CN119630407A

CN119630407A - Muscle targeting complexes and their use for the treatment of facial shoulder humerus muscular dystrophy

Info

Publication number: CN119630407A
Application number: CN202380056783.2A
Authority: CN
Inventors: 内尔松·赫西亚; 蒂莫西·威登; 斯特凡诺·扎诺蒂; 奥克萨娜·别斯克罗夫纳亚; 约亨·德克特; 汉斯-彼得·福恩洛克; 罗梅什·R·苏布拉马尼亚; 穆罕默德·T·卡塔纳尼; 科迪·A·德雅尔丹; 布伦丹·奎因; 约翰·纳吉姆; 马库斯·霍斯巴赫; 卡特林·胡尔奇; 邱奇峰; 斯科特·希尔德布兰德
Original assignee: Dyne Therapeutics Inc
Current assignee: Dyne Therapeutics Inc
Priority date: 2022-07-06
Filing date: 2023-07-05
Publication date: 2025-03-14
Also published as: WO2024011135A3; WO2024011135A2; KR20250034419A; AU2023303019A1; IL317871A

Abstract

Some aspects of the disclosure relate to oligonucleotides (e.g., RNAi oligonucleotides, e.g., siRNA) that are intended to target DUX4RNA and targeting complexes for delivering the oligonucleotides to cells (e.g., muscle cells) and uses thereof, particularly to uses in the treatment of diseases (e.g., FSHD).

Description

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

RELATED APPLICATIONS

The present application is in accordance with 35U.S. c. ≡119 (e) claims the benefit of U.S. provisional application No.63/367,783 entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY" filed on 7/6 at 2022 and U.S. provisional application No.63/477,160 entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY" filed on 23 at 12/2022, each of which is incorporated herein by reference in its entirety.

Technical Field

The present application relates to oligonucleotides intended for targeting DUX4 RNA and targeting complexes for delivering a molecular payload (e.g., an oligonucleotide) to a cell and uses thereof, in particular to the use of disease treatment.

Reference to electronic sequence Listing

The contents of the electronic sequence Listing (D082470078 WO00-SEQ-CBD. Xml; size: 339,190 bytes; and date of creation: 2023, 6, 30) are incorporated herein by reference in their entirety.

Background

Muscular dystrophy (muscular dystrophy, MD) is a group of diseases characterized by progressive weakness and reduced muscle mass. These diseases are caused by mutations in the genes encoding proteins required for healthy muscle tissue. Facial shoulder brachial muscular dystrophy (facioscapulohumeral muscular dystrophy, FSHD) is a dominant genetic type of MD that affects mainly the muscles of the face, shoulder blade and upper arm. Other symptoms of FSHD include abdominal muscle weakness, retinal abnormalities, hearing loss, joint pain and inflammation. FSHD is the most common of the nine types of MD affecting both adults and children, with a worldwide incidence of about 1 out of every 8,300. FSHD is caused by the abnormal production of double homeobox 4 (double homeobox, DUX 4), 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 testes and thymus of adults and in 2-cell stage embryos, after which it is inhibited by hypermethylation of the D4Z4 repeat that surrounds and compacts the DUX4 gene. Two types of FSHD, types 1 and 2, have been described. Type 1, accounting for about 95% of cases, is associated with a deletion of D4Z4 repeats in the subtelomere region of chromosome 4. Unaffected individuals typically have more than 10 repeats arranged in the subtelomere region of chromosome 4, whereas the most common form of FSHD (FSHD 1) is caused by array shrinkage to less than 10 repeats, associated with diversified expression of DUX4 in skeletal muscle and reduced epigenetic inhibition. Two allelic variants of chromosome 4q (4 qA and 4 qB) exist in the most distal unit of the D4Z4 repeat. The 4qA is in cis form with a functional polyadenylation consensus site. Contraction of the 4qA allele is pathogenic because the DUX4 transcript is polyadenylation and stable. About 5% of cases of FSHD type 2 is associated with mutations in the SMCHD1 gene on chromosome 18. Type 2 FSHD may also be associated with DNMT3B gene or LRIF a gene. Apart from supportive care and treatment of disease symptoms, there is no effective treatment for FSHD.

Disclosure of Invention

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

According to some aspects, the present disclosure provides complexes that target muscle cells (e.g., primary myoblasts) for the purpose of delivering molecular payloads (e.g., oligonucleotides described herein) to those cells. In some embodiments, the complexes provided herein are particularly useful for delivering a molecular payload that inhibits expression or activity of DUX4 (e.g., in a subject having or suspected of having FSHD). Thus, in some embodiments, the complexes provided herein comprise 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 the 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 engineered to deliver an oligonucleotide may release the oligonucleotide such that the oligonucleotide may inhibit DUX4 gene expression in a muscle cell. In some embodiments, the oligonucleotide in the ligation complex is released by endosomal cleavage of the covalent linker linking the oligonucleotide and the muscle targeting agent.

Some aspects of the disclosure provide complexes comprising a muscle targeting agent covalently linked to an oligonucleotide targeting double homologous box 4 (DUX 4) RNA (e.g., mRNA), wherein the oligonucleotide comprises an antisense strand of 18 to 25 nucleotides in length and comprises a complementary region of the target sequence shown in SEQ ID NOs 174 to 235, and wherein the complementary region is at least 16 contiguous nucleosides in length.

Some aspects of the disclosure provide complexes comprising a muscle targeting agent covalently linked to an oligonucleotide targeting double homologous box 4 (DUX 4) RNA (e.g., mRNA), wherein the oligonucleotide comprises an antisense strand of 18 to 25 nucleotides in length and comprises a region of complementarity to a target sequence set forth 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.

In some embodiments, the muscle targeting agent is an anti-transferrin receptor 1 (TRANSFERRIN RECEPTOR, tfr 1) antibody.

In some embodiments, the oligonucleotide is an RNAi oligonucleotide.

In some embodiments, the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs 236 to 266.

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

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

In some embodiments, the sense strand comprises 21 contiguous nucleosides complementary to the antisense strand.

In some embodiments, the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs 205 to 235.

In some embodiments, 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.

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

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

In some embodiments, the oligonucleotide comprises one or more modified nucleosides.

In some embodiments, one or more modified nucleosides is a2 'modified nucleotide, optionally wherein the one or more 2' modified nucleosides are selected from the group consisting of 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 '-O-N-methylacetamido (2' -O-NMA)).

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).

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

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

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.

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

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.

In some embodiments, one or more cytidine of the oligonucleotide is a 2 '-modified 5-methylcytidine, optionally wherein the 2' -modified 5-methylcytidine is a 2'-O-Me modified 5-methylcytidine or a 2' -F modified 5-methylcytidine.

In some embodiments, the antisense strand of an oligonucleotide described herein is selected from the modified forms of SEQ ID NOs 236 to 266 (e.g., MAS1 to MAS 4) set forth in Table 8. For example, in some embodiments, the antisense strand is selected from:

MAS1-236,MAS1-237,MAS1-238,MAS2-236,MAS2-237,MAS2-238,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,MAS2-257,MAS3-236,MAS3-237,MAS3-238-MAS3-239,MAS3-240,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,MAS3-259,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

In some embodiments, the sense strand of the oligonucleotides described herein is selected from modified versions (e.g., MS1 to MS 6) of SEQ ID NOS 205 to 235, listed in Table 8. For example, in some embodiments, the sense strand is selected from:

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,

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.

In some embodiments, the antisense strand is selected from modified forms of SEQ ID NOS 262, 253, 251, 248 and 252 set forth in Table 9 (e.g., VP-MAS5, VP-MAS6 and VP-MAS 7), and/or wherein the sense strand is selected from modified forms of SEQ ID NOS 231, 222, 220, 217 and 221 set forth in Table 9 (e.g., MS7 through MS 9).

In some embodiments, the oligonucleotide is an siRNA molecule selected from the group consisting of the sirnas listed in table 8.

In some embodiments, the oligonucleotide is an siRNA molecule selected from the group consisting of the sirnas listed in table 9.

In some embodiments, an anti-TfR 1 antibody comprises heavy chain complementarity determining region 1 (HEAVY CHAIN complementarity determining region, cdr-H1), heavy chain complementarity determining region 2 (HEAVY CHAIN complementarity determining region, cdr-H2), heavy chain complementarity determining region 3 (HEAVY CHAIN complementarity determining region, cdr-H3), light chain complementarity determining region 1 (LIGHT CHAIN complementarity determining region, cdr-L1), light chain complementarity determining region 2 (LIGHT CHAIN complementarity determining region, cdr-L2), light chain complementarity determining region 3 (LIGHT CHAIN complementarity determining region, cdr-L3) of any of the anti-TfR 1 antibodies listed in table 2.

In some embodiments, an anti-TfR 1 antibody comprises a heavy chain variable region (HEAVY CHAIN variable region, VH) and a light chain variable region (LIGHT CHAIN variable region, VL) of any of the anti-TfR 1 antibodies listed in table 3.

In some embodiments, the anti-TfR 1 antibody is a Fab, optionally wherein the Fab comprises the heavy and light chains of any of the anti-TfR 1 fabs listed in table 5.

In some embodiments, an anti-TfR 1 antibody comprises:

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

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

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

In some embodiments, an anti-TfR 1 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.

In some embodiments, the anti-TfR 1 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.

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

Also provided herein are methods of reducing DUX4 expression in a muscle cell 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.

Also provided herein are methods of treating facial shoulder humeral 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 a DUX4 protein.

Further aspects of the disclosure provide oligonucleotides comprising siRNA oligonucleotides selected from the group consisting of the siRNA oligonucleotides listed in table 8.

Further aspects of the disclosure provide oligonucleotides comprising siRNA oligonucleotides selected from the group consisting of the siRNA oligonucleotides listed in table 9.

Also provided herein are methods of producing a complex comprising an anti-transferrin receptor 1 (TfR 1) antibody covalently linked to an oligonucleotide, the method comprising (i) obtaining a compound comprising the structure of formula (B), wherein the oligonucleotide comprises a sense strand of an siRNA oligonucleotide, optionally wherein the siRNA oligonucleotide targets a 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-TfR 1 antibody to a compound comprising the structure of formula (D) with a muscle targeting agent to obtain a compound comprising the structure of formula (E), optionally wherein annealing in step (ii) is performed at 30 ℃, further optionally wherein the method further comprises separating the compound comprising the structure of formula (D) after step (iii) and before step (iv), further wherein the anti-TfR 1 antibody is covalently linked to the target of the DUX4 RNA.

Also provided herein are methods of producing a complex comprising an anti-transferrin receptor 1 (TfR 1) 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 a DUX4 RNA, (ii) annealing the antisense strand of the siRNA oligonucleotide to the sense strand, (iii) covalently linking the anti-TfR 1 antibody to the compound comprising a structure of formula (C) to obtain a compound comprising a structure of formula (F), and (iv) reacting the compound comprising a structure of formula (F) to the compound comprising a structure of formula (B), optionally wherein the annealing in step (ii) is performed at 30 ℃, further optionally wherein the anti-TfR 1 antibody is covalently linked to the 5' end of the sense strand of the siRNA oligonucleotide that targets the DUX4 RNA.

Drawings

FIG. 1 shows a non-limiting schematic diagram showing the effect of transfecting cells with siRNA. The graph shows the effect of siRNA on HPRT mRNA levels.

Figure 2 shows a non-limiting schematic showing the activity of muscle targeting complexes comprising siRNA. In vitro activity of the complexes was measured according to HPRT mRNA levels in cultured myocytes. The complex comprises an siRNA linked to a muscle targeting antibody.

Figures 3A to 3B show non-limiting schematic diagrams showing in vivo activity in mouse muscle tissue (gastrocnemius and heart) relative to vehicle-treated controls. (n=4C 57BL/6WT mice) the in vivo activity of the complexes was measured. The complex comprises an siRNA linked to a muscle targeting antibody.

Figures 4A to 4E show non-limiting schematic diagrams showing tissue selectivity of muscle targeting complexes comprising siRNA. The complex comprises an siRNA linked to a muscle targeting antibody.

FIG. 5 shows the integrated scores of mRNA levels of three DUX4 transcriptome markers (MBD 3L2, TRIM43 and ZCAN 4) in AB1080 immortalized FSHD patient-derived myotubes after incubation with siRNA conjugates comprising anti-TfR Fab 3M12 VH4/V kappa 3 covalently linked to siRNA-A, siRNA-B or siRNA-C. The anti-TfR Fab is covalently linked to the 3' end of each siRNA sense strand through a linker, and the corresponding antisense strand anneals to the sense strand.

FIG. 6 shows the combined scores of the mRNA levels of the DUX4 transcriptome markers MBD3L2, TRIM43 and ZCAN 4 in FSHD patient-derived C6 myotubes after transfection with siRNA 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 concentrations of 0.2nM or 20 nM. The siRNA numbers correspond to those in table 8.

Figures 7A through 7C show the resulting dose response curves 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 those in table 8. In cells treated with a range of concentrations (2 pM to 40 nM) of the indicated siRNA, the combined scores of DUX4 transcriptome markers MBD3L2, TRIM43 and ZSCAN4 were used to generate dose response curves.

FIG. 8 shows the combined scores of mRNA levels of three DUX4 transcriptome markers (MBD 3L2, TRIM43 and ZCAN 4) in FSHD patient-derived C6 myotubes after treatment with siRNA antibody complexes at concentrations of 10nM, 100nM or 1000 nM. The siRNA-antibody complex contained anti-TfR 1 Fab 3M12VH4/Vκ3 covalently linked to DUX 4-targeted siRNA. The SiRNAs tested were SiRNAs 142 through 148. The siRNA numbers correspond to those in table 9. Exon 1 of the targeted siRNA was used as a positive control.

Figures 9A to 9B show the combined scores of mRNA levels of three DUX4 mouse transcriptome markers (Wfdc, ord, serpin 6 c) in the mouse quadriceps (figure 9A) and gastrocnemius (figure 9B) relative to vehicle-treated controls. Tissues were harvested 4 weeks after a single intravenous injection of the siRNA complex covalently linked to anti-TfR 1 Fab 3M12 VH4/V.kappa.3 with a dose of 10mg/kg of the DUX 4-targeted siRNA complex. The sirnas tested were siRNA145, siRNA148, siRNA144, siRNA143 and siRNA142. The siRNA numbers correspond to those in table 9.

Figure 10 shows an example of a schematic illustrating how anti-TfR 1-siRNA complexes described herein can be prepared.

Detailed Description

Some aspects of the disclosure provide oligonucleotides that are directed to target DUX4 RNA. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA that can be used to reduce the level of DUX4 RNA and/or protein. In some embodiments, the oligonucleotides provided herein are intended to direct RNAi-mediated degradation of DUX4 RNA. In some embodiments, the oligonucleotides are intended to effectively engage the RNA-induced silencing complex (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 characteristics. In some embodiments, the oligonucleotides are intended to have desired binding affinity properties. In some embodiments, the oligonucleotides are intended to have a desired toxicity and/or immunogenicity profile. Abnormal (e.g., increased) expression of DUX4 RNA and/or protein is associated with features of the facial shoulder brachial muscular dystrophy (FSHD) pathology, including muscle atrophy, inflammation, and reduced differentiation potential, as well as oxidative stress. In some embodiments, the oligonucleotides described herein that reduce DUX4 RNA and/or protein levels are effective in treating FSHD.

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

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

I. Definition of the definition

Administration the term "administration" or variations thereof as used herein means providing a complex to a subject in a physiologically and/or (e.g., and) pharmacologically useful manner (e.g., to treat a disorder in a subject).

About the term "about" or "about" as used herein, as applied to one or more target values, refers to values similar to the stated reference values. In certain embodiments, the term "about" or "approximately" 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 or less) of the stated reference value, unless stated otherwise or otherwise apparent from the context (unless such a number exceeds 100% of the possible values).

Antibodies the term "antibody" as used herein refers to a polypeptide comprising at least one immunoglobulin variable domain or at least one epitope, e.g., paratope (paratope) that specifically binds 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, fab 'fragment, F (ab') 2 fragment, fv fragment, or scFv fragment. In some embodiments, the antibody is a nanobody derived from a camelidae antibody or a nanobody derived from a shark antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody comprises a framework with human germline sequences. In another embodiment, the antibody comprises a heavy chain constant domain selected from the group consisting of IgG, igG1, igG2A, igG, B, igG, C, igG3, igG4, igA1, igA2, igD, igM, and IgE constant domains. In some embodiments, the antibody comprises a heavy (H) chain variable region (abbreviated herein as VH) and/or a light (L) chain variable region (abbreviated herein as VL). In some embodiments, the antibody comprises a constant domain, such as an Fc region. Immunoglobulin constant domain refers to either a heavy chain constant domain or a light chain constant domain. The amino acid sequences of the human IgG heavy and light chain constant domains and their functional variations are known. With respect to heavy chains, in some embodiments, the heavy chains of the antibodies described herein may be alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chains. In some embodiments, the heavy chain of an antibody described herein can comprise a human alpha (α), delta (Δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. in a specific embodiment, an 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 E A et al, (1991) supra. In some embodiments, a VH domain comprises an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or at least 99% identical to any of the variable chain constant regions provided herein. In some embodiments, the antibody is modified, e.g., by glycosylation, phosphorylation, SUMO (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, glycosyl phosphatidyl inositol (GPI anchor attachment), and/or (e.g., and) phosphoglycosylation (phosphoglycosylation). In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules comprise mannose units, glucose units, N-acetylglucosamine units, N-acetylgalactosamine units, galactose units, fucose units, or phospholipid units. In some embodiments, an antibody is a construct comprising a polypeptide comprising one or more antigen binding fragments of the present disclosure linked to a linker polypeptide or immunoglobulin constant domain. The linker polypeptide comprises two or more amino acid residues linked by peptide bonds and is used to link one or more antigen binding portions. Some examples of linker polypeptides have been reported (see, e.g., ,Holliger,P.,et al.(1993)Proc.Natl.Acad.Sci.USA90:6444-6448;Poljak,R.J.,et al.(1994)Structure 2:1121-1123). additionally, antibodies 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 streptavidin core regions to make tetrameric scFv molecules (Kipriyanov, S.M., et al (1995) Human Antibodies and Hybridomas 6:93-101), and the use of cysteine residues, tag peptides and C-terminal polyhistidine tags to make bivalent and biotinylated scFv molecules (Kipriyanov, S.M., et al (1994) mol. Immunol.31:1047-1058).

