Muscle targeting complexes and their use for treatment of facial shoulder brachial muscular dystrophy

Abstract

Some aspects of the present disclosure relate to oligonucleotides (e.g., RNAi oligonucleotides, e.g., siRNAs) intended to target DUX4 RNA and targeting complexes for delivering the oligonucleotides to cells (e.g., muscle cells) and uses thereof, in particular to the treatment of diseases (e.g., FSHD).

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 367,783, filed on July 6, 2022, entitled “MUSCLETARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERALMUSCULAR DYSTROPHY,” and U.S. Provisional Application No. 63 / 477,160, filed on December 23, 2022, entitled “MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERALMUSCULAR DYSTROPHY,” under 35 U.S.C. §119(e); the contents of each of which are incorporated herein by reference in their entirety. Technical Field

[0003] The present application relates to oligonucleotides intended to target DUX4 RNA and targeting complexes for delivering molecular payloads (eg, oligonucleotides) to cells and uses thereof, particularly for the treatment of diseases.

[0004] References to Electronic Sequence Listings

[0005] The contents of the electronic sequence listing (D082470078WO00-SEQ-CBD.xml; size: 339,190 bytes; and creation date: June 30, 2023) are incorporated herein by reference in their entirety. Background Art

[0006] Muscular dystrophy (MD) is a group of diseases characterized by progressive weakness and loss of muscle mass. These diseases are caused by mutations in genes that encode proteins required for healthy muscle tissue. Facioscapulohumeral muscular dystrophy (FSHD) is a dominantly inherited type of MD that primarily affects the muscles of the face, scapula, and upper arms. Other symptoms of FSHD include abdominal muscle weakness, retinal abnormalities, hearing loss, and joint pain and inflammation. FSHD is the most common of the nine MD types that affect both adults and children, with a worldwide incidence of approximately 1 in 8,300 people. FSHD is caused by the abnormal production of double homeobox 4 (DUX4), a protein that regulates gene expression. The DUX4 gene encoding the DUX4 protein is located in the D4Z4 repeat region on chromosome 4 and is normally expressed in the adult testis and thymus and in the 2-cell stage embryo, after which it is suppressed by hypermethylation of the D4Z4 repeats that surround and compact the DUX4 gene. Two types of FSHD have been described, type 1 and type 2. Type 1, which accounts for approximately 95% of cases, is associated with the loss of D4Z4 repeats in the subtelomeric region of chromosome 4. Unaffected individuals usually have more than 10 repeats arranged in the subtelomeric region of chromosome 4, however the most common form of FSHD (FSHD1) is caused by a contraction of the array to less than 10 repeats, associated with a diverse expression of DUX4 in skeletal muscle and reduced epigenetic repression. Two allelic variants of chromosome 4q (4qA and 4qB) are present in the most distal unit of the D4Z4 repeat. 4qA is in cis form with a functional polyadenylation consensus site. Contraction of the 4qA allele is pathogenic because the DUX4 transcript is polyadenylated and stable. Type 2 FSHD, which accounts for approximately 5% of cases, is associated with mutations in the SMCHD1 gene on chromosome 18. Type 2 FSHD may also be associated with the DNMT3B gene or the LRIF1 gene. There is no effective treatment for FSHD other than supportive care and treatment to manage disease symptoms. Summary of the invention

[0007] Some aspects of the present disclosure provide oligonucleotides intended to target DUX4 RNA. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA, which can be used to reduce the level of DUX4 RNA and / or protein. In some embodiments, the present disclosure provides oligonucleotides complementary to exon 3 of DUX4 RNA, which can be used to reduce the level of DUX4 RNA. In some embodiments, the oligonucleotides provided herein are intended to guide RNAi-mediated degradation of DUX4 RNA. In some embodiments, the oligonucleotides are intended to effectively engage the RNA-induced silencing complex (RISC) for degrading DUX4 RNA and also have reduced off-target effects. In some embodiments, the oligonucleotides are intended to have desired bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are intended to have desired binding affinity properties. In some embodiments, the oligonucleotides are intended to have desired toxicity and / or immunogenicity profiles. Abnormal (e.g., increased) expression of DUX4 RNA and / or protein in muscle is associated with features of facioscapulohumeral muscular dystrophy (FSHD) pathology, including muscle atrophy, inflammation, and reduced differentiation potential and oxidative stress. In some embodiments, the oligonucleotides described herein that reduce DUX4 RNA and / or protein levels are effective in treating FSHD.

[0008] According to some aspects, the present disclosure provides a complex for the purpose of targeting muscle cells (e.g., primary myoblasts) for delivering molecular payloads (e.g., oligonucleotides described herein) to those cells. In some embodiments, the complex provided herein is particularly useful for delivering molecular payloads that inhibit the expression or activity of DUX4 (e.g., in subjects suffering from or suspected of suffering from FSHD). Therefore, in some embodiments, the complex provided herein comprises a muscle targeting agent (e.g., a muscle targeting antibody) that specifically binds to a receptor on the surface of a muscle cell for the purpose of delivering a molecular payload to a muscle cell. In some embodiments, the complex is taken up into the cell by receptor-mediated internalization, and then the molecular payload can be released to perform a function inside the cell. For example, a complex modified to deliver an oligonucleotide can release the oligonucleotide so that the oligonucleotide can inhibit DUX4 gene expression in a muscle cell. In some embodiments, the oligonucleotide is released by endosome cleavage of the covalent linker connecting the oligonucleotide and the muscle targeting agent in the complex.

[0009] Some aspects of the present disclosure provide complexes comprising a muscle targeting agent covalently linked to an oligonucleotide that targets a double homeobox 4 (DUX4) RNA (e.g., mRNA), wherein the oligonucleotide comprises an antisense strand that is 18 to 25 nucleotides in length and comprises a region of complementarity to a target sequence set forth in SEQ ID NOs: 174 to 235, and wherein the region of complementarity is at least 16 contiguous nucleosides in length.

[0010] Some aspects of the present disclosure provide complexes comprising a muscle targeting agent covalently linked to an oligonucleotide that targets a double homeobox 4 (DUX4) RNA (e.g., mRNA), wherein the oligonucleotide comprises an antisense strand that is 18 to 25 nucleotides in length and comprises a region of complementarity to the target sequence shown in SEQ ID NOs: 200, 191, 189, 186, 190, 174 to 185, 187, 188, 192 to 199, and 201 to 235, and wherein the region of complementarity is at least 16 contiguous nucleosides in length.

[0011] In some embodiments, the muscle targeting agent is an anti-transferrin receptor 1 (TfR1) antibody.

[0012] In some embodiments, the oligonucleotide is a RNAi oligonucleotide.

[0013] In some embodiments, the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 236-266.

[0014] In some embodiments, the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 262, 253, 251, 248, 252, 236-247, 249, 250, 254-261, 263-266.

[0015] In some embodiments, the oligonucleotide further comprises a sense strand comprising at least 18 consecutive nucleosides that are complementary to the antisense strand.

[0016] In some embodiments, the sense strand comprises 21 consecutive nucleosides that are complementary to the antisense strand.

[0017] In some embodiments, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 205-235.

[0018] In some embodiments, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 231, 222, 220, 217, 221, 205-216, 218, 219, 223, 230, 232-235.

[0019] In some embodiments, the muscle targeting agent is covalently linked to the 5' end or the 3' end of the sense strand.

[0020] In some embodiments, the antisense strand of the RNAi oligonucleotide further comprises a 5'-(E)-vinylphosphonate.

[0021] In some embodiments, an oligonucleotide comprises one or more modified nucleosides.

[0022] In some embodiments, one or more modified nucleosides are 2' modified nucleotides, optionally wherein one or more 2' modified nucleosides are selected from: 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamido (2'-O-NMA)).

[0023] In some embodiments, each 2' modified nucleotide is 2'-O-methyl (2'-O-Me) or 2'-fluoro (2'-F). In some embodiments, the 2' modified nucleotide is 2'-O-methyl (2'-O-Me). In some embodiments, the 2' modified nucleotide is 2'-fluoro (2'-F).

[0024] In some embodiments, an oligonucleotide comprises one or more phosphorothioate internucleoside linkages.

[0025] In some embodiments, one or more phosphorothioate internucleoside linkages are present on the antisense strand of the oligonucleotide.

[0026] In some embodiments, the two internucleoside linkages at the 3' end of the antisense strand are phosphorothioate internucleoside linkages. In some embodiments, the two internucleoside linkages at the 5' end of the antisense strand are phosphorothioate internucleoside linkages. In some embodiments, the two internucleoside linkages at the 3' end of the antisense strand and the two internucleoside linkages at the 5' end of the antisense strand are phosphorothioate internucleoside linkages.

[0027] In some embodiments, one or more phosphorothioate internucleoside linkages are present on the sense strand of the oligonucleotide.

[0028] In some embodiments, the two internucleoside linkages at the 3' end of the sense strand are phosphorothioate internucleoside linkages. In some embodiments, the two internucleoside linkages at the 5' end of the sense strand are phosphorothioate internucleoside linkages. In some embodiments, the two internucleoside linkages at the 3' end of the sense strand and the two internucleoside linkages at the 5' end of the sense strand are phosphorothioate internucleoside linkages.

[0029] In some embodiments, one or more cytidines of the oligonucleotide are 2'-modified 5-methylcytidine, optionally wherein the 2'-modified 5-methylcytidine is 2'-O-Me modified 5-methylcytidine or 2'-F modified 5-methylcytidine.

[0030] In some embodiments, the antisense strand of an oligonucleotide described herein is selected from modified forms of SEQ ID NOs: 236 to 266 listed in Table 8 (e.g., MAS1 to MAS4). For example, in some embodiments, the antisense strand is selected from:

[0031] MAS1-236, MAS1-237, MAS1-238, MAS2-236, MAS2-237, MAS2-238, MAS2-239, MAS2-240, MAS2-241, MAS2-2 42, MAS2-243, MAS2-244, MAS2-245, MAS2-246, MAS2-247, MAS2-248, MAS2-249, MAS2-250, MAS2-251, MAS2 -252, MAS2-253, MAS2-254, MAS2-255, MAS2-256, MAS2-257, MAS3-236, MAS3-237, MAS3-238-MAS3-239, MA S3-240, MAS3-241, MAS3-242, MAS3-243, MAS3-244, MAS3-245, MAS3-246, MAS3-247, MAS3-248, MAS3-249, MAS3-250, MAS3-251, MAS3-252, MAS3-253, MAS3-254, MAS3-255, MAS3-256, MAS3-257, MAS3-258, MAS3-2 59, MAS3-260, MAS3-261, MAS3-262, MAS3-263, MAS3-264, MAS3-265, MAS3-266, MAS4-236, MAS4-237, MAS4 -238, MAS4-239, MAS4-240, MAS4-241, MAS4-242, MAS4-243, MAS4-244, MAS4-245, MAS4-246, MAS4-247, MAS4-248, MAS4-249, MAS4-250, MAS4-251, MAS4-252, MAS4-253, MAS4-254, MAS4-255, MAS4-256, and MAS4-257

[0032] In some embodiments, the sense strand of the oligonucleotide described herein is selected from the modified forms of SEQ ID NOs: 205 to 235 listed in Table 8 (e.g., MS1 to MS6). For example, in some embodiments, the sense strand is selected from:

[0033] MS1-205, MS1-206, MS1-207, MS2-208, MS2-209, MS2-210, MS2-211, MS2-212, MS2-213, MS2-214, MS2-215, MS2- 216, MS2-217, MS2-218, MS2-219, MS2-220, MS2-221, MS2-222, MS2-223, MS2-224, MS2-225, MS2-226, MS3-205, MS3-206, MS3-207, MS3-208, MS3-209, MS3-210, MS3-211, MS3-212, MS3-213, MS3-214, MS3-215, MS3-216, MS3- 217, MS3-218, MS3-219, MS3-220, MS3-221, MS3-222, MS3-223, MS3-224, MS3-225, MS3-226, MS3-227, MS3-228, MS3-229, MS3-230, MS3-231, MS3-232, MS3-233, MS3-234, MS3-235, MS4-205, MS4-206, MS4-207, MS4-208, MS4- 209, MS4-210, MS4-211, MS4-212, MS4-213, MS4-214, MS4-215, MS4-216, MS4-217, MS4-218, MS4-219, MS4-220, MS4-221, MS4-222, MS4-223, MS4-224, MS4-225, MS4-226, MS5-205, MS5-206, MS5-207, MS5-208, MS5-209, MS5- 210, MS5-211, MS5-212, MS5-213, MS5-214, MS5-215, MS5-216cMS5-217, MS5-218, MS5-219, MS5-220, MS5-221,

[0034] MS5-222, MS5-223, MS5-224, MS5-225, MS5-226, MS6-205, MS6-206, MS6-207, MS6-208, MS6-209, MS6-210, MS6-211, MS6-212, MS6-213, MS6-214, MS6-215, MS6-216, MS6-217, MS6-218, MS6-219, MS6-220, MS6-221, MS6-222, MS6-223, MS6-224, MS6-225, and MS6-226.

[0035] In some embodiments, the antisense strand is selected from modified forms of SEQ ID NOs: 262, 253, 251, 248 and 252 listed in Table 9 (e.g., VP-MAS5, VP-MAS6 and VP-MAS7), and / or wherein the sense strand is selected from modified forms of SEQ ID NOs: 231, 222, 220, 217 and 221 listed in Table 9 (e.g., MS7 to MS9).

[0036] In some embodiments, the oligonucleotide is a siRNA molecule selected from the siRNAs listed in Table 8.

[0037] In some embodiments, the oligonucleotide is a siRNA molecule selected from the siRNAs listed in Table 9.

[0038] In some embodiments, the anti-TfR1 antibody comprises the heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), and light chain complementarity determining region 3 (CDR-L3) of any anti-TfR1 antibody listed in Table 2.

[0039] In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL) of any anti-TfR1 antibody listed in Table 3.

[0040] In some embodiments, the anti-TfR1 antibody is a Fab, optionally wherein the Fab comprises the heavy chain and light chain of any anti-TfR1 Fab listed in Table 5.

[0041] In some embodiments, the anti-TfR1 antibody comprises:

[0042] (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:27, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:28, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:29, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:30, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:31, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:32;

[0043] (ii) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:33, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:34, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:35, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:36, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:37, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:32; or

[0044] (ii) CDR-H1 comprising the amino acid sequence of SEQ ID NO:38, CDR-H2 comprising the amino acid sequence of SEQ ID NO:39, CDR-H3 comprising the amino acid sequence of SEQ ID NO:40, CDR-L1 comprising the amino acid sequence of SEQ ID NO:41, CDR-L2 comprising the amino acid sequence of SEQ ID NO:31, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:42.

[0045] In some embodiments, the anti-TfR1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0046] In some embodiments, the anti-TfR1 antibody is a Fab and comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0047] In some embodiments, the muscle targeting agent is covalently linked to the antisense oligonucleotide via a linker, optionally wherein the linker comprises a valine-citrulline sequence.

[0048] Also provided herein is a method of reducing DUX4 expression in a muscle cell, the method comprising contacting the muscle cell with an effective amount of a complex described herein to promote internalization of the oligonucleotide into the muscle cell. In some embodiments, reducing DUX4 expression comprises reducing DUX4 protein and / or mRNA levels.

[0049] Also provided herein are methods of treating facioscapulohumeral muscular dystrophy (FSHD), comprising administering to a subject in need thereof an effective amount of a complex provided herein. In some embodiments, the subject has abnormal production of DUX4 protein.

[0050] Other aspects of the present disclosure provide oligonucleotides comprising siRNA oligonucleotides selected from the siRNA oligonucleotides listed in Table 8.

[0051] Other aspects of the present disclosure provide oligonucleotides comprising siRNA oligonucleotides selected from the siRNA oligonucleotides listed in Table 9.

[0052] Also provided herein is a method for producing a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide, the method comprising: (i) obtaining a compound comprising the structure of formula (B), wherein the oligonucleotide comprises the sense strand of an siRNA oligonucleotide, optionally, wherein the siRNA oligonucleotide targets DUX4 RNA; (ii) annealing the antisense strand of the siRNA oligonucleotide to the sense strand; (iii) reacting the compound comprising the structure of formula (B) with a compound comprising the structure of formula (C) to obtain a compound comprising the structure of formula (D); and (iv) covalently linking the anti-TfR1 antibody to the compound comprising the structure of formula (D) with a muscle targeting agent to obtain a compound comprising the structure of formula (E), optionally, wherein the annealing in step (ii) is performed at 30° C., and optionally, wherein the method further comprises isolating the compound comprising the structure of formula (D) after step (iii) and before step (iv); and optionally, wherein the anti-TfR1 antibody is covalently linked to the 5' end of the sense strand of the siRNA oligonucleotide targeting DUX4 RNA.

[0053] Also provided herein is a method for producing a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide, the method comprising: (i) obtaining a compound comprising a structure of formula (B), wherein the oligonucleotide comprises a sense strand of an siRNA oligonucleotide, optionally wherein the siRNA oligonucleotide targets DUX4 RNA; (ii) annealing the antisense strand of the siRNA oligonucleotide to the sense strand; (iii) covalently linking the anti-TfR1 antibody to a compound comprising a structure of formula (C) to obtain a compound comprising a structure of formula (F); and (iv) reacting a compound comprising a structure of formula (F) with a compound comprising a structure of formula (B) to obtain a compound comprising a structure of formula (E), optionally wherein the annealing in step (ii) is performed at 30°C, and optionally wherein the anti-TfR1 antibody is covalently linked to the 5' end of the sense strand of the siRNA oligonucleotide targeting DUX4 RNA. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 A non-limiting schematic diagram showing the effect of transfecting cells with siRNA is shown. The figure shows the effect of siRNA on HPRT mRNA levels.

[0055] Figure 2 A non-limiting schematic diagram showing the activity of a muscle targeting complex comprising siRNA is shown. The in vitro activity of the complex was measured based on HPRT mRNA levels in cultured muscle cells. The complex comprises siRNA linked to a muscle targeting antibody.

[0056] Figures 3A to 3B A non-limiting schematic diagram showing in vivo activity in mouse muscle tissue (gastrocnemius and heart) relative to vehicle-treated controls is shown. (N=4 C57BL / 6WT mice) In vivo activity of the complexes was measured. The complexes comprised siRNA linked to a muscle-targeting antibody.

[0057] Figures 4A to 4E A non-limiting schematic diagram showing tissue selectivity of a muscle targeting complex comprising siRNA is shown. The complex comprises siRNA linked to a muscle targeting antibody.

[0058] Figure 5The composite score of mRNA levels of three DUX4 transcriptome markers (MBD3L2, TRIM43 and ZSCAN4) in AB1080 immortalized FSHD patient-derived myotubes after incubation with siRNA conjugates containing anti-TfR Fab 3M12VH4 / Vκ3 covalently linked to siRNA-A, siRNA-B or siRNA-C is shown. The anti-TfR Fab was covalently linked to the 3' end of each siRNA sense strand via a linker, and the corresponding antisense strand was annealed to the sense strand.

[0059] Figure 6 The composite score of mRNA levels of DUX4 transcriptome markers MBD3L2, TRIM43 and ZSCAN4 in FSHD patient-derived C6 myotubes after transfection with siRNAs selected from siRNA7, 49, 50, 53, 54, 136, 19, 114, 137, 138, 32, 35, 33, 36, 73, 74, 77, 76, 75, 78, 83, 68, 103, 104, 107, 106, 105, 108, 85, 86, 89, 88, 87, 90, 133, 135, 140, 97, 128, 132, 134, 47, 91, 92, 95, 94, 93 and 96 at a concentration of 0.2 nM or 20 nM is shown. The siRNA numbers correspond to the siRNA numbers in Table 8.

[0060] Figures 7A to 7C Shown are dose response curves generated for siRNAs 7, 136, 137, 74, 77, 78, 104, 107, 106, 86, 89, 133, 97, 128, 92, 95, and 94. The siRNA numbers correspond to the siRNA numbers in Table 8. Dose response curves were generated using a comprehensive score for the DUX4 transcriptome markers MBD3L2, TRIM43, and ZSCAN4 in cells treated with a range of concentrations (2 pM to 40 nM) of the indicated siRNAs.

[0061] Figure 8 The comprehensive score of mRNA levels of three DUX4 transcriptome markers (MBD3L2, TRIM43 and ZSCAN4) in FSHD patient-derived C6 myotubes after treatment with siRNA antibody complexes at concentrations of 10 nM, 100 nM or 1000 nM is shown. The siRNA-antibody complex comprises anti-TfR1 Fab 3M12VH4 / Vκ3 covalently linked to DUX4 targeting siRNA. The siRNAs tested are siRNAs 142 to 148. The siRNA numbers correspond to the siRNA numbers in Table 9. Exon 1 targeting siRNA is used as a positive control.

[0062] Figures 9A to 9B Shown is the mouse quadriceps ( Fig.9A ) and gastrocnemius ( Fig. 9B ) in the mRNA level of three DUX4 mouse transcriptome markers (Wfdc3, Sord, Serpinb6c). Tissues were collected 4 weeks after a single intravenous injection of siRNA at a dose of 10 mg / kg with a DUX4 targeting siRNA complex covalently linked to anti-TfR1 Fab 3M12 VH4 / Vκ3. The siRNAs tested were siRNA145, siRNA148, siRNA144, siRNA143, and siRNA142. The siRNA numbers correspond to the siRNA numbers in Table 9.

[0063] Fig.10 An example of a schematic diagram illustrating how to prepare the anti-TfR1-siRNA complex described herein is shown. DETAILED DESCRIPTION

[0064] Some aspects of the present disclosure provide oligonucleotides intended to target DUX4 RNA. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA, which can be used to reduce the level of DUX4 RNA and / or protein. In some embodiments, the oligonucleotides provided herein are intended to guide RNAi-mediated degradation of DUX4 RNA. In some embodiments, the oligonucleotides are intended to effectively engage RNA-induced silencing complexes (RISC) to degrade DUX4 RNA and also have reduced off-target effects. In some embodiments, the oligonucleotides are intended to have desired bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are intended to have desired binding affinity properties. In some embodiments, the oligonucleotides are intended to have desired toxicity and / or immunogenicity profiles. Abnormal (e.g., increased) expression of DUX4 RNA and / or protein is associated with features of facioscapulohumeral muscular dystrophy (FSHD) pathology, including muscle atrophy, inflammation, and reduced differentiation potential and oxidative stress. In some embodiments, the oligonucleotides described herein that reduce DUX4 RNA and / or protein levels are effective in treating FSHD.

[0065] In some aspects, the disclosure provides a complex comprising a muscle targeting agent covalently linked to an oligonucleotide for effectively delivering the oligonucleotide to a muscle cell. In some embodiments, the complex is particularly useful for delivering a molecular payload that inhibits the expression or activity of a target gene in a muscle cell (e.g., in an object suffering from or suspected of having a rare muscle disease). For example, in some embodiments, a complex for treating an object with FSHD is provided. In some embodiments, a complex for treating an object with FSHD1 is provided. In some embodiments, a complex for treating an object with FSHD2 is provided. In some embodiments, a complex for targeting DUX4 to treat an object with FSHD is provided. In some embodiments, the complex provided herein comprises an oligonucleotide that inhibits the expression of DUX4 in an object having one or more D4Z4 repeat deletions on chromosome 4. In some embodiments, the complex provided herein comprises an oligonucleotide that inhibits the expression of DUX4 in an object having a mutation in SMCHD1 or another DUX4 regulatory gene.

[0066] Additional aspects of the disclosure are provided below, including a description of defined terms.

[0067] I. Definitions

[0068] 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 acceptable manner.

[0069] Approximately: As used herein, the term "approximately" or "about", as applied to one or more target values, refers to a value similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values ​​that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value, unless otherwise specified or otherwise apparent from the context (unless such a number exceeds 100% of the possible value).

[0070] 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 constant domain or a 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 antibody described herein may be an alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibody described herein may comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In a specific embodiment, the antibody described herein comprises a human γ1CH1, CH2 and / or (e.g., and) CH3 domain. In some embodiments, the amino acid sequence of the VH domain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region, 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 that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or at least 99% identical to 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).

[0071] 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 and VL regions can be further subdivided into hypervariable regions, also known as "complementarity determining regions" ("complementarity determining regions, CDRs"), interspersed with more conservative regions called "framework regions" ("framework regions, FRs"). Each VH and VL is generally composed of three CDRs and four FRs, arranged in the following order from amino terminus to carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of the framework regions and CDRs can be precisely identified using methods known in the art, such as by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or (e.g., and) contact definitions (all of which are well known in the art). See, for example, 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 www.imgt.org,Lefranc,M.-P.et al.,Nucleic Acids Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[Epub],5:45-60(2005); al.,Nucleic Acids Res., 33: D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 37: D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res., 43: D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; Al-lazikani et al (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). 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).

[0072] 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 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 as L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate light chain regions 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 may still be consistent with the Kabat CDRs. CDR overlap, although can be shortened or extended based on prediction or based on experimental findings that specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding. The method used herein can utilize CDRs defined according to any one of these systems. Some examples of CDR definition systems are provided in Table 1.

[0073] Table 1. CDR definitions

[0074]

[0075] 1 the international ImMunoGeneTics information imgt.org, Lefranc, M.-P. et al., Nucleic Acids Res., 27: 209-212 (1999)

[0076] 2Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242

[0077] 3 Chothia et al., J. Mol. Biol. 196: 901-917 (1987))

[0078] CDR-grafted antibody: The term "CDR-grafted antibody" refers to an antibody comprising a heavy chain variable region sequence and a light chain variable region sequence from one species but in which the sequence of one or more CDR regions of VH and / or (e.g., and) VL is replaced by a CDR sequence from another species, such as an antibody having a murine heavy chain variable region and a light chain variable region in which one or more murine CDRs (e.g., CDR3) have been replaced by a human CDR sequence.

[0079] Chimeric antibody: The term "chimeric antibody" refers to an antibody that comprises heavy and light chain variable region sequences from one species and constant region sequences from another species, such as an antibody having murine heavy and light chain variable regions linked to human constant regions.

[0080] Complementary: The term "complementary" used herein refers to the ability to accurately pair between two nucleosides or two groups of nucleosides. In particular, complementarity is a term that characterizes the degree of hydrogen bond pairing that causes the binding between two nucleosides or two groups of nucleosides. For example, if the base at one position of an oligonucleotide can be hydrogen bonded with the base at the corresponding position of a target nucleic acid (e.g., mRNA), it is considered that the bases at this position are complementary to each other. Base pairing can include both standard Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, adenosine base (A) is complementary to thymidine base (T) or uracil base (U), cytosine base (C) is complementary to guanosine base (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 to any A, C, U or T. Inosine (I) is also considered in the art to be a universal base, and is considered complementary to any of A, C, U, or T.

[0081] Conservative amino acid substitution: As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods known to those of ordinary skill in the art for changing polypeptide sequences, such as those found in references compiling such methods: for example, Molecular Cloning: A Laboratory Manual, J, Sambrook, et al., eds., Fourth Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012, or Current Protocols in Molecular Biology, FM Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0082] 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 may 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 may 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 may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker.

