugate is prolonged when administered monthly and are consistent with previous results obtained in a mouse model that recapitulates the pathological development of DM1.

[0392] Additional embodiments

[0393] 1. A method for reducing DMPK expression in a subject, comprising administering to the subject a composition comprising an effective amount of a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to one or more oligonucleotides, wherein the effective amount provides the subject with 5 mg to 110 mg of the anti-TfR1 antibody / kg of the subject in the complex, wherein the antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a heavy chain complementary determining region 3 (CDR-H4) comprising the sequence shown in SEQ ID NO: 6, 8 or 13. NO: 6 or 16, wherein the oligonucleotide in the complex comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

[0394] 2. A method for treating myotonic dystrophy in a subject, comprising administering to the subject a composition comprising an effective amount of a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to one or more oligonucleotides, wherein the effective amount provides the subject with 5 mg to 110 mg of the anti-TfR1 antibody / kg of the subject in the complex, wherein the antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a heavy chain complementary determining region 3 (CDR-H4) comprising the sequence shown in SEQ ID NO: 6 or 8. NO: 6 or 16, wherein the oligonucleotide in the complex comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

[0395] 3. A method for reducing DMPK expression in a subject, comprising administering to the subject a composition comprising an effective amount of a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to one or more oligonucleotides, wherein the effective amount provides the subject with 1 mg to 12 mg of oligonucleotides / kg of the subject in the complex, wherein the antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a heavy chain complementary determining region 3 (CDR-H4) comprising the sequence shown in SEQ ID NO: 6, 8 or 13. NO: 6 or 16, wherein the oligonucleotide in the complex comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

[0396] 4. A method for treating myotonic dystrophy in a subject, comprising administering to the subject a composition comprising an effective amount of a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to one or more oligonucleotides, wherein the effective amount provides to the subject 1 mg to 12 mg of the oligonucleotides / kg of the subject in the complex, wherein the antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a heavy chain complementary determining region 3 (CDR-H4) comprising the sequence shown in SEQ ID NO: 6 or 8. NO: 6 or 16, wherein the oligonucleotide in the complex comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

[0397] 5. The method of any one of embodiments 1 to 4, wherein the oligonucleotide in the complex comprises a 5'-XYZ-3' configuration, wherein X and Z are flanking regions comprising one or more modified nucleosides, and Y is a spacer region comprising one or more 2'-deoxyribonucleosides.

[0398] 6. The method of any one of embodiments 1 to 5, wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of which:

[0399] Each R 1 Containing a group of formula (Ib):

[0400]

[0401] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage; and R 1 The oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0402] R 2 comprising an anti-TfR1 antibody; and

[0403] In each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A, optionally wherein the average value of n1 of the complexes in the composition is 0.5 to 5.

[0404] 7. The method of any one of embodiments 1 to 5, wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of which:

[0405] Each R 1 Containing a group of formula (Ic):

[0406]

[0407] Where R 2 comprising an anti-TfR1 antibody; and

[0408] In each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A, optionally wherein the average value of n1 of the complexes in the composition is 0.5 to 5.

[0409] 8. The method of any one of embodiments 1 to 7, wherein the anti-TfR1 antibody is a Fab fragment.

[0410] 9. The method of any one of embodiments 1 to 8, wherein the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 18.

[0411] 10. The method of any one of embodiments 1 to 9, wherein the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20.

[0412] 11. The method of any one of embodiments 1 to 10, wherein the administering occurs one or more times.