CDRs As used herein, the term "CDR" refers to complementarity determining regions within the variable sequences of an antibody. Typical antibody molecules comprise a heavy chain variable region (VH) and a light chain variable region (VL), which are typically involved in antigen binding. The VH and VL regions may be further subdivided into regions of higher variability, also known as "complementarity determining regions" ("complementarity determining region, CDRs") interspersed with regions that are more conserved, known as "framework regions" ("FR"). Each VH and VL is typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The framework regions and CDR ranges may be precisely identified using methods known in the art, such as by Kabat definition, IMGT definition, chothia definition, abM definition, and/or (e.g., and) contact definition (all of which are well known in the art). See, for example Kabat,E.A.,et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242;the international ImMunoGeneTics informationwww.imgt.org,Lefranc,M.-P.et al.,Nucleic Acids Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[Epub],5:45-60(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,33:D593-597(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,37:D1006-1012(2009);Lefranc,M.-P.et al.,Nucleic Acids Res.,43:D413-422(2015);Chothia et al.,(1989)Nature 342:877;Chothia,C.et al.(1987)J.Mol.Biol.196:901-917;Al-lazikani et al(1997)J.Molec.Biol.273:927-948; And Almagro, J.mol. Recognit.17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk/abs. As used herein, a CDR may refer to a CDR defined by any method known in the art. Two antibodies having the same CDR 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).

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

TABLE 1 CDR definition

¹ the international ImMunoGeneTics informationimgt.org,Lefranc,M.-P.et al.,Nucleic Acids Res.,27:209-212(1999)

²Kabat et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242

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

CDR-grafted antibodies (CDR-grafted anti-bodies) the term "CDR-grafted antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species but wherein the sequences of one or more CDR regions of VH and/or (e.g., and) VL are replaced by CDR sequences from another species, e.g., an antibody having murine heavy and light chain variable regions and wherein one or more murine CDRs (e.g., CDR 3) have been replaced by human CDR sequences.

Chimeric antibody the term "chimeric antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species and constant region sequences from another species, e.g., an antibody having murine heavy and light chain variable regions linked to human constant regions.

Complementarity the term "complementary" as used herein refers to the ability to pair precisely between two nucleosides or 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 groups of nucleosides. For example, bases at one position of an oligonucleotide are considered complementary to each other if the bases at that position are capable of hydrogen bonding with bases at the corresponding position of the target nucleic acid (e.g., mRNA). Base pairing can include both canonical Watson-Crick base pairing and non-Watson-Crick base pairing (e.g., wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairing, an adenosine base (a) is complementary to a thymidine base (T) or a uracil base (U), a cytosine base (C) is complementary to a guanosine base (G), and a universal base, such as 3-nitropyrrole or 5-nitroindole, can hybridize to any A, C, U or T and be considered complementary to any A, C, U or T. Inosine (I) is also known in the art as a universal base and is considered complementary to any A, C, U or T.

Conservative amino acid substitutions As used herein, "conservative amino acid substitutions" refers to amino acid substitutions that do not alter the relative charge or size characteristics of the protein in which they are made. Variants may be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as may be 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, f.m. ausubel, et al, eds., john Wiley & Sons, inc. Conservative substitutions of amino acids include substitutions between amino acids within the groups of (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.

Covalent linkage the term "covalent linkage" as used herein 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 disulfide bridge, that serves 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.

Cross-reactivity as used herein and in the case of a targeting agent (e.g., an antibody), the term "cross-reactivity" refers to the property of a substance that is capable of specifically binding with similar affinity or avidity to more than one antigen of similar type or class (e.g., antigens of multiple homologs, paralogs or orthologs). For example, in some embodiments, antibodies that are cross-reactive to similar types or classes of human and non-human primate antigens (e.g., human transferrin receptor and non-human primate transferrin receptor) are capable of binding with similar affinity or avidity to human and non-human primate antigens. In some embodiments, the antibodies are cross-reactive to human and rodent antigens of similar types or classes. In some embodiments, the antibodies are cross-reactive with a similar type or class of rodent antigens and non-human primate antigens. In some embodiments, the antibodies are cross-reactive with similar types or classes of human, non-human primate, and rodent antigens.

DUX4 the term "DUX4" as used herein refers to a gene encoding double homeobox 4, double homeobox 4 being a protein that is normally expressed during fetal development and in testes of adult males. In some embodiments, DUX4 may 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 facial shoulder brachial muscular dystrophy. In addition, a variety of human transcript variants have been characterized that encode different protein isoforms (e.g., as noted in GenBank RefSeq accession numbers: NM-001293798.2, NM-001306068.3, NM-001363820.1).

Facial shoulder muscular dystrophy (FSHD): the term "facial shoulder muscular dystrophy (FSHD)" as used herein refers to a genetic disease caused by mutations in the DUX4 gene, the SMCHD1 gene, the DNMT3B gene, or the LRIF gene, characterized by decreased muscle mass and muscle atrophy primarily in the facial, shoulder, and upper arm muscles. Two types of this disease, type 1 and type 2, have been described. Type 1 is associated with a deletion of the D4Z4 repeat region on chromosome 4 that contains the DUX4 gene. In some embodiments, type 1 is associated with a deletion of the D4Z4 repeat region comprising the DUX4 gene on chromosome 4 allelic variant 4 qA. Type 2 is associated with mutations in the SMCHD1 gene, DNMT3B gene, or LRIF gene (see, e.g., both Jia et al.,"Facioscapulohumeral muscular dystrophy type 2:an update on the clinical,genetic,and molecular findings"Neuromuscul Disord.(2021),31(11):1101-1112).1 -type and type 2 FSHD are characterized by abnormal production of DUX4 protein other than testis after fetal development, facial shoulder muscular dystrophy, the genetic basis of the disease, and related symptoms have been described in the art (see, e.g., Campbell,A.E.,et al.,"Facioscapulohumeral dystrophy:Activating an early embryonic transcriptional programin 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 human mendelian inheritance (Online MENDELIAN INHERITANCE IN MAN, OMIM) Entry #158900, type 2 FSHD is associated with OMIM Entry # 158901.

Framework the term "framework" or "framework sequence" as used herein refers to the remaining sequence of the variable region minus the CDRs. Since the exact definition of CDR sequences can be determined by different systems, the meaning of framework sequences accordingly has different interpretations. Six CDRs (CDR-L1, CDR-L2 and CDR-L3 of the light chain and CDR-H1, CDR-H2 and CDR-H3 of the heavy chain) also divide the framework on the light and heavy chains into four sub-regions (FR 1, FR2, FR3 and FR 4) on each chain, with CDR1 located between FR1 and FR2, CDR2 located between FR2 and FR3, and CDR3 located between FR3 and FR 4. In the case where a specific sub-region is not designated as FR1, FR2, FR3 or FR4, the framework regions mentioned by others represent the combined FR within the variable regions of a single naturally occurring immunoglobulin chain. As used herein, FR represents one of four subregions, and FRs represents two or more of the four subregions constituting the framework region. Human heavy and light chain acceptor sequences are known in the art. In one embodiment, acceptor sequences known in the art may be used in the antibodies disclosed herein.

Human antibodies the term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may comprise amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in CDRs, and in particular in CDR 3. 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.

Humanized antibody the term "humanized antibody" refers to an antibody that comprises heavy and light chain variable region sequences from a non-human species (e.g., mouse) but in which at least a portion of the VH and/or (e.g., and) VL sequences have been altered to be more "human-like" (i.e., more similar to human germline variable sequences). One type of humanized antibody is a CDR-grafted antibody in which human CDR sequences are introduced into non-human VH and VL sequences in place of the corresponding non-human CDR sequences. In one embodiment, humanized anti-TfR 1 antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining murine anti-TfR 1 monoclonal antibodies using conventional hybridoma techniques followed by humanization using in vitro genetic engineering, such as those disclosed in PCT publication No. wo 2005/123126 A2 of KASAIAN ET AL.

Internalization of cell surface receptor the term "internalized cell surface receptor" as used herein refers to a cell surface receptor that is internalized, for example, by a cell under external stimulus (e.g., ligand binding to receptor). In some embodiments, the internalized cell surface receptor is internalized by endocytosis. In some embodiments, the internalized cell surface receptor is internalized by clathrin-mediated endocytosis. However, in some embodiments, the internalized cell surface receptor is internalized by a clathrin-independent pathway, such as, for example, phagocytosis, megaloblastic, cell 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 following 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.

Isolated antibody As used herein, "isolated antibody" is intended to mean an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to a transferrin receptor is substantially free of antibodies that specifically bind to antigens other than a transferrin receptor). However, isolated antibodies that specifically bind to the transferrin receptor complex may have cross-reactivity with other antigens (e.g., transferrin receptor molecules from other species). In addition, the isolated antibodies may be substantially free of other cellular material and/or (e.g., and) chemicals.

Kabat numbering the terms "Kabat numbering", "Kabat definition" and "Kabat labeling" are used interchangeably herein. These terms are recognized in the art as referring to the system of numbering amino acid residues in the heavy and light chain variable regions of an antibody or antigen binding portion thereof that are more variable (i.e., hypervariable) than other amino acid residues (Kabat et al, (1971) ann.ny Acad, sci.190:382-391 and ,Kabat,E.A.,et al.(1991)Sequences of Proteins of Immunological Interest,Fifth Edition,U.S.Department of Health and Human Services,NIH Publication No.91-3242). for the heavy chain variable region, the hypervariable region of CDR1 is amino acids 31 to 35, the hypervariable region of CDR2 is amino acids 50 to 65, and the hypervariable region of CDR3 is amino acids 95 to 102. For the light chain variable region, the hypervariable region of CDR1 is amino acids 24 to 34, the hypervariable region of CDR2 is amino acids 50 to 56, and the hypervariable region of CDR3 is amino acids 89 to 97.

Molecular payload the term "molecular payload" as used herein refers to a molecule or substance that plays a role in regulating 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 functions to regulate transcription of a DNA sequence, regulate expression of a protein, or regulate activity of a protein. In some embodiments, the molecular payload is an oligonucleotide comprising a strand having a complementary region of the target gene.

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 the muscle cell may be a membrane protein, such as an integral membrane protein or a peripheral membrane protein. Generally, the muscle targeting agent specifically binds to an antigen on the muscle cell, which aids in internalizing the muscle targeting agent (and any associated molecular payloads) into the muscle cell. In some embodiments, the muscle targeting agent specifically binds to an internalized cell surface receptor on the muscle and is capable of internalizing into the muscle cell by receptor-mediated internalization. In some embodiments, the muscle targeting agent is a small molecule, protein, peptide, nucleic acid (e.g., aptamer), or antibody. In some embodiments, the muscle targeting agent is linked to the molecular payload.

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 aids in internalizing the muscle targeting antibody (and any associated molecular payloads) into the muscle cell. In some embodiments, the muscle targeting antibody specifically binds to an internalized cell surface receptor present on a muscle cell. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to a transferrin receptor.

Oligonucleotide As used herein, the term "oligonucleotide" refers to an oligonucleotide compound that is up to 200 nucleotides in length. Some examples of oligonucleotides include, but are not limited to, RNAi oligonucleotides (e.g., siRNA, shRNA), micrornas, spacer polymers, hybrid polymers, phosphodiamide morpholinos, peptide nucleic acids, aptamers, guide nucleic acids (e.g., cas9 guide RNAs), and the like. The oligonucleotide may be single-stranded or double-stranded. In some embodiments, the oligonucleotide may comprise one or more modified nucleosides (e.g., 2' -O-methyl sugar modification, purine or pyrimidine modification). In some embodiments, the oligonucleotide may comprise one or more modified internucleoside linkages. In some embodiments, the oligonucleotide may comprise one or more phosphorothioate linkages, which may be in Rp or Sp stereochemical conformation.

Recombinant antibodies the term "recombinant human antibody" as used herein is intended to include all human antibodies produced, expressed, produced 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 (Hoogenboom H.R.,(1997)TIB Tech.15:62-70;Azzazy H.,and Highsmith W.E.,(2002)Clin.Biochem.35:425-445;Gavilondo J.V.,and Larrick J.W.(2002)BioTechniques 29:128-145;Hoogenboom H.,and Chames P.(2000)Immunology Today 21:371-378), isolated from recombinant, combinatorial human antibody libraries are isolated from animals (e.g., mice) transgenic for human immunoglobulin genes (see e.g., Taylor,L.D.,et al.(1992)Nucl.Acids Res.20:6287-6295;Kellermann S-A.,and Green L.L.(2002)Current Opinion in Biotechnology 13:593-597;Little M.et al(2000)Immunology Today 21:364-370), or antibodies produced, expressed, produced or isolated by any other means involving splicing of human immunoglobulin gene sequences to other DNA sequences, such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences, however, in certain embodiments such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis when animals transgenic for human Ig sequences are used), and thus the amino acid sequences of VH and VL regions of recombinant antibodies are such sequences, which, although derived from and associated with human germline VH and VL sequences, may not be the human antibody germline library, one embodiment that naturally exists in vivo provides a human antibody germline library that binds human antibody such as that is known in human antibody library, such as that is known in wo 539, such as human antibody, 0079, and PCT 2005 is not fully known to bind human antibody library, such as human antibody, e.g., human antibody 0079.

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

Specific binding the term "specific binding" as used herein refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that allows the molecule to be used to distinguish the binding partner from a suitable control in a binding assay or other binding environment. 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 the extent that allows preferential targeting of certain cells (e.g., myocytes) by binding to an antigen as described herein, as compared to a suitable reference antigen or antigens. In some embodiments, an antibody specifically binds to a target if K _D that binds to the target is at least about 10^-4M、10^-5M、10^-6M、10^-7M、10^-8M、10^-9M、10^-10M、10^-11M、10^- ¹²M、10^-13M or less. In some embodiments, the antibody specifically binds to a transferrin receptor (e.g., an epitope of the top domain of the transferrin receptor).

Subject the term "subject" as used herein refers to a mammal. In some embodiments, the subject is a non-human primate or rodent. In some embodiments, the subject is a human. In some embodiments, the subject is a patient, e.g., a human patient having or suspected of having a disease. In some embodiments, the subject is a human patient suffering from or suspected of suffering from FSHD.

Transferrin receptor the term "transferrin receptor" (also referred to as TFRC, CD71, p90, TFR 1) as used herein refers to an internalized cell surface receptor that binds transferrin to promote uptake of iron 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 have been characterized that encode different isoforms of the receptor (e.g., as noted in GenBank RefSeq accession numbers: NP-001121620.1, NP-003225.2, NP-001300894.1, and NP-001300895.1).

2 '-Modified nucleoside the terms "2' -modified nucleoside" and "2 '-modified ribonucleoside" are used interchangeably herein and refer to a nucleoside having a modified sugar moiety at the 2' position. In some embodiments, the 2' -modified nucleoside is a 2' -4' bicyclic nucleoside in which the 2' and 4' positions of the sugar are bridged (e.g., by methylene, ethylene, or (S) -constrained ethyl bridging). 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-DMAEOEE), 2 '-O-N-methylacetamido (2' -O-NMA), locked nucleic acids (locked nucleic acid, LNA, methylene bridged nucleic acids), ethylene bridged nucleic acids (ethylene-bridged nucleic acid, ENA), and (S) -constrained ethyl bridged nucleic acids (cEt). In some embodiments, the 2 '-modified nucleosides described herein are high affinity modified nucleosides and oligonucleotides comprising 2' -modified nucleosides having increased affinity for a target sequence relative to an unmodified oligonucleotide. Some examples of structures of 2' -modified nucleosides are provided below:

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

II. Complex

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 a single antigenic site or binds at least two antigenic sites that may be present on the same or different antigens.

The complexes can be used to modulate the activity or function of at least one gene, protein, and/or (e.g., sum) nucleic acid. In some embodiments, the molecular payload present in the complex is responsible for the modulation of the gene, protein, and/or (e.g., sum) nucleic acid. The molecular payload may be a small molecule, protein, nucleic acid, oligonucleotide, or any molecular entity capable of modulating the activity or function of a gene, protein, and/or (e.g., and) nucleic acid in a cell. In some embodiments, the molecular payload is an oligonucleotide that targets DUX4 in a muscle cell.

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 DUX 4). In some embodiments, the complex comprises a muscle targeting agent, such as an anti-transferrin receptor antibody, covalently linked to a molecular payload (e.g., a DUX 4-targeted siRNA).

A. Muscle targeting agents

Some aspects of the present disclosure provide muscle targeting agents, for example, for delivering a molecular payload to a muscle cell. In some embodiments, such muscle targeting agents are capable of binding to a muscle cell, for example, by specifically binding to an antigen on the muscle cell, and delivering an associated molecular payload to the muscle cell. In some embodiments, the molecular payload binds (e.g., covalently binds) to the muscle targeting agent and internalizes into the muscle cell after the muscle targeting agent binds to the antigen on the muscle cell, e.g., by endocytosis. It should be understood that various types of muscle targeting agents may be used in accordance with the present disclosure. For example, a 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., microbubbles (microvesicle)), or a sugar moiety (e.g., a polysaccharide). Exemplary muscle targeting agents are described in further detail herein, however, it should be understood that the exemplary muscle targeting agents provided herein are not meant to be limiting.

Some aspects of the present disclosure provide muscle targeting agents that specifically bind to an antigen on a muscle (e.g., skeletal muscle, smooth muscle, or cardiac muscle). In some embodiments, any of the muscle targeting agents provided herein bind to (e.g., specifically bind to) an antigen on skeletal muscle cells, smooth muscle cells, and/or (e.g., and) cardiac muscle cells.

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 may be used to deliver a molecular payload into muscle tissue. Binding to the muscle surface recognition element is followed by endocytosis, which may allow even macromolecules (e.g., antibodies) to enter the muscle cells. As another example, a molecular payload conjugated to transferrin or an anti-TfR 1 antibody may be taken up by muscle cells by binding to transferrin receptor and then endocytosed, for example, by clathrin mediated endocytosis.