[0083] Cross-reactivity: As used herein and in the case of targeting agents (e.g., antibodies), the term "cross-reactivity" refers to the property that a substance can specifically bind to more than one antigen of a similar type or class (e.g., antigens of multiple homologs, paralogs, or orthologs) with similar affinity or avidity. For example, in some embodiments, antibodies that are cross-reactive to human and non-human primate antigens of similar types or classes (e.g., human transferrin receptor and non-human primate transferrin receptor) can bind to human antigens and non-human primate antigens with similar affinity or avidity. In some embodiments, antibodies have cross-reactivity to human antigens and rodent antigens of similar types or classes. In some embodiments, antibodies have cross-reactivity to rodent antigens and non-human primate antigens of similar types or classes. In some embodiments, antibodies have cross-reactivity to human antigens, non-human primate antigens, and rodent antigens of similar types or classes.

[0084] DUX4: The term "DUX4" as used herein refers to a gene encoding double homeobox 4, a protein that is normally expressed during fetal development and in the testes of adult males. In some embodiments, DUX4 can be a human gene (Gene ID: 100288687), a non-human primate gene (e.g., Gene ID: 750891, Gene ID: 100405864), or a rodent gene (e.g., Gene ID: 306226). In humans, expression of the DUX4 gene outside of fetal development and testes is associated with facioscapulohumeral muscular dystrophy. 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_001293798.2, NM_001306068.3, NM_001363820.1).

[0085] Facioscapulohumeral muscular dystrophy (FSHD): The term "facioscapulohumeral muscular dystrophy (FSHD)" as used herein refers to a genetic disease caused by mutations in the DUX4 gene, SMCHD1 gene, DNMT3B gene, or LRIF1 gene, which is characterized by decreased muscle mass and muscle atrophy primarily in the facial, scapula, and upper arm muscles. Two types of the disease have been described, type 1 and type 2. Type 1 is associated with a deletion of the D4Z4 repeat region on chromosome 4 that includes the DUX4 gene. In some embodiments, type 1 is associated with a deletion of the D4Z4 repeat region on chromosome 4 that includes the DUX4 gene, allelic variant 4qA. Type 2 is associated with mutations in the SMCHD1 gene, DNMT3B gene, or LRIF1 gene (see, e.g., Jia et al., "Facioscapulohumeral muscular dystrophy type 2: an update on the clinical, genetic, and molecular findings" Neuromuscul Disord. (2021), 31(11): 1101-1112). Both type 1 and type 2 FSHD are characterized by abnormal production of DUX4 protein outside the testis after fetal development. Facioscapulohumeral muscular dystrophy, the genetic basis of the disease, and associated symptoms have been described in the art (see, e.g., Campbell, AE, et al., "Facioscapulohumeral dystrophy: Activating an early embryonic transcriptional program in human skeletal muscle" Human Mol Genet. (2018); and Tawil, R. "Facioscapulohumeral muscular dystrophy" Handbook Clin. Neurol. (2018), 148: 541-548). Type 1 FSHD is associated with Online Mendelian Inheritance in Man (OMIM) Entry #158900. Type 2 FSHD is associated with OMIM Entry #158901.

[0086] 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. As used herein, FR represents one of the four subregions, and FRs represents 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.

[0087] 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.

[0088] Humanized antibody: The term "humanized antibody" refers to an antibody comprising a heavy chain variable region sequence and a light chain variable region sequence from a non-human species (e.g., a mouse) but in which at least a portion of the VH and / or (e.g., and) VL sequence has 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-TfR1 antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining murine anti-TfR1 monoclonal antibodies using conventional hybridoma technology followed by humanization using in vitro genetic engineering, such as those disclosed in PCT Publication No. WO 2005 / 123126 A2 by Kasaian et al.

[0089] Internalized cell surface receptor: The term "internalized cell surface receptor" as used herein refers to a cell surface receptor that is internalized by a cell, for example, under external stimulation (e.g., ligand binding to a receptor). In some embodiments, the internalized cell surface receptor is internalized by endocytosis. In some embodiments, the internalized cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalized cell surface receptor is internalized by a clathrin-independent pathway, such as phagocytosis, macropinocytosis, pits and raft-mediated uptake or constitutive clathrin-independent endocytosis. In some embodiments, the internalized cell surface receptor comprises an intracellular domain, a transmembrane domain and / or (e.g., and) an extracellular domain, which may optionally further comprise a ligand binding domain. In some embodiments, the cell surface receptor becomes internalized by the cell after ligand binding. In some embodiments, the ligand may be a muscle targeting agent or a muscle targeting antibody. In some embodiments, the internalized cell surface receptor is a transferrin receptor.

[0090] Separated antibody: "Separated antibody" as used herein is intended to refer to an antibody that is substantially free of other antibodies with different antigenic specificities (e.g., a separated antibody that specifically binds to transferrin receptor is substantially free of antibodies that specifically bind to antigens other than transferrin receptor). However, a separated antibody that specifically binds to transferrin receptor complex may have cross-reactivity with other antigens (e.g., transferrin receptor molecules from other species). In addition, the separated antibody may be substantially free of other cellular materials and / or (e.g., and) chemical substances.

[0091] 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 that are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or antigen-binding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci. 190:382-391 and Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, 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.

[0092] Molecular payload: The term "molecular payload" as used herein refers to a molecule or substance that plays a role in modulating a biological outcome. In some embodiments, the molecular payload is linked or otherwise associated with a muscle targeting agent. In some embodiments, the molecular payload is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular payload plays a role in modulating the transcription of a DNA sequence, modulating the expression of a protein, or modulating the activity of a protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a complementary region to a target gene.

[0093] Muscle targeting agent: The term "muscle targeting agent" as used herein refers to a molecule that specifically binds to an antigen expressed on a muscle cell. The antigen in or on a muscle cell can be a membrane protein, such as an integral membrane protein or a peripheral membrane protein. Generally speaking, a muscle targeting agent specifically binds to an antigen on a muscle cell, which helps internalize the muscle targeting agent (and any associated molecular payload) into the muscle cell. In some embodiments, a muscle targeting agent specifically binds to an internalizing cell surface receptor on the muscle and can be internalized into the muscle cell by receptor-mediated internalization. In some embodiments, a muscle targeting agent is a small molecule, a protein, a peptide, a nucleic acid (e.g., an aptamer), or an antibody. In some embodiments, a muscle targeting agent is connected to a molecular payload.

[0094] Muscle targeting antibody: The term "muscle targeting antibody" as used herein refers to a muscle targeting agent that is an antibody that specifically binds to an antigen present in or on a muscle cell. In some embodiments, the muscle targeting antibody specifically binds to an antigen on a muscle cell, which facilitates internalization of the muscle targeting antibody (and any associated molecular payload) into the muscle cell. In some embodiments, the muscle targeting antibody specifically binds to an internalizing cell surface receptor present on a muscle cell. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to the transferrin receptor.

[0095] 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.

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

[0097] 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.

[0098] 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).

[0099] Subject: As used herein, the term "subject" 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 having or suspected of having a disease. In some embodiments, the subject is a human patient having or suspected of having FSHD.

[0100] Transferrin receptor: The term "transferrin receptor" (also referred to as TFRC, CD71, p90, 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).

[0101] 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., 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-dimethylaminoethoxyethyl (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 nucleosides and oligonucleotides comprising 2'-modified nucleosides with increased affinity for the target sequence relative to unmodified oligonucleotides. Some examples of the structures of 2'-modified nucleosides are provided below:

[0102]

[0103] These examples are shown in the presence of a phosphate group, but any internucleoside linkage is contemplated between 2'-modified nucleosides.

[0104] II. Complex

[0105] Provided herein are complexes comprising a targeting agent (e.g., an antibody) covalently linked to a molecular payload. In some embodiments, the complex comprises a muscle targeting antibody covalently linked to an oligonucleotide. The complex may comprise an antibody that specifically binds to a single antigenic site or binds to at least two antigenic sites that may be present on the same or different antigens.

[0106] The complex can be used to regulate the activity or function of at least one gene, protein and / or (e.g., and) nucleic acid. In some embodiments, the molecular payload present in the complex is responsible for the regulation of genes, proteins and / or (e.g., and) nucleic acids. The molecular payload can be a small molecule, protein, nucleic acid, oligonucleotide or any molecular entity that can regulate the activity or function of genes, proteins and / or (e.g., and) nucleic acids in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets DUX4 in a muscle cell.

[0107] In some embodiments, the complex comprises a muscle targeting agent, e.g., an anti-transferrin receptor antibody, covalently linked to a molecular payload (e.g., an antisense oligonucleotide targeting DUX4). In some embodiments, the complex comprises a muscle targeting agent, e.g., an anti-transferrin receptor antibody, covalently linked to a molecular payload (e.g., an siRNA targeting DUX4).

[0108] A. Muscle Targeting Agents

[0109] Some aspects of the present disclosure provide muscle targeting agents, for example, for delivering molecular payloads to muscle cells. In some embodiments, such muscle targeting agents can bind to muscle cells, for example, by specifically binding to antigens on muscle cells, and the associated molecular payload is delivered to muscle cells. In some embodiments, the molecular payload is bound to the muscle targeting agent (e.g., covalently bound), and is internalized into the muscle cells after the muscle targeting agent binds to the antigen on the muscle cells, for example, by endocytosis. It should be understood that various types of muscle targeting agents can be used according to the present disclosure. For example, a muscle targeting agent may include or be composed of: nucleic acid (e.g., DNA or RNA), peptide (e.g., antibody), lipid (e.g., microvesicle (microvesicle)) or sugar moiety (e.g., polysaccharide). Exemplary muscle targeting agents are described in further detail herein, however, it should be understood that the exemplary muscle targeting agents provided herein are not meant to be restrictive.

[0110] Some aspects of the present disclosure provide muscle targeting agents that specifically bind to antigens on muscles (e.g., skeletal muscle, smooth muscle, or cardiac muscle). In some embodiments, any muscle targeting agent provided herein binds to (e.g., specifically binds to) antigens on skeletal muscle cells, smooth muscle cells, and / or (e.g., and) cardiac muscle cells.

[0111] By interacting with muscle-specific cell surface recognition elements (e.g., cell membrane proteins), both tissue localization and selective uptake into muscle cells can be achieved. In some embodiments, molecules that are substrates for muscle uptake transporters can be used to deliver molecular payloads into muscle tissue. Binding to muscle surface recognition elements is followed by endocytosis, which allows even large molecules (e.g., antibodies) to enter muscle cells. As another example, molecular payloads conjugated to transferrin or anti-TfR1 antibodies can be taken up by muscle cells by binding to transferrin receptors, and can then be internalized, for example, by clathrin-mediated endocytosis.

[0112] The use of muscle targeting agents can be used to concentrate molecular payloads (e.g., oligonucleotides) in muscle while reducing toxicity associated with effects in other tissues. In some embodiments, the muscle targeting agent concentrates the bound molecular payload in muscle cells compared to another cell type in the subject. In some embodiments, the muscle targeting agent concentrates the bound molecular payload in muscle cells (e.g., skeletal muscle, smooth muscle, or cardiomyocytes) in an amount that is at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 times higher than the amount in non-muscle cells (e.g., liver, neurons, blood, or adipocytes). In some embodiments, when the molecular payload is delivered to a subject in conjunction with a muscle targeting agent, its toxicity in the subject is reduced by at least 1%, 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, or 95%.

[0113] In some embodiments, a muscle recognition element (e.g., a myocyte antigen) may be required to achieve muscle selectivity. As an example, a muscle targeting agent may be a small molecule that is a substrate for a muscle-specific uptake transporter. As another example, a muscle targeting agent may be an antibody that enters myocytes via transporter-mediated endocytosis. As another example, a muscle targeting agent may be a ligand that binds to a cell surface receptor on myocytes. It should be understood that while transporter-based approaches provide a direct route for cell entry, receptor-based targeting may involve stimulated endocytosis to reach the desired site of action.

[0114] i. Muscle-targeted antibodies

[0115] In some embodiments, the muscle targeting agent is an antibody. Generally speaking, the high specificity of antibodies to their target antigens provides the potential for selective targeting of muscle cells (e.g., skeletal muscle, smooth muscle and / or (e.g., and) cardiomyocytes). This specificity can also limit off-target toxicity. Some examples of antibodies capable of targeting myocyte surface antigens have been reported and are within the scope of the present disclosure. For example, antibodies targeting the surface of muscle cells are described in: Arahata K., et al. "Immunostaining of skeletal and cardiac muscle surface membrane with antibody against Duchenne muscular dystrophy peptide" Nature 1988; 333: 861-3; Song KS, et al. "Expression of caveolin-3 in skeletal, cardiac, and smooth muscle cells. Caveolin-3 is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins" J Biol Chem 1996; 271: 15160-5; and Weisbart RH et al., "Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb" Mol Immunol. 2003 Mar, 39(13):78309; the entire contents of each of which are incorporated herein by reference.

[0116] a. Anti-Transferrin Receptor (TfR) antibody

[0117] Some aspects of the present disclosure are based on such recognition: substances (e.g., anti-transferrin receptor antibodies) that bind to transferrin receptors can target muscle cells. Transferrin receptors are internalized cell surface receptors that transfer transferrin across the cell membrane and participate in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor-binding proteins that can bind to transferrin receptors. Therefore, some aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to transferrin receptors. In some embodiments, the binding proteins that bind to transferrin receptors are internalized into muscle cells together with any bound molecular payloads. The antibodies that bind to transferrin receptors used herein can be interchangeably referred to as transferrin receptor antibodies, anti-transferrin receptor antibodies, or anti-TfR1 antibodies. Antibodies that bind to transferrin receptors (e.g., specifically bind) can be internalized into cells after binding to transferrin receptors, for example, by receptor-mediated endocytosis.

[0118] It should be understood that several known methods (e.g., library design using phage display) can be used to generate, synthesize and / or (e.g., and) derive anti-TfR1 antibodies. Exemplary methods have been characterized in the art and incorporated by reference (Díez, P. et al. "High-throughput phage-display screening in array format", Enzyme and microbial technology, 2015, 79, 34-41.; Christoph MH and Stanley, JR "Antibody Phage Display: Technique and Applications" J Invest Dermatol. 2014, 134: 2.; Engleman, Edgar (Ed.) "Human Hybridomas and Monoclonal Antibodies." 1985, Springer.). In other embodiments, anti-TfR1 antibodies have been previously characterized or disclosed.Antibodies that specifically bind to transferrin receptor are known in the art (see, e.g., U.S. Patent No. 4,364,934, filed December 4, 1979, “Monoclonal antibody to a human early thymocyte antigen and methods for preparing same”; U.S. Patent No. 8,409,573, filed June 14, 2006, “Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells”; U.S. Patent No. 9,708,406, filed May 20, 2014, “Anti-transferrin receptor antibodies and methods of use”; U.S. Patent No. 9,611,323, filed December 19, 2014, “Low affinity blood brain barrier receptor antibodies and uses therefor”; WO 2015 / 098989, filed December 24, 2014, “Novel anti-Transferrin receptor antibody that passes through blood-brain barrier”; Schneider C.et al. "Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9." J BiolChem.1982,257:14,8516-8522.; Lee et al. "Targeting Rat Anti-Mouse TransferrinReceptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse" 2000, J Pharmacol. Exp. Ther., 292:1048-1052).

[0119] In some embodiments, the anti-TfR1 antibodies described herein bind to transferrin receptor with high specificity and affinity. In some embodiments, the anti-TfR1 antibodies described herein specifically bind to any extracellular epitope of transferrin receptor or become exposed to the epitope of the antibody. In some embodiments, the anti-TfR1 antibodies provided herein specifically bind to transferrin receptors from humans, non-human primates, mice, rats, etc. In some embodiments, the anti-TfR1 antibodies provided herein bind to human transferrin receptors. In some embodiments, the anti-TfR1 antibodies described herein bind to the amino acid segments of human or non-human primate transferrin receptors (such as those provided in SEQ ID NO: 105 to 108). In some embodiments, the anti-TfR1 antibodies described herein bind to such amino acid segments: corresponding to amino acids 90 to 96 of human transferrin receptor (such as shown in SEQ ID NO: 105), which are not in the apical domain of transferrin receptor. In some embodiments, the humanized anti-TfR1 antibodies described herein bind to TfR1 but not to TfR2.

[0120] In some embodiments, the anti-TfR1 antibodies described herein (e.g., anti-TfR clone 8 in Table 2 below) bind to an epitope in TfR1, wherein the epitope comprises residues from amino acids 214 to 241 and / or amino acids 354 to 381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope comprising residues from amino acids 214 to 241 and amino acids 354 to 381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope comprising one or more residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1 shown in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein bind to an epitope comprising residues Y222, T227, K231, H234, T367, S368, S370, T376, and S378 of human TfR1 shown in SEQ ID NO:105.

[0121] In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope in TfR1, wherein the epitope comprises residues from amino acids 258 to 291 and / or from amino acids 358 to 381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope comprising residues from amino acids 258 to 291 and from amino acids 358 to 381 of SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope comprising one or more residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 shown in SEQ ID NO: 105. In some embodiments, the anti-TfR1 antibodies described herein (e.g., 3M12 and variants thereof in Table 2 below) bind to an epitope comprising residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 shown in SEQ ID NO:105.

[0122] An exemplary human transferrin receptor amino acid sequence corresponding to NCBI sequence NP_003225.2 (transferrin receptor protein 1 isoform 1, Homo sapiens) is as follows:

[0123]

[0124] An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001244232.1 (Transferrin receptor protein 1, Rhesus monkey (Macaca mulatta)) is as follows:

[0125] An exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence XP_005545315.1 (transferrin receptor protein 1, Macaca fascicularis) is as follows:

[0126]

[0127] An exemplary mouse transferrin receptor amino acid sequence corresponding to NCBI sequence NP_001344227.1 (transferrin receptor protein 1, Mus musculus) is as follows:

[0128]

[0129] In some embodiments, an anti-TfR1 antibody binds to the following receptor amino acid segment:

[0130]

[0131] And does not inhibit the binding interaction between transferrin receptor and transferrin and / or (eg, and) human hemochromatosis protein (also known as HFE).In some embodiments, the anti-TfR1 antibodies described herein do not bind to the epitope in SEQ ID NO:109.

[0132] Appropriate methods can be used to obtain and / or (e.g., and) produce antibodies, antibody fragments or antigen binding agents, for example, by using recombinant DNA protocols. In some embodiments, antibodies can also be produced by the production of hybridomas (see, for example, Kohler, G and Milstein, C. "Continuous cultures of fused cells secretingantibody of predefined specificity" Nature, 1975, 256: 495-497). The target antigen can be used as an immunogen in any form or entity (e.g., a recombinant or naturally occurring form or entity). Hybridomas are screened using standard methods (e.g., ELISA screening) to find at least one hybridoma that produces an antibody targeting a specific antigen. Antibodies can also be produced by screening a protein expression library (e.g., a phage display library) expressing an antibody. In some embodiments, phage display library design can also be used (see, e.g., U.S. Pat. No. 5,223,409, filed March 1, 1991, "Directed evolution of novel binding proteins"; WO 1992 / 18619, filed April 10, 1992, "Heterodimeric receptor libraries using phagemids"; WO 1991 / 17271, filed May 1, 1991, "Recombinant library screening methods"; WO 1992 / 20791, filed May 15, 1992, "Methods for producing members of specific binding pairs"; WO 1992 / 15679, filed February 28, 1992, and "Improved epitope displaying phage"). In some embodiments, the antigen of interest can be used to immunize non-human animals, such as rodents or goats. In some embodiments, antibodies are then obtained from non-human animals and optionally modified using a variety of methods (e.g., using recombinant DNA technology). Other examples and methods of antibody production are known in the art (see, e.g., Harlow et al. "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory, 1988.).

[0133] 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, there are about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by a chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, which may optionally lack an enzyme in the N- or O-glycosylation pathway, such as a glycosyltransferase. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, entitled “Modified antibody, antibody-conjugate and process for the preparation thereof”, published on May 1, 2014.

[0134] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VL domain and / or (e.g., and) a VH domain of any one of the anti-TfR1 antibodies selected from any one of Tables 2 to 7, and comprises a constant region comprising an amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b) of an immunoglobulin molecule. Some non-limiting examples of human constant regions are described in the art, for example, see Kabat EA et al., (1991), supra.

[0135] In some embodiments, a substance that binds to the transferrin receptor, such as an anti-TfR1 antibody, can target myocytes and / or (e.g., and) mediate the transport of substances across the blood-brain barrier. The transferrin receptor is an internalized cell surface receptor that transports transferrin across the cell membrane and participates in the regulation of intracellular iron levels and homeostasis. Some aspects of the present disclosure provide transferrin receptor-binding proteins that can bind to the transferrin receptor. An antibody that binds to the transferrin receptor (e.g., specifically binds) can be internalized into a cell after binding to the transferrin receptor, for example, by receptor-mediated endocytosis.

[0136] In some embodiments, the anti-TFR1 antibody is present in an amount of 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 binding affinity (e.g., as indicated by Kd) specifically binds to TfR1 (e.g., human or non-human primate TfR1). In some embodiments, the anti-TfR1 antibodies described herein bind to TfR1 with a KD in the sub-nanomolar range. In some embodiments, the anti-TfR1 antibodies described herein selectively bind to transferrin receptor 1 (TfR1) but not to transferrin receptor 2 (TfR2). In some embodiments, the anti-TfR1 antibodies described herein bind to human TfR1 and cynomolgus monkey TfR1 (e.g., with a Kd of 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 M, 10 -11 M, 10 -12 M, 10 -13 M or less), but does not bind to mouse TfR1. The affinity and binding kinetics of anti-TfR1 antibodies can be tested using any suitable method, including but not limited to biosensor technology (e.g., OCTET or BIACORE). In some embodiments, the binding of any anti-TfR1 antibody described herein does not compete with or inhibit the binding of transferrin to TfR1. In some embodiments, the binding of any anti-TfR1 antibody described herein does not compete with or inhibit the binding of HFE-β-2-microglobulin to TfR1.

[0137] Some non-limiting examples of anti-TfR1 antibodies are provided in Table 2.

[0138] Table 2. Some examples of anti-TfR1 antibodies

[0139]

[0140]

[0141]

[0142]

[0143] *The mutation positions are according to the Kabat numbering of the corresponding VH sequence containing the mutation

[0144] In some embodiments, an anti-TfR1 antibody of the present disclosure is a variant of any one of the anti-TfR1 antibodies provided in Table 2. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 that are identical to CDR-H1, CDR-H2, and CDR-H3 in any one of the anti-TfR1 antibodies provided in Table 2, and comprises a humanized heavy chain variable region and / or (e.g., and) a humanized light chain variable region.

[0145] Some examples of amino acid sequences of anti-TfR1 antibodies described herein are provided in Table 3.

[0146] Table 3. Variable regions of anti-TfR1 antibodies

[0147]

[0148]

[0149]

[0150] *The mutation positions are according to the Kabat numbering of the corresponding VH sequence containing the mutation

[0151] **CDRs according to the Kabat numbering system are in bold

[0152] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH, which contains CDR-H1, CDR-H2 and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid variations in the framework region compared to the corresponding VH provided in Table 3. Alternatively or additionally (e.g., supplementing), an anti-TfR1 antibody of the present disclosure comprises a VL, which contains CDR-L1, CDR-L2 and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) amino acid variations in the framework region compared to the corresponding VL provided in Table 3.

[0153] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH, which contains CDR-H1, CDR-H2 and CDR-H3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identity in the framework region compared to the corresponding VH provided in Table 3. As an alternative or supplement (e.g., supplement), an anti-TfR1 antibody of the present disclosure comprises a VL, which contains CDR-L1, CDR-L2 and CDR-L3 of any one of the anti-TfR1 antibodies provided in Table 3, and comprises an amino acid sequence having at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%) identity in the framework region compared to the corresponding VL provided in Table 3.

[0154] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:69 and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0155] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:71 and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0156] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:72 and a VL comprising the amino acid sequence of SEQ ID NO:70.

[0157] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73 and a VL comprising the amino acid sequence of SEQ ID NO:74.

[0158] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73 and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0159] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:74.

[0160] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a VL comprising the amino acid sequence of SEQ ID NO:75.

[0161] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77 and a VL comprising the amino acid sequence of SEQ ID NO:78.

[0162] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:79 and a VL comprising the amino acid sequence of SEQ ID NO:80.

[0163] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77 and a VL comprising the amino acid sequence of SEQ ID NO:80.

[0164] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:154 and a VL comprising the amino acid sequence of SEQ ID NO:155.

[0165] In some embodiments, the anti-TfR1 antibodies described herein are full-length IgG, which may include a heavy constant region and a light constant region from a human antibody. In some embodiments, the heavy chain of any anti-TfR1 antibody described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may have any suitable source, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from human IgG, such as IgG1, IgG2, or IgG4 (γ heavy chain). An example of a human IgG1 constant region is given below:

[0166]

[0167] In some embodiments, the heavy chain of any anti-TfR1 antibody described herein comprises a mutant human IgG1 constant region. For example, it is known that the introduction of a LALA mutation in the CH2 domain of human IgG1 (derived from a mutant of mAb b12, which has mutated to replace the lower hinge residues Leu234 Leu235 with Ala234 and Ala235) reduces Fcγ receptor binding (Bruhns, P., et al. (2009) and Xu, D. et al. (2000)). The mutant human IgG1 constant region is provided below (mutations are bold and underlined):

[0168]

[0169] In some embodiments, the light chain of any anti-TfR1 antibody described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some instances, CL is a kappa light chain. In other instances, CL is a lambda light chain. In some embodiments, CL is a kappa light chain, the sequence of which is provided below:

[0170]

[0171] Additional antibody heavy and light chain constant regions are known in the art, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0172] In some embodiments, the anti-TfR1 antibody described herein comprises a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof and a heavy chain constant region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibody described herein comprises a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof and a heavy chain constant region comprising no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to SEQ ID NO: 81 or SEQ ID NO: 82. In some embodiments, the anti-TfR1 antibody described herein comprises a heavy chain comprising any one VH listed in Table 3 or any variant thereof and a heavy chain constant region shown in SEQ ID NO: 81. In some embodiments, the anti-TfR1 antibody described herein comprises a heavy chain comprising any one VH listed in Table 3 or any variant thereof and a heavy chain constant region shown in SEQ ID NO: 82.

[0173] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof and a light chain constant region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof and a light chain constant region comprising no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof and a light chain constant region shown in SEQ ID NO: 83.

[0174] Some examples of IgG heavy and light chain amino acid sequences of the anti-TfR1 antibodies are provided in Table 4 below.