[0413] 12. The method of embodiment 11, wherein the effective amount per administration provides the subject with 10 mg to 110 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0414] 13. The method of embodiment 11, wherein the effective amount per administration provides the subject with 5 mg to 90 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0415] 14. The method described in embodiment 11, wherein the effective amount for each administration provides the subject with 10 mg to 20 mg of anti-TfR1 antibody / kg of the subject in the complex, optionally wherein the effective amount for each administration provides the subject with 13 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0416] 15. The method described in embodiment 11, wherein the effective amount for each administration provides the subject with 18 mg to 36 mg of anti-TfR1 antibody / kg of the subject in the complex, optionally wherein the effective amount for each administration provides the subject with 25 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0417] 16. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 36 mg to 72 mg of anti-TfR1 antibody / kg of the subject in the complex, optionally wherein the effective amount for each administration provides the subject with 50 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0418] 17. The method described in embodiment 11, wherein the effective amount for each administration provides the subject with 55 mg to 110 mg of anti-TfR1 antibody / kg of the subject in the complex, optionally wherein the effective amount for each administration provides the subject with 75 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0419] 18. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 6 mg to 12 mg of anti-TfR1 antibody / kg of the subject in the complex, optionally wherein the effective amount for each administration provides the subject with 8 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0420] 19. The method described in embodiment 11, wherein the effective amount for each administration provides the subject with 11 mg to 22 mg of anti-TfR1 antibody / kg of the subject in the complex, optionally wherein the effective amount for each administration provides the subject with 15 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0421] 20. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 22 mg to 44 mg of anti-TfR1 antibody / kg of the subject in the complex, optionally wherein the effective amount for each administration provides the subject with 30 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0422] 21. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 44 mg to 88 mg of anti-TfR1 antibody / kg of the subject in the complex, optionally wherein the effective amount for each administration provides the subject with 60 mg of anti-TfR1 antibody / kg of the subject in the complex.

[0423] 22. The method of embodiment 11, wherein the effective amount per administration provides the subject with 1 mg to 14 mg of oligonucleotide in the complex / kg of the subject.

[0424] 23. The method of embodiment 11, wherein the effective amount per administration provides the subject with 0.5 mg to 12 mg of oligonucleotide / kg of subject in the complex.

[0425] 24. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 1.2 mg to 2.4 mg of oligonucleotide in the complex / kg of subject, optionally wherein the effective amount for each administration provides the subject with 1.8 mg of oligonucleotide in the complex / kg of subject.

[0426] 25. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 2.2 mg to 4.5 mg of oligonucleotide in the complex / kg of subject, optionally wherein the effective amount for each administration provides the subject with 3.4 mg of oligonucleotide in the complex / kg of subject.

[0427] 26. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 4.5 mg to 9 mg of oligonucleotide in the complex / kg of the subject, optionally wherein the effective amount for each administration provides the subject with 6.8 mg of oligonucleotide in the complex / kg of the subject.

[0428] 27. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 7 mg to 14 mg of oligonucleotide in the complex / kg of subject, optionally wherein the effective amount for each administration provides the subject with 10.2 mg of oligonucleotide in the complex / kg of subject.

[0429] 28. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 0.7 mg to 1.5 mg of oligonucleotide in the complex / kg of the subject, optionally wherein the effective amount for each administration provides the subject with 1.1 mg of oligonucleotide in the complex / kg of the subject.

[0430] 29. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 1.4 mg to 2.7 mg of oligonucleotide in the complex / kg of the subject, optionally wherein the effective amount for each administration provides the subject with 2 mg of oligonucleotide in the complex / kg of the subject.

[0431] 30. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 2.7 mg to 5.4 mg of oligonucleotide in the complex / kg of subject, optionally wherein the effective amount for each administration provides the subject with 4.1 mg of oligonucleotide in the complex / kg of subject.

[0432] 31. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 5 mg to 11 mg of oligonucleotide in the complex / kg of the subject, optionally wherein the effective amount for each administration provides the subject with 8.2 mg of oligonucleotide in the complex / kg of the subject.

[0433] 32. The method of embodiment 11, wherein the effective amount for each administration provides the subject with 5 mg to 8 mg of oligonucleotide in the complex / kg of the subject, optionally wherein the effective amount for each administration provides the subject with 5 mg of oligonucleotide in the complex / kg of the subject.

[0434] 33. The method of any one of embodiments 1 to 32, wherein during the administration period, the composition is administered once every 4 weeks.

[0435] 34. The method of any one of embodiments 1 to 32, wherein during the administration period, the composition is administered once every 8 weeks.

[0436] 35. The method of any one of embodiments 1 to 32, wherein during the administration period, the composition is administered once every 12 weeks.