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 the muscle cells as compared to another cell type within the subject. In some embodiments, the muscle targeting agent concentrates the combined molecular payload in a muscle cell (e.g., skeletal muscle, smooth muscle, or cardiac muscle cell) 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 a non-muscle cell (e.g., liver, neuron, blood, or adipocyte). In some embodiments, when the molecular payload is delivered to a subject upon binding to 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%.

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

I. Muscle targeting antibodies

In some embodiments, the muscle targeting agent is an antibody. Generally, the high specificity of antibodies for their target antigens provides the potential for selective targeting of myocytes (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 a myocyte surface antigen 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 K.S.,et al."Expression of caveolin-3in skeletal,cardiac,and smooth muscle cells.Caveolin-3is a component of the sarcolemma and co-fractionates with dystrophin and dystrophin-associated glycoproteins"J Biol Chem 1996;271:15160-5; and Weisbart R.H.et al.,"Cell type specific targeted intracellular delivery into muscle of a monoclonal antibody that binds myosin IIb"Mol Immunol.2003Mar,39(13):78309; below, each of which is incorporated by reference in its entirety.

A. anti-transferrin receptor (Anti-TRANSFERRIN RECEPTOR, tfR) antibodies

Some aspects of the disclosure are based on the recognition that substances that bind to transferrin receptor (e.g., anti-transferrin receptor antibodies) are capable of targeting muscle cells. Transferrin receptors are internalized cell surface receptors that transduce transferrin across cell membranes and are involved in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to transferrin receptors. Accordingly, some aspects of the present disclosure provide binding proteins (e.g., antibodies) that bind to transferrin receptor. In some embodiments, the binding protein that binds to a transferrin receptor is internalized into a muscle cell along with any bound molecular payload. As used herein, an antibody that binds to a transferrin receptor may be interchangeably referred to as a transferrin receptor antibody, an anti-transferrin receptor antibody, or an anti-TfR 1 antibody. Antibodies that bind (e.g., specifically bind) to a transferrin receptor can be internalized into a cell after binding to the transferrin receptor, e.g., by receptor-mediated endocytosis.

It will be appreciated that several known methods (e.g., using phage display library design) can be used to generate, synthesize, and/or (e.g., and) derive anti-TfR 1 antibodies. Exemplary methods have been characterized in the art and (Díez,P.et al."High-throughput phage-display screening in array format",Enzyme and microbial technology,2015,79,34-41.;Christoph M.H.and Stanley,J.R."Antibody Phage Display:Technique and Applications"J Invest Dermatol.2014,134:2.;Engleman,Edgar(Ed.)"Human Hybridomas and Monoclonal Antibodies."1985,Springer.). are incorporated by reference in other embodiments, anti-TfR 1 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.9,708,406, "Anti-TRANSFERRIN RECEPTOR ANTIBODIES AND METHODS OF USE," US 9,611,323, "Low affinity blood brain barrier receptor antibodies and uses therefor," US 864, and US 534, 24, U.S. patent No.4,364,934,"Monoclonal antibody to a human early thymocyte antigen and methods for preparing same";2006, U.S. patent No.8,409,573,"Anti-CD71 monoclonal antibodies and uses thereof for treating malignant tumor cells";2014, U.S. patent 5, U.S. patent No. 20, U.S. patent No. 5, and U.S. patent No. 14, U.S. patent No. 5, and U.S. patent No. 20, filed on 12, and U.S. patent No. 24, respectively, of 1979, 12, and 2014, 12, 24, respectively) WO 2015/098989,"Novel anti-Transferrin receptor antibody that passes through blood-brain barrier";Schneider C.et al."Structural features of the cell surface receptor for transferrin that is recognized by the monoclonal antibody OKT9."J Biol Chem.1982,257:14,8516-8522.;Lee et al."Targeting Rat Anti-Mouse Transferrin Receptor Monoclonal Antibodies through Blood-Brain Barrier in Mouse"2000,J Pharmacol.Exp.Ther.,292:1048-1052).

In some embodiments, the anti-TfR 1 antibodies described herein bind to a transferrin receptor with high specificity and affinity. In some embodiments, an anti-TfR 1 antibody described herein specifically binds to any extracellular epitope of a transferrin receptor or an epitope that becomes exposed to the antibody. In some embodiments, an anti-TfR 1 antibody provided herein specifically binds to a transferrin receptor from human, non-human primate, mouse, rat, etc. In some embodiments, an anti-TfR 1 antibody provided herein binds to a human transferrin receptor. In some embodiments, an anti-TfR 1 antibody described herein binds to an amino acid segment of a human or non-human primate transferrin receptor (as provided in SEQ ID NOS: 105-108). In some embodiments, an anti-TfR 1 antibody described herein binds to an amino acid segment corresponding to amino acids 90 to 96 of a human transferrin receptor (as shown in SEQ ID NO: 105), which is not in the apical domain of the transferrin receptor. In some embodiments, the humanized anti-TfR 1 antibodies described herein bind to TfR1, but not to TfR 2.

In some embodiments, an anti-TfR 1 antibody described herein (e.g., anti-TfR clone 8 in table 2 below) binds to an epitope in TfR1, wherein the epitope comprises residues from amino acids 214 to 241 and/or from amino acids 354 to 381 of SEQ ID No. 105. In some embodiments, an anti-TfR 1 antibody described herein binds an epitope comprising residues in amino acids 214 to 241 and amino acids 354 to 381 of SEQ ID No. 105. In some embodiments, the anti-TfR 1 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-TfR 1 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.

In some embodiments, an anti-TfR 1 antibody described herein (e.g., 3M12 in table 2 below and variants thereof) binds 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, an anti-TfR 1 antibody described herein (e.g., 3M12 in table 2 below and variants thereof) binds to an epitope comprising residues from amino acids 258 to 291 and 358 to 381 of SEQ ID No. 105. In some embodiments, anti-TfR 1 antibodies described herein (e.g., 3M12 in table 2 below and variants thereof) 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-TfR 1 antibodies described herein (e.g., 3M12 in table 2 below and variants thereof) bind to an epitope comprising residues K261, S273, Y282, T362, S368, S370, and K371 of human TfR1 shown in SEQ ID No. 105.

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:

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:

an exemplary non-human primate transferrin receptor amino acid sequence corresponding to NCBI sequence xp_005545315.1 (transferrin receptor protein 1, cynomolgus monkey (Macaca fascicularis)) is as follows:

An exemplary mouse transferrin receptor amino acid sequence corresponding to NCBI sequence np_001344227.1 (transferrin receptor protein 1, mouse (Mus museuus)) is as follows:

in some embodiments, the anti-TfR 1 antibody binds to the following acceptor amino acid segment:

and does not inhibit the binding interactions between transferrin receptor and transferrin and/or (e.g., and) human blood pigmentation protein (human hemochromatosis protein, also known as HFE). In some embodiments, the anti-TfR 1 antibodies described herein do not bind to the epitope in SEQ ID NO. 109.

Antibodies, antibody fragments, or antigen binding agents can be obtained and/or (e.g., and) produced using appropriate methods, for example, by using recombinant DNA protocols. In some embodiments, antibodies may also be produced by the production of hybridomas (see, e.g., antigen of ,Kohler,G and Milstein,C."Continuous cultures of fused cells secreting antibody of predefined specificity"Nature,1975,256:495-497). mesh may be used as an immunogen in any form or entity (e.g., recombinant or naturally occurring form or entity), to find at least one hybridoma that produces an antibody that targets a particular antigen, may also be obtained by screening a protein expression library that expresses the antibody (e.g., phage display library) to produce Antibodies in some embodiments, phage display library design may also be used (see, e.g., U.S. Pat. No.5,223,409, "Directed evolution of novel binding proteins," filed 3/1/1991; in some embodiments, the antigen of interest may be used to immunize a non-human animal, such as a rodent or goat, in some embodiments, antibodies are then obtained from the non-human animal and optionally modified using a variety of methods (e.g., using recombinant DNA techniques), 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).

In some embodiments, the antibody is modified, e.g., by glycosylation, phosphorylation, SUMO methylation, 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, glycosyl phosphatidyl inositol (GPI anchor attachment), and/or (e.g., and) phosphoglycosylation. In some embodiments, one or more sugar or carbohydrate molecules are monosaccharides, disaccharides, oligosaccharides, or glycans. In some embodiments, one or more sugar or carbohydrate molecules are branched oligosaccharides or branched glycans. In some embodiments, one or more sugar or carbohydrate molecules 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 No. WO2014065661 entitled "Modified antibody, anti-body-conjugate and process for the preparation thereof" published on 5, month 1 of 2014.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VL domain and/or (e.g., and) a VH domain selected from any of the anti-TfR 1 antibodies of any of tables 2-7, and comprises a constant region comprising the amino acid sequence of a constant region of IgG, igE, igM, igD, igA or IgY immunoglobulin molecules, any class of immunoglobulin molecules (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or any subclass (e.g., igG2a and IgG2 b). Some non-limiting examples of human constant regions are described in the art, for example, see Kabat E a et al, supra (1991).

In some embodiments, a substance that binds to a transferrin receptor, such as an anti-TfR 1 antibody, is capable of targeting muscle cells and/or (e.g., and) mediating transport of the substance across the blood-brain barrier. Transferrin receptors are internalized cell surface receptors that transport transferrin across cell membranes and are involved in the regulation and homeostasis of intracellular iron levels. Some aspects of the present disclosure provide transferrin receptor binding proteins capable of binding to transferrin receptors. Antibodies that bind (e.g., specifically bind) to a transferrin receptor can be internalized into a cell after binding to the transferrin receptor, e.g., by receptor-mediated endocytosis.

In some embodiments, an anti-TFR 1 antibody specifically binds TFR1 (e.g., human or non-human primate TFR 1) with a binding affinity of at least about 10^-4M、10^-5M、10^-6M、10^-7M、10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13M or less (e.g., as shown by Kd). In some embodiments, an anti-TfR 1 antibody described herein binds TfR1 with a KD in the subnanomolar range. In some embodiments, an anti-TfR 1 antibody described herein selectively binds to transferrin receptor 1 (TfR 1) but not to transferrin receptor2 (TRANSFERRIN RECEPTOR2, tfR 2). In some embodiments, an anti-TfR 1 antibody described herein binds to human TfR1 and cynomolgus monkey TfR1 (e.g., kd of 10 ^-7M、10^-8M、10^-9M、10^-10M、10^-11M、10^-12M、10^-13 M or less), but not to mouse TfR 1. The affinity and binding kinetics of the anti-TfR 1 antibody may 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 of the anti-TfR 1 antibodies described herein does not compete or inhibit the binding of transferrin to TfR 1. In some embodiments, the binding of any of the anti-TfR 1 antibodies described herein does not compete or inhibit the binding of HFE- β -2-microglobulin to TfR 1.

Some non-limiting examples of anti-TfR 1 antibodies are provided in table 2.

TABLE 2 some examples of anti-TfR 1 antibodies

* The mutation position is numbered according to Kabat of the corresponding VH sequence comprising the mutation

In some embodiments, an anti-TfR 1 antibody of the present disclosure is a variant of any one of the anti-TfR 1 antibodies provided in table 2. In some embodiments, an anti-TfR 1 antibody of the 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-TfR 1 antibodies provided in table 2, and comprises a humanized heavy chain variable region and/or a humanized light chain variable region (e.g., and).

Some examples of the amino acid sequences of anti-TfR 1 antibodies described herein are provided in table 3.

TABLE 3 variable regions of anti-TfR 1 antibodies

* CDRs according to Kabat numbering system are bolded

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR 1 antibodies provided in table 3, and comprises one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework regions compared to the corresponding VH provided in table 3. Alternatively or additionally (e.g., complementary), an anti-TfR 1 antibody of the present disclosure comprises a VL that comprises CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR 1 antibodies provided in table 3, and comprises one or more (e.g., 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) amino acid variations in the framework regions compared to the corresponding VL provided in table 3.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising CDR-H1, CDR-H2, and CDR-H3 of any one of the anti-TfR 1 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 as compared to the corresponding VH provided in table 3. Alternatively or additionally (e.g., complementary), an anti-TfR 1 antibody of the present disclosure comprises a VL that comprises CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR 1 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 as compared to the corresponding VL provided in table 3.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:69 and a VL comprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:71 and a VL comprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:72 and a VL comprising the amino acid sequence of SEQ ID NO: 70.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:73 and a VL comprising the amino acid sequence of SEQ ID NO: 74.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID No. 73 and a VL comprising the amino acid sequence of SEQ ID No. 75.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO. 76 and a VL comprising the amino acid sequence of SEQ ID NO. 74.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID No. 76 and a VL comprising the amino acid sequence of SEQ ID No. 75.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:77 and a VL comprising the amino acid sequence of SEQ ID NO: 78.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID No. 79 and a VL comprising the amino acid sequence of SEQ ID No. 80.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID No. 77 and a VL comprising the amino acid sequence of SEQ ID No. 80.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO:154 and a VL comprising the amino acid sequence of SEQ ID NO: 155.

In some embodiments, an anti-TfR 1 antibody described herein is a full length IgG, which may comprise heavy and light constant regions from a human antibody. In some embodiments, the heavy chain of any anti-TfR 1 antibody described herein can comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can have any suitable origin, such as human, mouse, rat, or rabbit. In one specific example, the heavy chain constant region is from a human IgG such as IgG1, igG2, or IgG4 (gamma heavy chain). An example of a human IgG1 constant region is given below:

In some embodiments, the heavy chain of any anti-TfR 1 antibody described herein comprises a mutant human IgG1 constant region. For example, the introduction of LALA mutations (mutants derived from mAb b12, which have been mutated to replace the lower hinge residue Leu234 Leu235 with Ala234 and Ala 235) in the CH2 domain of human IgG1 is known to reduce fcγ receptor binding (Bruhns, p., et al (2009) and Xu, d.et al (2000)). The mutant human IgG1 constant regions (mutations are bolded and underlined) are provided below:

In some embodiments, the light chain of any anti-TfR 1 antibody described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, CL is a kappa light chain. In other examples, CL is a lambda light chain. In some embodiments, CL is a kappa light chain, the sequences of which are provided below:

Other antibody heavy and light chain constant regions are well known in the art, such as those provided in IMGT database (www.imgt.org) or www.vbase2.org/vbstat.

In some embodiments, an anti-TfR 1 antibody described herein comprises a heavy chain comprising any one of the VH 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, an anti-TfR 1 antibody described herein comprises a heavy chain comprising any of the VH 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 variations) compared to SEQ ID No. 81 or SEQ ID No. 82. In some embodiments, an anti-TfR 1 antibody described herein comprises a heavy chain comprising any one of the VH listed in table 3, or any variant thereof, and a heavy chain constant region shown in SEQ ID No. 81. In some embodiments, an anti-TfR 1 antibody described herein comprises a heavy chain comprising any of the VH's listed in Table 3 or any variant thereof and a heavy chain constant region as set forth in SEQ ID NO. 82.

In some embodiments, an anti-TfR 1 antibody described herein comprises a light chain comprising 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, or any variant thereof, and any one of the VLs listed in table 3. In some embodiments, an anti-TfR 1 antibody described herein comprises a light chain comprising a light chain constant region comprising any one of the VLs listed in table 3 or any variant thereof and 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) as compared to SEQ ID No. 83. In some embodiments, an anti-TfR 1 antibody described herein comprises a light chain comprising a light chain constant region as set forth in SEQ ID NO 83, or any of the VL's or any of the variants thereof set forth in Table 3.

Some examples of IgG heavy and light chain amino acid sequences of the anti-TfR 1 antibodies are provided in table 4 below.

TABLE 4 heavy and light chain sequences of some examples of anti-TfR 1 IgG

* The CDRs according to the Kabat numbering system are bolded, VH/VL sequences are underlined

In some embodiments, an anti-TfR 1 antibody of the 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. Alternatively or additionally (e.g., in addition), the anti-TfR 1 antibodies of the disclosure comprise 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) from the light chain shown in any of SEQ ID NOs 85, 89, 90, 93, 95, and 157.

In some embodiments, an anti-TfR 1 antibody described herein comprises 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., complementary), an anti-TfR 1 antibody described herein comprises 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, an anti-TfR 1 antibody described herein comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs 84, 86, 87, 88, 91, 92, 94 and 156. Alternatively or additionally (e.g., complementary), the anti-TfR 1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs 85, 89, 90, 93, 95, and 157.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 84 and a light chain comprising the amino acid sequence of SEQ ID No. 85.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 91 and a light chain comprising the amino acid sequence of SEQ ID NO. 89.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 91 and a light chain comprising the amino acid sequence of SEQ ID NO. 90.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 92 and a light chain comprising the amino acid sequence of SEQ ID No. 95.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 156 and a light chain comprising the amino acid sequence of SEQ ID NO. 157.

In some embodiments, the anti-TfR 1 antibody is a Fab fragment, fab 'fragment, or F (ab') ₂ fragment of an intact antibody (full length antibody). Antigen binding fragments of whole antibodies (full length antibodies) can be prepared by conventional methods (e.g., recombinantly or by digestion of the heavy chain constant region of full length IgG with enzymes such as papain). For example, the F (ab ') ₂ fragment can be produced by pepsin or papain digestion of an antibody molecule, and the Fab fragment can be produced by reduction of the disulfide bridge of the F (ab') ₂ fragment. In some embodiments, the heavy chain constant region in the Fab fragment of the anti-TfR 1 antibodies described herein comprises the following amino acid sequence:

In some embodiments, an anti-TfR 1 antibody described herein comprises a heavy chain comprising any one of the VH 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, an anti-TfR 1 antibody described herein comprises a heavy chain comprising any of the VH 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 variations) compared to SEQ ID NO: 96. In some embodiments, an anti-TfR 1 antibody described herein comprises a heavy chain comprising any one of the VH listed in table 3, or any variant thereof, and a heavy chain constant region shown in SEQ ID No. 96.

Some examples of Fab heavy and light chain amino acid sequences of the anti-TfR 1 antibodies are provided in table 5 below.