[0175] Table 4. Heavy chain sequences and light chain sequences of some examples of anti-TfR1 IgG

[0176]

[0177]

[0178]

[0179] *The mutation positions are according to the Kabat numbering of the corresponding VH sequence containing the mutation

[0180] **CDRs according to the Kabat numbering system are bold; VH / VL sequences are underlined

[0181] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain set forth in any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94, and 156. As an alternative or supplement (e.g., supplement), the anti-TfR1 antibody of the present disclosure comprises a light chain comprising no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variations) compared to the light chain shown in any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157.

[0182] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94 and 156. Alternatively or additionally (e.g., supplementing), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence of any one of SEQ ID NOs: 84, 86, 87, 88, 91, 92, 94 and 156. Alternatively or additionally (eg, supplementally), the anti-TfR1 antibody described herein comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157.

[0183] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:84 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0184] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:86 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0185] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:87 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0186] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0187] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:88 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0188] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0189] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:91 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0190] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0191] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:94 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0192] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:92 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0193] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:156 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0194] In some embodiments, the anti-TfR1 antibody is a Fab fragment, a Fab' fragment or a F(ab')2 fragment of a complete antibody (full-length antibody). The antigen-binding fragment of a complete antibody (full-length antibody) can be prepared by conventional methods (e.g., recombinantly prepared or prepared by digesting the heavy chain constant region of a full-length IgG with an enzyme such as papain). For example, a F(ab')2 fragment can be produced by digesting an antibody molecule with pepsin or papain, and a Fab fragment can be produced by reducing the disulfide bridges of a F(ab')2 fragment. In some embodiments, the heavy chain constant region in the Fab fragment of an anti-TfR1 antibody described herein comprises the following amino acid sequence:

[0195]

[0196] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof and a heavy chain constant region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof and a heavy chain constant region comprising no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to SEQ ID NO: 96. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising any one of the VHs listed in Table 3 or any variant thereof and a heavy chain constant region shown in SEQ ID NO: 96.

[0197] In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof and a light chain constant region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof and a light chain constant region comprising no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variation) compared to SEQ ID NO: 83. In some embodiments, the anti-TfR1 antibodies described herein comprise a light chain comprising any one of the VLs listed in Table 3 or any variant thereof and a light chain constant region shown in SEQ ID NO: 83.

[0198] Some examples of Fab heavy and light chain amino acid sequences of the anti-TfR1 antibodies are provided in Table 5 below.

[0199] Table 5. Heavy and light chain sequences of some examples of anti-TfR1 Fab

[0200]

[0201]

[0202]

[0203] *The mutation positions are according to the Kabat numbering of the corresponding VH sequence containing the mutation

[0204] **CDRs according to the Kabat numbering system are bold; VH / VL sequences are underlined

[0205] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the heavy chain shown in any one of SEQ ID NOs: 97 to 103, 158, and 159. As an alternative or supplement (e.g., supplement), the anti-TfR1 antibody of the present disclosure comprises a light chain comprising no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid variations) compared to the light chain shown in any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157.

[0206] In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to any one of SEQ ID NOs: 97 to 103, 158 and 159. Alternatively or additionally (e.g., supplementing), the anti-TfR1 antibodies described herein comprise a light chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157. In some embodiments, the anti-TfR1 antibodies described herein comprise a heavy chain comprising an amino acid sequence having at least 75% (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) identity to any one of SEQ ID NOs: 97 to 103, 158 and 159. Alternatively or additionally (eg, supplementally), the anti-TfR1 antibody described herein comprises a light chain comprising the amino acid sequence of any one of SEQ ID NOs: 85, 89, 90, 93, 95 and 157.

[0207] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:97 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0208] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:98 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0209] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:99 and a light chain comprising the amino acid sequence of SEQ ID NO:85.

[0210] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0211] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:100 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0212] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:89.

[0213] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90.

[0214] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO:93.

[0215] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:103 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0216] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO:95.

[0217] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:158 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0218] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:159 and a light chain comprising the amino acid sequence of SEQ ID NO:157.

[0219] Other known anti-TfR1 antibodies

[0220] Any other suitable anti-TfR1 antibody known in the art can be used as a muscle targeting agent in the complex disclosed herein. Some examples of known anti-TfR1 antibodies are listed in Table 6, including relevant references and binding epitopes. In some embodiments, the anti-TfR1 antibody comprises the complementary determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) of any anti-TfR1 antibody provided herein (e.g., the anti-TfR1 antibodies listed in Table 6).

[0221] Table 6 - List of anti-TfR1 antibody clones, including relevant references and binding epitope information.

[0222]

[0223]

[0224]

[0225]

[0226] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any anti-TfR1 antibody selected from Table 6. In some embodiments, an anti-TfR1 antibody comprises CDR-L1, CDR-L2, and CDR-L3 as provided for any anti-TfR1 antibody selected from Table 6. In some embodiments, an anti-TfR1 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 as provided for any anti-TfR1 antibody selected from Table 6.

[0227] In some embodiments, the anti-TfR1 antibodies of the present disclosure include any antibodies comprising a heavy chain variable domain and / or (e.g., and) a light chain variable domain of any anti-TfR1 antibody (e.g., any anti-TfR1 antibody selected from Table 6). In some embodiments, the anti-TfR1 antibodies of the present disclosure include any antibodies comprising a heavy chain variable and a light chain variable pair of any anti-TfR1 antibody (e.g., any anti-TfR1 antibody selected from Table 6).

[0228] Some aspects of the present disclosure provide anti-TfR1 antibodies having heavy chain variable (VH) and / or (e.g., and) light chain variable (VL) domain amino acid sequences homologous to any of those described herein. In some embodiments, the anti-TfR1 antibody comprises a heavy chain variable sequence or light chain variable sequence having at least 75% (e.g., 80%, 85%, 90%, 95%, 98% or 99%) identity with a heavy chain variable sequence and / or any light chain variable sequence of any anti-TfR1 antibody (e.g., any anti-TfR1 antibody selected from Table 6). In some embodiments, homologous heavy chain variable and / or (e.g., and) light chain variable amino acid sequences do not vary within any CDR sequence provided herein. For example, in some embodiments, the degree of sequence variation (e.g., 75%, 80%, 85%, 90%, 95%, 98% or 99%) may occur in a heavy chain variable and / or (e.g., and) light chain variable sequence that does not include any CDR sequence provided herein. In some embodiments, any anti-TfR1 antibody provided herein comprises a heavy chain variable sequence and a light chain variable sequence comprising a framework sequence that is at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identical to the framework sequence of any anti-TfR1 antibody (e.g., any anti-TfR1 antibody selected from Table 6).

[0229] Examples of transferrin receptor antibodies that can be used according to the present disclosure are described in International Application Publication No. WO2016 / 081643, which is incorporated herein by reference. The amino acid sequence of this antibody is provided in Table 7.

[0230] Table 7. Heavy and light chain CDRs of examples of known anti-TfR1 antibodies

[0231]

[0232]

[0233]

[0234] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises CDR-H1, CDR-H2, and CDR-H3 identical to those shown in Table 7. Alternatively or additionally (e.g., supplementally), the anti-TfR1 antibody of the present disclosure comprises CDR-L1, CDR-L2, and CDR-L3 identical to those shown in Table 7.

[0235] In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-L3 comprising no more than 3 amino acid variations (e.g., no more than 3, 2, or 1 amino acid variations) compared to a CDR-L3 as shown in Table 7. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-L3 comprising one amino acid variation compared to a CDR-L3 as shown in Table 7. In some embodiments, an anti-TfR1 antibody of the present disclosure comprises a CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or a CDR-L3 of QHFAGTPL (SEQ ID NO: 127) (according to the Contact definition system). In some embodiments, the anti-TfR1 antibody of the present disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 that are identical to the CDR-H1, CDR-H2, and CDR-H3 shown in Table 7, and comprises CDR-L3 of QHFAGTPLT (SEQ ID NO: 126) (according to the Kabat and Chothia definition system) or CDR-L3 of QHFAGTPLT (SEQ ID NO: 127) (according to the Contact definition system).

[0236] In some embodiments, the anti-TfR1 antibodies of the present disclosure comprise heavy chain CDRs that collectively have at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the heavy chain CDRs as shown in Table 7. Alternatively or additionally (e.g., supplementally), the anti-TfR1 antibodies of the present disclosure comprise light chain CDRs that collectively have at least 80% (e.g., 80%, 85%, 90%, 95%, or 98%) identity with the light chain CDRs as shown in Table 7.

[0237] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 124. Alternatively or additionally (eg, supplemented), the anti-TfR1 antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO:125.

[0238] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 128. Alternatively or additionally (eg, supplemented), the anti-TfR1 antibody of the present disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO:129.

[0239] In some embodiments, the anti-TfR1 antibody of the present disclosure comprises a VH comprising no more than 25 amino acid variations (e.g., no more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VH shown in SEQ ID NO: 128. As an alternative or supplement (e.g., supplement), the anti-TfR1 antibody of the present disclosure comprises a VL comprising no more than 15 amino acid variations (e.g., no more than 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid variations) compared to the VL shown in SEQ ID NO: 129.

[0240] In some embodiments, the anti-TfR1 antibody of the present disclosure is a full-length IgG1 antibody, which may include a heavy constant region and a light constant region from a human antibody. In some embodiments, the heavy chain of any anti-TfR1 antibody as described herein may include a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, such as human, mouse, rat, or rabbit. In a specific example, the heavy chain constant region is from human IgG, such as IgG1, IgG2, or IgG4 (γ heavy chain). An example of a human IgG1 constant region is given below:

[0241]

[0242] In some embodiments, the light chain of any anti-TfR1 antibody described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some instances, CL is a kappa light chain. In other instances, CL is a lambda light chain. In some embodiments, CL is a kappa light chain, the sequence of which is provided below:

[0243]

[0244] In some embodiments, the anti-TfR1 antibody described herein is a chimeric antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 132. Alternatively or additionally (eg, supplemented), the anti-TfR1 antibody described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 133.

[0245] In some embodiments, the anti-TfR1 antibody described herein is a fully human antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO: 134. Alternatively or additionally (eg, supplemented), the anti-TfR1 antibody described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 135.

[0246] In some embodiments, the anti-TfR1 antibody is an antigen-binding fragment (Fab) of a complete antibody (full-length antibody). In some embodiments, the anti-TfR1 Fab described herein comprises a heavy chain containing an amino acid sequence of SEQ ID NO: 136. As an alternative or supplement (e.g., supplement), the anti-TfR1 Fab described herein comprises a light chain containing an amino acid sequence of SEQ ID NO: 133. In some embodiments, the anti-TfR1 Fab described herein comprises a heavy chain containing an amino acid sequence of SEQ ID NO: 137. As an alternative or supplement (e.g., supplement), the anti-TfR1 Fab described herein comprises a light chain containing an amino acid sequence of SEQ ID NO: 135.

[0247] The anti-TfR1 antibodies described herein may be in any antibody form, including but not limited to complete (ie, full-length) antibodies, antigen-binding fragments thereof (eg, Fab, Fab', F(ab')2, Fv), single-chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the anti-TfR1 antibodies described herein are scFvs. In some embodiments, the anti-TfR1 antibodies described herein are scFv-Fabs (eg, scFvs fused to a portion of a constant region). In some embodiments, the anti-TfR1 antibodies described herein are scFvs fused to a constant region (eg, a human IgG1 constant region shown in SEQ ID NO: 81).

[0248] In some embodiments, conservative mutations may be introduced into the antibody sequence (e.g., CDR or framework sequence) at positions where the residues are unlikely to be involved in interacting with the target antigen (e.g., transferrin receptor) (e.g., as determined based on the crystal structure). In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the anti-TfR1 antibodies described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG1) and / or (e.g., and) the hinge region, numbered according to the Kabat numbering system (e.g., the EU index in Kabat)), to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or (e.g., and) antigen-dependent cellular toxicity.

[0249] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Pat. No. 5,677, 425. The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, for example, to facilitate assembly of light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.

[0250] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle targeting antibodies described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG1) and / or (e.g., and) the hinge region, numbered according to the Kabat numbering system (e.g., the EU index in Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Mutations in the Fc region of an antibody that decrease or increase the affinity of an antibody for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those of skill in the art. Some examples of mutations in antibody Fc receptors that can be made to alter the affinity of the antibody for the Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0251] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., reduce or increase) the half-life of the anti-TfR1 antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for example, mutations that will alter (e.g., reduce or increase) the half-life of an antibody in vivo.

[0252] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, Fc or hinge-Fc domain fragment) to reduce the half-life of the anti-TfR1 antibody in vivo. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341 to 447 of human IgG1) (numbered according to the EU index in Kabat (Kabat EA et al., (1991) supra)). In some embodiments, the constant region of the IgG1 of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, the positions being numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as a "YTE mutant") has been shown to exhibit a 4-fold increase in half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions at amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436, as numbered according to the EU index as in Kabat.

[0253] In some embodiments, one, two or more amino acid substitutions are introduced into the IgG constant domain Fc region to change the effector function of the anti-TfR1 antibody. The effector ligand to which affinity is changed can be, for example, an Fc receptor or the C1 component of complement. This method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, the deletion or inactivation of the constant region domain (by point mutation or otherwise) can reduce the Fc receptor binding of circulating antibodies, thereby improving tumor localization. For descriptions of mutations that delete or inactivate the constant domain and thereby improve tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions can be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields R Let al., (2001) J Biol Chem 276: 6591-604).

[0254] In some embodiments, one or more amino groups in the constant region of the anti-TfR1 antibodies described herein may be replaced with different amino acid residues so that the antibody has altered C1q binding and / or (e.g., and) reduced or eliminated complement dependent cytotoxicity (CDC). This method is described in more detail in U.S. Patent No. 6,194,551 (Idusogie et al). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibodies described herein are changed to change the ability of the antibody to fix complement. This method is further described in International Publication No. WO 94 / 29351. In some embodiments, the Fc region of the antibodies described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or (e.g., and) increase the affinity of the antibody for Fcγ receptors. This method is further described in International Publication No. WO 00 / 42072.

[0255] In some embodiments, the heavy chain and / or (e.g., and) light chain variable domain sequences of antibodies provided herein can be used to produce, for example, CDR grafted, chimeric, humanized or composite human antibodies or antigen-binding fragments, as described elsewhere herein. As understood by those of ordinary skill in the art, any variant (CDR grafted, chimeric, humanized or composite antibody) derived from any antibody provided herein can be used in the compositions and methods described herein, and will maintain the ability to specifically bind to transferrin receptor, so that relative to the original antibody from which it is derived, the variant (CDR grafted, chimeric, humanized or composite antibody) has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to transferrin receptor.

[0256] In some embodiments, the antibodies provided herein include mutations that confer desired properties on the antibodies. For example, to avoid potential complications due to known Fab-arm exchanges with natural IgG4 mAbs, the antibodies provided herein may include stability 'Adair' mutations (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), wherein the 228th (EU numbering, according to Kabat numbering, the 241st residue) serine is converted to proline, thereby generating an IgG1-like hinge sequence. Therefore, any antibody may include stability 'Adair' mutations.

[0257] 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, there are about 1 to 10, about 1 to 5, about 5 to 10, about 1 to 4, about 1 to 3, or about 2 sugar molecules. In some embodiments, the glycosylated antibody is fully or partially glycosylated. In some embodiments, the antibody is glycosylated by a chemical reaction or by enzymatic means. In some embodiments, the antibody is glycosylated in vitro or in a cell, which may optionally lack an enzyme in the N- or O-glycosylation pathway, such as a glycosyltransferase. In some embodiments, the antibody is functionalized with a sugar or carbohydrate molecule as described in International Patent Application Publication WO2014065661, entitled “Modified antibody, antibody-conjugate and process for the preparation thereof”, published on May 1, 2014.

[0258] In some embodiments, any of the anti-TfR1 antibodies described herein may include a signal peptide (e.g., an N-terminal signal peptide) in the heavy chain sequence and / or (e.g., and) light chain sequence. In some embodiments, the anti-TfR1 antibodies described herein include any of the VH sequences and VL sequences described herein, any of the IgG heavy chain sequences and light chain sequences, or any of the F(ab') heavy chain sequences and light chain sequences, and further include a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide includes the amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

[0259] In some embodiments, the antibodies provided herein may have one or more post-translational modifications. In some embodiments, the N-terminal cyclization also referred to as pyroglutamic acid formation (pyro-Glu) 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 the antibodies designated as having a sequence comprising N-terminal glutamic acid or glutamine residues encompass antibodies that have undergone pyroglutamic acid formation caused by post-translational modifications. In some embodiments, pyroglutamic acid formation occurs in the heavy chain sequence. In some embodiments, pyroglutamic acid formation occurs in the light chain sequence.

[0260] b. Other muscle-targeted antibodies

[0261] In some embodiments, the muscle targeting antibody is an antibody that specifically binds hemojuvelin, caveolin-3, Duchenne muscular dystrophy peptide, myosin IIb or CD63. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to myogenic precursor proteins. Some exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin / cadherin-15, caveolin-1, CD34, FoxK1, integrin α7, integrin α7β1, MYF-5, MyoD, myogenic protein, NCAM-1 / CD56, Pax3, Pax7, and Pax9. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to skeletal muscle proteins. Some exemplary skeletal muscle proteins include, but are not limited to, alpha-Sarcoglycan, beta-Sarcoglycan, calpain inhibitor, creatine kinase MM / CKMM, eIF5A, enolase 2 / neuron-specific enolase, epsilon-Sarcoglycan, FABP3 / H-FABP, GDF-8 / myostatin, GDF-11 / GDF-8, integrin alpha 7, integrin alpha 7 beta 1, integrin beta 1 / CD29, MCAM / CD146, MyoD, myogenin, myosin light chain kinase inhibitor, NCAM-1 / CD56, and troponin I. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to a smooth muscle protein. Some exemplary smooth muscle proteins include, but are not limited to, α-smooth muscle actin, VE-cadherin, calmodulin binding protein / CALD1, calmodulin 1, desmin, histamine H2 R, motilin R / GPR38, transglutamin / TAGLN, and vimentin. However, it should be understood that antibodies to other targets are within the scope of the present disclosure, and the exemplary list of targets provided herein is not meant to be limiting.

[0262] c. Antibody characteristics / changes

[0263] In some embodiments, conservative mutations may be introduced into the antibody sequence (e.g., CDR or framework sequence) at positions where the residues are unlikely to be involved in interacting with the target antigen (e.g., transferrin receptor) (e.g., as determined based on the crystal structure). In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle-targeted antibodies described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG1) and / or (e.g., and) the hinge region, numbered according to the Kabat numbering system (e.g., the EU index in Kabat)) to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding and / or (e.g., and) antigen-dependent cellular toxicity.

[0264] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Pat. No. 5,677, 425. The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, for example, to facilitate assembly of light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.

[0265] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the muscle targeting antibodies described herein (e.g., in the CH2 domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG1) and / or (e.g., and) the hinge region, numbered according to the Kabat numbering system (e.g., the EU index in Kabat)) to increase or decrease the affinity of the antibody for an Fc receptor on the surface of an effector cell (e.g., an activated Fc receptor). Mutations in the Fc region of an antibody that decrease or increase the affinity of the antibody for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those of skill in the art. Some examples of mutations in antibody Fc receptors that can be made to alter the affinity of the antibody for the Fc receptor are described in, e.g., Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0266] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to alter (e.g., reduce or increase) the half-life of the antibody in vivo. See, e.g., International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, and U.S. Pat. Nos. 5,869,046, 6,121,022, 6,277,375, and 6,165,745, for example, mutations that will alter (e.g., reduce or increase) the half-life of the antibody in vivo.

[0267] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, Fc or hinge-Fc domain fragment) to reduce the half-life of the anti-transferrin receptor antibody in vivo. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or its FcRn binding fragment (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231 to 340 of human IgG1) and / or (e.g., and) the third constant (CH3) domain (residues 341 to 447 of human IgG1) (numbered according to the EU index in Kabat (Kabat EA et al., (1991) supra)). In some embodiments, the constant region of the IgG1 of the antibodies described herein comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, the positions being numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as a "YTE mutant") has been shown to show a 4-fold increase in half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem 281: 23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three, or more amino acid substitutions at amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436, as numbered according to the EU index as in Kabat.

[0268] In some embodiments, one, two or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to change the effector function of the anti-transferrin receptor antibody. The effector ligand to which the affinity is changed can be, for example, an Fc receptor or the C1 component of complement. This method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some embodiments, the deletion or inactivation of the constant region domain (by point mutation or other means) can reduce the Fc receptor binding of the circulating antibody, thereby improving tumor localization. For descriptions of mutations that delete or inactivate the constant domain and thereby improve tumor localization, see, for example, U.S. Patent Nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions can be introduced into the Fc region of the antibody described herein to remove potential glycosylation sites on the Fc region, which can reduce Fc receptor binding (see, for example, Shields RL et al., (2001) J Biol Chem 276: 6591-604).

[0269] In some embodiments, one or more amino groups in the constant region of the muscle-targeted antibodies described herein can be replaced with different amino acid residues so that the antibody has altered C1q binding and / or (e.g., and) reduced or eliminated complement-dependent cytotoxicity (CDC). This approach is described in more detail in U.S. Patent No. 6,194,551 (Idusogie et al). In some embodiments, one or more amino acid residues in the N-terminal region of the CH2 domain of the antibodies described herein are changed to change the ability of the antibody to fix complement. This approach is further described in International Publication No. WO94 / 29351. In some embodiments, the Fc region of the antibodies described herein is modified to improve the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or (e.g., and) improve the affinity of the antibody for Fcγ receptors. This approach is further described in International Publication No. WO 00 / 42072.

[0270] In some embodiments, the heavy chain and / or (e.g., and) light chain variable domain sequences of antibodies provided herein can be used to produce, for example, CDR grafted, chimeric, humanized or composite human antibodies or antigen-binding fragments, as described elsewhere herein. As understood by those of ordinary skill in the art, any variant (CDR grafted, chimeric, humanized or composite antibody) derived from any antibody provided herein can be used in the compositions and methods described herein, and will maintain the ability to specifically bind to transferrin receptor, so that relative to the original antibody from which it is derived, the variant (CDR grafted, chimeric, humanized or composite antibody) has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more binding to transferrin receptor.

[0271] In some embodiments, the antibodies provided herein include mutations that confer desired properties on the antibodies. For example, to avoid potential complications due to known Fab-arm exchanges with natural IgG4 mAbs, the antibodies provided herein may include stability 'Adair' mutations (Angal S., et al., "A single amino acid substitution abolishes the heterogeneity of chimeric mouse / human (IgG4) antibody," Mol Immunol 30, 105-108; 1993), wherein the 228th (EU numbering, according to Kabat numbering, the 241st residue) serine is converted to proline, thereby generating an IgG1-like hinge sequence. Therefore, any antibody may include stability 'Adair' mutations.

[0272] As provided herein, the antibodies of the present disclosure may optionally include a constant region or a portion thereof. For example, the VL domain may be connected to a light chain constant domain, such as Cκ or Cλ, at its C-terminal end. Similarly, the VH domain or a portion thereof may be connected to all or a portion of a heavy chain such as IgA, IgD, IgE, IgG and IgM, and any isotype subclass. The antibody may include a suitable constant region (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, No. 91-3242, National Institutes of Health Publications, Bethesda, Md. (1991)). Therefore, antibodies within the scope of the present disclosure may include VH and VL domains or antigen binding portions thereof in combination with any suitable constant region.

[0273] ii. Muscle Targeting Peptides

[0274] Some aspects of the present disclosure provide muscle targeting peptides as muscle targeting agents.Short peptide sequences (eg, peptide sequences 5 to 20 amino acids in length) that bind to specific cell types have been described. For example, cell targeting peptides have been described in: Vines E., et al., A. "Cell-penetrating and cell-targeting peptides in drug delivery" Biochim Biophys Acta 2008, 1786: 126-38; Jarver P., et al., "Invivo biodistribution and efficacy of peptide mediated delivery" Trends Pharmacol Sci 2010; 31: 528-35; Samoylova TI, et al., "Elucidation of muscle-binding peptides by phage display screening" Muscle Nerve 1999; 22: 460-6; ​​U.S. Patent No. 6,329,501, which was issued on December 11, 2001, and is entitled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE"; and Samoylov AM, et al., "Recognition of cell-specific binding of Phage display derived peptides using an acoustic wavesensor." Biomol Eng 2002; 18: 269-72; the entire contents of each of which are incorporated herein by reference. By designing peptides to interact with specific cell surface antigens (e.g., receptors), selectivity for desired tissues such as muscle can be achieved. Skeletal muscle targeting has been studied and is capable of delivering a range of molecular payloads. These methods can be highly selective for muscle tissue without many of the practical disadvantages of large antibodies or viral particles. Therefore, in some embodiments, the muscle targeting agent is a muscle targeting peptide of 4 to 50 amino acids in length.In some embodiments, the muscle targeting peptide is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length. Any of several methods, such as phage display, can be used to generate muscle targeting peptides.

[0275] In some embodiments, the muscle targeting peptide can bind to an internalized cell surface receptor, such as a transferrin receptor, that is overexpressed or relatively highly expressed in muscle cells compared to certain other cells. In some embodiments, the muscle targeting peptide can target the transferrin receptor (e.g., bind to it). In some embodiments, the peptide targeting the transferrin receptor can include a segment of a naturally occurring ligand (e.g., transferrin). In some embodiments, the peptide targeting the transferrin receptor is as described in U.S. Patent No. 6,743,893, filed on November 30, 2000, "RECEPTOR-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR". In some embodiments, the peptide targeting the transferrin receptor is as described in Kawamoto, M. et al, "A novel transferrin receptor-targeted hybrid peptide disintegrates cancer cell membrane to induce rapid killing of cancer cells." BMC Cancer. 2011Aug 18; 11: 359. In some embodiments, the peptide targeting transferrin receptor is as described in U.S. Patent No. 8,399,653, filed May 20, 2011, “TRANSFERRIN / TRANSFERRIN RECEPTOR-MEDIATED SIRNA DELIVERY.”

[0276] As discussed above, some examples of muscle targeting peptides have been reported. For example, muscle-specific peptides were identified using a phage display library presenting surface heptapeptides. As an example, a peptide having the amino acid sequence ASSLNIA (SEQ ID NO: 167) binds to C2C12 mouse myotubes in vitro and binds to mouse muscle tissue in vivo. Therefore, in some embodiments, the muscle targeting agent comprises the amino acid sequence ASSLNIA (SEQ ID NO: 167). The peptide exhibits increased specificity for binding to cardiac and skeletal muscle tissue after intravenous injection in mice, as well as reduced binding to the liver, kidney and brain. Additional muscle-specific peptides have been identified using phage display. For example, a 12 amino acid peptide was identified by a phage display library for muscle targeting in the case of DMD treatment. See Yoshida D., et al., "Targeting of salicylate to skin and muscle following topical injections in rats." Int JPharm 2002; 231: 177-84; the entire contents of which are incorporated herein by reference. Here, a 12 amino acid peptide having the sequence SKTFNTHPQSTP (SEQ ID NO: 168) was identified and this muscle-targeted peptide showed improved binding to C2C12 cells relative to the ASSLNIA (SEQ ID NO: 167) peptide.