[0437] 36. The method of any one of embodiments 33 to 35, wherein the administration period is less than 10 years.

[0438] 37. The method of any one of embodiments 33 to 35, wherein the administration period is for the remainder of the subject's life.

[0439] 38. The method of any one of embodiments 1 to 32, wherein the composition is administered once every 4 weeks during a first administration period and then administered once every 8 weeks during a second administration period.

[0440] 39. The method of embodiment 38, wherein the first administration period is 8 to 16 weeks, and / or wherein the second administration period is 16 weeks to the remainder of the subject's life.

[0441] 40. The method of any one of embodiments 1 to 39, wherein the composition is in the form of an aqueous solution and further comprises tris(hydroxymethyl)aminomethane and sucrose.

[0442] 41. The method of embodiment 40, wherein tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 25 mM, sucrose is present in the aqueous solution at a concentration of 10 w / v%, and the pH of the aqueous solution is 7.5.

[0443] 42. The method of embodiment 40 or embodiment 41, wherein the complex is present in the composition at a concentration of 10 mg / mL to 50 mg / mL.

[0444] 43. The method of any one of embodiments 1 to 42, wherein the administration reduces DMPK expression in the subject's muscle cells, optionally wherein reducing DMPK expression comprises reducing the amount of DMPK RNA in the muscle cells, optionally wherein the amount of DMPK RNA in the nucleus of the muscle cells is reduced.

[0445] 44. The method of embodiment 43, wherein reducing DMPK expression in the muscle cells comprises reducing the amount of DMPK protein in the muscle cells.

[0446] 45. The method of any one of embodiments 1 to 44, wherein the subject has a mutant DMPK allele comprising a disease-associated CTG repeat.

[0447] 46. ​​The method of embodiment 45, wherein the administration results in at least a 30% decrease in DMPK mRNA in the subject's muscle cells, optionally wherein the administration results in a decrease in DMPK mRNA in the nuclei of the subject's muscle cells.

[0448] 47. The method of any one of embodiments 1 to 46, wherein the subject is a human.

[0449] 48. The method of any one of embodiments 1 to 46, wherein the subject is a rodent.

[0450] 49. The method of any one of embodiments 1 to 46, wherein the subject is a non-human primate.

[0451] 50. The method of any one of embodiments 1 to 49, wherein the complex is administered systemically, optionally wherein the complex is administered intravenously, and further optionally wherein the complex is administered by infusion.

[0452] 51. A method for reducing DMPK expression or treating myotonic dystrophy in a subject, comprising intravenously administering an effective amount of a composition comprising a complex comprising formula (I): [R 1 ] n1 -R 2 The structure of which:

[0453] Each R 1 Containing a group of formula (Ia):

[0454]

[0455] Where R 3 comprising an oligonucleotide comprising a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21) and comprising a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dv*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage;

[0456] R 2 comprising an anti-TfR1 Fab comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; and

[0457] In each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via a connection point A, optionally wherein the average value of n1 of the complexes in the composition is 0.5 to 5;

[0458] wherein the effective amount provides to the subject 5 mg to 110 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount provides to the subject 8 mg to 60 mg or 13 mg to 75 mg anti-TfR1 antibody / kg subject in the complex.

[0459] 52. A method for reducing DMPK expression or treating myotonic dystrophy in a subject, comprising intravenously administering an effective amount of a composition comprising a complex comprising formula (I): [R 1 ] n1 -R 2 The structure of which:

[0460] Each R 1 Containing a group of formula (Ib):

[0461]

[0462] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage, and R 1 The oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0463] R 2 comprising an anti-TfR1 Fab comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; and

[0464] In each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via a connection point A, optionally wherein the average value of n1 of the complexes in the composition is 0.5 to 5;

[0465] wherein the effective amount provides to the subject 5 mg to 110 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount provides to the subject 8 mg to 60 mg or 13 mg to 75 mg anti-TfR1 antibody / kg subject in the complex.