TABLE 5 heavy and light chain sequences of some examples of anti-TfR 1 Fab

In some embodiments, an anti-TfR 1 antibody of the 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 97 to 103, 158, and 159. Alternatively or additionally (e.g., complementary), an anti-TfR 1 antibody of the 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 of SEQ ID NOs 85, 89, 90, 93, 95, and 157.

In some embodiments, an anti-TfR 1 antibody described herein comprises 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., complementary), an anti-TfR 1 antibody described herein comprises 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, an anti-TfR 1 antibody described herein comprises a heavy chain comprising the amino acid sequence of any one of SEQ ID NOs 97 to 103, 158 and 159. Alternatively or additionally (e.g., complementary), the anti-TfR 1 antibodies described herein comprise a light chain comprising the amino acid sequence of any one of SEQ ID NOs 85, 89, 90, 93, 95, and 157.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 97 and a light chain comprising the amino acid sequence of SEQ ID No. 85.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 101 and a light chain comprising the amino acid sequence of SEQ ID No. 89.

In some embodiments, an anti-TfR 1 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.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 102 and a light chain comprising the amino acid sequence of SEQ ID NO. 93.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 103 and a light chain comprising the amino acid sequence of SEQ ID No. 95.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 102 and a light chain comprising the amino acid sequence of SEQ ID No. 95.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 158 and a light chain comprising the amino acid sequence of SEQ ID NO. 157.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 159 and a light chain comprising the amino acid sequence of SEQ ID No. 157.

Other known anti-TfR 1 antibodies

Any other suitable anti-TfR 1 antibody known in the art may be used as a muscle targeting agent in the complexes disclosed herein. Some examples of known anti-TfR 1 antibodies, including related references and binding epitopes, are listed in table 6. In some embodiments, an anti-TfR 1 antibody comprises the complementarity determining regions (CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3) of any anti-TfR 1 antibody provided herein (e.g., an anti-TfR 1 antibody listed in Table 6).

Table 6-list of anti-TfR 1 antibody clones, including relevant references and binding epitope information.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises one or more CDR-H (e.g., CDR-H1, CDR-H2, and CDR-H3) amino acid sequences from any one of the anti-TfR 1 antibodies selected from table 6. In some embodiments, the anti-TfR 1 antibody comprises CDR-L1, CDR-L2, and CDR-L3 as provided for any one of the anti-TfR 1 antibodies selected from table 6. In some embodiments, an anti-TfR 1 antibody comprises a CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2 and CDR-L3 as provided for any one of the anti-TfR 1 antibodies selected from Table 6.

In some embodiments, an anti-TfR 1 antibody of the present disclosure includes any antibody comprising a heavy chain variable domain and/or (e.g., and) a light chain variable domain of any anti-TfR 1 antibody (e.g., any anti-TfR 1 antibody selected from table 6). In some embodiments, the anti-TfR 1 antibodies of the present disclosure include any antibody comprising a variable pair of heavy and light chains of any anti-TfR 1 antibody (e.g., any anti-TfR 1 antibody selected from table 6).

Some aspects of the disclosure provide anti-TfR 1 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, an anti-TfR 1 antibody comprises a heavy chain variable sequence or a light chain variable sequence having at least 75% (e.g., 80%, 85%, 90%, 95%, 98%, or 99%) identity to the heavy chain variable sequence and/or to any light chain variable sequence of any anti-TfR 1 antibody (e.g., any anti-TfR 1 antibody selected from table 6). In some embodiments, the cognate heavy chain variable and/or (e.g., and) light chain variable amino acid sequence is unchanged 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%) can occur in heavy chain variable and/or (e.g., and) light chain variable sequences that do not include any CDR sequences provided herein. In some embodiments, any anti-TfR 1 antibody provided herein comprises a heavy chain variable sequence and a light chain variable sequence comprising a framework sequence having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% identity to the framework sequence of any anti-TfR 1 antibody (e.g., any anti-TfR 1 antibody selected from table 6).

Examples of transferrin receptor antibodies that can be used in accordance with the present disclosure are described in international application publication WO 2016/081643, which is incorporated herein by reference. The amino acid sequences of the antibodies are provided in table 7.

TABLE 7 heavy and light chain CDRs for examples of known anti-TfR 1 antibodies

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises the same CDR-H1, CDR-H2 and CDR-H3 as the CDR-H1, CDR-H2 and CDR-H3 shown in Table 7. Alternatively or additionally (e.g., complementary), the anti-TfR 1 antibodies of the present disclosure comprise CDR-L1, CDR-L2, and CDR-L3 that are identical to CDR-L1, CDR-L2, and CDR-L3 shown in table 7.

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

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises 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., complementary), an anti-TfR 1 antibody of the disclosure comprises 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.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO. 124. Alternatively or additionally (e.g., complementary), an anti-TfR 1 antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 125.

In some embodiments, an anti-TfR 1 antibody of the disclosure comprises a VH comprising the amino acid sequence of SEQ ID NO: 128. Alternatively or additionally (e.g., complementary), an anti-TfR 1 antibody of the disclosure comprises a VL comprising the amino acid sequence of SEQ ID NO: 129.

In some embodiments, an anti-TfR 1 antibody of the 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 set forth in SEQ ID No. 128. Alternatively or additionally (e.g., in addition), the anti-TfR 1 antibodies of the disclosure comprise 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) from the VL shown in SEQ ID NO: 129.

In some embodiments, the anti-TfR 1 antibodies of the present disclosure are full length IgG1 antibodies, which may comprise heavy and light constant regions from a human antibody. In some embodiments, the heavy chain of any anti-TfR 1 antibody as described herein may comprise a heavy chain constant region (CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region can have any suitable origin, such as human, mouse, rat, or rabbit. In one specific example, the heavy chain constant region is from a human IgG such as IgG1, igG2, or IgG4 (gamma heavy chain). An example of a human IgG1 constant region is given below:

in some embodiments, an anti-TfR 1 antibody described herein is a chimeric antibody comprising a heavy chain comprising the amino acid sequence of SEQ ID NO. 132. Alternatively or additionally (e.g., complementary), the anti-TfR 1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID NO: 133.

In some embodiments, an anti-TfR 1 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 (e.g., complementary), the anti-TfR 1 antibodies described herein comprise a light chain comprising the amino acid sequence of SEQ ID No. 135.

In some embodiments, the anti-TfR 1 antibody is an antigen-binding fragment (Fab) of an intact antibody (full length antibody). In some embodiments, an anti-TfR 1 Fab described herein comprises a heavy chain comprising the amino acid sequence of SEQ ID NO. 136. Alternatively or additionally (e.g., complementary), anti-TfR 1 Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID NO: 133. In some embodiments, an anti-TfR 1 Fab described herein comprises a heavy chain that comprises the amino acid sequence of SEQ ID NO. 137. Alternatively or additionally (e.g., complementary), an anti-TfR 1 Fab described herein comprises a light chain comprising the amino acid sequence of SEQ ID No. 135.

The anti-TfR 1 antibodies described herein may be in any antibody format, including, but not limited to, whole (i.e., full length) antibodies, antigen binding fragments thereof (e.g., fab ', F (ab') 2, fv), single chain antibodies, bispecific antibodies, or nanobodies. In some embodiments, the anti-TfR 1 antibodies described herein are scFv. In some embodiments, an anti-TfR 1 antibody described herein is an scFv-Fab (e.g., an scFv fused to a portion of a constant region). In some embodiments, an anti-TfR 1 antibody described herein is a scFv fused to a constant region (e.g., a human IgG1 constant region shown in SEQ ID NO: 81).

In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., CDR or framework sequence) at positions where the residues are unlikely to be involved in an interaction with a target antigen (e.g., transferrin receptor), e.g., as determined based on crystal structure. In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., at residues 231 to 340 of human IgG 1) and/or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG 1) and/or (e.g., and) the hinge region of an anti-TfR 1 antibody described herein, numbered according to the Kabat numbering system (e.g., 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 cytotoxicity.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH 1 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. patent No.5,677,425. The number of cysteine residues in the hinge region of the CH1 domain can be altered, for example, to facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or to facilitate linker conjugation.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., at residues 231 to 340 of human IgG 1) and/or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG 1) and/or (e.g., and) the hinge region of the muscle-targeting antibodies described herein, numbered according to the Kabat numbering system (e.g., the EU index in Kabat) to increase or decrease the affinity of the antibody for Fc receptors (e.g., activated Fc receptors) on the surface of effector cells. Mutations in the Fc region of antibodies that reduce or increase the affinity of the antibody for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments 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 an antibody for an Fc receptor are described in, for example, smith P et al, (2012) PNAS 109:6181-6186, U.S. Pat. No.6,737,056, and international publications nos. WO 02/060919, WO 98/23289 and WO 97/34631, which are incorporated herein by reference.

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 anti-TfR 1 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 would alter (e.g., reduce or increase) the half-life of an antibody in vivo.

In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into the IgG constant domain or FcRn binding fragment thereof (preferably, fc or hinge-Fc domain fragment) to reduce the half-life of the anti-TfR 1 antibody in vivo. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn binding fragment thereof (preferably, fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH 2) domain (residues 231 to 340 of human IgG 1) and/or (e.g., and) the third constant (CH 3) domain (residues 341 to 447 of human IgG 1) (numbered according to EU index (Kabat E Aet al., 1991) supra) in Kabat). In some embodiments, the constant region of 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 numbered according to the EU index as in Kabat. See U.S. Pat. No.7,658,921, which is incorporated herein by reference. Mutant IgG of this type (known as "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 W F et al, (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436, numbered according to the EU index as in Kabat.

In some embodiments, one, two, or more amino acid substitutions are introduced into the Fc region of an IgG constant domain to alter the effector function of an anti-TfR 1 antibody. The effector ligand for which affinity is altered may be, for example, an Fc receptor or the C1 component of complement. Such a process is described in more detail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation (by point mutation or otherwise) of the constant region domains may reduce Fc receptor binding of circulating antibodies, thereby improving tumor localization. For a description of mutations that delete or inactivate constant domains and thereby improve tumor localization, see, e.g., U.S. Pat. nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, e.g., SHIELDS R LET al, (2001) J Biol Chem 276:6591-604).

In some embodiments, one or more amino groups in the constant regions of an anti-TfR 1 antibody described herein may be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or (e.g., and) reduced or eliminated complement dependent cytotoxicity (complement dependent cytotoxicity, CDC). Such a process is described in more detail in U.S. Pat. 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 an antibody described herein are altered, thereby altering the ability of the antibody to fix complement. Such a process is further described in International publication No. WO 94/29351. In some embodiments, the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (antibody dependent cellular cytotoxicity, ADCC) and/or (e.g., and) to increase the affinity of the antibody for fcγ receptors. Such a method is further described in International publication No. WO 00/42072.

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

In some embodiments, the antibodies provided herein comprise mutations that confer a desired property to the antibody. For example, to avoid potential complications due to Fab-arm exchange known to occur with native IgG4 mabs, the antibodies provided herein may comprise a stable 'Adair' mutation (Angal S.,et al.,"Asingle amino acid substitution abolishes the heterogeneity of chimeric mouse/human(IgG4)antibody,"Mol Immunol 30,105-108;1993), in which serine at position 228 (EU numbering, residue 241 according to Kabat numbering) is converted to proline, thereby producing an IgG 1-like hinge sequence. Thus, any antibody may comprise a stable 'Adair' mutation.

In some embodiments, any of the anti-TfR 1 antibodies described herein may comprise a signal peptide (e.g., an N-terminal signal peptide) in the heavy chain sequence and/or (e.g., and) the light chain sequence. In some embodiments, an anti-TfR 1 antibody described herein comprises 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 comprises a signal peptide (e.g., an N-terminal signal peptide). In some embodiments, the signal peptide comprises amino acid sequence MGWSCIILFLVATATGVHS (SEQ ID NO: 104).

In some embodiments, the antibodies provided herein can have one or more post-translational modifications. In some embodiments, N-terminal cyclization, also known as pyroglutamic acid formation (pyro-Glu), can occur at the N-terminal glutamic acid (Glu) and/or glutamine (Gln) residues of the antibody during production. Thus, it is understood that antibodies designated as having a sequence comprising an N-terminal glutamic acid or glutamine residue encompass antibodies that have undergone pyroglutamic acid formation resulting from post-translational modification. In some embodiments, pyroglutamic acid formation occurs in the heavy chain sequence. In some embodiments, pyroglutamic acid formation occurs in the light chain sequence.

B. Other muscle targeting antibodies

In some embodiments, the muscle targeting antibody is an antibody that specifically binds to hemojuin (hemojuvelin), caveolin-3, duchenne muscular dystrophy peptide (Duchenne muscular dystrophy peptide), myosin IIb, or CD 63. In some embodiments, the muscle targeting antibody is an antibody that specifically binds to a myogenic precursor protein. Some exemplary myogenic precursor proteins include, but are not limited to, ABCG2, M-cadherin/cadherin-15, nidogen-1, CD34, foxK1, integrin alpha 7 beta 1, MYF-5, myoD, myogenin, NCAM-1/CD56, pax3, pax7, and Pax9. In some embodiments, the muscle targeting antibody is an antibody that specifically binds skeletal muscle protein. Some exemplary skeletal muscle proteins include, but are not limited to, alpha-actin (alpha-Sarcoglycan), beta-actin, calpain inhibitors, creatine kinase MM/CKMM, eIF5A, enolase 2/neuron-specific enolase, epsilon-actin, FABP3/H-FABP, GDF-8/myosin, GDF-11/GDF-8, integrin alpha 7 beta 1, integrin beta 1/CD29, MCAM/CD146, myoD, myogenin, myosin light chain kinase inhibitors, NCAM-1/CD56, and troponin I. In some embodiments, the muscle targeting antibody is an antibody that specifically binds smooth muscle protein. Some exemplary smooth muscle proteins include, but are not limited to, alpha-smooth muscle actin, VE-cadherin, calmodulin binding protein/CALD 1, calmodulin 1, desmin (Desmin), histamine H2R, motilin R/GPR38, transferrin/TAGLN, and vimentin. However, it is to be understood that antibodies to other targets are within the scope of the present disclosure, and that the exemplary list of targets provided herein is not meant to be limiting.

C. Antibody characterization/alteration

In some embodiments, conservative mutations may be introduced into an antibody sequence (e.g., CDR or framework sequence) at positions where the residues are unlikely to be involved in an interaction with a target antigen (e.g., transferrin receptor), e.g., as determined based on crystal structure. In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., at residues 231 to 340 of human IgG 1) and/or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG 1) and/or (e.g., and) the hinge region of a muscle-targeting antibody described herein, according to the Kabat numbering system (e.g., 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 cytotoxicity.

In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., at residues 231 to 340 of human IgG 1) and/or (e.g., and) the CH3 domain (residues 341 to 447 of human IgG 1) and/or (e.g., and) the hinge region of the muscle-targeting antibodies described herein, numbered according to the Kabat numbering system (e.g., the EU index in Kabat) to increase or decrease the affinity of the antibody for Fc receptors (e.g., activated Fc receptors) on the surface of effector cells. Techniques for reducing or increasing the affinity of an antibody for an Fc receptor by mutation in the Fc region of the antibody and introducing such mutation into the Fc receptor or fragment thereof are known to those skilled in the art. Some examples of mutations in antibody Fc receptors that can be made to alter the affinity of an antibody for an Fc receptor are described in, for example, smith P et al, (2012) PNAS109:6181-6186, U.S. Pat. No.6,737,056, and international publications nos. WO 02/060919, WO 98/23289 and WO 97/34631, which are incorporated herein by reference.

In some embodiments, one, two, or more amino acid mutations (i.e., substitutions, insertions, or deletions) are introduced into an 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 mutations that alter (e.g., reduce or increase) the half-life of an antibody in vivo.

In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn binding fragment thereof (preferably, fc or hinge-Fc domain fragment) to 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 FcRn binding fragment thereof (preferably, fc or hinge-Fc domain fragment) to increase the half-life of the antibody in vivo. In some embodiments, the antibody may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH 2) domain (residues 231 to 340 of human IgG 1) and/or (e.g., and) the third constant (CH 3) domain (residues 341 to 447 of human IgG 1) (numbered according to EU index (Kabat E Aet al., 1991) supra) in Kabat). In some embodiments, the constant region of 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 numbered according to the EU index as in Kabat. See U.S. Pat. No.7,658,921, which is incorporated herein by reference. Mutant IgG of this type (known as "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 W F et al, (2006) J Biol Chem 281:23514-24). In some embodiments, the antibody comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251 to 257, 285 to 290, 308 to 314, 385 to 389, and 428 to 436, numbered according to the EU index as in Kabat.

In some embodiments, one, two, or more amino acid substitutions are introduced into the IgG constant domain Fc region to alter the effector function of the anti-transferrin receptor antibody. The effector ligand for which affinity is altered may be, for example, an Fc receptor or the C1 component of complement. Such a process is described in more detail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, deletion or inactivation (by point mutation or otherwise) of the constant region domains may reduce Fc receptor binding of circulating antibodies, thereby improving tumor localization. For a description of mutations that delete or inactivate constant domains and thereby improve tumor localization, see, e.g., U.S. Pat. nos. 5,585,097 and 8,591,886. In some embodiments, one or more amino acid substitutions may be introduced into the Fc region of an antibody described herein to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, e.g., SHIELDS R L ET al, (2001) J Biol Chem 276:6591-604).

In some embodiments, one or more amino groups in the constant regions of the muscle-targeting antibodies described herein can be replaced with a different amino acid residue such that the antibody has altered C1q binding and/or (e.g., and) reduced or eliminated Complement Dependent Cytotoxicity (CDC). Such a process is described in more detail in U.S. Pat. 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 an antibody described herein are altered, thereby altering the ability of the antibody to fix complement. Such a process is further described in International publication No. WO 94/29351. In some embodiments, the Fc region of an antibody described herein is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or (e.g., and) increase the affinity of the antibody for fcγ receptors. Such a method is further described in International publication No. WO 00/42072.