[0277] Another method for identifying peptides that are selective for muscle (e.g., skeletal muscle) relative to other cell types includes in vitro selection, which has been described in Ghosh D., et al., "Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting" J Virol 2005; 79: 13667-72; the entire contents of which are incorporated herein by reference. Non-specific cell binders were selected by pre-incubating a random 12-mer peptide phage display library with a mixture of non-muscle cell types. After several rounds of selection, the 12 amino acid peptide TARGEHKEEELI (SEQ ID NO: 169) appeared most frequently. Therefore, in some embodiments, the muscle targeting agent comprises the amino acid sequence TARGEHKEEELI (SEQ ID NO: 169).

[0278] The muscle targeting agent can be an amino acid-containing molecule or peptide. The muscle targeting peptide can correspond to a protein sequence that preferentially binds to a protein receptor present in a muscle cell. In some embodiments, the muscle targeting peptide comprises a highly propensity hydrophobic amino acid, such as valine, such that the peptide preferentially targets a muscle cell. In some embodiments, the muscle targeting peptide is previously uncharacterized or disclosed. These peptides can be conceived, generated, synthesized, and / or (e.g., and) derived using any of several methods (e.g., phage display peptide libraries, single bead single compound peptide libraries, or position scanning synthetic peptide combinatorial libraries). Some exemplary methods have been characterized in the art and incorporated by reference (Gray, BP and Brown, KC "Combinatorial Peptide Libraries: Mining for Cell-Binding Peptides" Chem Rev. 2014, 114:2, 1020--1081.; Samoylova, TI and Smith, BF "Elucidation of muscle-binding peptides by phage display screening." Muscle Nerve, 1999, 22:4.460-6.).In some embodiments, muscle targeting peptides have been previously disclosed (see, e.g., Writer MJ et al. "Targeted gene delivery to human airway epithelial cells with synthetic vectors incorporating novel targeting peptides selected by phage display." J. Drug Targeting. 2004; 12: 185; Cai, D. "BDNF-mediated enhancement of inflammation and injury in the aging heart." Physiol Genomics. 2006, 24: 3, 191-7.; Zhang, L. "Molecular profiling of heart endothelial cells." Circulation, 2005, 112: 11, 1601-11.; McGuire, MJ et al. "In vitro selection of a peptide with high selectivity for cardiomyocytes in vivo." J Mol Biol. 2004, 342: 1, 171-82.). Some exemplary muscle targeting peptides include the following group of amino acid sequences:.

[0279]

[0280] In some embodiments, the muscle targeting peptide may comprise about 2 to 25 amino acids, about 2 to 20 amino acids, about 2 to 15 amino acids, about 2 to 10 amino acids, or about 2 to 5 amino acids. The muscle targeting peptide may comprise naturally occurring amino acids such as cysteine, alanine, or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, homo-amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the muscle targeting peptide may be linear; in other embodiments, the muscle targeting peptide may be cyclic, such as bicyclic (see, e.g., Silvana, M Get al. Mol. Therapy, 2018, 26: 1, 132-–147.).

[0281] iii. Muscle-targeted receptor ligands

[0282] The muscle targeting agent can be a ligand, such as a ligand that binds to a receptor protein. The muscle targeting ligand can be a protein, such as transferrin that binds to an internalized cell surface receptor expressed by a muscle cell. Therefore, in some embodiments, the muscle targeting agent is transferrin or a derivative thereof that binds to a transferrin receptor. The muscle targeting ligand can alternatively be a small molecule, such as a lipophilic small molecule that preferentially targets muscle cells relative to other cell types. Some exemplary lipophilic small molecules that can target muscle cells include compounds comprising: cholesterol, cholesterol radicals, stearic acid, palmitic acid, oleic acid, oleyl, linolene, linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerol, alkyl chains, trityl groups, and alkoxy acids.

[0283] iv. Muscle-targeting aptamers

[0284] Muscle targeting agents can be aptamers, such as RNA aptamers, which preferentially target muscle cells relative to other cell types. In some embodiments, muscle targeting aptamers are previously uncharacterized or disclosed. Any of several methods (e.g., systematic evolution of ligands by exponential enrichment) can be used to conceive, produce, synthesize and / or (e.g., and) derive these aptamers. Some exemplary methods have been characterized in the art and incorporated by reference (Yan, A C and Levy, M. "Aptamers and aptamer targeted delivery" RNA biology, 2009, 6: 3, 316-20.; Germer, K. et al. "RNA aptamers and their therapeutic and diagnostic applications." Int. J. Biochem. Mol. Biol. 2013; 4: 27–40.). In some embodiments, muscle targeting aptamers have been previously disclosed (see, e.g., Phillippou, S. et al. "Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers." Mol Ther Nucleic Acids. 2018, 10: 199-214.; Thiel, W Het al. "Smooth Muscle Cell-targeted RNA Aptamer Inhibits NeointimalFormation." Mol Ther. 2016, 24: 4, 779-87.). Exemplary muscle targeting aptamers include A01B RNA aptamers and RNA Apt 14. In some embodiments, the aptamer is a nucleic acid-based aptamer, an oligonucleotide aptamer, or a peptide aptamer. In some embodiments, the aptamer can be about 5 kDa to 15 kDa, about 5 kDa to 10 kDa, about 10 kDa to 15 kDa, about 1 to 5 Da, about 1 kDa to 3 kDa, or less.

[0285] v. Other muscle targeting agents

[0286] One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use substrates of muscle transporter proteins (e.g., transporter proteins expressed on the sarcolemma). In some embodiments, a muscle targeting agent is a substrate of an influx transporter that is specific for muscle tissue. In some embodiments, the influx transporter is specific for skeletal muscle tissue. Two major classes of transporters are expressed on the skeletal muscle sarcolemma: (1) the adenosine triphosphate (ATP) binding cassette (ABC) superfamily, which facilitates efflux from skeletal muscle tissue and (2) the solute carrier (SLC) superfamily, which facilitates substrate influx into skeletal muscle. In some embodiments, a muscle targeting agent is a substrate that binds to the ABC superfamily or the SLC superfamily of transporters. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a naturally occurring substrate. In some embodiments, the substrate that binds to the ABC or SLC superfamily of transporters is a non-naturally occurring substrate, for example, a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

[0287] In some embodiments, the muscle targeting agent is any muscle targeting agent (e.g., antibody, nucleic acid, small molecule, peptide, aptamer, lipid, sugar moiety) of the SLC superfamily of targeted transporters described herein. In some embodiments, the muscle targeting agent is a substrate of the SLC superfamily of transporters. SLC transporters are equilibrium-type or use proton or sodium ion gradients generated across the membrane to drive transport of substrates. Some exemplary SLC transporters with high skeletal muscle expression include, but are not limited to, SATT transporter (ASCT1; SLC1A4), GLUT4 transporter (SLC2A4), GLUT7 transporter (GLUT7; SLC2A7), ATRC2 transporter (CAT-2; SLC7A2), LAT3 transporter (KIAA0245; SLC7A6), PHT1 transporter (PTR4; SLC15A4), OATP-J transporter (OATP5A1; SLC21A15), OCT3 transporter (EMT; SLC22A3), OCTN2 transporter (FLJ46769; SLC22A5), ENT transporter (ENT1; SLC29A1 and ENT2; SLC29A2), PAT2 transporter (SLC36A2), and SAT2 transporter (KIAA1382; SLC38A2). These transporters can facilitate substrate influx into skeletal muscle, providing an opportunity for muscle targeting.

[0288] In some embodiments, the muscle targeting agent is a substrate of the equilibrative nucleoside transporter 2 (ENT2) transporter. ENT2 has one of the highest mRNA expressions in skeletal muscle relative to other transporters. Although human ENT2 (hENT2) is expressed in most body organs such as the brain, heart, placenta, thymus, pancreas, prostate, and kidney, it is particularly abundant in skeletal muscle. Human ENT2 promotes the absorption of its substrate according to its concentration gradient. ENT2 plays a role in maintaining nucleoside homeostasis by transporting a wide range of purine and pyrimidine nucleobases. The hENT2 transporter has a low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except inosine. Therefore, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2', 3'-dideoxyinosine, and calofarabine. In some embodiments, any muscle targeting agent provided herein is associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular payload.

[0289] In some embodiments, the muscle targeting agent is a substrate of an organic cation / carnitine transporter (OCTN2), which is a sodium ion-dependent high affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, meldonate, acetylcarnitine, or any derivative thereof that binds to OCTN2. In some embodiments, carnitine, meldonate, acetylcarnitine, or a derivative thereof is covalently linked to a molecular payload (e.g., an oligonucleotide payload).

[0290] The muscle targeting agent can be a protein that is present in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein can be hemojuvelin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), which is a protein involved in iron overload and homeostasis. In some embodiments, hemojuvelin can be full-length or fragments, or mutants having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity with a functional hemojuvelin protein. In some embodiments, the hemojuvelin mutant can be a soluble fragment, can lack N-terminal signaling, and / or (e.g., and) lack a C-terminal anchoring domain. In some embodiments, hemojuvelin can be annotated with GenBank RefSeq Accession Nos. NM_001316767.1, NM_145277.4, NM_202004.3, NM_213652.3, or NM_213653.3. It is understood that hemojuvelin can be of human, non-human primate, or rodent origin.

[0291] B. Molecular Payload

[0292] Some aspects of the present disclosure provide molecular payloads, for example, oligonucleotides intended to target DUX4 RNA to regulate the expression or activity of DUX4. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA, which can be used to reduce the level of DUX4 mRNA and / or protein associated with facioscapulohumeral muscular dystrophy (FSHD) pathological features, including muscle atrophy, inflammation, and reduced differentiation potential and oxidative stress. In some embodiments, the oligonucleotides provided herein are intended to guide RNAi-mediated degradation of DUX4 RNA. In some embodiments, the oligonucleotides are intended to effectively engage RNA-induced silencing complexes (RISC) to degrade DUX4 RNA and also have reduced off-target effects. In some embodiments, the oligonucleotides are intended to have desired bioavailability and / or serum stability properties. In some embodiments, the oligonucleotides are intended to have desired binding affinity properties. In some embodiments, the oligonucleotides are intended to have desired toxicity and / or immunogenicity profiles.

[0293] In some embodiments, the oligonucleotide comprises a strand having a complementary region to DUX4 RNA.Exemplary oligonucleotides are described in further detail herein, however, it is to be understood that the exemplary oligonucleotides provided herein are not meant to be limiting.

[0294] i. Oligonucleotides

[0295] In some embodiments, the oligonucleotides provided herein are intended to cause RNAi-mediated degradation of DUX4 mRNA. In some embodiments, the oligonucleotides provided herein comprise an antisense strand complementary to DUX4 mRNA. In some embodiments, the oligonucleotides provided herein further comprise a sense strand (e.g., siRNA) forming a double-stranded oligonucleotide. 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.

[0296] As described herein, any suitable oligonucleotide may be used as a molecular payload. Some examples of oligonucleotides that can be used to target DUX4 are provided in the following: U.S. Patent No. 9,988,628, which was published on February 2, 2017, and is entitled “AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; U.S. Patent No. 9,469,851, which was published on October 30, 2014, and is entitled “RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4”; U.S. Patent Application Publication 20120225034, which was published on September 6, 2012, and is entitled “AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY”; PCT Patent Application Publication No. WO 2013 / 120038, which was published on August 15, 2013, and is entitled “MORPHOLINO TARGETING DUX4FOR TREATING FSHD";Chen et al., "Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular DystrophyTherapeutics," Molecular Therapy, 2016, 24: 8, 1405-1411.;And Ansseau et al., "Antisense Oligonucleotides Used to Target the DUX4 mRNA as TherapeuticApproaches in Facioscapulohumeral Muscular Dystrophy(FSHD)," Genes, 2017, 8, 93; The contents of each are incorporated herein in their entirety. In some embodiments, the oligonucleotide is an antisense oligonucleotide, a morpholino, a siRNA, a shRNA, or other oligonucleotide that hybridizes to a target DUX4 gene or mRNA. In some embodiments, the oligonucleotide is a siRNA oligonucleotide.

[0297] In some embodiments, the oligonucleotides described herein have a complementary region to the following sequences: human DUX4, corresponding to the following NCBI sequence NM_001293798.2 (SEQ ID NO: 160) or NCBI sequence: NM_001306068.3 (SEQ ID NO: 161) and / or (e.g., and) mouse DUX4, corresponding to the following NCBI sequence NM_001081954.1 (SEQ ID NO: 162). Other non-limiting exemplary human DUX4 mRNAs include NCBI sequence: NM_033178, GenBank accession number FJ439133,

[0298] AF117653, HM101229, HM101230, HM101232, HM101233, HM101234, HM101235, HM101240, HM101241, HM10 1242, HM101243, HM101244, HM101245, HM101246, HM101247, HM101248, HM101249, HM101250, HM101251 and HM190160, HM190161, HM190162, HM190163, HM190164, HM190165, HM190166, HM190167, HM190168, HM 190169, HM190170, HM190171, HM190172, HM190173, HM190174, HM190175, HM190176, HM190177, HM19 0178, HM190179, HM190180, HM190181, HM190182, HM190183, HM190184, HM190185, HM190186, HM1901 87, HM190188, HM190189, HM190190, HM190191, HM190192, HM190193, HM190194, HM190195, HM190196,

[0299] Each of which is incorporated herein by reference. In some embodiments, the oligonucleotide may have a complementary region to a hypomethylated compact D4Z4 repeat, such as Daxinger, et al., "Genetic and Epigenetic Contributors to FSHD," published in Curr Opin Genet Dev, Lim JW, et al., DICER / AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet. 2015 Sep 1; 24(17): 4817-4828, the contents of each of which are incorporated in their entirety.

[0300] In some embodiments, the oligonucleotide may have a complementary region to the sequence shown below, which is an exemplary human DUX4 gene sequence (NM_001293798.2) (SEQ ID NO: 160):

[0301]

[0302] In some embodiments, the oligonucleotide may have a complementary region to the sequence shown below, which is an exemplary human DUX4 gene sequence (NM_001306068.3) (SEQ ID NO: 161):

[0303]

[0304] In some embodiments, the oligonucleotide may have a complementary region to the sequence shown below, which is an exemplary mouse DUX4 gene sequence (SEQ ID NO: 162) (NM_001081954.1):

[0305]

[0306]

[0307] In some embodiments, the oligonucleotides may have complementary regions to DUX4 gene sequences of multiple species, such as selected from human, mouse, and non-human species. In some embodiments, the non-human species is cynomolgus monkey.

[0308] ii. Oligonucleotide size / sequence

[0309] Oligonucleotide can have multiple different lengths, for example this depends on form.In some embodiments, the length of oligonucleotide is 7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30,35,40,45,50,75 or more nucleotide.In some embodiments, the length of oligonucleotide is 8 to 50 nucleotide, length is 8 to 40 nucleotide, length is 8 to 32 nucleotide, length is 10 to 15 nucleotide, length is 10 to 20 nucleotide, length is 15 to 25 nucleotide, length is 21 to 23 nucleotide, etc. In some embodiments, the oligonucleotide is 8 to 32 nucleotides, 15 to 29 nucleotides, 15 to 27 nucleotides, 15 to 20 nucleotides, 20 to 25 nucleotides, 21 to 27 nucleotides, 23 to 27 nucleotides, 25 to 30 nucleotides, or 25 to 32 nucleotides in length.

[0310] In some embodiments, when the binding of the complementary nucleic acid sequence of the oligonucleotide to the target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) thereby causing loss of activity (e.g., inhibition of translation) or expression (e.g., degradation of the target mRNA), and has a sufficient degree of complementarity to avoid non-specific binding of the sequence to non-target sequences under the following conditions, for the purposes of the present disclosure, the complementary nucleic acid sequence of the oligonucleotide can specifically hybridize with the target nucleic acid or has specificity for the target nucleic acid: under conditions where it is desired to avoid non-specific binding, such as physiological conditions in the case of in vivo assays or therapeutic treatments, and physiological conditions in the case of in vitro assays, under conditions where the assay is performed under appropriate stringent conditions. Thus, in some embodiments, the oligonucleotide can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to the contiguous nucleotides of the target nucleic acid. In some embodiments, the complementary nucleotide sequence does not need to be 100% complementary to its targeted to specifically hybridize with the target nucleic acid or have specificity to the target nucleic acid. In some embodiments, the oligonucleotide comprises one or more nuclear bases mismatched with respect to the target nucleic acid. In some embodiments, the activity associated with the target is reduced due to such mismatch, but the amount of activity reduction associated with the non-target is greater (that is, the selectivity of the target nucleic acid is improved and the off-target effect is reduced). In some embodiments, the target nucleic acid is a pre-mRNA molecule or an mRNA molecule.

[0311] In some embodiments, the oligonucleotide comprises a complementary region to a target nucleic acid, the length of the complementary region being in the range of 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides. In some embodiments, the oligonucleotide comprises a complementary region to a target nucleic acid, the length of the complementary region being in the range of 8 to 32, 15 to 29, 15 to 27, 21 to 27, 23 to 27 nucleotides. In some embodiments, the oligonucleotide comprises a complementary region to a target nucleic acid, the length of the complementary region being in the range of 15 to 29, 15 to 27, 15 to 20, 20 to 25, 21 to 27, 23 to 27, 25 to 27, or 25 to 32 nucleotides. In some embodiments, the length of the complementary region of the oligonucleotide and the target nucleic acid is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50 nucleotides. In some embodiments, the complementary region is complementary to at least 8 continuous nucleotides of the target nucleic acid. In some embodiments, the complementary region is complementary to at least 12 continuous nucleotides of the target nucleic acid. In some embodiments, the complementary region is complementary to at least 16 continuous nucleotides of the target nucleic acid. In some embodiments, compared with the continuous nucleotide portion of the target nucleic acid, the oligonucleotide can include 1, 2 or 3 base mispairings. In some embodiments, an oligonucleotide can have up to 3 mismatches over 15 bases, or up to 2 mismatches over 10 bases.

[0312] In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides comprising a sequence of any one of SEQ ID NOs: 236 to 266. In some embodiments, the oligonucleotide comprises a sequence comprising any one of SEQ ID NOs: 236 to 266. In some embodiments, the oligonucleotide comprises a sequence that shares at least 70%, 75%, 80%, 85%, 90%, 95%, or 97% sequence identity with at least 12 or at least 15 consecutive nucleotides of any one of SEQ ID NOs: 236 to 266.

[0313] In some embodiments, the oligonucleotide comprises a region of complementarity to a target sequence as set forth in any one of SEQ ID NOs: 174 to 235. In some embodiments, the oligonucleotide comprises a region of complementarity that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or 100% complementary to at least 12 or at least 15 consecutive nucleotides of a target sequence as set forth in any one of SEQ ID NOs: 174 to 235. In some embodiments, the length of the region of complementarity is 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 19, or at least 20 nucleotides. In some embodiments, the length of the region of complementarity is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides. In some embodiments, the length of the region of complementarity is in the range of 8 to 20, 10 to 20, or 15 to 20 nucleotides. In some embodiments, the complementary region is fully complementary to all or part of its target sequence.In some embodiments, the complementary region comprises 1, 2, 3 or more mismatches.

[0314] In some embodiments, the oligonucleotide is complementary to the target sequence of any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 8) (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary). In some embodiments, the oligonucleotide is complementary to the target sequence of any one of the oligonucleotides provided herein (e.g., the oligonucleotides listed in Table 9) (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary). In some embodiments, such a target sequence is 100% complementary to the oligonucleotides listed in Table 8. In some embodiments, such a target sequence is 100% complementary to the oligonucleotides listed in Table 9. In some embodiments, the oligonucleotide is complementary to the target sequence of any one of the oligonucleotides provided herein (e.g., an oligonucleotide comprising any one of SEQ ID NOs: 236 to 266) (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary). In some embodiments, the oligonucleotide is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary) to the target sequence of any one of the oligonucleotides provided herein (e.g., an oligonucleotide comprising any one of SEQ ID NOs: 248, 251 to 253, and 262). In some embodiments, such a target sequence is 100% complementary to an oligonucleotide described herein (e.g., an oligonucleotide comprising any one of SEQ ID NOs: 236 to 266). In some embodiments, such a target sequence is 100% complementary to an oligonucleotide described herein (e.g., an oligonucleotide comprising any one of SEQ ID NOs: 248, 251 to 253, and 262).

[0315] 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 a thymine nucleoside.

[0316] In some embodiments, one or more thymine bases (T) in any of the oligonucleotides provided herein may independently and optionally be uracil bases (U), and / or any one or more U may independently and optionally be T. In some embodiments, one or more thymine bases (T) in any of the oligonucleotides listed in Table 8 may independently and optionally be uracil bases (U), and / or any one or more U may independently and optionally be T. In some embodiments, one or more thymine bases (T) in any of the oligonucleotides listed in Table 9 may independently and optionally be uracil bases (U), and / or any one or more U may independently and optionally be T.

[0317] b. Oligonucleotide modification:

[0318] The oligonucleotides described herein may be modified, for example, to include a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide or nucleoside, and / or (e.g., and) a combination thereof. In addition, in some embodiments, the oligonucleotide may exhibit one or more of the following properties: not mediating alternative splicing; not immunostimulatory; having nuclease resistance; having increased cellular uptake compared to unmodified oligonucleotides; being nontoxic to cells or mammals; increasing internal expulsion of endosomes in cells; minimizing TLR stimulation; or avoiding pattern recognition receptors. Any modified chemical composition (chemistry) or form of the oligonucleotides described herein may be combined with each other. For example, one, two, three, four, five or more different types of modifications may be included in the same oligonucleotide.

[0319] In some embodiments, certain nucleotide or nucleoside modifications may be used that render the oligonucleotides into which they are incorporated more resistant to nuclease digestion than native oligodeoxynucleotide or oligoribonucleotide molecules; these modified oligonucleotides survive intact longer than unmodified oligonucleotides. Some specific examples of modified oligonucleotides include those containing modified backbones, such as modified internucleoside linkages, such as phosphorothioate linkages, phosphotriester linkages, methylphosphonate linkages, short-chain alkyl linkages or cycloalkyl sugar inter-linkages or short-chain heteroatom linkages or heterocyclic sugar inter-linkages. Thus, the oligonucleotides of the present disclosure may be stabilized to resist nucleolytic degradation, for example, by incorporating modifications such as nucleotide or nucleoside modifications.

[0320] In some embodiments, the length of the oligonucleotide can be as many as 50 or as many as 100 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30, 2 to 40, 2 to 45 or more nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The length of the oligonucleotide can be 8 to 30 nucleotides, wherein 2 to 10, 2 to 15, 2 to 16, 2 to 17, 2 to 18, 2 to 19, 2 to 20, 2 to 25, 2 to 30 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. The length of the oligonucleotide can be 8 to 15 nucleotides, wherein 2 to 4, 2 to 5, 2 to 6, 2 to 7, 2 to 8, 2 to 9, 2 to 10, 2 to 11, 2 to 12, 2 to 13, 2 to 14 nucleotides or nucleosides of the oligonucleotide are modified nucleotides / nucleosides. Optionally, the oligonucleotide can have every nucleotide or nucleoside except 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 modified nucleotides / nucleosides. Oligonucleotide modifications are further described herein.

[0321] c. Modified Nucleosides

[0322] In some embodiments, the oligonucleotides described herein comprise at least one nucleoside modified at the 2' position of the sugar. In some embodiments, the oligonucleotide comprises at least one 2'-modified nucleoside. In some embodiments, all nucleosides in the oligonucleotide are 2'-modified nucleosides.

[0323] In some embodiments, the oligonucleotides described herein comprise one or more non-bicyclic 2'-modified nucleosides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA) modified nucleosides.

[0324] In some embodiments, the oligonucleotides described herein comprise one or more 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the oligonucleotides described herein comprise at least two 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the oligonucleotides described herein comprise at least four 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the oligonucleotides described herein comprise at least six 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the oligonucleotides described herein comprise one or more 2'-O-methyl (2'-O-Me) modified nucleosides.

[0325] In some embodiments, the oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleosides, wherein the ribose ring comprises a bridging portion connecting two atoms in the ring, for example, the 2'-O atom is connected to the 4'-C atom by a methylene (LNA) bridge, an ethylene (ENA) bridge, or a (S)-constrained ethyl (cEt) bridge. Some examples of LNAs are described in International Patent Application Publication WO / 2008 / 043753, which was published on April 17, 2008, and is entitled "RNA Antagonist Compounds For The Modulation Of PCSK9", the contents of which are incorporated herein by reference in their entirety. Some examples of ENAs are provided in International Patent Publication No. WO 2005 / 042777, published on May 12, 2005, and entitled "APP / ENA Antisense"; Morita et al., Nucleic Acid Res., Suppl 1:241-242, 2001; Surono et al., Hum. Gene Ther., 15:749-757, 2004; Koizumi, Curr. Opin. Mol. Ther., 8:144-149, 2006, and Horie et al., Nucleic Acids Symp. Ser (Oxf), 49:171-172, 2005; the disclosures of which are incorporated herein by reference in their entirety. Some examples of cEt are provided in: US Patents 7,101,993, 7,399,845, and 7,569,686, each of which is incorporated herein by reference in its entirety.

[0326] In some embodiments, the oligonucleotide comprises a modified nucleoside disclosed in one of the following U.S. patents or patent application publications: U.S. Patent 7,399,845, issued on July 15, 2008, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,741,457, issued on June 22, 2010, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 8,022,193, issued on September 20, 2011, and entitled “6-Modified Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,569,686, issued on August 4, 2009, and entitled “Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs”; U.S. Patent 7,335,765, issued on February 26, 2008, and entitled “Novel Nucleoside And Oligonucleotide Analogues”; U.S. Patent 7,314,923, issued on January 1, 2008, and entitled “Novel Nucleoside And Oligonucleotide Analogues”; U.S. Patent 7,816,333, issued on October 19, 2010, and entitled “Oligonucleotide Analogues And Methods Utilizing The Same” and U.S. Publication No. 2011 / 0009471, now U.S. Patent 8,957,201, issued on February 17, 2015, and entitled “Oligonucleotide Analogues And Methods Utilizing The Same”, the entire contents of each of which are incorporated herein by reference for all purposes.

[0327] In some embodiments, the oligonucleotide comprises at least one modified nucleoside that results in an increase in Tm of the oligonucleotide by 1° C., 2° C., 3° ​​C., 4° C., or 5° C. compared to an oligonucleotide that does not have at least one modified nucleoside. An oligonucleotide may have multiple modified nucleosides that result in an overall increase in Tm of the oligonucleotide by 2° C., 3° ​​C., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 15° C., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., or more compared to an oligonucleotide that does not have a modified nucleoside.