[0466] 53. A method for reducing DMPK expression or treating myotonic dystrophy in a subject, comprising intravenously administering an effective amount of a composition comprising a complex comprising formula (I): [R 1 ] n1 -R 2 The structure of which:

[0467] Each R1 Containing a group of formula (Ic):

[0468]

[0469] wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, * represents phosphorothioate internucleoside linkage, and the oligonucleotide in R1 comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21);

[0470] R 2 comprising an anti-TfR1 Fab comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 20; and

[0471] In each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via a connection point A, optionally wherein the average value of n1 of the complexes in the composition is 0.5 to 5;

[0472] wherein the effective amount provides to the subject 5 mg to 110 mg anti-TfR1 antibody / kg subject in the compl...

Claims

1. A method for reducing DMPK expression and / or treating myotonic dystrophy in a subject, comprising administering to the subject a composition comprising an effective amount of a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to one or more oligonucleotides, wherein the effective amount provides the subject with 5 mg to 110 mg of the anti-TfR1 antibody / kg of the subject in the complex, wherein the antibody comprises: a heavy chain complementary determining region 1 (CDR-H1) comprising the sequence shown in SEQ ID NO: 1, 7 or 12, a heavy chain complementary determining region 2 (CDR-H2) comprising the sequence shown in SEQ ID NO: 2, 8 or 13, a heavy chain complementary determining region 3 (CDR-H3) comprising the sequence shown in SEQ ID NO: 3, 9 or 14, a light chain complementary determining region 1 (CDR-L1) comprising the sequence shown in SEQ ID NO: 4, 10 or 15, a light chain complementary determining region 2 (CDR-L2) comprising the sequence shown in SEQ ID NO: 5 or 11, and a heavy chain complementary determining region 3 (CDR-H4) comprising the sequence shown in SEQ ID NO: 6 or 8. NO: 6 or 16, wherein the oligonucleotide in the complex comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21).

2. The method of claim 1, wherein the oligonucleotide in the complex comprises a 5'-XYZ-3' configuration, wherein X and Z are flanking regions comprising one or more modified nucleosides, and Y is a spacer region comprising one or more 2'-deoxyribonucleosides.

3. The method of claim 1 or claim 2, wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of which: Each R 1 Containing a group of formula (Ia): Where R 3 comprising an oligonucleotide comprising a nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21) and comprising a structure of +C*+A*oG*oC*dG*dC*dC*dC*dA*dC*dC*dA*oG*oU*+C*+A (SEQ ID NO: 21), wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents a phosphorothioate internucleoside linkage; R 2 comprising an anti-TfR1 antibody; and In each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A, optionally wherein the average value of n1 of the complexes in the composition is 0.5 to 5.

4. The method of claim 1 or claim 2, wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of which: Each R 1 Containing a group of formula (Ib): wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage, and R 1 The oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); R 2 comprising an anti-TfR1 antibody; and In each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A, optionally wherein the average value of n1 of the complexes in the composition is 0.5 to 5.

5. The method of claim 1 or claim 2, wherein each complex comprises formula (I): [R 1 ] n1 -R 2 The structure of which: Each R 1 Containing a group of formula (Ic): Where R 2 comprising an anti-TfR1 antibody; and In each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A, optionally wherein the average value of n1 of the complexes in the composition is 0.5 to 5.

6. The method of claim 1 or claim 2, wherein each complex comprises a structure of formula (Id): wherein +N represents LNA (2'-4' methylene bridge) ribonucleoside, dN represents 2'-deoxyribonucleoside, oN represents 2'-MOE modified ribonucleoside, oC represents 5-methyl-2'-MOE-cytidine, +C represents 5-methyl-2'-4'-bicyclo-cytidine (2'-4' methylene bridge), oU represents 5-methyl-2'-MOE-uridine, and * represents phosphorothioate internucleoside linkage, and R 1 The oligonucleotide comprises the nucleobase sequence of CAGCGCCCACCAGUCA (SEQ ID NO: 21); R 2 comprising an anti-TfR1 antibody; and In each complex, n1 is independently an integer of 1 or greater, which represents R 1 The number of instances of R 1 Each instance of is covalently linked to a different lysine of the anti-TfR1 antibody via attachment point A, optionally wherein the average value of n1 of the complexes in the composition is 0.5 to 5.