In some embodiments, the antibodies provided herein comprise mutations that confer a desired property to the antibody. For example, to avoid potential complications due to Fab-arm exchange known to occur with native IgG4 mabs, the antibodies provided herein may comprise a stable 'Adair' mutation (Angal S.,et al.,"A single amino acid substitution abolishes the heterogeneity of chimeric mouse/human(IgG4)antibody,"Mol Immunol 30,105-108;1993), in which serine at position 228 (EU numbering, residue 241 according to Kabat numbering) is converted to proline, thereby producing an IgG 1-like hinge sequence. Thus, any antibody may comprise a stable 'Adair' mutation.

As provided herein, the antibodies of the present disclosure may optionally comprise a constant region or a portion thereof. For example, a VL domain may be linked at its C-terminal end to a light chain constant domain, such as ck or cλ. Similarly, VH domains or portions thereof may be linked to all or a portion of heavy chains such as IgA, igD, igE, igG and IgM, as well as any isotype subclasses. Antibodies may include suitable constant regions (see, e.g., ,Kabat et al.,Sequences of Proteins of Immunological Interest,No.91-3242,National Institutes of Health Publications,Bethesda,Md.(1991)). thus, antibodies within the scope of the disclosure may comprise VH and VL domains, or antigen-binding portions thereof, in combination with any suitable constant region.

Muscle targeting peptides

Some aspects of the present disclosure provide muscle targeting peptides as muscle targeting agents. Short peptide sequences (e.g., peptide sequences 5 to 20 amino acids in length) have been described that bind to specific cell types. For example, cell-targeted peptides are described below in U.S. Pat. No. ：Vines e.,et al.,A."Cell-penetrating and cell-targeting peptides in drug delivery"Biochim Biophys Acta 2008,1786:126-38;Jarver P.,et al.,"In vivo biodistribution and efficacy of peptide mediated delivery"Trends Pharmacol Sci 2010;31:528-35;Samoylova T.I.,et al.,"Elucidation of muscle-binding peptides by phage display screening"Muscle Nerve 1999;22:460-6;, 6,329,501, entitled "METHODS AND COMPOSITIONS FOR TARGETING COMPOUNDS TO MUSCLE" on month 12 and 11 of 2001, and Samoylov A.M.,et al.,"Recognition of cell-specific binding of phage display derived peptides using an acoustic wave sensor."Biomol Eng 2002;18:269-72;, each of which is incorporated herein by reference in its entirety. By designing the peptide to interact with a particular cell surface antigen (e.g., receptor), selectivity for a desired tissue, 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 to muscle tissue without many of the practical disadvantages of large antibodies or viral particles. Thus, in some embodiments, the muscle targeting agent is a muscle targeting peptide that is 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 (e.g., phage display) can be used to produce muscle targeting peptides.

In some embodiments, the muscle targeting peptide may bind to an internalized cell surface receptor, such as a transferrin receptor, that is overexpressed or relatively highly expressed in muscle cells as compared to certain other cells. In some embodiments, the muscle targeting peptide can target (e.g., bind to) a transferrin receptor. In some embodiments, a peptide that targets a transferrin receptor can comprise a segment of a naturally occurring ligand (e.g., transferrin). In some embodiments, the peptide that targets the transferrin RECEPTOR is as described in U.S. patent No.6,743,893, "receiver-MEDIATED UPTAKE OF PEPTIDES THAT BIND THE HUMAN TRANSFERRIN RECEPTOR," filed 11/30/2000. In some embodiments, the peptide that targets 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 that targets the transferrin receptor is as described in U.S. patent No.8,399,653, "TRANSFERRIN/TRANSFERRIN RECEPTOR-MEDIATED SIRNADELIVERY," filed 5, 20, 2011.

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

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

The muscle targeting agent may be an amino acid containing molecule or peptide. The muscle targeting peptide may 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-prone hydrophobic amino acid, such as valine, such that the peptide preferentially targets muscle cells. In some embodiments, the muscle targeting peptide is not previously characterized or disclosed. These peptides can be contemplated, generated, synthesized, and/or (e.g., and) derivatized using any of a number of methods, such as phage display peptide libraries, single-bead single-compound peptide libraries, or positionally scanned synthetic peptide combinatorial libraries. Some exemplary methods have been characterized in the art and (Gray,B.P.and Brown,K.C."Combinatorial Peptide Libraries:Mining for Cell-Binding Peptides"Chem Rev.2014,114:2,1020-–1081.;Samoylova,T.I.and Smith,B.F."Elucidation of muscle-binding peptides by phage display screening."Muscle Nerve,1999,22:4.460-6.). incorporated by reference in some embodiments, muscle targeting peptides have been previously disclosed (see, e.g., Writer M.J.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,M.J.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 comprise the following sets of amino acid sequences:

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. Muscle targeting peptides 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, homoamino 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, and in other embodiments, the muscle targeting peptide may be cyclic, e.g., bicyclic (see, e.g., silvana, m.g. et al mol. Therapy,2018,26:1, 132-147.).

Muscle targeting receptor ligands

The muscle targeting agent may be a ligand, for example a ligand that binds to a receptor protein. The muscle targeting ligand may be a protein, such as transferrin, which binds to internalized cell surface receptors expressed by muscle cells. Thus, in some embodiments, the muscle targeting agent is transferrin or a derivative thereof that binds to a transferrin receptor. The muscle targeting ligand may alternatively be a small molecule, such as a lipophilic small molecule that preferentially targets muscle cells over other cell types. Some exemplary lipophilic small molecules that can target muscle cells include compounds comprising cholesterol, cholesteryl, stearic acid, palmitic acid, oleic acid, oleyl, linolen (linolene), linoleic acid, myristic acid, sterols, dihydrotestosterone, testosterone derivatives, glycerol, alkyl chains, trityl groups, and alkoxy acids.

Muscle targeting aptamer

The muscle targeting agent may be an aptamer, e.g., an RNA aptamer, that preferentially targets muscle cells over other cell types. In some embodiments, the muscle targeting aptamer is previously uncharacterized or disclosed. These aptamers can be conceived, generated, synthesized, and/or (e.g., and) derived using any of several methods (e.g., by systematic evolution of exponentially enriched ligands). Some exemplary methods have been characterized in the art and incorporated by reference (Yan,A.C.and Levy,M."Aptamers and aptamer targeted delivery"RNAbiology,2009,6:3,316-20.;Germer,K.et al."RNAaptamers and their therapeutic and diagnostic applications."Int.J.Biochem.Mol.Biol.2013;4:27–40.). in some embodiments, muscle targeting aptamers have been previously disclosed (see, e.g., Phillippou,S.et al."Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers."Mol Ther Nucleic Acids.2018,10:199-214.;Thiel,W.H.et al."Smooth Muscle Cell-targeted RNAAptamer Inhibits Neointimal Formation."Mol Ther.2016,24:4,779-87.). exemplary muscle targeting aptamers include a01B RNA aptamer and RNAApt, in some embodiments, in some embodiments, the aptamer may be about 5kDa to 15kDa, about 5kDa to 10kDa, about 10kDa to 15kDa, about 1 to 5Da, about 1kDa to 3kDa, or less.

V. other muscle targeting agents

One strategy for targeting muscle cells (e.g., skeletal muscle cells) is to use substrates for muscle transporter proteins (e.g., transporter proteins expressed on the myomembrane). In some embodiments, the muscle targeting agent is a substrate for an influx transporter specific for muscle tissue. In some embodiments, the inflow transporter is specific for skeletal muscle tissue. Two major classes of transporters are expressed on skeletal muscle membranes, (1) adenosine triphosphate (adenosine triphosphate, ATP) binding cassette (adenosine triphosphate binding cassette, ABC) superfamily, which promotes efflux from skeletal muscle tissue and (2) solute carrier (solute carrier, SLC) superfamily, which can promote substrate influx into skeletal muscle. In some embodiments, the 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, e.g., a synthetic derivative thereof that binds to the ABC or SLC superfamily of transporters.

In some embodiments, the muscle targeting agent is any muscle targeting agent (e.g., an 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 balanced or use proton or sodium ion gradients generated across the membrane to drive substrate transport. Some exemplary SLC transporters with high skeletal muscle expression include, but are not limited to, SATT transporter (ASCT 1; SLC1A 4), GLUT4 transporter (SLC 2A 4), GLUT7 transporter (GLUT 7; SLC2A 7), ATRC2 transporter (CAT-2; SLC7A 2), LAT3 transporter (KIAA 0245; SLC7A 6), PHT1 transporter (PTR 4; SLC15A 4), OATP-J transporter (OATP 5A1; SLC21A 15), OCT3 transporter (EMT; SLC22A 3), OCTN2 transporter (FLJ 46769; SLC22A 5), ENT transporter (ENT 1; SLC29A1 and ENT2; SLC29A 2), PAT2 transporter (SLC 36A 2) and SAT2 transporter (KIAA 2; SLC38A 2) 138138138138. These transporters may facilitate substrate flow into skeletal muscle, providing opportunities for muscle targeting.

In some embodiments, the muscle targeting agent is a substrate for an equilibrium nucleoside transporter 2 (equilibrative nucleoside transporter, ent 2) transporter. ENT2 has one of the highest mRNA expression in skeletal muscle relative to other transporters. Although human ENT2 (hENT 2) is expressed in most body organs such as brain, heart, placenta, thymus, pancreas, prostate and kidney, it is particularly abundant in skeletal muscle. Human ENT2 promotes its substrate absorption 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 low affinity for all nucleosides (adenosine, guanosine, uridine, thymidine, and cytidine) except inosine. Thus, in some embodiments, the muscle targeting agent is an ENT2 substrate. Exemplary ENT2 substrates include, but are not limited to, inosine, 2',3' -dideoxyinosine, and clofarabine (calofarabine). In some embodiments, any of the muscle targeting agents provided herein are associated with a molecular payload (e.g., an oligonucleotide payload). In some embodiments, the muscle targeting agent is covalently linked to the molecular payload. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular payload.

In some embodiments, the muscle targeting agent is a substrate for an organic cation/carnitine transporter (OCTN 2), which is a sodium ion dependent high affinity carnitine transporter. In some embodiments, the muscle targeting agent is carnitine, mildronate (mildronate), acetyl carnitine, or any derivative thereof that binds to OCTN 2. In some embodiments, carnitine, mildronate, acetyl carnitine, or derivatives thereof, is covalently linked to a molecular payload (e.g., an oligonucleotide payload).

The muscle targeting agent may be a protein, which is a protein that exists in at least one soluble form that targets muscle cells. In some embodiments, the muscle targeting protein may be a hemojuin (also known as repulsive guidance molecule C or hemochromatosis type 2 protein), a protein involved in iron overload and homeostasis. In some embodiments, the hemojuin may be full length or a fragment, or a mutant having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to a functional hemojuin 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 may be annotated with GenBank RefSeq accession No. nm_001316767.1, nm_145277.4, nm_202004.3, nm_213652.3, or nm_ 213653.3. It is understood that the hemojuvelin may be of human, non-human primate or rodent origin.

B. Molecular payload

Some aspects of the disclosure provide molecular payloads, e.g., oligonucleotides that are intended to target DUX4RNA to modulate expression or activity of DUX 4. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4RNA that are useful for reducing the level of DUX4 mRNA and/or protein associated with facial shoulder brachial muscular dystrophy (FSHD) pathology features, including muscle atrophy, inflammation, and reduced differentiation potential, as well as oxidative stress. In some embodiments, the oligonucleotides provided herein are intended to direct RNAi-mediated degradation of DUX4 RNA. In some embodiments, the oligonucleotides are intended to effectively engage the RNA-induced silencing complex (RISC) to degrade DUX4RNA and also have reduced off-target effects. In some embodiments, the oligonucleotides are intended to have desired bioavailability and/or serum stability characteristics. In some embodiments, the oligonucleotides are intended to have desired binding affinity properties. In some embodiments, the oligonucleotides are intended to have a desired toxicity and/or immunogenicity profile.

In some embodiments, the oligonucleotide comprises a strand having a complementary region to the 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.

I. oligonucleotides

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) that forms a double-stranded oligonucleotide. It will be appreciated that in some embodiments, one form of the oligonucleotide (e.g., antisense oligonucleotide) may be suitably adapted to another form (e.g., siRNA oligonucleotide) by incorporating a functional sequence (e.g., antisense strand sequence) from one form to another.

Any suitable oligonucleotide may be used as the molecular payload, as described herein. Some examples of oligonucleotides that can be used to target DUX4 are provided in U.S. patent No. 9,988,628, which is disclosed on month 2, 2017, under the heading "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY", U.S. patent No. 9,469,851, which is disclosed on month 10, 30, 2014, under the heading "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX4", U.S. patent application publication No. 20120225034, which is disclosed on month 9, 6, 2012, under the heading "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY", PCT patent application publication No. WO 2013/120038, which is disclosed on month 8, 15, 2013, under the heading "MORPHOLINO TARGETING DUX4FOR TREATING FSHD";Chen et al.,"Morpholino-mediated Knockdown of DUX4 Toward Facioscapulohumeral Muscular Dystrophy Therapeutics,"Molecular Therapy,2016,24：8,1405-1411.; and Ansseau et al.,"Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in Facioscapulohumeral Muscular Dystrophy(FSHD),"Genes,2017,8,93;, each of which is incorporated herein in its entirety. In some embodiments, the oligonucleotide is an antisense oligonucleotide, morpholino, siRNA, shRNA, or other oligonucleotide that hybridizes to a target DUX4 gene or mRNA. In some embodiments, the oligonucleotide is an siRNA oligonucleotide.

In some embodiments, the oligonucleotides described herein have a complementary region of the indicated sequence corresponding to human DUX4, corresponding to 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 NCBI sequence NM-001081954.1 (SEQ ID NO: 162). Other non-limiting exemplary human DUX4 mRNA includes NCBI sequence NM-033178, genBank accession No. FJ439133,

AF117653,HM101229,HM101230,HM101232,HM101233,HM101234,HM101235,HM101240,HM101241,HM101242,HM101243,HM101244,HM101245,HM101246,HM101247,HM101248,HM101249,HM101250,HM101251 and HM190160,HM190161,HM190162,HM190163,HM190164,HM190165,HM190166,HM190167,HM190168,HM190169,HM190170,HM190171,HM190172,HM190173,HM190174,HM190175,HM190176,HM190177,HM190178,HM190179,HM190180,HM190181,HM190182,HM190183,HM190184,HM190185,HM190186,HM190187,HM190188,HM190189,HM190190,HM190191,HM190192,HM190193,HM190194,HM190195,HM190196,

Each of which is incorporated herein by reference. In some embodiments, the oligonucleotide may have a complementary region of hypomethylated compact D4Z4 repeats, such as Daxinger, et al, "GENETIC AND EPIGENETIC Contributors to FSHD," Curr Opin Genet Dev,Lim J-W,et al.,DICER/AGO-dependent epigenetic silencing of D4Z4 repeats enhanced by exogenous siRNA suggests mechanisms and therapies for FSHD Hum Mol Genet.2015Sep 1;24(17)：4817-4828, disclosed in 2015, each of which is incorporated in its entirety.

In some embodiments, the oligonucleotide may have a complementary region of the sequence shown below, which is an exemplary human DUX4 gene sequence (NM-001293798.2) (SEQ ID NO: 160):

In some embodiments, the oligonucleotide may have a complementary region of the sequence shown below, which is an exemplary human DUX4 gene sequence (NM-001306068.3) (SEQ ID NO: 161):

In some embodiments, the oligonucleotide may have a complementary region of the sequence shown below, which is an exemplary mouse DUX4 gene sequence (SEQ ID NO: 162) (NM-001081954.1):

In some embodiments, the oligonucleotides may have complementary regions of DUX4 gene sequences of a variety of species, for example selected from human, mouse, and non-human species. In some embodiments, the non-human species is cynomolgus monkey.

Oligonucleotide size/sequence

Oligonucleotides may be of a variety of different lengths, for example, depending on the format. In some embodiments, the 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 nucleotides in length. In some embodiments, the oligonucleotide is 8 to 50 nucleotides in length, 8 to 40 nucleotides in length, 8 to 32 nucleotides in length, 10 to 15 nucleotides in length, 10 to 20 nucleotides in length, 15 to 25 nucleotides in length, 21 to 23 nucleotides in length, and the like. 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.

In some embodiments, when the binding of the complementary nucleic acid sequence of an oligonucleotide to a target molecule (e.g., mRNA) interferes with the normal function of the target (e.g., mRNA) resulting in 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 the non-target sequence, the complementary nucleic acid sequence of the oligonucleotide may specifically hybridize or be specific to the target nucleic acid for purposes of this disclosure under conditions in which non-specific binding is desired to be avoided, e.g., under physiological conditions in the case of in vivo assays or therapeutic treatments, and under physiological conditions in the case of in vitro assays, under conditions in which assays are performed under suitably stringent conditions. Thus, in some embodiments, an oligonucleotide can be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to consecutive nucleotides of a target nucleic acid. In some embodiments, the complementary nucleotide sequence need not be 100% complementary to the target nucleic acid to which it is targeted to specifically hybridize or be specific for the target nucleic acid. In some embodiments, the oligonucleotide comprises one or more nucleobases mismatched relative to the target nucleic acid. In some embodiments, the activity associated with the target is reduced due to such mismatches, but the amount of activity associated with the non-target is reduced more (i.e., the selectivity for the target nucleic acid is increased and the off-target effect is reduced). In some embodiments, the target nucleic acid is a pre-mRNA molecule or an mRNA molecule.

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

In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive nucleotides comprising the 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 sharing 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.

In some embodiments, the oligonucleotide comprises a region complementary to the target sequence set forth in any one of SEQ ID NOS.174 to 235. In some embodiments, the oligonucleotide comprises a region of complementarity having at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% complementarity to at least 12 or at least 15 consecutive nucleotides of the target sequence set forth in any one of SEQ ID NOS.174 to 235. In some embodiments, the length of the complementary region 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 complementary region is 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides in length. In some embodiments, the length of the complementary region 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.