[0328] Oligonucleotides may include a mixture of different types of nucleosides. For example, oligonucleotides may include a mixture of 2'-deoxyribonucleosides or ribonucleosides and 2'-fluorine modified nucleosides. Oligonucleotides may include a mixture of deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. Oligonucleotides may include a mixture of 2'-fluorine modified nucleosides and 2'-O-methyl modified nucleosides. Oligonucleotides may include a mixture of bridged nucleosides and 2'-fluorine or 2'-O-methyl modified nucleosides. Oligonucleotides may include a mixture of non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). Oligonucleotides may include a mixture of 2'-fluorine modified nucleosides and 2'-O-Me modified nucleosides. Oligonucleotides may include a mixture of 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluorine or 2'-O-Me modified nucleosides. The oligonucleotide may comprise a mixture of non-bicyclic 2'-modified nucleosides (eg, 2'-MOE, 2'-Fluoro, or 2'-0-Me) and 2'-4' bicyclic nucleosides (eg, LNA, ENA, cEt).

[0329] Oligonucleotides may include different types of alternative nucleosides. For example, oligonucleotides may include alternative 2'-deoxyribonucleosides or ribonucleosides and 2'-fluorine modified nucleosides. Oligonucleotides may include alternative deoxyribonucleosides or ribonucleosides and 2'-O-Me modified nucleosides. Oligonucleotides may include alternative 2'-fluorine modified nucleosides and 2'-O-Me modified nucleosides. Oligonucleotides may include alternative bridging nucleosides and 2'-fluorine or 2'-O-methyl modified nucleosides. Oligonucleotides may include alternative non-bicyclic 2'-modified nucleosides (e.g., 2'-O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). Oligonucleotides may include alternative 2'-4' bicyclic nucleosides and 2'-MOE, 2'-fluorine or 2'-O-Me modified nucleosides. Oligonucleotides may comprise alternative non-bicyclic 2'-modified nucleosides (eg, 2'-MOE, 2'-Fluoro, or 2'-0-Me) and 2'-4' bicyclic nucleosides (eg, LNA, ENA, cEt).

[0330] In some embodiments, the oligonucleotides described herein comprise a 5'-vinylphosphonate modification, one or more abasic residues, and / or one or more inverted abasic residues. In some embodiments, the oligonucleotides described herein are siRNA oligonucleotides comprising a sense strand and an antisense strand, wherein the antisense strand or the sense strand comprises a 5'-vinylphosphonate (e.g., a 5'-(E)-vinylphosphonate modification). In some embodiments, the oligonucleotides described herein are siRNA oligonucleotides comprising a sense strand and an antisense strand, wherein the antisense strand comprises a 5'-vinylphosphonate (e.g., a 5'-(E)-vinylphosphonate) modification).

[0331] d. Internucleoside Linkages / Backbone

[0332] In some embodiments, the oligonucleotide may include a phosphorothioate linkage or other modified internucleoside linkage. In some embodiments, the oligonucleotide includes a phosphorothioate internucleoside linkage. In some embodiments, the oligonucleotide includes a phosphorothioate internucleoside linkage between at least two nucleosides. In some embodiments, the oligonucleotide includes a phosphorothioate internucleoside linkage between all nucleosides. For example, in some embodiments, the oligonucleotide includes a modified internucleoside linkage at the first, second and / or (e.g., and) third internucleoside linkage at the 5' or 3' end of the nucleotide sequence.

[0333] Phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates and other alkylphosphonates including 3' alkylenephosphonates, as well as chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thiophosphoramidates, thioalkylphosphonates, thioalkylphosphotriesters and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with opposite polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat. No.

[0334] 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405 ,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0335] In some embodiments, the oligonucleotide may have a heteroatom backbone, such as a methylene (methylimino) or MMI backbone; an amide backbone (see De Mesmaeker et al. Ace. Chem. Res. 1995, 28: 366-374); a morpholino backbone (see Summerton and Weller, U.S. Pat. No. 5,034,506); or a peptide nucleic acid (PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, and the nucleotides are directly or indirectly bound to the aza nitrogen atoms of the polyamide backbone, see Nielsen et al., Science 1991, 254, 1497).

[0336] e. Stereospecific oligonucleotides

[0337] In some embodiments, the internucleotide phosphorus atom of the oligonucleotide is chiral, and the properties of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atom. In some embodiments, P-chiral oligonucleotide analogs can be synthesized in a stereocontrolled manner using appropriate methods (e.g., as described in Oka N, Wada T. Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorusatoms. Chem Soc Rev. 2011 Dec; 40 (12): 5829-43). In some embodiments, oligonucleotides containing phosphorothioate are provided, which include nucleoside units linked together by substantially all Sp or substantially all Rp phosphorothioate sugar interlinkages. In some embodiments, such phosphorothioate oligonucleotides having substantially chiral pure sugar interlinkages are prepared by enzymatic or chemical synthesis, as described, for example, in U.S. Pat. No. 5,587,261, issued on December 12, 1996, the contents of which are incorporated herein by reference in their entirety. In some embodiments, chiral controlled oligonucleotides provide a selective cleavage pattern of a target nucleic acid. For example, in some embodiments, chirality-controlled oligonucleotides provide single-site cleavage within the complementary sequence of a nucleic acid, as described, for example, in U.S. Patent Application Publication No. 20170037399 A1, published on February 2, 2017, entitled “CHIRAL DESIGN,” the contents of which are incorporated herein by reference in their entirety.

[0338] f. Morpholino

[0339] In some embodiments, the oligonucleotide can be a morpholino-based compound. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R. Corey, Biochemistry, 2002, 41 (14), 4503-4510); Genesis, Vol. 30, No. 3, 2001; Heasman, J., Dev. Biol., 2002, 243, 209-214; Nasevicius et al., Nat. Genet., 2000, 26, 216-220; Lacerra et al., Proc. Natl. Acad. Sci., 2000, 97, 9591-9596; and U.S. Pat. No. 5,034,506, issued July 23, 1991. In some embodiments, the morpholino-based oligomeric compound is a phosphorodiamidate morpholino oligomer (PMO) (e.g., as described in Iverson, Curr. Opin. Mol. Ther., 3:235-238, 2001; and Wang et al., J. Gene Med., 12:354-364, 2010; the disclosures of which are incorporated herein by reference in their entireties).

[0340] h. Gap polymer

[0341] In some embodiments, the oligonucleotides described herein are spaced polymers. Spaced polymer oligonucleotides generally have a formula 5'-XYZ-3', wherein X and Z are used as flanking regions around spacer Y. In some embodiments, the flanking region X of formula 5'-XYZ-3' is also referred to as X region, flanking sequence X, 5' wing region X, or 5' wing segment. In some embodiments, the flanking region Z of formula 5'-XYZ-3' is also referred to as Z region, flanking sequence Z, 3' wing region Z, or 3' wing segment. In some embodiments, the spacer Y of formula 5'-XYZ-3' is also referred to as Y region, Y segment, or spacer segment Y. In some embodiments, each nucleoside in spacer Y is a 2'-deoxyribonucleoside, and 5' wing region X or 3' wing region Z do not contain any 2'-deoxyribonucleoside.

[0342] In some embodiments, the Y region is a continuous extension of nucleotides, for example, a region of 6 or more DNA nucleotides, which can recruit RNA enzymes (e.g., RNA enzyme H). In some embodiments, the spacer binds to the target nucleic acid, at which point the RNA enzyme is recruited and can subsequently cut the target nucleic acid. In some embodiments, the flanks of the Y region 5' and 3' are X and Z regions comprising high-affinity modified nucleosides, for example, 1 to 6 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 sequences X and Z can be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides. The flanking sequences X and Z can have similar lengths or different lengths. In some embodiments, the spacer segment Y can be a nucleotide sequence of 5 to 20 nucleotides, 5 to 15 nucleotides, or 6 to 10 nucleotides in length.

[0343] In some embodiments, in addition to DNA nucleotides, the spacer of the spacer oligonucleotide may include modified nucleotides known to be acceptable for efficient RNase H action, such as C4'-substituted nucleotides, non-cyclic nucleotides and arabinose (arabino) configuration nucleotides. In some embodiments, the spacer comprises one or more unmodified internucleoside linkages. In some embodiments, one or two flanking regions each independently comprise one or more thiophosphate internucleoside linkages (e.g., thiophosphate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides. In some embodiments, the spacer and two flanking regions each independently comprise modified internucleoside linkages (e.g., thiophosphate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five or more nucleotides.

[0344] Suitable methods can be used to generate gap polymers. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of gap polymers include, but are not limited to:

[0345] U.S. Patent Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; 5,700,922; 5,898,031; 7,015,3 15; 7,101,993; 7,399,845; 7,432,250; 7,569,686; 7,683,036; 7,750,131; 8,580,756; 9,045,754; 9,428,534; 9,695,418; 10,017,764; 10,260,069; 9,428,534; 8,580,756;

[0346] U.S. Patent Publication Nos. US20050074801, US20090221685, US20090286969, US20100197762, and US20110112170; PCT Publication Nos. WO2004069991, WO2005023825, WO2008049085, and WO2009090182; and EP Patent No. EP 2,149,605, each of which is incorporated herein by reference in its entirety.

[0347] In some embodiments, the length of the gap polymer is 10 to 40 nucleosides. For example, the length of the gap polymer can be 10 to 40, 10 to 35, 10 to 30, 10 to 25, 10 to 20, 10 to 15, 15 to 40, 15 to 35, 15 to 30, 15 to 25, 15 to 20, 20 to 40, 20 to 35, 20 to 30, 20 to 25, 25 to 40, 25 to 35, 25 to 30, 30 to 40, 30 to 35, or 35 to 40 nucleosides. In some embodiments, the gapmer is 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleosides in length.

[0348] 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.

[0349] In some embodiments, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) are independently 1 to 20 nucleosides in length. For example, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) can independently be 1 to 20, 1 to 15, 1 to 10, 1 to 7, 1 to 5, 1 to 3, 1 to 2, 2 to 5, 2 to 7, 3 to 5, 3 to 7, 5 to 20, 5 to 15, 5 to 10, 10 to 20, 10 to 15, or 15 to 20 nucleosides in length. In some embodiments, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) 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 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) are the same length. In some embodiments, the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) are different lengths. In some embodiments, the 5' wing region (X in the 5'-XYZ-3' formula) of the gapmer is longer than the 3' wing region (Z in the 5'-XYZ-3' formula) of the gapmer. In some embodiments, the 5' wing region (X in the 5'-XYZ-3' formula) of the gapmer is shorter than the 3' wing region (Z in the 5'-XYZ-3' formula) of the gapmer.

[0350] In some embodiments, the gapmer comprises the following 5'-XYZ-3':

[0351] 5-10-5，4-12-4，3-14-3，2-16-2，1-18-1，3-10-3，2-10-2，1-10-1，2-8-2，4-6-4，3-6-3，2-6-2，4-7-4，3-7-3，2-7-2，4-8-4，3-8-3，2-8-2，1-8-1，2-9-2，1-9-1，2-10-2，1-10-1，1-12-1，1-16-1，2-15-1，1-15-2，1-14-3，3-14-1，2-14-2，1-13-4，4-13-1，2-13-3，3-13-2，1-12-5，5-12-1，2-12-4，4-12-2，3-12-3，1-11-6，6-11-1，2-11-5，5-11-2，3-11-4，4-11-3，1-17-1，2-16-1，1-16-2，1-15-3，3-15-1，2-15-2，1-14-4，4-14-1，2-14-3，3-14-2，1-13-5，5-13-1，2-13-4，4-13-2，3-13-3，1-12-6，6-12-1，2-12-5，5-12-2，3-12-4，4-12-3，1-11-7，7-11-1，2-11-6，6-11-2，3-11-5，5-11-3，4-11-4，1-18-1，1-17-2，2-17-1，1-16-3，1-16-3，2-16-2，1-15-4，4-15-1，2-15-3，3-15-2，1-14-5，5-14-1，2-14-4，4-14-2，3-14-3，1-13-6，6-13-1，2-13-5，5-13-2，3-13-4，4-13-3，1-12-7，7-12-1，2-12-6，6-12-2，3-12-5，5-12-3，1-11-8，8-11-1，2-11-7，7-11-2，3-11-6，6-11-3，4-11-5，5-11-4，1-18-1，1-17-2，2-17-1，1-16-3，3-16-1，2-16-2，1-15-4，4-15-1，2-15-3，3-15-2，1-14-5，2-14-4，4-14-2，3-14-3，1-13-6，6-13-1，2-13-5，5-13-2，3-13-4，4-13-3，1-12-7，7-12-1，2-12-6，6-12-2，3-12-5，5-12-3，1-11-8，8-11-1，2-11-7，7-11-2，3-11-6，6-11-3，4-11-5，5-11-4，1-19-1，1-18-2，2-18-1，1-17-3，3-17-1，2-17-2，1-16-4，4-16-1，2-16-3，3-16-2，1-15-5，2-15-4，4-15-2，3-15-3，1-14-6，6-14-1，2-14-5，5-14-2，3-14-4，4-14-3，1-13-7，7-13-1，2-13-6，6-13-2，3-13-5，5-13-3，4-13-4，1-12-8，8-12-1，2-12-7，7-12-2，3-12-6，6-12-3，4-12-5，5-12-4，2-11-8，8-11-2，3-11-7，7-11-3，4-11-6，6-1 1-4，5-11-5，1-20-1，1-19-2，2-19-1，1-18-3，3-18-1，2-18-2，1-17-4，4-17-1，2-17-3，3-17-2，1-16-5，2-16-4，4-16-2，3-16-3，1-15-6，6-15-1，2-15-5，5-15-2，3-15-4，4-15-3，1-14-7，7-14-1，2-14-6，6-14-2，3-14-5，5-14-3，4-14-4，1-13-8，8-13-1，2-13-7，7-13-2，3-13-6，6-13-3，4-13-5，5-13-4，2-12-8，8-12-2，3-12-7，7-12-3，4-12-6，6-12-4，5-12-5，3-11-8，8-11-3，4-11-7，7-11-4，5-11-6，6-11-5，1-21-1，1-20-2，2-20-1，1-20-3，3-19-1，2-19-2，1-18-4，4-18-1，2-18-3，3-18-2，1-17-5，2-17-4，4-17-2，3-17-3，1-16-6，6-16-1，2-16-5，5-16-2，3-16-4，4-16-3，1-15-7，7-15-1，2-15-6，6-15-2，3-15-5，5-15-3，4-15-4，1-14-8，8-14-1，2-14-7，7-14-2，3-14-6，6-14-3，4-14-5，5-14-4，2-13-8，8-13-2，3-13-7，7-13-3，4-13-6，6-13-4，5-13-5，1-12-10，10-12-1，2-12-9，9-12-2，3-12-8，8-12-3，4-12-7，7-12-4，5-12-6，6-12-5，4-1 1-8，8-1 1-4，5-11-7, 7-11-5, 6-1 1-6, 1-22-1, 1-21-2, 2-21-1, 1-21-3, 3-20-1, 2-20-2, 1-19-4, 4-19-1, 2-19-3, 3-19-2, 1-18-5, 2-18-4, 4-18-2, 3-18-3, 1-17-6, 6-17-1, 2-17-5, 5-17-2, 3-17-4, 4-17-3, 1-16-7, 7-16-1, 2-16-6, 6-16-2, 3-16-5, 5-16-3, 4-16-4, 1-15-8, 8- 15-1, 2-15-7, 7-15-2, 3-15-6, 6-15-3, 4-15-5, 5-15-4, 2-14-8, 8-14-2, 3-14-7, 7-14-3, 4-14-6, 6-14-4, 5-14-5, 3-13-8, 8-13-3, 4-13-7, 7-13-4, 5-13-6, 6-13-5, 4-12-8, 8-12-4, 5-12-7, 7-12-5, 6-12-6, 5-11-8, 8-11-5, 6-11-7, or 7-11-6,

[0352] The numbers indicate the number of nucleosides in the X, Y, and Z regions of the 5'-XYZ-3' gapmer.

[0353] In some embodiments, one or more nucleosides in the 5' wing of the gapmer (X in the 5'-XYZ-3' formula) or the 3' wing of the gapmer (Z in the 5'-XYZ-3' formula) are modified nucleosides (e.g., high affinity modified nucleosides). In some embodiments, modified nucleosides (e.g., high affinity modified nucleosides) are 2'-modified nucleosides. In some embodiments, 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA)).

[0354] In some embodiments, one or more nucleosides in the 5' wing region (X in the 5'-XYZ-3' formula) of the gap polymer are high affinity modified nucleosides. In some embodiments, each nucleoside in the 5' wing region (X in the 5'-XYZ-3' formula) of the gap polymer is a high affinity modified nucleoside. In some embodiments, one or more nucleosides in the 3' wing region (Z in the 5'-XYZ-3' formula) of the gap polymer are high affinity modified nucleosides. In some embodiments, each nucleoside in the 3' wing region (Z in the 5'-XYZ-3' formula) of the gap polymer is a high affinity modified nucleoside. In some embodiments, one or more nucleosides in the 5' wing region (X in the 5'-XYZ-3' formula) of the gap polymer are high affinity modified nucleosides, and one or more nucleosides in the 3' wing region (Z in the 5'-XYZ-3' formula) of the gap polymer are high affinity modified nucleosides. In some embodiments, every nucleoside in the 5' wing (X in the 5'-XYZ-3' formula) of the gapmer is a high affinity modified nucleoside, and every nucleoside in the 3' wing (Z in the 5'-XYZ-3' formula) of the gapmer is a high affinity modified nucleoside.

[0355] In some embodiments, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) comprises the same high affinity nucleoside as the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula). For example, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) can comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me). In another example, the 5' wing region of the gapmer (X in the 5'-XYZ-3' formula) and the 3' wing region of the gapmer (Z in the 5'-XYZ-3' formula) can comprise one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, each nucleoside in the 5'-wing (X in the 5'-XYZ-3' formula) of the gapmer and the 3'-wing (Z in the 5'-XYZ-3' formula) of the gapmer is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me). In some embodiments, each nucleoside in the 5'-wing (X in the 5'-XYZ-3' formula) of the gapmer and the 3'-wing (Z in the 5'-XYZ-3' formula) of the gapmer is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt).

[0356] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 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 each nucleoside of X and Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 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 each nucleoside of X and Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside of Y is a 2'-deoxyribonucleoside. In some embodiments, the 5'-wing region (X in the 5'-XYZ-3' formula) of the gapmer comprises a different high affinity nucleoside than the 3'-wing region (Z in the 5'-XYZ-3' formula) of the gapmer. For example, the 5' wing region (X in the 5'-XYZ-3' formula) of the gapmer may include one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 3' wing region (Z in the 5'-XYZ-3' formula) of the gapmer may include one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In another example, the 3' wing region (Z in the 5'-XYZ-3' formula) of the gapmer may include one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me), and the 5' wing region (X in the 5'-XYZ-3' formula) of the gapmer may include one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0357] In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 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 each nucleoside in X is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), each nucleoside in Z is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), and each nucleoside in Y is a 2'-deoxyribonucleoside. In some embodiments, the gapmer comprises a 5'-XYZ-3' configuration, wherein X and Z are independently 1 to 7 (e.g., 1, 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 each nucleoside in X is a 2'-4' bicyclic nucleoside (e.g., LNA or cEt), each nucleoside in Z is a non-bicyclic 2'-modified nucleoside (e.g., 2'-MOE or 2'-O-Me), and each nucleoside in Y is a 2'-deoxyribonucleoside.

[0358] In some embodiments, the 5'-wing region (X in the 5'-XYZ-3' formula) of the gapmer comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, the 3'-wing region (Z in the 5'-XYZ-3' formula) of the gapmer comprises one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt). In some embodiments, both the 5'-wing of the gapmer (X in the 5'-XYZ-3' formula) and the 3'-wing of the gapmer (Z in the 5'-XYZ-3' formula) comprise one or more non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and one or more 2'-4' bicyclic nucleosides (e.g., LNA or cEt).

[0359] In some embodiments, the gapmer 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 being 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, the gapmer 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, the gapmer 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 in X and at least one but not all (e.g., 1, 2, 3, 4, 5, or 6) of positions 1, 2, 3, 4, 5, 6, or 7 (the 5'-most position is position 1) in 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.

[0360] Some non-limiting examples of gapmer configurations having a mixture of non-bicyclic 2'-modified nucleosides (e.g., 2'-MOE or 2'-O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA or cEt) in the 5'-wing (X in the 5'-XYZ-3' formula) of the gapmer and / or the 3'-wing (Z in the 5'-XYZ-3' formula) of the gapmer include:

[0361] BBB-(D)n-BBBAA;KKK-(D)n-KKKAA;LLL-(D)n-LLLAA;BBB-(D)n-BBBEE;KK K-(D)n-KKKEE;LLL-(D)n-LLLEE;BBB-(D)n-BBBAA;KKK-(D)n-KKKAA;LLL- (D)n-LLLAA;BBB-(D)n-BBBEE;KKK-(D)n-KKKEE;LLL-(D)n-LLLEE;BBB-(D )n-BBBAAA;KKK-(D)n-KKKAAA;LLL-(D)n-LLLAAA;BBB-(D)n-BBBEEE;KKK- (D)n-KKKEEE;LLL-(D)n-LLLEEE;BBB-(D)n-BBBAAA;KKK-(D)n-KKKAAA;LLL-(D)n-LLLAAA;BBB-(D)n-BBBEEE;KKK-(D)n-KKKEE-LLLEE-LEED); FATHER-(D)n-FATHER;BROTHER-(D)n-BROTHER;LALA-(D)n-ALAL;BEBE-(D)n-EBEB;KEKE-(D)n-EKEK;LELE-(D)n-ELEL;BABA-(D)n-ABAB;UNBROTHER-(D)n-ALAK; -(D)n-ALAL;BEBE-(D)n-EBEB;CAKE-(D)n-EKEK;LELE-(D)n-ELEL;ABAB-(D)n-ABAB;AKAK-(D)n-AKAK;ALAL-(D)n-ALAL;EBEB-(D)n-EKEB-(D)EB-(D) n-EKEK;ELEL-(D)n-ELEL;ABAB-(D)n-ABAB;AKAK-(D)n-AKAC;ALAL-(D)n-ALAL;EBEB-(D)n-EBEB;EKEK-(D)n-EKEK;ELEL-(D)n-ELEL;AABB-D) AA;BBAA-(D)n-AABB;AAKK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;AABB-(D)n-BBAAK-AKAK-Ad) ALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;BBB -(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-B BE;KKK-(D)n-KKE;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D) n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;ABBB-(D)n-BBBA;AK KK-(D)n-KKKA;ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK-(D)n-KKKE;ELLL -(D)n-LLLE;ABBB-(D)n-BBBA;AKKK-(D)n-KKKA;ALLL-(D)n-LLLA;EBBB-(D)n-BBBE;EKKK-(D)n-KKKE;ELLL-(D)n-LLLE;ABBB-(D)n-AKK-A )n-KKKAA;ALLL-(D)n-LLLAA;EBBB-(D)n-BBBEE;EKKK-(D)n-KKKEE;ELLL-(D)n-LLLEE;ABBB-(D)n-BBBAA;AKKK-(D)n-KKKAA;ALLL-LLLAA(ABBLA) -(D)n-BBBEE;EKKK-(D)n-KKKEE;ELLL-(D)n-LLLEE;AABBB-(D)n-BBB;AAK KK-(D)n-KKK;AALLL-(D)n-LLL;EEBBB-(D)n-BBB;EEKKK-(D)n-KKK;EELLL -(D)n-LLL;AABBB-(D)n-BBB;AACCK-(D)n-KKK;AALLL-(D)n-LLL;EEBBB-( D)n-BBB;EEKKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBBA;AAKKK-(D )n-KKKA;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE;AABBB-(D)n-BBBA;AAKKK-(D)n-KKKA;ALLLL-(D)LL-LLA) B-(D)n-BBBE?AKKAAKK-(D)n-BBB ;ALLALLL-(D)n-LLL;EBBEBB-(D)n-BBB;EKKEKK-(D)n-KKK;ELLELL-(D)n-LLL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALL-(D)n-LLL;EBBEBB-(D)n-BBB;EKKEKK-(D)n-KK K; ELLELL-(D)n-LLL; EEEK-(D)n-EEEEEEEE; EEK-(D)n-EEEEEEEE; EK-(D)n-EEEEEEEEEE; EK-(D)n-EEEKK; K-(D)n-EEEKEKE; K-(D)n-EEEKEKEE; K-(D)n-EEKEK; EEKEKE;EK-(D)n-EEEKEK;EEK-(D)n-KEEKE;EK-(D)n-EEKEK;EK-(D)n-KEEK;EEK-(D)n -EEEKEK; EK-(D)n-KEEEKEE; EK-(D)n-EEKEKE; EK-(D)n-EEEKEKE; and EK-(D)n-EEEEKEK. ;

[0362] "A" nucleotides include 2'-modified nucleosides; "B" represents 2'-4' bicyclic nucleosides; "K" represents constrained ethyl nucleosides (cEt); "L" represents LNA nucleosides; and "E" represents 2'-MOE modified ribonucleosides; "D" represents 2'-deoxyribonucleosides; "n" represents the length of the spacer segment (Y in the 5'-XYZ-3' configuration) and is an integer from 1 to 20.

[0363] In some embodiments, any of the internucleoside linkages described herein comprise one or more modified nucleoside linkages (e.g., phosphorothioate linkages) in each of the X, Y, and Z regions. In some embodiments, each internucleoside linkage in any of the internucleoside linkages described herein is a phosphorothioate linkage. In some embodiments, each of the X, Y, and Z regions independently comprises a mixture of phosphorothioate linkages and phosphodiester linkages. In some embodiments, each internucleoside linkage in the spacer Y is a phosphorothioate linkage, the 5' wing region X comprises a mixture of phosphorothioate linkages and phosphodiester linkages, and the 3' wing region Z comprises a mixture of phosphorothioate linkages and phosphodiester linkages.

[0364] i. RNA interference (RNAi)

[0365] In some embodiments, the oligonucleotide provided herein is a small interfering RNA (siRNA), which is also referred to as short interfering RNA or silencing RNA. siRNA is a class of double-stranded RNA molecules, usually about 20 to 25 base pairs in length, which is used for degradation by RNA interference (RNAi) pathway targeting nucleic acid (e.g., mRNA) in cells. The specificity of siRNA molecules can be determined by the combination of the antisense strand of the molecule with its target RNA. The length of effective siRNA molecules is usually less than 30 to 35 base pairs to prevent non-specific RNA interference pathways in cells triggered by interferon responses, although longer siRNAs may also be effective. In some embodiments, the length of siRNA molecules is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more base pairs. In some embodiments, the siRNA molecules are 8 to 30 base pairs in length, 10 to 15 base pairs in length, 10 to 20 base pairs in length, 15 to 25 base pairs in length, 19 to 21 base pairs in length, 21 to 23 base pairs in length. In some embodiments, the siRNA molecules are 8 to 32 base pairs in length, 8 to 29 base pairs in length, 8 to 27 base pairs in length, 15 to 32 base pairs in length, 15 to 29 base pairs in length, 15 to 27 base pairs in length, 21 to 31 base pairs in length, 21 to 29 base pairs in length, 21 to 27 base pairs in length, 21 to 23 base pairs in length, 23 to 32 base pairs in length, 23 to 29 base pairs in length, or 23 to 27 base pairs in length.