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

8. The method of any one of claims 1 to 7, wherein the anti-TfR1 antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 18, Optionally, the anti-TfR1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO:

20.

9. The method of any one of claims 1 to 8, wherein the administering occurs one or more times.

10. The method of claim 9, wherein each administration of an effective amount provides to the subject: (a) 10 mg to 110 mg of anti-TfR1 antibody / kg of subject in the complex; (b) 5 mg to 90 mg of anti-TfR1 antibody / kg of subject in the complex; (c) 10 mg to 20 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount per administration provides 13 mg anti-TfR1 antibody / kg subject in the complex to the subject; (d) 18 mg to 36 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount per administration provides 25 mg anti-TfR1 antibody / kg subject in the complex to the subject; (e) 36 mg to 72 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount per administration provides 50 mg anti-TfR1 antibody / kg subject in the complex to the subject; (f) 55 mg to 110 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount per administration provides 75 mg anti-TfR1 antibody / kg subject in the complex to the subject; (g) 6 mg to 12 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount per administration provides 8 mg anti-TfR1 antibody / kg subject in the complex to the subject; (h) 11 mg to 22 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount per administration provides 15 mg anti-TfR1 antibody / kg subject in the complex to the subject; (i) 22 mg to 44 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount per administration provides 30 mg anti-TfR1 antibody / kg subject in the complex to the subject; or (j) 44 mg to 88 mg anti-TfR1 antibody / kg subject in said complex, optionally wherein the effective amount per administration provides to said subject 60 mg anti-TfR1 antibody / kg subject in said complex.

11. The method of claim 9, wherein each administration of an effective amount provides to the subject: (a) 20 mg to 43 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount per administration provides to the subject 29 mg anti-TfR1 antibody / kg subject in the complex; or (j) 26 mg to 53 mg anti-TfR1 antibody / kg subject in the complex, optionally wherein the effective amount per administration provides to the subject 37 mg anti-TfR1 antibody / kg subject in the complex.

12. The method of any one of claims 1 to 11, wherein during the administration period, the composition is administered once every 4 weeks, once every 8 weeks, or once every 12 weeks.

13. The method of claim 12, wherein the administration period is less than 10 years.

14. The method of claim 12, wherein the administration period is for the remainder of the subject's life.

15. The method of any one of claims 1 to 11, wherein the composition is administered once every 4 weeks during a first administration period and then administered once every 8 weeks during a second administration period.

16. The method of claim 15, wherein the first administration period is from 8 to 16 weeks, and / or wherein the second administration period is from 16 weeks to the remainder of the subject's life.

17. The method of any one of claims 1 to 16, wherein the composition is in the form of an aqueous solution and further comprises tris(hydroxymethyl)aminomethane and sucrose.

18. The method of claim 17, wherein the tris(hydroxymethyl)aminomethane is present in the aqueous solution at a concentration of 25 mM, the sucrose is present in the aqueous solution at a concentration of 10 w / v%, and the pH of the aqueous solution is 7.

5.

19. The method of claim 17 or claim 18, wherein the complex is present in the composition at a concentration of 10 mg / mL to 50 mg / mL.

20. The method of any one of claims 1 to 19, wherein the administering reduces DMPK expression in a muscle cell of the subject, optionally wherein reducing DMPK expression comprises reducing the amount of DMPK RNA in the muscle cell, optionally wherein the amount of DMPK RNA in the nucleus of the muscle cell is reduced.

21. The method of claim 20, wherein reducing DMPK expression in the muscle cells comprises reducing the amount of DMPK protein in the muscle cells.

22. The method of any one of claims 1 to 21, wherein the subject is a human.

23. The method of any one of claims 1 to 22, wherein the complex is administered systemically, optionally wherein the complex is administered intravenously, and further optionally wherein the complex is administered by infusion.

24. The method of any one of claims 1 to 23, wherein the composition further comprises one or more anti-TfR1 antibodies that are not covalently linked to the oligonucleotide.

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