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

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

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

B. oligonucleotide modification:

The oligonucleotides described herein can be modified, e.g., comprise a modified sugar moiety, a modified internucleoside linkage, a modified nucleotide or nucleoside, and/or (e.g., and) combinations thereof. In addition, in some embodiments, the oligonucleotides may exhibit one or more of the following properties of not mediating alternative splicing, not immunostimulatory, nuclease resistance, improved cellular uptake compared to unmodified oligonucleotides, non-toxicity to cells or mammals, improved endosomal excretion within cells, minimizing TLR stimulation, or avoiding pattern recognition receptors. Any of the modified chemical compositions (chemistry) or forms of the oligonucleotides described herein may be combined with one another. For example, one, two, three, four, five or more different types of modifications may be included within the same oligonucleotide.

In some embodiments, certain nucleotide or nucleoside modifications may be used that render the oligonucleotides incorporating them more resistant to nuclease digestion than the native oligodeoxynucleotide or oligoribonucleotide molecule, and these modified oligonucleotides survive longer than the unmodified oligonucleotides intact. Some specific examples of modified oligonucleotides include those containing modified backbones (backbones), such as modified internucleoside linkages, e.g., phosphorothioate linkages, phosphotriester linkages, methylphosphonate linkages, short chain alkyl linkages or cycloalkyl intersugar linkages, or short chain heteroatom linkages or heterocyclic intersugar linkages. Thus, the oligonucleotides of the present disclosure may be stabilized against nucleolytic degradation, for example, by incorporating modifications such as nucleotides or nucleoside modifications.

In some embodiments, the length of the oligonucleotide may be up to 50 or up to 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 may 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 may 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 may have each nucleotide or nucleoside other than 1,2,3, 4,5, 6,7, 8, 9 or 10 modified nucleotides/nucleosides. Oligonucleotide modifications are further described herein.

C. Modified nucleosides

In some embodiments, an oligonucleotide described herein comprises at least one nucleoside modified at the 2' position of a 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.

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 '-O-N-methylacetamido (2' -O-NMA) modified nucleosides.

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, an oligonucleotide described herein comprises 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.

In some embodiments, the oligonucleotides described herein comprise one or more 2'-4' bicyclic nucleosides, wherein the ribose ring comprises a bridging moiety connecting two atoms in the ring, e.g., connecting the 2'-O atom to the 4' -C atom by methylene (LNA) bridging, ethylene (ENA) bridging, or (S) -constrained ethyl (cEt) bridging. Some examples of LNAs are described in international patent application publication WO/2008/043753, published on month 17 of 2008, and titled "RNA Antagonist Compounds For The Modulation Of PCSK9", the contents of which are incorporated herein by reference in their entirety. Some examples of ENA are provided in International patent publication No. WO 2005/042777, published on month 5 and 12 of 2005, and titled "APP/ENA ANTISENSE", morita et al, nucleic Acids Res, journal 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 cets are provided in U.S. patent 7,101,993, 7,399,845, and 7,569,686, each of which is incorporated by reference herein in its entirety.

In some embodiments, the oligonucleotides comprise modified nucleosides disclosed in one of the following U.S. patent or patent application publications, U.S. patent 7,399,845, which is entitled to "6-Modified Bicyclic Nucleic Acid Analogs" at 15 months of 2008, U.S. patent 7,741,457, which is entitled to "6-Modified Bicyclic Nucleic Acid Analogs" at 22 months of 2010, U.S. patent 8,022,193, which is entitled to "6-Modified Bicyclic Nucleic Acid Analogs" at 20 months of 2011, U.S. patent 7,569,686, which is entitled to "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs" at 4 months of 2009, and entitled to "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs", U.S. patent 7,335,765, which is entitled to "Novel Nucleoside And Oligonucleotide Analogues" at 26 months of 2008, U.S. patent 7,314,923, which is entitled to "1 month of 2008, and entitled to" Novel Nucleoside And Oligonucleotide Analogues ", U.S. patent 7,816,333, which is entitled to" Oligonucleotide Analogues And Methods Utilizing The Same "and U.S. publication No. 2011/0009471, which is entitled to" 8,957,201, month 2, and 2015, each of which is entitled to "2015, and all of which is entitled to be individually incorporated herein by reference.

In some embodiments, the oligonucleotide comprises at least one modified nucleoside that results in an increase in Tm of 1 ℃,2 ℃,3 ℃,4 ℃, or 5 ℃ compared to an oligonucleotide without the at least one modified nucleoside. The oligonucleotide can have a plurality of modified nucleosides that result in an overall increase in Tm of 2 ℃,3 ℃,4 ℃,5 ℃, 6 ℃, 7 ℃, 8 ℃, 9 ℃,10 ℃,15 ℃, 20 ℃, 25 ℃, 30 ℃, 35 ℃,40 ℃, 45 ℃ or higher as compared to an oligonucleotide without the modified nucleoside.

The oligonucleotides may comprise a mixture of different kinds of nucleosides. For example, the oligonucleotide may comprise a2 '-deoxyribonucleoside or a mixture of ribonucleosides and 2' -fluoro modified nucleosides. The oligonucleotide may comprise deoxyribonucleosides or a mixture of ribonucleosides and 2' -O-Me modified nucleosides. The oligonucleotide may comprise a mixture of 2 '-fluoro modified nucleosides and 2' -O-methyl modified nucleosides. The oligonucleotide may comprise a mixture of bridged nucleosides and 2 '-fluoro or 2' -O-methyl modified nucleosides. The oligonucleotide may comprise a mixture of non-bicyclic 2 '-modified nucleosides (e.g., 2' -O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). The oligonucleotide may comprise a mixture of 2 '-fluoro modified nucleosides and 2' -O-Me modified nucleosides. The oligonucleotide may comprise a mixture of 2' -4' bicyclic nucleosides and 2' -MOE, 2' -fluoro, or 2' -O-Me modified nucleosides. The oligonucleotide may comprise a mixture of non-bicyclic 2 '-modified nucleosides (e.g., 2' -MOE, 2 '-fluoro, or 2' -O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

The oligonucleotides may comprise different kinds of substituted nucleosides. For example, the oligonucleotide may comprise a substituted 2 '-deoxyribonucleoside or ribonucleoside and a 2' -fluoro modified nucleoside. The oligonucleotides may comprise alternative deoxyribonucleosides or ribonucleosides and 2' -O-Me modified nucleosides. The oligonucleotides may comprise alternative 2 '-fluoro modified nucleosides and 2' -O-Me modified nucleosides. The oligonucleotide may comprise alternative bridged nucleosides and 2 '-fluoro or 2' -O-methyl modified nucleosides. The oligonucleotides may comprise alternative non-bicyclic 2 '-modified nucleosides (e.g., 2' -O-MOE) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt). The oligonucleotides may comprise alternative 2' -4' bicyclic nucleosides and 2' -MOE, 2' -fluoro or 2' -O-Me modified nucleosides. The oligonucleotides may comprise alternative non-bicyclic 2 '-modified nucleosides (e.g., 2' -MOE, 2 '-fluoro, or 2' -O-Me) and 2'-4' bicyclic nucleosides (e.g., LNA, ENA, cEt).

In some embodiments, the oligonucleotides described herein comprise 5' -vinylphosphonate modifications, 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 sense strand comprises a 5 '-vinylphosphonate (e.g., 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., 5' - (E) -vinylphosphonate modification).

D. Internucleoside linkage/backbone

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

Phosphorus-containing linkages that may be used include, but are not limited to, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methylphosphonates and other alkylphosphonates comprising 3 '-alkylene phosphonates, as well as chiral phosphonates, phosphinates, phosphoramidates comprising 3' -phosphoramidates and aminoalkyl phosphoramidates, phosphorothioate alkyl phosphonates, phosphorothioate alkyl phosphotriesters and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with opposite polarity where adjacent pairs of nucleoside units are linked at 3'-5' to 5'-3' or 2'-5' to 5'-2', see U.S. patent 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;5,405,939;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.

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 (peptide nucleic acid, PNA) backbone (in which the phosphodiester backbone of the oligonucleotide is replaced with a polyamide backbone, the nucleotides being directly or indirectly bound to the aza nitrogen atoms of the polyamide backbone, see NIELSEN ET al., science 1991,254,1497).

E. Stereospecific oligonucleotides

In some embodiments, the internucleotide phosphorus atoms of the oligonucleotide are chiral, and the properties of the oligonucleotide are adjusted based on the configuration of the chiral phosphorus atoms. In some embodiments, the P-chiral oligonucleotide analogs can be synthesized in a stereotactic manner using appropriate methods (e.g., as described in Oka N,Wada T.Stereocontrolled synthesis of oligonucleotide analogs containing chiral internucleotidic phosphorus atoms.Chem Soc Rev.2011Dec;40(12)：5829-43). In some embodiments, phosphorothioate-containing oligonucleotides are provided comprising nucleoside units linked together by substantially all Sp or substantially all Rp phosphorothioate sugar-to-sugar linkages. In some embodiments, such phosphorothioate oligonucleotides with substantially chiral pure intersaccharide linkages are prepared by enzymatic or chemical synthesis, as described, for example, in U.S. patent 5,587,261 issued 12/1996, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the chiral control oligonucleotide provides a selective cleavage pattern for a target nucleic acid. For example, in some embodiments, the chirally controlled oligonucleotides provide single site cleavage within the complementary sequence of the nucleic acid, as described, for example, in U.S. patent application publication No. 20170037399 A1, published 2 nd 2017, titled "CHIRAL DESIGN", the contents of which are incorporated herein by reference in their entirety.

F. Morpholino compounds

In some embodiments, the oligonucleotide may be a morpholino-based compound. Morpholino-based oligomeric compounds are described in Dwaine A. Braasch and David R.Corey, biochemistry,2002,41 (14), 4503-4510), genesis, volume 30, stage 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 7/23 1991. In some embodiments, the morpholino-based oligomeric compound is a diamide morpholino phosphate 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 entirety).

H. Spacer polymers

In some embodiments, the oligonucleotides described herein are spacer polymers. The spacer oligonucleotide generally has the formula 5'-X-Y-Z-3', wherein X and Z act as flanking regions around spacer Y. In some embodiments, flanking region X of the formula 5'-X-Y-Z-3' is also referred to as the X region, flanking sequence X, 5 'flanking region X or 5' flanking region. In some embodiments, flanking region Z of the formula 5'-X-Y-Z-3' is also referred to as the Z region, flanking sequence Z, 3 'flanking region Z or 3' flanking region. In some embodiments, spacer Y of formula 5'-X-Y-Z-3' is also referred to as a Y region, Y segment or spacer Y. In some embodiments, each nucleoside in spacer Y is a2 '-deoxyribonucleoside, and neither the 5' wing region X nor the 3 'wing region Z comprises any 2' -deoxyribonucleoside.

In some embodiments, the Y region is a contiguous extension of nucleotides, e.g., a region of 6 or more DNA nucleotides, that is capable of recruiting an rnase (e.g., rnase H). In some embodiments, spacer and target nucleic acid binding, at which point RNase recruits and can then cut the target nucleic acid. In some embodiments, both the 5 'and 3' regions of Y are flanked by X and Z regions comprising high affinity modified nucleosides, e.g., 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, flanking sequences X and Z may be 1 to 20 nucleotides, 1 to 8 nucleotides, or 1 to 5 nucleotides in length. Flanking sequences X and Z may have similar lengths or different lengths. In some embodiments, the spacer segment Y may be a nucleotide sequence of 5 to 20 nucleotides, 5 to 15 twelve nucleotides, or 6 to 10 nucleotides in length.

In some embodiments, the spacer region of the spacer oligonucleotide may comprise modified nucleotides, such as C4' -substituted nucleotides, acyclic nucleotides, and arabinose (arabino) configured nucleotides, that are known to be acceptable for efficient rnase H action, in addition to DNA nucleotides. In some embodiments, the spacer comprises one or more unmodified internucleoside linkages. In some embodiments, one or both flanking regions each independently comprise one or more phosphorothioate internucleoside linkages (e.g., phosphorothioate internucleoside linkages or other linkages) between at least two, at least three, at least four, at least five, or more nucleotides. In some embodiments, the spacer region and the two flanking regions each independently comprise a modified internucleoside linkage (e.g., phosphorothioate internucleoside linkage or other linkage) between at least two, at least three, at least four, at least five or more nucleotides.

Spacer polymers can be produced using suitable methods. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of spacer polymers include, but are not limited to:

U.S. patent No.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;7,015,315;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;

U.S. patent publication nos. US20050074801, US20090221685, US20090286969, US20100197762 and US20110112170, PCT publication nos. WO2004069991, WO2005023825, WO2008049085 and WO2009090182, and EP patent No. EP2,149,605, each of which is incorporated herein by reference in its entirety.

In some embodiments, the spacer is 10 to 40 nucleosides in length. For example, the spacer 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 length. In some embodiments, the spacer 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.

In some embodiments, the spacer region Y in the spacer is 5 to 20 nucleosides in length. For example, the length of spacer Y may 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 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 spacer Y is a 2' -deoxyribonucleoside. In some embodiments, all nucleosides in spacer Y are 2' -deoxyribonucleosides. In some embodiments, one or more nucleosides in spacer Y are modified nucleosides (e.g., 2' modified nucleosides, such as those described herein). In some embodiments, one or more cytosines in spacer Y are optionally 5-methyl-cytosine. In some embodiments, each cytosine in spacer Y is a 5-methyl-cytosine.

In some embodiments, the 5 'wing region of the spacer (X in the 5' -X-Y-Z-3 'formula) and the 3' wing region of the spacer (Z in the 5'-X-Y-Z-3' formula) are independently 1 to 20 nucleosides in length. For example, the 5 'wing region of the spacer (X in the 5' -X-Y-Z-3 'formula) and the 3' wing region of the spacer (Z in the 5'-X-Y-Z-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 long. In some embodiments, the 5 'wing region of the spacer (X in the 5' -X-Y-Z-3 'formula) and the 3' wing region of the spacer (Z in the 5'-X-Y-Z-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 region of the spacer (X in the 5' -X-Y-Z-3 ') and the 3' wing region of the spacer (Z in the 5 '-X-Y-Z-3') have the same length. In some embodiments, the 5 'wing region of the spacer (X in the 5' -X-Y-Z-3 'formula) and the 3' wing region of the spacer (Z in the 5'-X-Y-Z-3' formula) have different lengths. In some embodiments, the 5 'wing region of the spacer (X in the 5' -X-Y-Z-3 'formula) is longer than the 3' wing region of the spacer (Z in the 5'-X-Y-Z-3' formula). In some embodiments, the 5 'wing region of the spacer (X in the 5' -X-Y-Z-3 'formula) is shorter than the 3' wing region of the spacer (Z in the 5'-X-Y-Z-3' formula).

In some embodiments, the spacer polymer comprises the following 5'-X-Y-Z-3':

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

The numbers represent the number of nucleosides in the X, Y '-X-Y-Z-3' spacer and in the Z region.

In some embodiments, one or more nucleosides in the 5 'wing region of the spacer (X in the 5' -X-Y-Z-3 'formula) or the 3' wing region of the spacer (Z in the 5'-X-Y-Z-3' formula) are modified nucleosides (e.g., high affinity modified nucleosides). In some embodiments, the modified nucleoside (e.g., a high affinity modified nucleoside) is a 2' -modified nucleoside. In some embodiments, the 2 '-modified nucleoside is a 2' -4 'bicyclic nucleoside or a non-bicyclic 2' -modified nucleoside. In some embodiments, the high affinity modified nucleoside is a 2' -4' bicyclic nucleoside (e.g., LNA, cEt, or ENA) or a non-bicyclic 2' -modified nucleoside (e.g., 2' -fluoro (2 ' -F), 2' -O-methyl (2 ' -O-Me), 2' -O-methoxyethyl (2 ' -MOE), 2' -O-aminopropyl (2 ' -O-AP), 2' -O-dimethylaminoethyl (2 ' -O-DMAOE), 2' -O-dimethylaminopropyl (2 ' -O-DMAP), 2' -O-dimethylaminoethoxyethyl (2 ' -O-DMAEOE), or 2' -O-N-methylacetamido (2 ' -O-NMA)).

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

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

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

In some embodiments, the spacer comprises a 5'-X-Y-Z-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 spacer comprises a 5'-X-Y-Z-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.

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

In some embodiments, the spacer comprises a 5' -X-Y-Z-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, or 7 (most 5' positions) in X are non-bicyclic 2' -modified nucleosides (e.g., 2' -MOE or 2' -O-Me), wherein the remaining nucleosides in both X and Z are 2' -4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, the spacer comprises a 5' -X-Y-Z-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, or 7 (most 5' positions) in Z are non-bicyclic 2' -modified nucleosides (e.g., 2' -MOE or 2' -O-Me), wherein the remaining nucleosides in both X and Z are 2' -4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a 2' deoxyribonucleoside. In some embodiments, the spacer comprises a 5' -X-Y-Z-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, or 6) in X and at least one but not all (e.g., 1,2,3, 5, 6, or 7 (the most 5' position is position 1) in Z are non-bicyclic 2' -modified nucleosides (e.g., 2' -MOE or 2' -O-Me), wherein the remaining nucleosides in both X and Z are 2' -4' bicyclic nucleosides (e.g., LNA or cEt), and wherein each nucleoside in Y is a deoxyribonucleoside in 2' ribose.