[0366] After selecting an appropriate target RNA sequence, an appropriate method can be used to design and prepare siRNA molecules comprising a nucleotide sequence complementary to all or part of the target sequence (ie, an antisense sequence) (see, e.g., PCT Publication No. WO 2004 / 016735; and U.S. Patent Publication Nos. 2004 / 0077574 and 2008 / 0081791).

[0367] The siRNA molecule can be double-stranded (i.e., a dsRNA molecule comprising an antisense strand and a complementary sense strand) or single-stranded (i.e., an ssRNA molecule comprising only an antisense strand). The siRNA molecule can comprise a duplex, an asymmetric duplex, a hairpin, or an asymmetric hairpin secondary structure having a self-complementary sense strand and an antisense strand. In some embodiments, the oligonucleotide described herein is a siRNA comprising an antisense strand and a sense strand.

[0368] In some embodiments, the antisense strand of the siRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In some embodiments, the antisense strand is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 19 to 21 nucleotides in length, 21 to 23 nucleotides in length. In some embodiments, the antisense strand is 8 to 32 nucleotides in length, 8 to 29 nucleotides in length, 8 to 27 nucleotides in length, 15 to 32 nucleotides in length, 15 to 29 nucleotides in length, 15 to 27 nucleotides in length, 21 to 31 nucleotides in length, 21 to 29 nucleotides in length, 21 to 27 nucleotides in length, 21 to 23 nucleotides in length, 23 to 32 nucleotides in length, 23 to 29 nucleotides in length, or 23 to 27 nucleotides in length.

[0369] In some embodiments, the sense strand of the siRNA molecule is 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50 or more nucleotides in length. In some embodiments, the sense strand is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 30 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 19 to 21 nucleotides in length, 21 to 23 nucleotides in length. In some embodiments, the sense strand is 8 to 32 nucleotides in length, 8 to 29 nucleotides in length, 8 to 27 nucleotides in length, 15 to 32 nucleotides in length, 15 to 29 nucleotides in length, 15 to 27 nucleotides in length, 21 to 31 nucleotides in length, 21 to 29 nucleotides in length, 21 to 27 nucleotides in length, 21 to 23 nucleotides in length, 23 to 32 nucleotides in length, 23 to 29 nucleotides in length, or 23 to 27 nucleotides in length.

[0370] In some embodiments, the siRNA molecule comprises an antisense strand comprising a complementary region to a target region in DUX4 mRNA. In some embodiments, the complementary region is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% complementary to the target region in DUX4 mRNA. In some embodiments, the target region is a region of contiguous nucleotides in DUX4 mRNA. In some embodiments, the complementary nucleotide sequence does not need to be 100% complementary to the nucleotide sequence of its target to be specifically hybridizable with the target RNA sequence or to be specific to the target RNA sequence.

[0371] In some embodiments, the siRNA molecule comprises an antisense strand comprising a complementary region to a DUX4 mRNA sequence, and the complementary region is 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, or 5 to 40 nucleotides in length. In some embodiments, the complementary region is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 67, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides in length. In some embodiments, the complementary region is complementary to 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 21, at least 22, at least 23, at least 24, at least 25 or more consecutive nucleotides of the DUX4 mRNA sequence. In some embodiments, the complementary region comprises a nucleotide sequence containing no more than 1, 2, 3, 4, or 5 base mismatches compared to the complementary portion of the DUX4 mRNA sequence. In some embodiments, the complementary region comprises a nucleotide sequence having at most 3 mismatches in 15 bases or at most 2 mismatches in 10 bases.

[0372] In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary) to a target RNA sequence as set forth in any one of SEQ ID NOs: 174 to 235. In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is complementary (e.g., at least 85%, at least 90%, at least 95%, or 100% complementary) to a target RNA sequence as set forth in any one of SEQ ID NOs: 186, 189 to 191, and 200. In some embodiments, the siRNA molecule comprises an antisense strand that is 18 to 25 nucleotides in length and comprises a complementary region of at least 15 nucleotides (e.g., at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, or at least 19 nucleotides) of a target RNA sequence as set forth in any one of SEQ ID NOs: 174 to 235. In some embodiments, the siRNA molecule comprises an antisense strand having a length of 18 to 25 nucleotides and comprises a complementary region of at least 15 nucleotides (e.g., at least 15 nucleotides, at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, or at least 19 nucleotides) of the target RNA sequence shown in any one of SEQ ID NOs: 186, 189 to 191, and 200.

[0373] In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to an oligonucleotide set forth in any one of SEQ ID NOs: 236 to 266. In some embodiments, the siRNA molecule comprises an antisense strand comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% identical to an oligonucleotide set forth in any one of SEQ ID NOs: 248, 251 to 253, and 262. In some embodiments, the siRNA molecule comprises an antisense strand that is 18 to 25 nucleotides in length and comprises 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, or at least 19 consecutive nucleotides of an oligonucleotide set forth in any one of SEQ ID NOs: 236 to 266. In some embodiments, the siRNA molecule comprises an antisense strand that is 18 to 25 nucleotides in length and comprises 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, or at least 19 consecutive nucleotides of the oligonucleotide set forth in any one of SEQ ID NOs: 248, 251 to 253, and 262.

[0374] Double-stranded siRNA can comprise sense and antisense RNA chains of the same or different lengths. Double-stranded siRNA molecules can also be assembled into stem-loop structures by single oligonucleotides, wherein the self-complementary sense and antisense regions of siRNA molecules are connected by the following: nucleic acid-based or non-nucleic acid-based joints, and circular single-stranded RNA, which has two or more loop structures and a stem comprising self-complementary sense strands and antisense strands, wherein circular RNA can be processed in vivo or in vitro to produce active siRNA molecules that can mediate RNAi. Therefore, small hairpin RNA (small hairpin RNA, shRNA) molecules are also considered herein. Except for the reverse complementary (sense) sequence separated by spacer or loop sequence, these molecules also comprise specific antisense sequences. The cutting of spacer or loop provides single-stranded RNA molecules and their reverse complements, so that they can be annealed to form dsRNA molecules (optionally, with the other processing steps that can cause the addition or removal of one, two, three or more nucleotides from the 3' end and / or (for example, and) 5' end of any one or two chains). The spacer may be of sufficient length to allow the antisense and sense sequences to anneal and form a double-stranded structure (or stem) prior to cleavage of the spacer (and optionally, subsequent processing steps that may result in the addition or removal of one, two, three, four or more nucleotides from the 3' end and / or (e.g., and) the 5' end of either or both strands). The spacer sequence may be an unrelated nucleotide sequence located between two complementary nucleotide sequence regions that, when annealed into a double-stranded nucleic acid, comprises the shRNA.

[0375] The total length of the siRNA molecule can vary from about 14 to about 100 nucleotides depending on the type of siRNA molecule being designed. Generally speaking, about 14 to about 50 of these nucleotides are complementary to the RNA target sequence, i.e., constitute the specific antisense sequence of the siRNA molecule. For example, when the siRNA is a double-stranded or single-stranded siRNA, the length can vary from about 14 to about 50 nucleotides, and when the siRNA is a shRNA or a circular molecule, the length can vary from about 40 nucleotides to about 100 nucleotides.

[0376] The siRNA molecule may include a 3' overhang at one end of the molecule. The other end may be a flat end or may also have an overhang (5' or 3'). When the siRNA molecule includes an overhang at both ends of the molecule, the length of the overhang may be the same or different. In one embodiment, the siRNA molecule of the present disclosure includes a 3' overhang of about 1 to about 3 (e.g., 1, 2, 3) nucleotides at both ends of the molecule. In some embodiments, the siRNA molecule includes a 3' overhang of about 1 to about 3 nucleotides on the sense strand. In some embodiments, the siRNA molecule includes a 3' overhang of about 1 to about 3 (e.g., 1, 2, 3) nucleotides on the antisense strand. In some embodiments, the siRNA molecule includes a 3' overhang of about 1 to about 3 (e.g., 1, 2, 3) nucleotides on both the sense strand and the antisense strand.

[0377] In some embodiments, the siRNA molecule comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the siRNA molecule comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleoside linkages. In some embodiments, the modified nucleotide is a modified sugar moiety (e.g., a 2' modified nucleotide). In some embodiments, the siRNA molecule comprises one or more 2' modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, each nucleotide of the siRNA molecule is a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the siRNA molecule comprises one or more 2'-O-methyl modified nucleotides. In some embodiments, the siRNA molecule comprises one or more 2'-F modified nucleotides. In some embodiments, the siRNA molecule comprises one or more 2'-O-methyl and 2'-F modified nucleotides. In some embodiments, the siRNA molecule comprises one or more modified nucleosides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the siRNA molecule comprises one or more modified nucleosides and / or (e.g., and) one or more modified internucleoside linkages. In some embodiments, the modified nucleoside is a modified sugar moiety (e.g., a 2' modified nucleoside). In some embodiments, the siRNA molecule comprises one or more 2' modified nucleosides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, each nucleoside of the siRNA molecule is a modified nucleotide (e.g., a 2'-modified nucleoside). In some embodiments, the siRNA molecule comprises one or more 2'-O-methyl modified nucleosides. In some embodiments, the siRNA molecule comprises one or more 2'-F modified nucleosides.In some embodiments, the siRNA molecule comprises one or more 2'-O-methyl and 2'-F modified nucleosides.

[0378] In some embodiments, the siRNA molecule comprises a phosphorothioate or other modified internucleotide linkage. In some embodiments, the siRNA molecule comprises a phosphorothioate or other modified internucleoside linkage. In some embodiments, the siRNA molecule comprises a phosphorothioate internucleoside linkage. In some embodiments, the siRNA molecule comprises a phosphorothioate internucleotide linkage. In some embodiments, the siRNA molecule comprises a phosphorothioate internucleoside linkage between at least two nucleotides. In some embodiments, the siRNA molecule comprises a phosphorothioate internucleoside linkage between at least two nucleosides. In some embodiments, the siRNA molecule comprises a phosphorothioate internucleotide linkage between at least two nucleotides. In some embodiments, the siRNA molecule comprises a phosphorothioate internucleoside linkage between all nucleotides. In some embodiments, the siRNA molecule comprises a phosphorothioate internucleoside linkage between all nucleosides. For example, in some embodiments, the first, second and / or (e.g., and) third internucleoside linkage of the siRNA molecule at the 5' or 3' end of the siRNA molecule comprises a modified internucleoside linkage. In some embodiments, the siRNA molecule comprises phosphorothioate internucleotide linkages between all nucleotides. For example, in some embodiments, the siRNA molecule comprises a modified internucleotide linkage at the first, second, and / or (e.g., and) third internucleotide linkage at the 5' or 3' terminus of the siRNA molecule. For example, in some embodiments, the siRNA molecule comprises a modified internucleotide linkage at the first, second, and / or (e.g., and) third internucleoside linkage at the 5' or 3' terminus of the siRNA molecule.

[0379] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, the modified internucleoside linkage is a phosphorus-containing linkage. In some embodiments, the phosphorus-containing linkage that can be used includes, but is not limited to: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkylphosphonates including 3'alkylenephosphonates, and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with opposite polarities where adjacent pairs of nucleoside units are linked at 3'-5' to 5'-3' or 2'-5' to 5'-2'; see

[0380] U.S. Patents No. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5, and 5,625,050.

[0381] Any modified chemical composition or form of the siRNA molecules described herein can be combined with each other. For example, one, two, three, four, five or more different types of modifications can be included in the same siRNA molecule.

[0382] In some embodiments, the antisense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the antisense strand comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleotide linkages. In some embodiments, the modified nucleotides comprise a modified sugar moiety (e.g., a 2' modified nucleotide). In some embodiments, the antisense strand comprises one or more 2' modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, each nucleotide of the antisense strand is a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the antisense strand comprises one or more 2'-O-methyl modified nucleotides. In some embodiments, the antisense strand comprises one or more 2'-F modified nucleotides. In some embodiments, the antisense strand comprises one or more 2'-O-methyl and 2'-F modified nucleotides. In some embodiments, the antisense strand comprises one or more modified nucleosides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the antisense strand comprises one or more modified nucleosides and / or (e.g., and) one or more modified internucleoside linkages. In some embodiments, the modified nucleosides comprise modified sugar moieties (e.g., 2' modified nucleosides). In some embodiments, the antisense strand comprises one or more 2' modified nucleosides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE) or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, each nucleoside of the antisense strand is a modified nucleoside (e.g., a 2'-modified nucleoside). In some embodiments, the antisense strand described herein comprises one or more 2'-F modified nucleosides. In some embodiments, the antisense strand described herein comprises at least two 2'-F modified nucleosides. In some embodiments, the antisense strand described herein comprises at least four 2'-F modified nucleosides.In some embodiments, the antisense strand described herein comprises at least six 2'-fluoro (2'-F) modified nucleosides. In some embodiments, the antisense strand described herein comprises one or more 2'-O-methyl modified nucleosides. In some embodiments, the antisense strand comprises one or more 2'-O-methyl and 2'-F modified nucleosides.

[0383] In some embodiments, the antisense strand comprises a phosphorothioate or other modified internucleotide linkage. In some embodiments, the antisense strand comprises a phosphorothioate or other modified internucleoside linkage. In some embodiments, the antisense strand comprises a phosphorothioate internucleoside linkage. In some embodiments, the antisense strand comprises a phosphorothioate internucleoside linkage. In some embodiments, the antisense strand comprises a phosphorothioate internucleoside linkage between at least two nucleotides. In some embodiments, the antisense strand comprises a phosphorothioate internucleoside linkage between at least two nucleosides. In some embodiments, the antisense strand comprises a phosphorothioate internucleoside linkage between at least two nucleotides. In some embodiments, the antisense strand comprises a phosphorothioate internucleoside linkage between all nucleotides. In some embodiments, the antisense strand comprises a phosphorothioate internucleoside linkage between all nucleosides. For example, in some embodiments, the antisense strand comprises a modified internucleoside linkage at the first, second and / or (e.g., and) third internucleoside linkage at the 5' or 3' end of the siRNA molecule. In some embodiments, the two internucleoside linkages at the 3' end of the antisense strand are phosphorothioate internucleoside linkages. In some embodiments, the antisense strand comprises phosphorothioate internucleoside linkages between all nucleotides. For example, in some embodiments, the antisense strand comprises modified internucleoside linkages at the first, second and / or (e.g., and) third internucleoside linkages at the 5' or 3' end of the siRNA molecule. In some embodiments, the two internucleoside linkages at the 3' end of the antisense strand are phosphorothioate internucleoside linkages. For example, in some embodiments, the antisense strand comprises modified internucleoside linkages at the first, second and / or (e.g., and) third internucleoside linkages at the 5' or 3' end of the siRNA molecule. In some embodiments, the two internucleoside linkages at the 3' end of the antisense strand are phosphorothioate internucleoside linkages.

[0384] In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, the modified internucleoside linkage is a phosphorus-containing linkage. In some embodiments, the phosphorus-containing linkage that can be used includes, but is not limited to: phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkylphosphonates including 3'alkylenephosphonates, and chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with opposite polarities in which adjacent pairs of nucleoside units are linked at 3'-5' to 5'-3' or 2'-5' to 5'-2'; see

[0385] U.S. Patents No. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5, and 5,625,050.

[0386] Any modified chemical composition or form of the antisense strand described herein can be combined with each other. For example, one, two, three, four, five or more different types of modifications can be included in the same antisense strand.

[0387] In some embodiments, the sense strand comprises one or more modified nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the sense strand comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleotide linkages. In some embodiments, the modified nucleotides comprise a modified sugar moiety (e.g., a 2' modified nucleotide). In some embodiments, the sense strand comprises one or more 2' modified nucleotides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, each nucleotide of the sense strand is a modified nucleotide (e.g., a 2'-modified nucleotide). In some embodiments, the sense strand comprises one or more modified nucleosides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more). In some embodiments, the sense strand comprises one or more modified nucleotides and / or (e.g., and) one or more modified internucleoside linkages. In some embodiments, the modified nucleoside comprises a modified sugar moiety (e.g., a 2' modified nucleoside). In some embodiments, the sense strand comprises one or more 2' modified nucleosides, such as 2'-deoxy, 2'-fluoro (2'-F), 2'-O-methyl (2'-O-Me), 2'-O-methoxyethyl (2'-MOE), 2'-O-aminopropyl (2'-O-AP), 2'-O-dimethylaminoethyl (2'-O-DMAOE), 2'-O-dimethylaminopropyl (2'-O-DMAP), 2'-O-dimethylaminoethoxyethyl (2'-O-DMAEOE), or 2'-ON-methylacetamido (2'-O-NMA). In some embodiments, each nucleoside of the sense strand is a modified nucleoside (e.g., a 2'-modified nucleoside). In some embodiments, the sense strand comprises one or more phosphorodiamidate morpholino. In some embodiments, the sense strand is a phosphorodiamidate morpholino oligomer (PMO). In some embodiments, the sense strand comprises one or more 2'-O-methyl modified nucleotides. In some embodiments, the sense strand comprises one or more 2'-F modified nucleotides. In some embodiments, the sense strand comprises one or more 2'-O-methyl and 2'-F modified nucleotides.

[0388] In some embodiments, the sense strand described herein comprises one or more 2'-F modified nucleosides. In some embodiments, the sense strand described herein comprises at least two 2'-F modified nucleosides. In some embodiments, the sense strand described herein comprises at least four 2'-F modified nucleosides. In some embodiments, the sense strand described herein comprises at least six 2'-F modified nucleosides. In some embodiments, the sense strand described herein comprises one or more 2'-O-methyl modified nucleosides. In some embodiments, the sense strand comprises one or more 2'-O-methyl and 2'-F modified nucleosides.

[0389] In some embodiments, the sense strand comprises a phosphorothioate or other modified internucleotide linkage. In some embodiments, the sense strand comprises a phosphorothioate or other modified internucleoside linkage. In some embodiments, the sense strand comprises a phosphorothioate internucleoside linkage. In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage. In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage. In some embodiments, the sense strand comprises a phosphorothioate internucleoside linkage between at least two nucleotides. In some embodiments, the sense strand comprises a phosphorothioate internucleoside linkage between at least two nucleosides. In some embodiments, the sense strand comprises a phosphorothioate internucleotide linkage between at least two nucleotides. In some embodiments, the sense strand comprises a phosphorothioate internucleoside linkage between all nucleotides. In some embodiments, the sense strand comprises a phosphorothioate internucleoside linkage between all nucleosides. For example, in some embodiments, the sense strand comprises a modified internucleoside linkage at the first, second, and / or (e.g., and) third internucleoside linkage at the 5' or 3' end of the sense strand. In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between all nucleotides. For example, in some embodiments, the sense strand comprises a modified internucleotide linkage at the first, second, and / or (e.g., and) third internucleotide linkage at the 5' or 3' terminus of the sense strand. For example, in some embodiments, the sense strand comprises a modified internucleotide linkage at the first, second, and / or (e.g., and) third internucleoside linkage at the 5' or 3' terminus of the sense strand. In some embodiments, the sense strand comprises a phosphodiester internucleoside linkage. In some embodiments, the sense strand does not comprise a phosphorothioate internucleoside linkage. In some embodiments, the modified internucleotide linkage is a phosphorus-containing linkage. In some embodiments, the modified internucleoside linkage is a phosphorus-containing linkage. In some embodiments, phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methylphosphonates, and other alkylphosphonates including 3' alkylenephosphonates, as well as chiral phosphonates, phosphinates, phosphoramidates including 3'-aminophosphoramidates and aminoalkylphosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphotriesters, and boranophosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with opposite polarity where adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'; see U.S. Pat.

[0390] 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405 ,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

[0391] Any modified chemical composition or form of the sense strand described herein can be combined with each other. For example, one, two, three, four, five or more different types of modifications can be included within the same sense strand.

[0392] In some embodiments, the antisense strand or sense strand of the siRNA molecule includes a modification that enhances or reduces RNA-induced silencing complex (RISC) loading. In some embodiments, the antisense strand of the siRNA molecule includes a modification that enhances RISC loading. In some embodiments, the sense strand of the siRNA molecule includes a modification that reduces RISC loading and reduces off-target effects. In some embodiments, the antisense strand of the siRNA molecule includes a modification that reduces RISC loading and reduces off-target effects. In some embodiments, the antisense strand of the siRNA molecule includes a 2'-O-methoxyethyl (2'-MOE) modification. Adding 2'-O-methoxyethyl (2'-MOE) groups at the cleavage site improves the specificity and silencing activity of siRNA by promoting the directional RNA-induced silencing complex (RISC) loading of the modified strand, as described in Song et al., (2017) Mol Ther Nucleic Acids 9: 242-250, which is incorporated herein by reference in its entirety. In some embodiments, the antisense strand of the siRNA molecule comprises a 2'-OMe-phosphorodithioate modification that increases RISC loading as described in Wu et al., (2014) Nat Commun 5:3459, which is incorporated herein by reference in its entirety.

[0393] In some embodiments, the sense strand of the siRNA molecule comprises a 5'-morpholino, which reduces the RISC load of the sense strand and improves antisense strand selection and RNAi activity, as described in Kumar et al., (2019) Chem Commun (Camb) 55(35): 5139-5142, which is incorporated herein by reference in its entirety. In some embodiments, the sense strand of the siRNA molecule is modified with a synthetic RNA-like high-affinity nucleotide analog locked nucleic acid (LNA), which reduces the RISC load of the sense strand and further enhances the incorporation of the antisense strand into RISC, as described in Elman et al., (2005) Nucleic Acids Res. 33(1): 439-447, which is incorporated herein by reference in its entirety. In some embodiments, the sense strand of the siRNA molecule comprises a 5' unlocked nucleic acid (UNA) modification, which reduces the RISC loading of the sense strand and improves the silencing efficacy of the antisense strand, as described in Snead et al., (2013) Mol Ther Nucleic Acids 2(7):e103, which is incorporated herein by reference in its entirety. In some embodiments, the sense strand of the siRNA molecule comprises a 5-nitroindole modification, which reduces the RNAi efficacy of the sense strand and reduces off-target effects, as described in Zhang et al., (2012) Chembiochem 13(13):1940-1945, which is incorporated herein by reference in its entirety. In some embodiments, the sense strand comprises a 2'-O' methyl (2'-O-Me) modification, which reduces the RISC load and off-target effects of the sense strand, as described in Zheng et al., FASEB (2013) 27 (10): 4017-4026, which is incorporated herein by reference in its entirety. In some embodiments, the sense strand of the siRNA molecule is completely replaced by morpholino, 2'-MOE or 2'-O-Me residues and is not recognized by RISC, as described in Kole et al., (2012) Nature reviews. Drug Discovery 11 (2): 125-140, which is incorporated herein by reference in its entirety. In some embodiments, the antisense strand of the siRNA molecule comprises a 2'-MOE modification and the sense strand comprises a 2'-O-Me modification (see, e.g., Song et al., (2017) Mol Ther Nucleic Acids 9: 242-250). In some embodiments, at least one (eg, at least 2, at least 3, at least 4, at least 5, at least 10) siRNA molecules are (eg, covalently) linked to a muscle targeting agent.In some embodiments, the muscle targeting agent may comprise or consist of a nucleic acid (e.g., DNA or RNA), a peptide (e.g., an antibody), a lipid (e.g., a microvesicle), or a sugar moiety (e.g., a polysaccharide). In some embodiments, the muscle targeting agent is an antibody. In some embodiments, the muscle targeting agent is an anti-transferrin receptor antibody (e.g., any one of the anti-TfR1 antibodies provided in Tables 2 to 7). In some embodiments, the muscle targeting agent may be connected to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be covalently linked to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be connected to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be covalently linked to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be internally linked to the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be covalently linked to the internal sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent may be connected to the 5' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the 5' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the 3' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the 3' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the 3' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the interior of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the interior of the antisense strand of the siRNA molecule.

[0394] Some non-limiting examples of siRNAs described herein are provided in Table 8.

[0395] Table 8. Non-limiting examples of siRNA

[0396]

[0397]

[0398]

[0399]

[0400]

[0401]

[0402]

[0403]

[0404]

[0405]

[0406]

[0407]

[0408]

[0409]

[0410]

[0411]

[0412]

[0413]

[0414]

[0415] "m" indicates a 2'-O-methyl (2'-O-Me) modified nucleoside; "f" indicates a 2'-fluoro (2'-F) modified nucleoside; "*" indicates a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides indicates a phosphodiester internucleoside linkage.

[0416] Each uracil base (U) in any of the oligonucleotides and / or target sequences provided in Table 8 can be independently and optionally replaced with a thymine base (T), and / or each T can be independently and optionally replaced with a U. The target sequences listed in Table 8 include T, but binding of the oligonucleotides described herein to RNA and / or DNA is contemplated.

[0417] ^The target sequence starts at NM_001306068.3 (SEQ ID NO: 161)

[0418] The siRNA oligonucleotides provided in Table 8 may have different modification patterns, and the siRNA oligonucleotides have a sense strand and an antisense strand with the same nucleobase sequence as shown in SEQ ID NO. The modified sense strand and antisense strand are represented by "MS#" or "MAS#", respectively. The siRNA oligonucleotides provided in Table 8 include an antisense strand without 5'-(E)-vinylphosphonate modification. However, siRNA oligonucleotides comprising an antisense strand with 5'-(E)-vinylphosphonate modification are also contemplated and are represented herein by the same MAS# with "VP" before MAS# (i.e., VP-MAS#). The siRNA oligonucleotides provided in Table 8 can be conjugated to a compound of formula NH2-(CH2)6- at the 5' or 3' nucleoside of the sense strand.

[0419] Table 9. Non-limiting examples of additional siRNAs

[0420]

[0421]

[0422] "m" indicates a 2'-O-methyl (2'-O-Me) modified nucleoside; "f" indicates a 2'-fluoro (2'-F) modified nucleoside; "*" indicates a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides indicates a phosphodiester internucleoside linkage. "VP" indicates 5'-(E)-vinylphosphonate.

[0423] Each uracil base (U) in any of the oligonucleotides and / or target sequences provided in Table 9 can be independently and optionally replaced with a thymine base (T), and / or each T can be independently and optionally replaced with a U. The target sequences listed in Table 9 include T, but binding of the oligonucleotides described herein to RNA and / or DNA is contemplated.

[0424] ^The target sequence starts at NM_001306068.3 (SEQ ID NO: 161) The siRNA oligonucleotides provided in Table 9 may have different modification patterns, and the siRNA oligonucleotides have a sense strand and an antisense strand of the same nucleobase sequence as shown in SEQ ID NO. The modified sense strand and antisense strand are represented by "MS#" or "MAS#", respectively. The siRNA oligonucleotides provided in Table 9 include an antisense strand that includes a 5'-(E)-vinylphosphonate modification represented by "VP" before MAS# (i.e., VP-MAS#). siRNA oligonucleotides comprising an antisense strand without a 5'-(E)-vinylphosphonate modification are also contemplated and are represented herein by the same MAS# without "VP". The siRNA oligonucleotides provided in Table 9 can be conjugated to a compound of formula NH2-(CH2)6- at the 5' or 3' (e.g., 5') nucleoside of the sense strand.