Some non-limiting examples of spacer 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 region of the spacer (X in the 5' -X-Y-Z-3 ') and/or the 3' wing region of the spacer (Z in the 5' -X-Y-Z-3 ') include:

BBB-(D)n-BBBAA;KKK-(D)n-KKKAA;LLL-(D)n-LLLAA;BBB-(D)n-BBBEE;KKK-(D)n-KKKEE;LLL-(D)n-LLLEE;BBB-(D)n-BBBAA;KKK-(D)n-KKKAA;LLL-(D)n-LLLAA;BBB-(D)n-BBBEE;KKK-(D)n-KKKEE;LLL-(D)n-LLLEE;BBB-(D)n-BBBAAA;KKK-(D)n-KKKAAA;LLL-(D)n-LLLAAA;BBB-(D)n-BBBEEE;KKK-(D)n-KKKEEE;LLL-(D)n-LLLEEE;BBB-(D)n-BBBAAA;KKK-(D)n-KKKAAA;LLL-(D)n-LLLAAA;BBB-(D)n-BBBEEE;KKK-(D)n-KKKEEE;LLL-(D)n-LLLEEE;BABA-(D)n-ABAB;KAKA-(D)n-AKAK;LALA-(D)n-ALAL;BEBE-(D)n-EBEB;KEKE-(D)n-EKEK;LELE-(D)n-ELEL;BABA-(D)n-ABAB;KAKA-(D)n-AKAK;LALA-(D)n-ALAL;BEBE-(D)n-EBEB;KEKE-(D)n-EKEK;LELE-(D)n-ELEL;ABAB-(D)n-ABAB;AKAK-(D)n-AKAK;ALAL-(D)n-ALAL;EBEB-(D)n-EBEB;EKEK-(D)n-EKEK;ELEL-(D)n-ELEL;ABAB-(D)n-ABAB;AKAK-(D)n-AKAK;ALAL-(D)n-ALAL;EBEB-(D)n-EBEB;EKEK-(D)n-EKEK;ELEL-(D)n-ELEL;AABB-(D)n-BBAA;BBAA-(D)n-AABB;AAKK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;AABB-(D)n-BBAA;AAKK-(D)n-KKAA;AALL-(D)n-LLAA;EEBB-(D)n-BBEE;EEKK-(D)n-KKEE;EELL-(D)n-LLEE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;LLL-(D)n-LLE;BBB-(D)n-BBA;KKK-(D)n-KKA;LLL-(D)n-LLA;BBB-(D)n-BBE;KKK-(D)n-KKE;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;AKKK-(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-BBBAA;AKKK-(D)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-(D)n-LLLAA;EBBB-(D)n-BBBEE;EKKK-(D)n-KKKEE;ELLL-(D)n-LLLEE;AABBB-(D)n-BBB;AAKKK-(D)n-KKK;AALLL-(D)n-LLL;EEBBB-(D)n-BBB;EEKKK-(D)n-KKK;EELLL-(D)n-LLL;AABBB-(D)n-BBB;AAKKK-(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;AALLL-(D)n-LLLA;EEBBB-(D)n-BBBE;EEKKK-(D)n-KKKE;EELLL-(D)n-LLLE;ABBAABB-(D)n-BB;AKKAAKK-(D)n-KK;ALLAALLL-(D)n-LL;EBBEEBB-(D)n-BB;EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL;ABBAABB-(D)n-BB;AKKAAKK-(D)n-KK;ALLAALL-(D)n-LL;EBBEEBB-(D)n-BB;EKKEEKK-(D)n-KK;ELLEELL-(D)n-LL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALLL-(D)n-LLL;EBBEBB-(D)n-BBB;EKKEKK-(D)n-KKK;ELLELL-(D)n-LLL;ABBABB-(D)n-BBB;AKKAKK-(D)n-KKK;ALLALL-(D)n-LLL;EBBEBB-(D)n-BBB;EKKEKK-(D)n-KKK;ELLELL-(D)n-LLL;EEEK-(D)n-EEEEEEEE;EEK-(D)n-EEEEEEEEE;EK-(D)n-EEEEEEEEEE;EK-(D)n-EEEKK;K-(D)n-EEEKEKE;K-(D)n-EEEKEKEE;K-(D)n-EEKEK;EK-(D)n-EEEEKEKE;EK-(D)n-EEEKEK;EEK-(D)n-KEEKE;EK-(D)n-EEKEK;EK-(D)n-KEEK;EEK-(D)n-EEEKEK;EK-(D)n-KEEEKEE;EK-(D)n-EEKEKE;EK-(D)n-EEEKEKE; And EK- (D) n-EEEEKEK.

The "A" nucleotide comprises a 2' -modified nucleoside, the "B" represents a 2' -4' bicyclic nucleoside, the "K" represents a constrained ethyl nucleoside (cEt), the "L" represents an LNA nucleoside, and the "E" represents a 2' -MOE modified ribonucleoside, the "D" represents a 2' -deoxyribonucleoside, and the "n" represents the length of the spacer segment (Y in the 5' -X-Y-Z-3' configuration) and is an integer from 1 to 20.

In some embodiments, any of the spacer polymers 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 spacer polymers described herein is a phosphorothioate linkage. In some embodiments, the X, Y and Z regions each independently comprise a mixture of phosphorothioate linkages and phosphodiester linkages. In some embodiments, each internucleoside linkage in spacer Y is a phosphorothioate linkage, 5 'wing region X comprises a mixture of phosphorothioate linkages and phosphodiester linkages, and 3' wing region Z comprises a mixture of phosphorothioate linkages and phosphodiester linkages.

RNA interference (RNAi)

In some embodiments, the oligonucleotides provided herein are small interfering RNAs (SMALL INTERFERING RNA, SIRNA), also referred to as short interfering RNAs or silencing RNAs. siRNA is a class of double stranded RNA molecules, typically about 20 to 25 base pairs in length, that target nucleic acids (e.g., mRNA) for degradation via an RNA interference (RNAi) pathway in a cell. The specificity of an siRNA molecule can be determined by the binding of the antisense strand of the molecule to its target RNA. Effective siRNA molecules are typically less than 30 to 35 base pairs in length to prevent triggering of non-specific RNA interference pathways in cells by an interferon response, although longer sirnas may also be effective. In some embodiments, 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 base pairs in length. In some embodiments, the siRNA molecule is 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 molecule is 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.

After selection of the appropriate target RNA sequence, siRNA molecules comprising nucleotide sequences (i.e., antisense sequences) that are complementary to all or part of the target sequence can be designed and prepared using appropriate methods (see, e.g., PCT publication No. WO 2004/016735; and U.S. patent publications Nos. 2004/007574 and 2008/0081791).

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

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.

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.

In some embodiments, the siRNA molecule comprises an antisense strand comprising a region complementary 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 the DUX4 mRNA. In some embodiments, the target region is a region of contiguous nucleotides in the DUX4 mRNA. In some embodiments, the complementary nucleotide sequence need not be 100% complementary to the nucleotide sequence of its target to specifically hybridize to or be specific for the target RNA sequence.

In some embodiments, the siRNA molecule comprises an antisense strand comprising a complementary region of a DUX4mRNA sequence, and the complementary region is in the range of 8 to 15, 8 to 30, 8 to 40, or 10 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 DUX4mRNA sequence. In some embodiments, the complementary region comprises a nucleotide sequence that contains no more than 1,2,3, 4, or 5 base mismatches as compared to the complementary portion of the DUX4mRNA sequence. In some embodiments, the complementary region comprises a nucleotide sequence having up to 3 mismatches at 15 bases or up to 2 mismatches at 10 bases.

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 set forth in any one of SEQ ID NOS.174-235. 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 set forth in any one of SEQ ID NOS 186, 189-191, and 200. In some embodiments, the siRNA molecule comprises an antisense strand that is 18 to 25 nucleotides in length and comprises a region of complementarity 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 set forth in any one of SEQ ID NOS.174 to 235. In some embodiments, the siRNA molecule comprises an antisense strand of 18 to 25 nucleotides in length, and comprises a region of complementarity 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 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 comprising a nucleotide sequence having at least 85%, at least 90%, at least 95% or 100% identity 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 having at least 85%, at least 90%, at least 95% or 100% identity 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 the oligonucleotide set forth in any one of SEQ ID NOs 236 to 266. In some embodiments, the siRNA molecule comprises an antisense strand of 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 248, 251 to 253, and 262.

Double stranded siRNA may comprise sense and antisense RNA strands of the same length or different lengths. Double stranded siRNA molecules can also be assembled from a single oligonucleotide into a stem-loop structure, wherein the self-complementary sense and antisense regions of the siRNA molecule are joined by a nucleic acid-based or non-nucleic acid-based linker, and a circular single stranded RNA having two or more loop structures and a stem comprising the self-complementary sense and antisense strands, wherein the circular RNA can be processed in vivo or in vitro to produce an active siRNA molecule capable of mediating RNAi. Thus, small hairpin RNA (SMALL HAIRPIN RNA, SHRNA) molecules are also contemplated herein. In addition to the reverse complement (sense) sequences, which are typically separated by a spacer or loop sequence, these molecules also contain specific antisense sequences. Cleavage of the spacer or loop provides single stranded RNA molecules and their reverse complements such that they can be annealed to form dsRNA molecules (optionally with additional processing steps that can result in the addition or removal of one, two, three, or more nucleotides from the 3 'end and/or (e.g., and) the 5' end of either or both strands). The spacer may be of sufficient length to allow the antisense and sense sequences to anneal and form a duplex 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 to a double stranded nucleic acid comprises shRNA.

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

The siRNA molecule may comprise a 3' overhang at one end of the molecule. The other end may be blunt or also have a protruding end (5 'or 3'). When the siRNA molecule comprises overhangs at both ends of the molecule, the length of the overhangs may be the same or different. In one embodiment, the siRNA molecules of the present disclosure comprise a 3' overhang of about 1 to about 3 (e.g., 1,2, 3) nucleotides on both ends of the molecule. In some embodiments, the siRNA molecule comprises a 3' overhang of about 1 to about 3 nucleotides on the sense strand. In some embodiments, the siRNA molecule comprises 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 comprises a 3' overhang of about 1 to about 3 (e.g., 1,2, 3) nucleotides on both the sense and antisense strands.

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 '-O-N-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 '-O-N-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.

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

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, aminoalkyl phosphotriesters, methylphosphonates and other alkylphosphonates comprising 3 'alkylene phosphonates, as well as chiral phosphonates, phosphinates, phosphoramidates comprising 3' -phosphoramidates and aminoalkyl phosphoramidates, thiocarbonyl phosphoramidates, thiocarbonylalkyl phosphonates, thiocarbonylalkyl phosphotriesters and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with opposite polarity in which adjacent pairs of nucleoside units are linked at 3'-5' to 5'-3' or 2'-5' to 5'-2'

U.S. patent 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;5,405,939;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.

Any of the modified chemical compositions or forms of the siRNA molecules described herein can be combined with one another. For example, one, two, three, four, five or more different types of modifications can be included within the same siRNA molecule.

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 nucleotide comprises 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 '-O-N-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 nucleoside comprises a modified sugar moiety (e.g., a 2' modified nucleoside). 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 '-O-N-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.

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

Any of the modified chemical compositions or forms of the antisense strands described herein can be combined with one another. For example, one, two, three, four, five or more different types of modifications may be included within the same antisense strand.

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 nucleotide comprises 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 '-O-N-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 '-O-N-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 phosphodiamide morpholinos. In some embodiments, the sense strand is a Phosphodiamide 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.

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.

In some embodiments, the sense strand comprises phosphorothioate or other modified internucleotide linkages. In some embodiments, the sense strand comprises phosphorothioate or other modified internucleoside linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages. In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between at least two nucleotides. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between at least two nucleosides. In some embodiments, the sense strand comprises phosphorothioate internucleotide linkages between at least two nucleotides. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages between all nucleotides. In some embodiments, the sense strand comprises phosphorothioate internucleoside linkages 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 linkages of 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 linkages of 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 phosphorothioate internucleoside linkages. 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, aminoalkyl phosphotriesters, methylphosphonates and other alkylphosphonates comprising 3 'alkylene phosphonates, as well as chiral phosphonates, phosphinates, phosphoramidates comprising 3' -phosphoramidates and aminoalkyl phosphoramidates, thiocarbonyl phosphoramidates, thiocarbonylalkyl phosphonates, thiocarbonylalkyl phosphotriesters and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those with opposite polarity in which adjacent pairs of nucleoside units are linked at 3'-5' to 5'-3' or 2'-5' to 5'-2'

Any of the modified chemical compositions or forms of the sense strands described herein may be combined with one another. For example, one, two, three, four, five or more different types of modifications may be included within the same sense strand.

In some embodiments, the antisense strand or sense strand of the siRNA molecule comprises a modification that increases or decreases RNA-induced silencing complex (RISC) loading. In some embodiments, the antisense strand of the siRNA molecule comprises a modification that enhances RISC loading. In some embodiments, the sense strand of the siRNA molecule comprises a modification that reduces RISC loading and reduces off-target effects. In some embodiments, the antisense strand of the siRNA molecule comprises a2 '-O-methoxyethyl (2' -MOE) modification. The addition of a2 '-O-methoxyethyl (2' -MOE) group at the cleavage site improves both the specificity and silencing activity of the siRNA by promoting targeted RNA-induced silencing complex (RISC) loading of the modified strand, as described in Song et al, (2017) Mol Ther Nucleic Acids 9:9:242-250, which is incorporated herein by reference in its entirety. In some embodiments, the antisense strand of the siRNA molecule comprises a 2' -OMe-dithiophosphate modification that increases RISC loading, as described in Wu et al, (2014) Nat Commun5:3459, which is incorporated herein by reference in its entirety.

In some embodiments, the sense strand of the siRNA molecule comprises 5' -morpholino, which reduces RISC loading 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) that reduces RISC loading 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' unlocking nucleic acid (unlocked nucleic acid, UNA) modification that reduces RISC loading of the sense strand and improves 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 that reduces RNAi potency and reduces off-target effects of the sense strand, as described in Zhang et al, (2012) Chembiochem (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 that reduces RISC loading 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 with a morpholino, 2'-MOE, or 2' -O-Me residue and is not recognized by RISC, as described in Kole et al, (2012) Nature reviews. Drug Discovery11 (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 (e.g., at least 2, at least 3, at least 4, at least 5, at least 10) siRNA molecules is linked (e.g., covalently) 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-TfR 1 antibodies provided in tables 2-7). In some embodiments, the muscle targeting agent can be linked to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the 5' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be attached to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the 3' end of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be linked internally to the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the interior of the sense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be 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 5' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be 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 linked internally to the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be covalently linked to the antisense strand of the siRNA molecule.

Some non-limiting examples of sirnas described herein are provided in table 8.

TABLE 8 non-limiting examples of siRNA

"M" means a 2 '-O-methyl (2' -O-Me) modified nucleoside, "F" means a 2 '-fluoro (2' -F) modified nucleoside, "x" means phosphorothioate internucleoside linkage, and no "x" means phosphodiester internucleoside linkage between two nucleosides.

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 comprise T, but binding of the oligonucleotides described herein to RNA and/or DNA is contemplated.

The position of the start of the target sequence is in NM-001306068.3 (SEQ ID NO: 161)

The siRNA oligonucleotides provided in table 8, which have sense and antisense strands of the same nucleobase sequence as shown in SEQ ID NOs, can have different modification patterns. The modified sense and antisense strands are denoted by "ms#" or "mas#" respectively. The siRNA oligonucleotides provided in table 8 comprise antisense strands without 5' - (E) -vinylphosphonate modification. However, siRNA oligonucleotides comprising antisense strands with 5' - (E) -vinylphosphonate modifications are also contemplated and are represented herein by the same MAS # (i.e., VP-MAS #) with "VP" prior to MAS #. The siRNA oligonucleotides provided in Table 8 can be conjugated to a compound of formula NH ₂-(CH₂)₆ -at the 5 'or 3' nucleoside of the sense strand.

TABLE 9 non-limiting examples of additional siRNAs

"M" means a2 '-O-methyl (2' -O-Me) modified nucleoside, "F" means a2 '-fluoro (2' -F) modified nucleoside, "x" means phosphorothioate internucleoside linkage, and no "x" means phosphodiester internucleoside linkage between two nucleosides. "VP" means 5' - (E) -vinylphosphonate.

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 comprise T, but binding of the oligonucleotides described herein to RNA and/or DNA is contemplated.

The position of the start of the target sequence is in NM-001306068.3 (SEQ ID NO: 161)The siRNA oligonucleotides provided in table 9, which have sense and antisense strands of the same nucleobase sequence as shown in SEQ ID NOs, can have different modification patterns. The modified sense and antisense strands are denoted by "ms#" or "mas#" respectively. The siRNA oligonucleotides provided in Table 9 comprise antisense strands comprising a 5' - (E) -vinylphosphonate modification represented by a "VP" preceding MAS# (i.e., VP-MAS#). siRNA oligonucleotides comprising antisense strands without 5' - (E) -vinylphosphonate modifications 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 NH ₂-(CH₂)₆ -at the 5' or 3' (e.g., 5 ') nucleoside of the sense strand.

In some embodiments, the oligonucleotides described herein comprise an antisense strand of 18 to 25 nucleotides in length (e.g., 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides) and comprise a region of complementarity to a target sequence set forth in any one of SEQ ID NOS: 174 to 235, wherein the region of complementarity is at least 16 nucleotides in length (e.g., 16, 17, 18, or 19 nucleotides). In some embodiments, the antisense strand is 23 nucleotides in length and comprises a region of complementarity to the target sequence set forth in any one of SEQ ID NOS.174 through 235, wherein the region of complementarity is 20 nucleotides in length. 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.

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 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 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 the nucleotide sequence of any one of SEQ ID NOs 205 to 235.

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.

In some embodiments, the oligonucleotides described herein are double-stranded oligonucleotides (e.g., siRNAs) comprising an antisense strand comprising a nucleotide sequence of any one of SEQ ID NOS: 236 to 266 and a sense strand that hybridizes to the antisense strand and comprises a 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, the one or more modified nucleosides are selected from 2'-O-Me and 2' -F modified nucleosides.

In some embodiments, the oligonucleotides described herein are double-stranded oligonucleotides (e.g., siRNAs) comprising an antisense strand comprising a nucleotide sequence of any one of SEQ ID NOS: 236 to 266 and a sense strand that hybridizes to the antisense strand and comprises a 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 group consisting of 2' -O-Me and 2' -F modified nucleosides.

In some embodiments, the oligonucleotides described herein are double-stranded oligonucleotides (e.g., siRNAs) comprising an antisense strand and a sense strand, said antisense strand comprising a nucleotide sequence of any one of SEQ ID NOS: 236 to 266, said sense strand hybridizing to the antisense strand and comprising a 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 the group consisting of 2' -O-Me and 2' -F modified nucleosides, and wherein the antisense strand and/or (e.g., and) the sense strand each comprise 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.