[0425] In some embodiments, the oligonucleotides described herein comprise an antisense strand of 18 to 25 nucleosides (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleosides) in length, and comprise a complementary region to a target sequence as set forth in any one of SEQ ID NOs: 174 to 235, wherein the length of the complementary region is at least 16 nucleotides (e.g., 16, 17, 18, or 19 nucleotides). In some embodiments, the antisense strand is 23 nucleotides in length and comprises a complementary region to a target sequence as set forth in any one of SEQ ID NOs: 174 to 235, wherein the length of the complementary region is 20 nucleotides. In some embodiments, the complementary region is fully complementary to all or part of its target sequence. In some embodiments, the complementary region comprises 1, 2, 3, or more mismatches.

[0426] In some embodiments, the oligonucleotides described herein comprise an antisense strand comprising at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) of a nucleotide sequence of any one of SEQ ID NOs: 236 to 266. In some embodiments, the oligonucleotides described herein further comprise a sense strand comprising at least 15 consecutive nucleosides (e.g., at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20) of a nucleotide sequence of any one of SEQ ID NOs: 205 to 235.

[0427] In some embodiments, the oligonucleotides described herein comprise an antisense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 236 to 266. In some embodiments, the oligonucleotides described herein further comprise a sense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 205 to 235.

[0428] In some embodiments, the oligonucleotide described herein is a double-stranded oligonucleotide (e.g., siRNA) comprising an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 236 to 266, the sense strand hybridizing to the antisense strand and comprising the nucleotide sequence of any one of SEQ ID NOs: 205 to 235, wherein the antisense strand and / or (e.g., and) comprises one or more modified nucleosides (e.g., 2'-modified nucleosides). In some embodiments, one or more modified nucleosides are selected from 2'-O-Me and 2'-F modified nucleosides.

[0429] In some embodiments, the oligonucleotide described herein is a double-stranded oligonucleotide (e.g., siRNA) comprising an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 236 to 266, the sense strand hybridizing to the antisense strand and comprising the nucleotide sequence of any one of SEQ ID NOs: 205 to 235, wherein each nucleoside in the antisense strand and / or (e.g., and) each nucleoside in the sense strand is a 2'-modified nucleoside selected from the following: 2'-O-Me and 2'-F modified nucleosides.

[0430] In some embodiments, the oligonucleotide described herein is a double-stranded oligonucleotide (e.g., siRNA) comprising an antisense strand and a sense strand, the antisense strand comprising the nucleotide sequence of any one of SEQ ID NOs: 236 to 266, the sense strand hybridizing to the antisense strand and comprising the nucleotide sequence of any one of SEQ ID NOs: 205 to 235, wherein each nucleoside in the antisense strand and each nucleoside in the sense strand is a 2'-modified nucleoside selected from 2'-O-Me and 2'-F modified nucleosides, and wherein the antisense strand and / or (e.g., and) the sense strand each comprises one or more phosphorothioate internucleoside linkages. In some embodiments, the sense strand does not comprise any phosphorothioate internucleoside linkages (all internucleoside linkages in the sense strand are phosphodiester internucleoside linkages), and the antisense strand comprises 1, 2, or 3 phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 2 phosphorothioate internucleoside linkages and the antisense strand comprises 4 phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises 4 phosphorothioate internucleoside linkages and the antisense strand comprises 4 phosphorothioate internucleoside linkages.

[0431] In some embodiments, the antisense strand comprises 2 phosphorothioate internucleoside linkages, optionally, wherein the two internucleoside linkages at the 3' end of the antisense strand are phosphorothioate internucleoside linkages, and the remaining internucleoside linkages in the antisense strand are phosphodiester internucleoside linkages. In some embodiments, the antisense strand comprises 4 phosphorothioate internucleoside linkages, optionally, wherein the two internucleoside linkages at the 3' end of the antisense strand and the two internucleoside linkages at the 5' end are phosphorothioate internucleoside linkages, and the remaining internucleoside linkages in the antisense strand are phosphodiester internucleoside linkages. In some embodiments, the sense strand comprises 2 phosphorothioate internucleoside linkages, optionally, wherein the two internucleoside linkages at the 5' end of the sense strand are phosphorothioate internucleoside linkages, and the remaining internucleoside linkages in the sense strand are phosphodiester internucleoside linkages. In some embodiments, the sense strand comprises 4 phosphorothioate internucleoside linkages. In some embodiments, two internucleoside linkages at the 5' terminus and two internucleoside linkages at the 3' terminus of the sense strand are phosphorothioate internucleoside linkages, and the remaining internucleoside linkages in the sense strand are phosphodiester internucleoside linkages.

[0432] In some embodiments, the antisense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MAS1"):

[0433] fN*fN*mNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, antisense strands MAS1-236, MAS1-237, and MAS1-238 contain such a structure.

[0434] In some embodiments, the antisense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MAS2"):

[0435] fN*fN*mNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN,

[0436] Wherein "mN" indicates a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" indicates a 2'-fluoro (2'-F) modified nucleoside; "*" indicates a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides indicates a phosphodiester internucleoside linkage. For example, in Table 8, the antisense strands MAS2-236, MAS2-237, MAS2-238,

[0437] MAS2-239, MAS2-240, MAS2-241, MAS2-242, MAS2-243, MAS2-244, MAS2-245, MAS2-246, MAS2-247, MAS2-248, MAS2-249, MAS2-250, MAS2-251, MAS2-252, MAS2-253, MAS2-254, MAS2-255, MAS2-256, and MAS2-257

[0438] Contains this structure.

[0439] In some embodiments, the antisense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MAS3"):

[0440] fN*fN*mNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmN*mN*mN,

[0441] Where "mN" indicates a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" indicates a 2'-fluoro (2'-F) modified nucleoside; "*" indicates a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides indicates a phosphodiester internucleoside linkage. For example, in Table 8, the antisense strand

[0442] MAS3-236, MAS3-237, MAS3-238, MAS3-239, MAS3-240, MAS3-241, MAS3-242, MAS3-2 43, MAS3-244, MAS3-245, MAS3-246, MAS3-247, MAS3-248, MAS3-249, MAS3-250, MAS3 -251, MAS3-252, MAS3-253, MAS3-254, MAS3-255, MAS3-256, MAS3-257, MAS3-258, MA S3-259, MAS3-260, MAS3-261, MAS3-262, MAS3-263, MAS3-264, MAS3-265, and MAS3-266

[0443] Contains this structure.

[0444] In some embodiments, the antisense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MAS4"):

[0445] fN*fN*mNmNINmNfNfNmNmNmNmNfNmNfNmNmNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the antisense strand

[0446] MAS4-236, MAS4-237, MAS4-238, MAS4-239, MAS4-240, MAS4-241, MAS4-242, MAS4-243, MAS4-244, MAS4-245, MAS4-246, MAS4-247, MAS4-248, MAS4-249, MAS4-250, MAS4-251, MAS4-252, MAS4-253, MAS4-254, MAS4-255, MAS4-256, and MAS4-257

[0447] Contains this structure.

[0448] In some embodiments, the antisense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MAS5"):

[0449] mN*fN*mNmNfNmNmNmNmNmNmNmNmfNmNfNmNmNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0450] In some embodiments, the MAS5 structure further comprises a 5' vinylphosphonate (eg, 5'-(E)-vinylphosphonate) modification (5' to 3', referred to herein as "VP-MAS5"):

[0451] VP-mN*fN*mNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage; and VP represents a 5'-(E)-vinylphosphonate. For example, in Table 9, the antisense strand VP-MAS5-248 comprises this structure.

[0452] In some embodiments, the antisense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MAS6"):

[0453] mN*fN*mNmNmNfNmNININmNmNmNmNINmNfNmNmNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0454] In some embodiments, the MAS6 structure further comprises a 5' vinylphosphonate (eg, 5'-(E)-vinylphosphonate) modification (5' to 3', referred to herein as "VP-MAS6"):

[0455] VP-mN*fN*mNmNfNmNfNmNmNmNmNfNmNmNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage; and VP represents 5'-(E)-vinylphosphonate. For example, in Table 9, antisense strands VP-MAS6-251, VP-MAS6-253, and VP-MAS6-248 comprise this structure.

[0456] In some embodiments, the antisense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MAS7"):

[0457] mN*fN*mNmNmNmNmNmNmNmNmNmNmfNmNmNmNmNmNmNmNmNm*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0458] In some embodiments, the MAS7 structure further comprises a 5' vinylphosphonate (eg, 5'-(E)-vinylphosphonate) modification (5' to 3', referred to herein as "VP-MAS7"):

[0459] VP-mN*fN*mNmNmNmNmNmNmNmNmNmNmfNmNmNmNmNmNmNmNmNm*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage; and VP represents 5'-(E)-vinylphosphonate. For example, in Table 9, antisense strands VP-MAS7-252 and VP-MAS7-262 contain this structure.

[0460] In some embodiments, the sense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MS1"):

[0461] mN*mN*fNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, sense strands MS1-205, MS1-206, and MS1-207 contain such a structure.

[0462] In some embodiments, the sense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MS2"):

[0463] mN*mN*mNmNfNmNfNmNfNfNmNmNmNmNfNmNmNmNmNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the sense strand

[0464] MS2-208, MS2-209, MS2-210, MS2-211, MS2-212, MS2-213, MS2-214, MS2-215, MS2-216, MS2- 217, MS2-218, MS2-219, MS2-220, MS2-221, MS2-222, MS2-223, MS2-224, MS2-225, and MS2-226

[0465] Contains this structure.

[0466] In some embodiments, the sense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MS3"):

[0467] mN*mN*mNmNfNmNmNmNfNfNmNmNfNmNmNmNmNfNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the sense strand

[0468] MS3-205, MS3-206, MS3-207, MS3-208, MS3-209, MS3-210, MS3-211, MS3-212, MS3-213, MS3-214, MS3-215, MS3-216, MS3-217, MS3-218, MS3-219, MS3-220, MS3-221, MS3-222, MS3-223, MS3-224, MS3-225, MS3-226, MS3-227, MS3-228, MS3-229, MS3-230, MS3-231, MS3-232, MS3-233, MS3-234, and MS3-235 contain this structure.

[0469] In some embodiments, the sense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MS4"):

[0470] mN*mN*mNmNmNmfNmNfNfNmNmNmNmNmNmNmNmNmNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the sense strand

[0471] MS4-205, MS4-206, MS4-207, MS4-208, MS4-209, MS4-210, MS4-211, MS4-212, MS4-213, MS4-214, MS4-215, MS4-216, MS4-217, MS4-218, MS4-219, MS4-220, MS4-221, MS4-222, MS4-223, MS4-224, MS4-225, and MS4-226 contain this structure.

[0472] In some embodiments, the sense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MS5"):

[0473] mN*mN*mNmNmNmfNmNfNfNmNmNmNmNfNfNmNmNmNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the sense strand

[0474] MS5-205, MS5-206, MS5-207, MS5-208, MS5-209, MS5-210, MS5-211, MS5-212, MS5-213, MS5-214, MS5-215, MS5-216, MS5-217, MS5-218, MS5-219, MS5-220, MS5-221, MS5-222, MS5-223, MS5-224, MS5-225, and MS5-226 contain this structure.

[0475] In some embodiments, the sense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MS6"):

[0476] mN*mN*mNmNmNmfNmNfNfNmNmNmNmNfNmNmNfNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 8, the sense strand

[0477] MS6-205, MS6-206, MS6-207, MS6-208, MS6-209, MS6-210, MS6-211, MS6-212, MS6-213, MS6-214, MS6-215, MS6-216, MS6-217, MS6-218, MS6-219, MS6-220, MS6-221, MS6-222, MS6-223, MS6-224, MS6-225, and MS6-226 contain this structure.

[0478] In some embodiments, the sense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MS7"):

[0479] mN*mN*mNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 9, sense strands MS7-220, MS7-222, and MS7-217 contain such a structure.

[0480] In some embodiments, the sense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MS8"):

[0481] mN*mN*mNmNfNmNmNmNfNfNmNmNmNfNmNmNmNmNfN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 9, sense strands MS8-221 and MS8-231 contain such a structure.

[0482] In some embodiments, the sense strand of an oligonucleotide described herein comprises the structure (5' to 3', referred to herein as "MS9"):

[0483] mN*mN*mNmNfNmNfNmNfNfNmNmNmNfNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. For example, in Table 9, sense strand MS9-217 comprises such a structure.

[0484] In some embodiments, the antisense strand of an oligonucleotide described herein is selected from modified forms of SEQ ID NOs: 236 to 266 listed in Table 8 (e.g., MAS1 to MAS4). For example, in some embodiments, the antisense strand is selected from:

[0485] MAS1-236, MAS1-237, MAS1-238, MAS2-236, MAS2-237, MAS2-238, MAS2-239, MAS2-240, MAS2-241, MAS2-2 42, MAS2-243, MAS2-244, MAS2-245, MAS2-246, MAS2-247, MAS2-248, MAS2-249, MAS2-250, MAS2-251, MAS2 -252, MAS2-253, MAS2-254, MAS2-255, MAS2-256, MAS2-257, MAS3-236, MAS3-237, MAS3-238, MAS3-239, MA S3-240, MAS3-241, MAS3-242, MAS3-243, MAS3-244, MAS3-245, MAS3-246, MAS3-247, MAS3-248, MAS3-249, MAS3-250, MAS3-251, MAS3-252, MAS3-253, MAS3-254, MAS3-255, MAS3-256, MAS3-257, MAS3-258, MAS3-25 9. MAS3-260, MAS3-261, MAS3-262, MAS3-263, MAS3-264, MAS3-265, MAS3-266, MAS4-236, MAS4-237, MAS4- 238, MAS4-239, MAS4-240, MAS4-241, MAS4-242, MAS4-243, MAS4-244, MAS4-245, MAS4-246, MAS4-247, MAS4-248, MAS4-249, MAS4-250, MAS4-251, MAS4-252, MAS4-253, MAS4-254, MAS4-255, MAS4-256, and MAS4-257.

[0486] In some embodiments, the antisense strand of an oligonucleotide described herein is selected from a modified form of SEQ ID NOs: 236 to 266 listed in Table 8 (e.g., MAS5 to MAS7). In some embodiments, the antisense strand of an oligonucleotide described herein is selected from a modified form of SEQ ID NOs: 251, 253, 262, 248, and 252 listed in Table 9 (e.g., MAS5, MAS6, MAS7, VP-MAS5, VP-MAS6, and VP-MAS7). For example, in some embodiments, the antisense strand is selected from VP-MAS6-251, VP-MAS7-252, VP-MAS6-253, VP-MAS7-262, VP-MAS6-248, and VP-MAS5-248.

[0487] In some embodiments, the sense strand of the oligonucleotide described herein is selected from the modified forms of SEQ ID NOs: 205 to 235 listed in Table 8 (e.g., MS1 to MS6). For example, in some embodiments, the sense strand is selected from:

[0488] MS1-205, MS1-206, MS1-207, MS2-208, MS2-209, MS2-210, MS2-211, MS2-212, MS2-213, MS2-214, MS2-215, MS2-216, MS2-217, MS2-218, MS2-219 , MS2-220, MS2-221, MS2-222, MS2-223, MS2-224, MS2-225, MS2-226, MS3-205, MS3-206, MS3-207, MS3-208, MS3-209, MS3-210, MS3-211, MS3-21 2. MS3-213, MS3-214, MS3-215, MS3-216, MS3-217, MS3-218, MS3-219, MS3-220, MS3-221, MS3-222, MS3-223, MS3-224, MS3-225, MS3-226, MS3-2 27, MS3-228, MS3-229, MS3-230, MS3-231, MS3-232, MS3-233, MS3-234, MS3-235, MS4-205, MS4-206, MS4-207, MS4-208, MS4-209, MS4-210, MS4- 211, MS4-212, MS4-213, MS4-214, MS4-215, MS4-216, MS4-217, MS4-218, MS4-219, MS4-220, MS4-221, MS4-222, MS4-223, MS4-224, MS4-225, MS4 -226, MS5-205, MS5-206, MS5-207, MS5-208, MS5-209, MS5-210, MS5-211, MS5-212, MS5-213, MS5-214, MS5-215, MS5-216, MS5-217, MS5-218, MS 5-219, MS5-220, MS5-221, MS5-222, MS5-223, MS5-224, MS5-225, MS5-226, MS6-205, MS6-206, MS6-207, MS6-208, MS6-209, MS6-210, MS6-211, MS6-212, MS6-213, MS6-214, MS6-215, MS6-216, MS6-217, MS6-218, MS6-219, MS6-220, MS6-221, MS6-222, MS6-223, MS6-224, MS6-225, and MS6-226.

[0489] In some embodiments, the sense strand of an oligonucleotide described herein is selected from a modified form of SEQ ID NOs: 205 to 235 listed in Table 8 (e.g., MS7 to MS9). In some embodiments, the sense strand of an oligonucleotide described herein is selected from a modified form of SEQ ID NOs: 220, 222, 231, 217, and 221 listed in Table 9 (e.g., MS7 to MS9). For example, in some embodiments, the sense strand is selected from MS7-220, MS8-221, MS7-222, MS8-231, MS9-217, and MS7-217.

[0490] In some embodiments, the oligonucleotides described herein may include an antisense strand and a sense strand, the antisense strand including any antisense strand structure described herein (e.g., MAS1 to MAS4, regardless of its nucleobase sequence), and the sense strand including any sense strand structure described herein (e.g., MS1 to MS6, regardless of its nucleobase sequence). In some embodiments, the oligonucleotides described herein may include an antisense strand and a sense strand, the antisense strand including any antisense strand structure described herein (e.g., MAS5, MAS6, MAS7, VP-MAS5, VP-MAS6, and VP-MAS7, regardless of its nucleobase sequence), and the sense strand including any sense strand structure described herein (e.g., MS7 to MS9, regardless of its nucleobase sequence).

[0491] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS3):

[0492] fN*fN*mNmNmNmNmNmNmNmNmNmNmfNmNmNmNmNmNmNmNmN*mN*mN, the sense strand comprises the structure (5' to 3', MS3): mN*mN*mNmNfNmNmNmNfNfNfNmNmNfNmNmNmNmNfNmNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0493] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS4):

[0494] fN*fN*mNmNfNmNfNmNmNmNmNfNmNfNmNmNmNmNmNm*mN*mN, the sense strand comprising the structure (5' to 3'; MS4):

[0495] mN*mN*mNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0496] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS4):

[0497] fN*fN*mNmNfNmNfNmNmNmNmNfNmNfNmNmNmNmNmNmN*m*mN, the sense strand comprising the structure (5' to 3', MS6):

[0498] mN*mN*mNmNmNmNfNmNfNfNmNmNmNmNmNfNmNmNfNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0499] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS2):

[0500] fN*fN*mNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmN*m*mN, the sense strand comprising the structure (5' to 3', MS5):

[0501] mN*mN*mNmNmNmNfNmNfNfNmNmNmNmNfNfNmNmNmNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0502] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS2):

[0503] fN*fN*mNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmN*m*mN, the sense strand comprising the structure (5' to 3', MS4):

[0504] mN*mN*mNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0505] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS1):

[0506] fN*fN*mNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmN*fN*mN, the sense strand comprising the structure (5' to 3'; MS1):

[0507] mN*mN*fNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfNmNfN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0508] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS4):

[0509] fN*fN*mNmNfNmNfNmNmNmNmNfNmNfNmNmNmNmNmNmN*mN*mN, the sense strand comprising the structure (5' to 3'; MS2):

[0510] mN*mN*mNmNfNmNfNmNfNfNmNmNmNmNfNmNmNmNmNmN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0511] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS5):

[0512] mN*fN*mNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmNmN*m*mN, the sense strand comprising the structure (5' to 3', MS7):

[0513] mN*mN*mNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNm*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0514] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS6):

[0515] mN*fN*mNmNmNfNmNfNmNmNmNmNfNmNfNmNmNmNmNmNm*mN*mN, the sense strand comprising the structure (5' to 3', MS7):

[0516] mN*mN*mNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNm*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0517] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS 7):

[0518] mN*fN*mNmNmNmNmNmNmNmNmNmNmfNmNmNmNmNmNmNmNmNm*mN*mN, the sense strand comprises the structure (5' to 3', MS8):

[0519] mN*mN*mNmNfNmNmNmNfNfNmNmNmNfNmNmNmNmNmNfN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0520] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', MAS6):

[0521] mN*fN*mNmNmNfNmNfNmNmNmNmNfNmNfNmNmNmNmNmNm*mN*mN, the sense strand comprising the structure (5' to 3', MS9):

[0522] mN*mN*mNmNfNmNfNmNfNfNmNmNmNfNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. In some embodiments, the oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', VP-MAS5):

[0523] VP-mN*fN*mNmNmNfNmNmNmNmNmNmNmfNmNfNmNmNmNmNmNmN*m*mN, the sense strand comprising the structure (5' to 3', MS7):

[0524] mN*mN*mNmNmNmNfNmNfNfNfNmNmNmN mNmNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0525] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', VP-MAS6):

[0526] VP-mN*fN*mNmNmNfNmNfNmNmNmNfNmNmNmNfNmNmNmNmNmN*m*mN, the sense strand comprising the structure (5' to 3', MS7):

[0527] mN*mN*mNmNmNmNfNmNfNfNmNmNmNmNmNmNmNmNm*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0528] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', VP-MAS7):

[0529] v P -mN*fN*mNmNmNmNmNmNmNmNmNmNmfNmNmNmNmNmNmNmNmN*mN*mN, the sense strand comprising the structure (5' to 3', MS8):

[0530] mN*mN*mNmNfNmNmNmNfNfNmNmNmNfNmNmNmNmNmNfN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage.

[0531] In some embodiments, an oligonucleotide described herein comprises an antisense strand and a sense strand, the antisense strand comprising the structure (5' to 3', VP-MAS6):

[0532] VP-mN*fN*mNmNmNfNmNfNmNmNmNfNmNmNfNmNmNmNmNmN*m*mN, the sense strand comprising the structure (5' to 3', MS9):

[0533] mN*mN*mNmNfNmNfNmNfNfNmNmNmNfNmNmNmNmN*mN*mN, wherein "mN" represents a 2'-O-methyl (2'-O-Me) modified nucleoside; "fN" represents a 2'-fluoro (2'-F) modified nucleoside; "*" represents a phosphorothioate internucleoside linkage; and the absence of "*" between two nucleosides represents a phosphodiester internucleoside linkage. In some embodiments, the oligonucleotide described herein is a siRNA selected from the siRNAs listed in Table 8. In some embodiments, the oligonucleotide described herein is a siRNA selected from the siRNAs listed in Table 9.

[0534] In some embodiments, any oligonucleotide described herein (e.g., an siRNA selected from the siRNA in Table 8) can be in salt form, for example, as a sodium salt, potassium salt, magnesium salt. In some embodiments, any oligonucleotide described herein (e.g., an siRNA selected from the siRNA in Table 9) can be in salt form, for example, as a sodium salt, potassium salt, magnesium salt.

[0535] In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of any oligonucleotide described herein (e.g., oligonucleotides listed in Table 8) is optionally conjugated to an amine group via a spacer. In some embodiments, the 5' or 3' nucleoside (e.g., terminal nucleoside) of any oligonucleotide described herein (e.g., oligonucleotides listed in Table 9) 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, a phosphodiester linkage is present between the spacer and the 5' or 3' nucleoside of the oligonucleotide. In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any oligonucleotide described herein (e.g., an oligonucleotide listed in Table 8) is conjugated to a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(R A )-, -S-, -C(=O)-, -C(=O)O-, -C(=O)NR A -,-NR A C(=O)-,-NR A C(=O)R A -,-C(=O)R A -,-NR A C(=O)O-,-NR A C(=O)N(R A )-, -OC(=O)-, -OC(=O)O-, -OC(=O)N(R A )-,-S(O)2NR A , -NR A S(O)2-, or a combination thereof, each R A are independently hydrogen or substituted or unsubstituted alkyl. In some embodiments, the 5' or 3' nucleoside (e.g., the terminal nucleoside) of any oligonucleotide described herein (e.g., an oligonucleotide listed in Table 9) is conjugated to a spacer that is a substituted or unsubstituted aliphatic, substituted or unsubstituted heteroaliphatic, substituted or unsubstituted carbocyclylene, substituted or unsubstituted heterocyclylene, substituted or unsubstituted arylene, substituted or unsubstituted heteroarylene, -O-, -N(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 -,-NRA C(=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.

[0536] In some embodiments, the 5' or 3' nucleoside of any of the oligonucleotides described herein (eg, the sense strand or antisense strand of the oligonucleotides listed in Table 8) is in the form of -NH2-(CH2) n -compound conjugation, wherein n is an integer from 1 to 12. In some embodiments, the 5' or 3' nucleoside of any one of the oligonucleotides described herein (e.g., the oligonucleotides listed in Table 9, sense strand or antisense strand) is conjugated to a compound of formula -NH2-(CH2) n -compound conjugation, wherein n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11 or 12. In some specific embodiments, n is 6. In some embodiments, in the formula NH2-(CH2) n - There is a phosphodiester linkage between the compound and the 5' or 3' nucleoside of an oligonucleotide (e.g., an oligonucleotide listed in Table 8, sense strand or antisense strand), wherein n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9, 10, 11 or 12. In some specific embodiments, n is 6.

[0537] In some embodiments, the 5' nucleoside of the sense strand of any one of the oligonucleotides described herein (eg, the oligonucleotides listed in Table 8) is in the formula -NH2-(CH2) n -compound conjugation, wherein n is an integer from 1 to 12 (eg, 6).

[0538] In some embodiments, the 5' nucleoside of the sense strand of any one of the oligonucleotides described herein (eg, the oligonucleotides listed in Table 9) is in the formula -NH2-(CH2) n -compound conjugation, wherein n is an integer from 1 to 12 (eg, 6).

[0539] In some embodiments, the 3' nucleoside of the sense strand of any one of the oligonucleotides described herein (eg, the oligonucleotides listed in Table 8) is in the formula -NH2-(CH2) n -compound conjugation, wherein n is an integer from 1 to 12 (eg, 6).

[0540] In some embodiments, the 3' nucleoside of the sense strand of any one of the oligonucleotides described herein (eg, the oligonucleotides listed in Table 9) is in the formula -NH2-(CH2) n -compound conjugation, wherein n is an integer from 1 to 12 (eg, 6).

[0541] In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to the oligonucleotide by reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the oligonucleotide (e.g., the 5' phosphate of the sense strand or the antisense strand). In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to the oligonucleotide by reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the sense strand of the oligonucleotide (e.g., the oligonucleotides listed in Table 8). In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to the oligonucleotide by reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the sense strand of the oligonucleotide (e.g., the oligonucleotides listed in Table 9). In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to an oligonucleotide by a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the antisense strand of an oligonucleotide (e.g., an oligonucleotide listed in Table 8). In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to an oligonucleotide by a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 5' phosphate of the antisense strand of an oligonucleotide (e.g., an oligonucleotide listed in Table 9). In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to an oligonucleotide by a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3' phosphate of an oligonucleotide (e.g., the 3' phosphate of the sense strand or the antisense strand). In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to an oligonucleotide by a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3' phosphate of the sense strand of an oligonucleotide (e.g., an oligonucleotide listed in Table 8). In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to an oligonucleotide by a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3' phosphate of the sense strand of an oligonucleotide (e.g., an oligonucleotide listed in Table 9). In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to an oligonucleotide by a reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3' phosphate of the antisense strand of an oligonucleotide (e.g., an oligonucleotide listed in Table 8). In some embodiments, the compound of formula NH2-(CH2)6- is conjugated to an oligonucleotide by reaction between 6-amino-1-hexanol (NH2-(CH2)6-OH) and the 3' phosphate of the antisense strand of an oligonucleotide (e.g., an oligonucleotide listed in Table 9). In some embodiments, the oligonucleotide is conjugated to a targeting agent, such as a muscle targeting agent, such as an anti-TfR1 antibody, via, for example, an amine group.

[0542] In some embodiments, the oligonucleotides described herein (eg, siRNA molecules listed in Table 8 or Table 9) have reduced off-target effects, for example, compared to other known DUX4-targeting siRNAs.

[0543] C. Connector

[0544] The complex described herein generally comprises a linker that covalently links any anti-TfR1 antibody described herein to a molecular payload. The linker comprises at least one covalent bond. In some embodiments, the linker may be a single bond that covalently links an anti-TfR1 antibody to a molecular payload, such as a disulfide bond or a disulfide bridge. However, in some embodiments, the linker may covalently link any anti-TfR1 antibody described herein to a molecular payload via multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker. The linker is generally stable in vitro and in vivo, and may be stable in certain cellular environments. In addition, generally speaking, the linker does not negatively affect the functional properties of the anti-TfR1 antibody or molecular payload. Some examples and methods of linker synthesis are known in the art (see, e.g., Kline, T. et al. "Methods to Make Homogenous Antibody Drug Conjugates." Pharmaceutical Research, 2015, 32: 11, 3480-3493.; Jain, N. et al. "Current ADC Linker Chemistry" Pharm Res. 2015, 32: 11, 3526-3540.; McCombs, JR and Owen, SC "Antibody Drug Conjugates: Design and Selection of Linker, Payload and Conjugation Chemistry" AAPS J. 2015, 17: 2, 339-351.).

[0545] The joint will generally include two different reactive substances that allow connection to both the anti-TfR1 antibody and the molecular payload. In some embodiments, the two different reactive substances can be nucleophiles and / or electrophiles. In some embodiments, the joint includes two different electrophiles or nucleophiles, which are specific to two different nucleophiles or electrophiles. In some embodiments, the joint is covalently linked to the anti-TfR1 antibody by conjugating to the lysine residues or cysteine ​​residues of the anti-TfR1 antibody. In some embodiments, the joint is covalently linked to the cysteine ​​residues of the anti-TfR1 antibody by a maleimide-containing joint, wherein optionally, the maleimide-containing joint includes maleimidohexanoyl or maleimidomethylcyclohexane-1-carboxylate groups. In some embodiments, the joint is covalently linked to the cysteine ​​residues of the anti-TfR1 antibody or the sulfhydryl-functionalized molecular payload by a 3-aryl propionitrile functional group. In some embodiments, the joint is covalently linked to the lysine residues of the anti-TfR1 antibody. In some embodiments, the linker is independently covalently attached to the anti-TfRl antibody and / or (eg, and) the molecular payload via an amide bond, a carbamate bond, a hydrazide, a triazole, a thioether, and / or a disulfide bond.

[0546] i. Cleavable Linkers

[0547] The cleavable linker can be a protease-sensitive linker, a pH-sensitive linker or a glutathione-sensitive linker. These linkers are usually only cleavable intracellularly and are preferably stable in an extracellular environment, such as the extracellular space of a muscle cell.

[0548] Protease sensitive joints can be cut by protease enzyme activity. These joints usually contain peptide sequences, and the length can be 2 to 10 amino acids, about 2 to 5 amino acids, about 5 to 10 amino acids, about 10 amino acids, about 5 amino acids, about 3 amino acids or about 2 amino acids. In some embodiments, the peptide sequence may include naturally occurring amino acids (e.g., cysteine, alanine) or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include β-amino acids, high amino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids and other amino acids known in the art. In some embodiments, protease sensitive joints include valine-citrulline or alanine-citrulline sequences. In some embodiments, protease sensitive joints can be cut by lysosomal proteases (e.g., cathepsin B) and / or (e.g., and) endosomal proteases.

[0549] A pH sensitive linker is a covalent linkage that is easily degraded in a high or low pH environment. In some embodiments, the pH sensitive linker can be cleaved at a pH ranging from 4 to 6. In some embodiments, the pH sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH sensitive linker is cleaved in an endosome or lysosome.

[0550] In some embodiments, the glutathione-sensitive linker comprises a disulfide portion. In some embodiments, the glutathione-sensitive linker is cleaved by a disulfide exchange reaction with an intracellular glutathione substance. In some embodiments, the disulfide portion further comprises at least one amino acid, such as a cysteine ​​residue.

[0551] In some embodiments, the linker comprises a valine-citrulline sequence (e.g., as described in U.S. Pat. No. 6,214,345, which is incorporated herein by reference). In some embodiments, prior to conjugation, the linker comprises the following structure:

[0552]

[0553] In some embodiments, after conjugation, the linker comprises the following structure:

[0554]

[0555] In some embodiments, prior to conjugation, the linker comprises a structure of Formula (A):

[0556]

[0557] wherein n is any number from 0 to 10. In some embodiments, n is 3.

[0558] In some embodiments, the linker comprises a structure of Formula (H):

[0559]

[0560] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and / or (e.g., and) m is 4.

[0561] In some embodiments, the linker comprises the structure of Formula (I):

[0562]

[0563] wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and / or (e.g., and) m is 4.

[0564] ii. Non-cleavable linker

[0565] In some embodiments, a non-cleavable linker can be used. Generally speaking, a non-cleavable linker is not easily degraded in a cell or physiological environment. In some embodiments, a non-cleavable linker comprises an optionally substituted alkyl group, wherein the substitution may include halogens, hydroxyls, oxygen species, and other common substitutions. In some embodiments, the linker may comprise an optionally substituted alkyl group, an optionally substituted alkylene group, an optionally substituted arylene group, a heteroarylene group, a peptide sequence comprising at least one non-natural amino acid, a truncated polysaccharide, one or more sugars that cannot be degraded by an enzyme, an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, biotin, a biphenyl, a repeating unit of polyethylene glycol or an equivalent compound, an acidic ester, an amide, a sulfonamide, and / or an alkoxy-amine linker. In some embodiments, a sortase-mediated connection can be used to covalently link an anti-TfR1 antibody comprising an LPXT sequence to a polypeptide comprising (G) n Molecular payload of the sequence (see, for example, ProftT. Sortase-mediated protein ligation: an emerging biotechnology tool for protein modification and immobilization. Biotechnol Lett. 2010, 32(1): 1-10.).

[0566] In some embodiments, the linker can comprise a substituted alkylene, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O, and S; an optionally substituted heterocyclylene further comprising at least one heteroatom selected from N, O, and S; an imino group, an optionally substituted nitrogen species, an optionally substituted oxygen species O, an optionally substituted sulfur species, or a poly(alkylene oxide), such as polyethylene oxide or polypropylene oxide. In some embodiments, the linker can be a non-cleavable N-gamma-maleimidobutyryl-oxysuccinimide ester (GMBS) linker.

[0567] iii. Linker conjugation

[0568] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload via a phosphate, thioether, ether, carbon-carbon, carbamate, or amide bond. In some embodiments, the linker is covalently linked to the oligonucleotide via a phosphate or phosphorothioate group, such as a terminal phosphate of the oligonucleotide backbone. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody via a lysine or cysteine ​​residue present on the anti-TfR1 antibody.

[0569] In some embodiments, the linker or a portion thereof is covalently linked to an anti-TfR1 antibody and / or (e.g., and) a molecular payload to form a triazole by a cycloaddition reaction between an azide and an alkyne, wherein the azide or alkyne may be located on the anti-TfR1 antibody, the molecular payload, or the linker. In some embodiments, the alkyne may be a cycloalkyne, such as a cyclooctyne. In some embodiments, the alkyne may be a bicyclononyne (also referred to as bicyclo[6.1.0]nonyne or BCN) or a substituted bicyclononyne. In some embodiments, the cyclooctane is as described in International Patent Application Publication WO2011136645, which was published on November 3, 2011, entitled "Fused Cyclooctyne Compounds And Their Use In Metal-freeClick Reactions". In some embodiments, the azide may be a sugar or carbohydrate molecule comprising an azide. In some embodiments, the azide may be 6-azido-6-deoxygalactose or 6-azido-N-acetylgalactosamine. In some embodiments, the sugar or carbohydrate molecule comprising an azide is as described in International Patent Application Publication No. WO2016170186, published on October 27, 2016, entitled “Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”. In some embodiments, a cycloaddition reaction is performed between an azide and an alkyne to form a triazole, wherein the azide or alkyne may be located on an anti-TfR1 antibody, a molecular payload, or a linker as described in International Patent Application Publication WO2014065661, published on May 1, 2014, entitled “Modified antibody, antibody-conjugate and process for the preparation thereof”; or International Patent Application Publication WO2016170186, published on October 27, 2016, entitled “Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase”.

[0570] In some embodiments, the linker comprises a spacer, such as a polyethylene glycol spacer or an acyl / carbamoylsulfonamide spacer, such as HydraSpace TM Spacer. In some embodiments, the spacer is as described in Verkade, JMMet al., "A Polar Sulfamide Spacer Significantly Enhances the Manufacturability, Stability, and Therapeutic Index of Antibody-Drug Conjugates", Antibodies, 2018, 7, 12.

[0571] In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload by a Diels-Alder reaction between a dienophile and a diene / heterodiene, wherein the dienophile or diene / heterodiene may be located on the anti-TfR1 antibody, molecular payload, or linker. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload by other pericyclic reactions such as ene reactions. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload by amide, thioamide, or sulfonamide bonding reactions. In some embodiments, the linker is covalently linked to the anti-TfR1 antibody and / or (e.g., and) molecular payload by a condensation reaction to form an oxime, hydrazone, or semicarbazide group present between the linker and the anti-TfR1 antibody and / or (e.g., and) molecular payload.

[0572] In some embodiments, the linker is covalently linked to an anti-TfR1 antibody and / or (e.g., and) a molecular payload by a conjugate addition reaction between a nucleophile (e.g., an amine or hydroxyl) and an electrophile (e.g., a carboxylic acid, a carbonic acid, or an aldehyde). In some embodiments, prior to the reaction between the linker and the anti-TfR1 antibody or the molecular payload, the nucleophile may be present on the linker and the electrophile may be present on the anti-TfR1 antibody or the molecular payload. In some embodiments, prior to the reaction between the linker and the anti-TfR1 antibody or the molecular payload, the electrophile may be present on the linker and the nucleophile may be present on the anti-TfR1 antibody or the molecular payload. In some embodiments, the electrophile can be an azide, a pentafluorophenyl group, a silicon center, a carbonyl group, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimide ester, a sulfosuccinimide ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl group, an activated phosphorus center, and / or an activated sulfur center. In some embodiments, the nucleophile can be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl group, an optionally substituted heterocyclyl group, a hydroxyl group, an amino group, an alkylamino group, an anilino group, and / or a thiol group.

[0573] In some embodiments, the linker comprises a valine-citrulline sequence covalently linked to a reactive chemical moiety (e.g., an azide moiety or a BCN moiety for click chemistry). In some embodiments, the linker comprising a valine-citrulline sequence covalently linked to a reactive chemical moiety (e.g., an azide moiety for click chemistry) comprises the structure of formula (A):

[0574]

[0575] wherein n is any number from 0 to 10. In some embodiments, n is 3.

[0576] In some embodiments, a linker comprising a structure of formula (A) (e.g., optionally via another chemical moiety) is covalently linked to a molecular payload (e.g., an oligonucleotide). In some embodiments, the molecular payload is a double-stranded siRNA oligonucleotide, and a linker comprising a structure of formula (A) (e.g., optionally via another chemical moiety) is covalently linked to the sense strand of the siRNA oligonucleotide (e.g., at the 5' end). In some embodiments, the molecular payload is a double-stranded siRNA oligonucleotide, and a linker comprising a structure of formula (A) (e.g., optionally via another chemical moiety) is covalently linked to the sense strand of the siRNA oligonucleotide (e.g., at the 3' end). In some embodiments, a linker comprising a structure of formula (A) is covalently linked to an oligonucleotide, for example, by nucleophilic substitution with an amine-L1-oligonucleotide to form a carbamate bond, thereby generating a compound comprising a structure of formula (B):

[0577]

[0578] Wherein n is any number from 0 to 10. In some embodiments, n is 3. In some embodiments, the oligonucleotide comprising the compound of formula (B) structure comprises the sense strand of the siRNA oligonucleotide. In some embodiments, the antisense strand is annealed to the sense strand of the siRNA oligonucleotide (e.g., at an annealing temperature of 25°C to 105°C). In some embodiments, the annealing step is performed at the following annealing temperature: 25°C to 150°C (e.g., 25°C to 150°C, 25°C to 100°C, 25°C to 75°C, 25°C to 50°C, 25°C to 40°C, 25°C to 30°C, 30°C to 150°C, 30°C to 100°C, 30°C to 75°C, 30°C to 50°C, 30°C to 40°C, 75°C to 150°C, 75°C to 125°C, or 75°C to 100°C). In some embodiments, the annealing step is performed at an annealing temperature of 100°C. For example, the annealing reaction mixture can be incubated at 100° C. (e.g., 3, 4, 5, 6, 7, 8, 9, 10 minutes or longer) and allowed to cool at room temperature to anneal the sense and antisense strands. In some embodiments, the annealing step is performed at an annealing temperature of 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., 37° C., 38° C., 39° C., 40° C. In some embodiments, the annealing step is performed at an annealing temperature of 30° C. For example, the annealing reaction mixture can be incubated at 30° C. (e.g., 1 to 24 hours, e.g., 1 to 24 hours, 5 to 20 hours, 5 to 15 hours, 5 to 10 hours, 1 to 5 hours, 10 to 24 hours, 10 to 20 hours, 10 to 15 hours, 15 to 24 hours, 15 to 20 hours, or 20 to 24 hours, or overnight) to anneal the sense strand and the antisense strand. In some embodiments, the oligonucleotide comprising a compound of formula (B) structure comprises an siRNA oligonucleotide comprising a sense strand and an antisense strand, wherein the 5' end of the sense strand is covalently linked to L1. In some embodiments, the oligonucleotide of formula (B) comprises an siRNA oligonucleotide comprising a sense strand and an antisense strand, wherein the 3' end of the sense strand is covalently linked to L1.

[0579] In some embodiments, the compound of formula (B) is further covalently linked to an additional moiety via a triazole, wherein the triazole is formed by a click reaction between an alkyne provided on the bicyclononyne and an azide of formula (A) or formula (B). In some embodiments, the compound comprising the bicyclononyne comprises the structure of formula (C):

[0580]

[0581] wherein m is any number from 0 to 10. In some embodiments, m is 4. In some embodiments, in the click reaction between the compound comprising the structure of formula (B) and the compound comprising the structure of formula (C), the compound of formula (C) is in molar excess (e.g., the molar ratio of (C) to (B) is 3:1 or 4:1). In some embodiments, in the click reaction between the compound comprising the structure of formula (B) and the compound comprising the structure of formula (C), the compound of formula (B) is in molar excess (e.g., the molar ratio of (C) to (B) is 0.9:1 or 0.85:1).

[0582] In some embodiments, the azide of the compound of structure (B) forms a triazole via a click reaction with the alkyne of the compound of structure (C), thereby forming a compound comprising the structure of formula (D):

[0583]

[0584] Wherein n is any number from 0 to 10, and wherein m is any number from 0 to 10. In some embodiments, n is 3 and m is 4. In some embodiments, the oligonucleotide of formula (D) comprises an siRNA oligonucleotide comprising a sense strand and an antisense strand, wherein the 5' end of the sense strand is covalently linked to L1. In some embodiments, the oligonucleotide of formula (D) comprises an siRNA oligonucleotide comprising a sense strand and an antisense strand, wherein the 3' end of the sense strand is covalently linked to L1. In some embodiments, the compound comprising the structure of formula (D) is separated before further covalently linking to the lysine of the anti-TfR1 antibody. In some embodiments, when the compound comprising the structure of formula (D) is separated before further covalently linking to the lysine of the anti-TfR1 antibody, in the click reaction between the compound comprising the structure of formula (B) and the compound comprising the structure of formula (C), the molar ratio between the compound comprising the structure of formula (C) and the compound comprising the structure of formula (B) is greater than 1, for example 3:1 or 4:1.

[0585] In some embodiments, the compound comprising the structure of formula (D) is not separated before further covalently linking to the lysine of the anti-TfR1 antibody. In some embodiments, when the compound comprising the structure of formula (D) is not separated before further covalently linking to the lysine of the anti-TfR1 antibody, in the click reaction between the compound comprising the structure of formula (B) and the compound comprising the structure of formula (C), the molar ratio between the compound comprising the structure of formula (C) and the compound comprising the structure of formula (B) is less than 1, for example 0.9:1 or 0.85:1.

[0586] In some embodiments, the compound...

Claims

1. A complex comprising a muscle targeting agent covalently linked to an oligonucleotide targeting double homeobox 4 (DUX4) RNA, wherein the oligonucleotide comprises an antisense strand having a length of 18 to 25 nucleotides and comprising a region of complementarity to the target sequence shown in SEQ ID NO: 200, 191, 189, 186, 190, 174 to 185, 187, 188, 192 to 199 and 201 to 235, and wherein the length of the region of complementarity is at least 16 consecutive nucleosides.

2. The complex of claim 1, wherein the muscle targeting agent is an anti-transferrin receptor 1 (TfR1) antibody.

3. The complex of claim 1 or claim 2, wherein the oligonucleotide is a RNAi oligonucleotide.

4. The complex of any one of claims 1 to 3, wherein the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 262, 253, 251, 262, 248, 252, 236 to 247, 249, 250, 254 to 261, 263 to 266.

5. The complex of any one of claims 1 to 4, wherein the oligonucleotide further comprises a sense strand comprising at least 18 consecutive nucleosides complementary to the antisense strand, optionally wherein the sense strand comprises 21 consecutive nucleosides complementary to the antisense strand.

6. The complex of claim 5, wherein the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs: 231, 222, 220, 217, 221, 205 to 216, 218, 219, 223, 230, 232 to 235.

7. The complex of claim 5 or claim 6, wherein the muscle targeting agent is covalently linked to the 5' end or the 3' end of the sense strand.

8. The complex of any one of claims 1 to 7, wherein the antisense strand further comprises 5'-(E)-vinylphosphonate.

9. The complex of any one of claims 1 to 8, wherein the oligonucleotide comprises one or more modified nucleosides.

10. The complex of claim 9, wherein the one or more modified nucleosides are 2' modified nucleotides, optionally wherein the one or more 2' modified nucleosides are selected from: 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-dimethylaminoethoxyethyl (2'-O-DMAEOE), 2'-ON-methylacetamido (2'-O-NMA)). The complex of claim 10 , wherein each 2′-modified nucleotide is 2′-O-methyl (2′-O-Me) or 2′-fluoro (2′-F).

12. The complex of any one of claims 1 to 11, wherein the oligonucleotide comprises one or more phosphorothioate internucleoside linkages.

13. The complex of claim 12, wherein the one or more phosphorothioate internucleoside linkages are present on the antisense strand of the oligonucleotide, optionally wherein the two internucleoside linkages at the 3' end of the antisense strand are phosphorothioate internucleoside linkages, and / or wherein the two internucleoside linkages at the 5' end of the antisense strand are phosphorothioate internucleoside linkages.

14. The complex of claim 12 or claim 13, wherein the one or more phosphorothioate internucleoside linkages are present on the sense strand of the oligonucleotide, optionally wherein the two internucleoside linkages at the 3' end of the sense strand are phosphorothioate internucleoside linkages, and / or wherein the two internucleoside linkages at the 5' end of the sense strand are phosphorothioate internucleoside linkages.

15. The complex of any one of claims 6 to 14, wherein the antisense strand is selected from modified forms of SEQ ID NOs: 236 to 266 listed in Table 8 (e.g., MAS1 to MAS4), and / or wherein the sense strand is selected from modified forms of SEQ ID NOs: 205 to 235 listed in Table 8 (e.g., MS1 to MS6).

16. The complex of any one of claims 6 to 14, wherein the antisense strand is selected from modified forms of SEQ ID NOs: 262, 253, 251, 248 and 252 listed in Table 9 (e.g., VP-MAS5, VP-MAS6 and VP-MAS7), and / or wherein the sense strand is selected from modified forms of SEQ ID NOs: 231, 222, 220, 217 and 221 listed in Table 9 (e.g., MS7 to MS9).

17. The complex of claims 1 to 15, wherein the oligonucleotide is a siRNA molecule selected from the siRNAs listed in Table 8.

18. The complex of claim 1 to 14 or 16, wherein the oligonucleotide is a siRNA molecule selected from the siRNAs listed in Table 9.

19. The complex of any one of claims 2 to 18, wherein the anti-TfR1 antibody comprises the heavy chain complementary determining region 1 (CDR-H1), heavy chain complementary determining region 2 (CDR-H2), heavy chain complementary determining region 3 (CDR-H3), light chain complementary determining region 1 (CDR-L1), light chain complementary determining region 2 (CDR-L2), and light chain complementary determining region 3 (CDR-L3) of any anti-TfR1 antibody listed in Table 2.

20. The complex of any one of claims 2 to 18, wherein the anti-TfR1 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL) of any anti-TfR1 antibody listed in Table 3.

21. The complex of any one of claims 2 to 18, wherein the anti-TfR1 antibody is a Fab, optionally wherein the Fab comprises the heavy chain and light chain of any anti-TfR1 Fab listed in Table 5.

22. The complex of any one of claims 2 to 21, wherein the anti-TfR1 antibody comprises: (i) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:27, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:28, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:29, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:30, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:31, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:32; (ii) a CDR-H1 comprising the amino acid sequence of SEQ ID NO:33, a CDR-H2 comprising the amino acid sequence of SEQ ID NO:34, a CDR-H3 comprising the amino acid sequence of SEQ ID NO:35, a CDR-L1 comprising the amino acid sequence of SEQ ID NO:36, a CDR-L2 comprising the amino acid sequence of SEQ ID NO:37, and a CDR-L3 comprising the amino acid sequence of SEQ ID NO:32; or (ii) CDR-H1 comprising the amino acid sequence of SEQ ID NO:38, CDR-H2 comprising the amino acid sequence of SEQ ID NO:39, CDR-H3 comprising the amino acid sequence of SEQ ID NO:40, CDR-L1 comprising the amino acid sequence of SEQ ID NO:41, CDR-L2 comprising the amino acid sequence of SEQ ID NO:31, and CDR-L3 comprising the amino acid sequence of SEQ ID NO:

42.

23. The complex of claim 22, wherein the anti-TfR1 antibody comprises VH and VL, the VH comprising the amino acid sequence of SEQ ID NO: 76, and the VL comprising the amino acid sequence of SEQ ID NO:

75. 24 . The complex of claim 23 , wherein the anti-TfR1 antibody is a Fab and comprises a heavy chain and a light chain, the heavy chain comprising the amino acid sequence of SEQ ID NO: 101, and the light chain comprising the amino acid sequence of SEQ ID NO:

90.

25. The complex of any one of claims 1 to 24, wherein the muscle targeting agent is covalently linked to the antisense oligonucleotide via a linker, optionally wherein the linker comprises a valine-citrulline sequence.

26. A method of reducing DUX4 expression in a muscle cell, the method comprising contacting the muscle cell with an effective amount of the complex of any one of claims 1 to 25 to promote internalization of the oligonucleotide into the muscle cell.

27. The method of claim 26, wherein reducing DUX4 expression comprises reducing DUX4 protein and / or mRNA levels.

28. A method of treating facioscapulohumeral muscular dystrophy (FSHD), the method comprising administering to a subject in need thereof an effective amount of the complex of any one of claims 1 to 25, wherein the subject has abnormal production of DUX4 protein.

29. An oligonucleotide comprising a siRNA oligonucleotide selected from the group consisting of the siRNA oligonucleotides listed in Table 8.

30. An oligonucleotide comprising a siRNA oligonucleotide selected from the group consisting of the siRNA oligonucleotides listed in Table 9.

31. A method for producing a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide, the method comprising: (i) obtaining a compound comprising the structure of formula (B), wherein the oligonucleotide comprises the sense strand of a siRNA oligonucleotide, optionally wherein the siRNA oligonucleotide targets DUX4 RNA; (ii) annealing the antisense strand of the siRNA oligonucleotide to the sense strand; (iii) reacting the compound comprising the structure of formula (B) with the compound comprising the structure of formula (C) to obtain a compound comprising the structure of formula (D); and (iv) covalently linking the anti-TfR1 antibody to the compound comprising the structure of formula (D) with a muscle targeting agent to obtain a compound comprising the structure of formula (E), Optionally, wherein the annealing in step (ii) is performed at 30°C, Optionally, the method further comprises isolating the compound comprising the structure of formula (D) after step (iii) and before step (iv); Optionally, the anti-TfR1 antibody is covalently linked to the 5' end of the sense strand of the siRNA oligonucleotide targeting DUX4 RNA.

32. A method for producing a complex comprising an anti-transferrin receptor 1 (TfR1) antibody covalently linked to an oligonucleotide, the method comprising: (i) obtaining a compound comprising the structure of formula (B), wherein the oligonucleotide comprises the sense strand of a siRNA oligonucleotide, optionally wherein the siRNA oligonucleotide targets DUX4 RNA; (ii) annealing the antisense strand of the siRNA oligonucleotide to the sense strand; (iii) covalently linking an anti-TfR1 antibody to a compound comprising the structure of formula (C) to obtain a compound comprising the structure of formula (F); and (iv) reacting the compound comprising the structure of formula (F) with the compound comprising the structure of formula (B) to obtain a compound comprising the structure of formula (E), Optionally, wherein the annealing in step (ii) is performed at 30°C, Optionally, the anti-TfR1 antibody is covalently linked to the 5' end of the sense strand of the siRNA oligonucleotide targeting DUX4 RNA.

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