In some embodiments, the antisense strand comprises 2 phosphorothioate internucleoside linkages, optionally, wherein two internucleoside linkages at the 3' terminus 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 two internucleoside linkages at the 3 'end and two internucleoside linkages at the 5' 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 sense strand comprises 2 phosphorothioate internucleoside linkages, optionally, wherein 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, the two internucleoside linkages at the 5 'end and the two internucleoside linkages at the 3' 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 antisense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MAS 1"):

fN MNFNMNFNMNFNMNFNMNFNMNFNMNFNMNFNMNFNMN fN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no ". X" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, antisense strands MAS1-236, MAS1-237 and MAS1-238 comprise such structures.

In some embodiments, the antisense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MAS 2"):

fN*fN*mNmNmNfNmNmNmNmNmNmNmNfNmNfNmNmNmNmNmN*mN*mN,

Wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "x" represents phosphorothioate internucleoside linkage, and no "x" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, antisense strands MAS2-236, MAS2-237, MAS2-238,

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

Including such a structure.

In some embodiments, the antisense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MAS 3"):

fN*fN*mNmNmNmNmNmNmNmNmNmNmNfNmNmNmNmNmNmNmN*mN*mN,

wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "x" represents phosphorothioate internucleoside linkage, and no "x" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, the antisense strand

MAS3-236,MAS3-237,MAS3-238,MAS3-239,MAS3-240,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,MAS3-259,MAS3-260,MAS3-261,MAS3-262,MAS3-263,MAS3-264,MAS3-265, And MAS3-266

Including such a structure.

In some embodiments, the antisense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MAS 4"):

fN is MNMNMNINMNFNFNMNMNMNMNFNMNFNMNMNMNMNMN mN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, the antisense strand

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

Including such a structure.

In some embodiments, the antisense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MAS 5"):

mN, fN, MNMNMNFNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMN mN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, ", represents phosphorothioate internucleoside linkages, and no". X "represents phosphodiester internucleoside linkages between two nucleosides.

In some embodiments, the MAS5 structure further comprises 5 'vinylphosphonate (e.g., 5' - (E) -vinylphosphonate) modifications (5 'to 3', referred to herein as "VP-MAS 5"):

VP-mN at MNMNMNFNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMN mN at wherein "mN" represents a 2' -O-methyl (2 ' -O-Me) modified nucleoside, "fN" represents a 2' -fluoro (2 ' -F) modified nucleoside, ". Times." represents phosphorothioate internucleoside linkages, no ". Times." represents phosphodiester internucleoside linkages between the two nucleosides, and VP represents 5' - (E) -vinylphosphonate. For example, in Table 9, antisense strand VP-MAS5-248 comprises this structure.

In some embodiments, the antisense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MAS 6"):

mN, fN, MNMNMNFNMNININMNMNMNMNINMNFNMNMNMNMNMN mN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, ", represents phosphorothioate internucleoside linkages, and no". X "represents phosphodiester internucleoside linkages between two nucleosides.

In some embodiments, the MAS6 structure further comprises 5 'vinylphosphonate (e.g., 5' - (E) -vinylphosphonate) modifications (5 'to 3', referred to herein as "VP-MAS 6"):

VP-mN at MNMNMNFNMNFNFNMNMNMNMNFNMNFNMNMNMNMNMN mN at wherein "mN" represents a 2' -O-methyl (2 ' -O-Me) modified nucleoside, "fN" represents a 2' -fluoro (2 ' -F) modified nucleoside, ". Times." represents phosphorothioate internucleoside linkages, no ". Times." represents phosphodiester internucleoside linkages between the two nucleosides, and VP represents 5' - (E) -vinylphosphonate. For example, in Table 9, antisense strands VP-MAS6-251, VP-MAS6-253 and VP-MAS6-248 comprise such structures.

In some embodiments, the antisense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MAS 7"):

mN, fN, MNMNMNMNMNMNMNMNMNMNMNFNMNMNMNMNMNMNMN mN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, ", represents phosphorothioate internucleoside linkages, and no". X "represents phosphodiester internucleoside linkages between two nucleosides.

In some embodiments, the MAS7 structure further comprises 5 'vinylphosphonate (e.g., 5' - (E) -vinylphosphonate) modifications (5 'to 3', referred to herein as "VP-MAS 7"):

VP-mN at MNMNMNMNMNMNMNMNMNMNMNFNMNMNMNMNMNMNMN mN at wherein "mN" represents a 2' -O-methyl (2 ' -O-Me) modified nucleoside, "fN" represents a 2' -fluoro (2 ' -F) modified nucleoside, ". Times." represents phosphorothioate internucleoside linkages, no ". Times." represents phosphodiester internucleoside linkages between the two nucleosides, and VP represents 5' - (E) -vinylphosphonate. For example, in Table 9, antisense strands VP-MAS7-252 and VP-MAS7-262 comprise this structure.

In some embodiments, the sense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MS 1"):

mN FNMNFNMNFNMNFNMNFNMNFNMNFNMNFNMNFNMNFN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, sense strands MS1-205, MS1-206 and MS1-207 contain such structures.

In some embodiments, the sense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MS 2"):

mN MNMNFNMNFNMNFNFNFNMNMNMNMNFNMNMNMNMNMN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, the sense strand

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

Including such a structure.

In some embodiments, the sense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MS 3"):

mN MNMNFNMNMNMNFNFNFNMNMNFNMNMNMNMNFNMNMN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, the sense strand

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 contains this structure.

In some embodiments, the sense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MS 4"):

mN MNMNMNMNFNMNFNFNFNMNMNMNMNMNMNMNMNMNMN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, the sense strand

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 MSs 4-226 contain such structures.

In some embodiments, the sense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MS 5"):

mN MNMNMNMNFNMNFNFNFNMNMNMNMNFNFNMNMNMNMN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, the sense strand

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 contains such a structure.

In some embodiments, the sense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MS 6"):

mN MNMNMNMNFNMNFNFNFNMNMNMNMNFNMNMNFNMNMN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 8, the sense strand

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 contains this structure.

In some embodiments, the sense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MS 7"):

mN MNMNMNMNFNMNFNFNFNMNMNMNMNMNMNMNMN mN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 9, sense strands MS7-220, MS7-222 and MS7-217 contain such structures.

In some embodiments, the sense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MS 8"):

mN MNMNFNMNMNMNFNFNFNMNMNFNMNMNMNMNFN mN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 9, sense strands MS8-221 and MS8-231 contain such structures.

In some embodiments, the sense strand of the oligonucleotides described herein comprises the structure (5 'to 3', referred to herein as "MS 9"):

mN MNMNFNMNFNMNFNFNFNMNMNMNMNFNMNMNMN mN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. For example, in Table 9, sense strand MS9-217 contains this structure.

MAS1-236,MAS1-237,MAS1-238,MAS2-236,MAS2-237,MAS2-238,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,MAS2-257,MAS3-236,MAS3-237,MAS3-238,MAS3-239,MAS3-240,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,MAS3-259,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.

In some embodiments, the antisense strand of an oligonucleotide described herein is selected from the modified forms of SEQ ID NOs 236 to 266 (e.g., MAS5 to MAS 7) set forth in Table 8. In some embodiments, the antisense strand of an oligonucleotide described herein is selected from the modified forms of SEQ ID NOs 251, 253, 262, 248 and 252 set forth in Table 9 (e.g., MAS5, MAS6, MAS7, VP-MAS5, VP-MAS6 and VP-MAS 7). For example, in some embodiments, the antisense strand is selected from the group consisting of VP-MAS6-251, VP-MAS7-252, VP-MAS6-253, VP-MAS7-262, VP-MAS6-248, and VP-MAS5-248.

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,MS5-216,MS5-217,MS5-218,MS5-219,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.

In some embodiments, the sense strand of the oligonucleotides described herein is selected from modified versions of SEQ ID NOs 205 to 235 (e.g., MS7 to MS 9) set forth in Table 8. In some embodiments, the sense strand of the oligonucleotides described herein is selected from the modified forms of SEQ ID NOs 220, 222, 231, 217, and 221 (e.g., MS7 to MS 9) set forth in Table 9. For example, in some embodiments, the sense strand is selected from the group consisting of MS7-220, MS8-221, MS7-222, MS8-231, MS9-217, and MS7-217.

In some embodiments, an oligonucleotide described herein may comprise an antisense strand and a sense strand, the antisense strand comprising any one of the antisense strand structures described herein (e.g., MAS 1-MAS 4, independent of its nucleobase sequence), and the sense strand comprising any one of the sense strand structures described herein (e.g., MS 1-MS 6, independent of its nucleobase sequence). In some embodiments, an oligonucleotide described herein may comprise an antisense strand and a sense strand, the antisense strand comprising any one of the antisense strand structures described herein (e.g., MAS5, MAS6, MAS7, VP-MAS5, VP-MAS6, and VP-MAS7, independent of their nucleobase sequences), and the sense strand comprising any one of the sense strand structures described herein (e.g., MS 7-MS 9, independent of their nucleobase sequences).

In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', MAS 3):

fN is MNMNMNMNMNMNMNMNMNMNMNFNMNMNMNMNMNMNMN is mN, the sense strand comprises structures (5 'to 3', MS 3) mN is MNMNFNMNMNMNFNFNFNMNMNFNMNMNMNMNFNMNMN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, ". X" represents phosphorothioate internucleoside linkage, and no ". X" represents phosphodiester internucleoside linkage between two nucleosides.

In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', MAS 4):

fN MNMNMNFNMNFNFNMNMNMNMNFNMNFNMNMNMNMNMN mN, the sense strand comprising the structure (5 'to 3'; MS 4):

mN MNMNMNMNFNMNFNFNFNMNMNMNMNMNMNMNMNMNMN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides.

fN MNMNMNFNMNFNFNMNMNMNMNFNMNFNMNMNMNMNMN mN, the sense strand comprising the structure (5 'to 3', MS 6):

mN MNMNMNMNFNMNFNFNFNMNMNMNMNFNMNMNFNMNMN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides.

In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', MAS 2):

fN MNMNMNFNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMN mN, the sense strand comprising the structure (5 'to 3', MS 5):

mN MNMNMNMNFNMNFNFNFNMNMNMNMNFNFNMNMNMNMN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides.

fN MNMNMNFNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMN mN, the sense strand comprising the structure (5 'to 3', MS 4):

In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', MAS 1):

fN MNFNMNFNMNFNMNFNMNFNMNFNMNFNMNFNMNFNMN fN mN, the sense strand comprising the structure (5 'to 3'; MS 1):

mN FNMNFNMNFNMNFNMNFNMNFNMNFNMNFNMNFNMNFN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides.

fN MNMNMNFNMNFNFNMNMNMNMNFNMNFNMNMNMNMNMN mN, the sense strand comprising the structure (5 'to 3'; MS 2):

mN MNMNFNMNFNMNFNFNFNMNMNMNMNFNMNMNMNMNMN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "×" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides.

In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', MAS 5):

mN, fN, MNMNMNFNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMN, mN, the sense strand comprising the structure (5 'to 3', MS 7):

mN MNMNMNMNFNMNFNFNFNMNMNMNMNMNMNMNMN mN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides.

In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', MAS 6):

mN, fN, MNMNMNFNMNFNFNMNMNMNMNFNMNFNMNMNMNMNMN, mN, the sense strand comprising the structure (5 'to 3', MS 7):

In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', MAS 7):

mN, fN, MNMNMNMNMNMNMNMNMNMNMNFNMNMNMNMNMNMNMN, mN, the sense strand comprising the structure (5 'to 3', MS 8):

mN MNMNFNMNMNMNFNFNFNMNMNFNMNMNMNMNFN mN, wherein "mN" represents a 2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a 2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides.

mN, fN, MNMNMNFNMNFNFNMNMNMNMNFNMNFNMNMNMNMNMN, mN, the sense strand comprising the structure (5 'to 3', MS 9):

mN MNMNFNMNFNMNFNFNFNMNMNMNMNFNMNMNMN mN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', VP-MAS 5):

VP-mN, fN, MNMNMNFNMNMNMNMNMNMNMNFNMNFNMNMNMNMNMN mN, the sense strand comprising the structure (5 'to 3', MS 7):

mN MNMNMNMNFNMNFNFNFNMNMNMN MNMNMNMNMN mN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides.

In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', VP-MAS 6):

VP-mN, fN, MNMNMNFNMNFNFNMNMNMNMNFNMNFNMNMNMNMNMN mN, the sense strand comprising the structure (5 'to 3', MS 7):

In some embodiments, the oligonucleotides described herein comprise an antisense strand and a sense strand, the antisense strand comprising the structure (5 'to 3', VP-MAS 7):

v _P -mN, fN MNMNMNMNMNMNMNMNMNMNMNFNMNMNMNMNMNMNMN mN, the sense strand comprising the structure (5 'to 3', MS 8):

VP-mN, fN, MNMNMNFNMNFNFNMNMNMNMNFNMNFNMNMNMNMNMN mN, the sense strand comprising the structure (5 'to 3', MS 9):

mN MNMNFNMNFNMNFNFNFNMNMNMNMNFNMNMNMN mN, wherein "mN" represents a2 '-O-methyl (2' -O-Me) modified nucleoside, "fN" represents a2 '-fluoro (2' -F) modified nucleoside, "" represents phosphorothioate internucleoside linkage, and no "×" represents phosphodiester internucleoside linkage between two nucleosides. In some embodiments, the oligonucleotides described herein are siRNA selected from the group consisting of the sirnas listed in table 8. In some embodiments, the oligonucleotides described herein are siRNA selected from the group consisting of the sirnas listed in table 9.

In some embodiments, any of the oligonucleotides described herein (e.g., siRNA selected from the sirnas in table 8) can be in salt form, e.g., as sodium, potassium, magnesium salts. In some embodiments, any of the oligonucleotides described herein (e.g., siRNA selected from the sirnas in table 9) can be in salt form, e.g., as sodium, potassium, magnesium salts.

In some embodiments, a 5 'or 3' nucleoside (e.g., a terminal nucleoside) of any one of the oligonucleotides described herein (e.g., an oligonucleotide set forth in table 8) is conjugated to an amine group, optionally through a spacer. In some embodiments, a 5 'or 3' nucleoside (e.g., a terminal nucleoside) of any one of the oligonucleotides described herein (e.g., an oligonucleotide set forth in table 9) is conjugated to an amine group, optionally through 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 a spacer and a 5 'or 3' nucleoside of an oligonucleotide. In some embodiments, a 5 'or 3' nucleoside (e.g., a terminal nucleoside) of any of the oligonucleotides described herein (e.g., the oligonucleotides listed in table 8) is conjugated to a spacer that is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene 、-O-,-N(R^A)-,-S-,-C(＝O)-,-C(＝O)O-,-C(＝O)NR^A-,-NR^AC(＝O)-,-NR^AC(＝O)R^A-,-C(＝O)R^A-,-NR^AC(＝O)O-,-NR^AC(＝O)N(R^A)-,-OC(＝O)-,-OC(＝O)O-,-OC(＝O)N(R^A)-,-S(O)₂NR^A,-NR^AS(O)₂-、, or a combination thereof, each R ^A is independently hydrogen or a substituted or unsubstituted alkyl. In some embodiments, a 5 'or 3' nucleoside (e.g., a terminal nucleoside) of any of the oligonucleotides described herein (e.g., the oligonucleotides listed in table 9) is conjugated to a spacer that is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene 、-O-,-N(R^A)-,-S-,-C(＝O)-,-C(＝O)O-,-C(＝O)NR^A-,-NR^AC(＝O)-,-NR^AC(＝O)R^A-,-C(＝O)R^A-,-NR^AC(＝O)O-,-NR^AC(＝O)N(R^A)-,-OC(＝O)-,-OC(＝O)O-,-OC(＝O)N(R^A)-,-S(O)₂NR^A-,-NR^AS(O)₂-、, or a combination thereof, each R ^A is independently hydrogen or a substituted or unsubstituted alkyl.

In some embodiments, a 5 'or 3' nucleoside of any one of the oligonucleotides described herein (e.g., an oligonucleotide, sense strand, or antisense strand listed in table 8) is conjugated to a compound of formula-NH ₂-(CH₂)_n -, wherein n is an integer from 1 to 12. In some embodiments, a 5 'or 3' nucleoside of any one of the oligonucleotides described herein (e.g., an oligonucleotide, sense strand, or antisense strand listed in table 9) is conjugated to a compound of formula-NH ₂-(CH₂)_n -, wherein n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9,10, 11, or 12. In some embodiments, n is 6. In some embodiments, there is a phosphodiester linkage between a compound of formula NH ₂-(CH₂)_n -and a 5 'or 3' nucleoside of an oligonucleotide (e.g., an oligonucleotide, sense strand, or antisense strand listed in table 8), wherein n is an integer from 1 to 12. In some embodiments, n is 6, 7, 8, 9,10, 11, or 12. In some embodiments, n is 6.

In some embodiments, the 5' nucleoside of the sense strand of any one of the oligonucleotides described herein (e.g., an oligonucleotide set forth in table 8) is conjugated to a compound of formula-NH ₂-(CH₂)_n -wherein n is an integer from 1 to 12 (e.g., 6).

In some embodiments, the 5' nucleoside of the sense strand of any one of the oligonucleotides described herein (e.g., an oligonucleotide set forth in table 9) is conjugated to a compound of formula-NH ₂-(CH₂)_n -wherein n is an integer from 1 to 12 (e.g., 6).

In some embodiments, the 3' nucleoside of the sense strand of any one of the oligonucleotides described herein (e.g., an oligonucleotide set forth in table 8) is conjugated to a compound of formula-NH ₂-(CH₂)_n -wherein n is an integer from 1 to 12 (e.g., 6).

In some embodiments, the 3' nucleoside of the sense strand of any one of the oligonucleotides described herein (e.g., an oligonucleotide set forth in table 9) is conjugated to a compound of formula-NH ₂-(CH₂)_n -wherein n is an integer from 1 to 12 (e.g., 6).

In some embodiments, the compound of formula NH ₂-(CH₂)₆ -is conjugated to the oligonucleotide by a reaction between 6-amino-1-hexanol (NH ₂-(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 NH ₂-(CH₂)₆ -is conjugated to an oligonucleotide (e.g., an oligonucleotide listed in table 8) by a reaction between 6-amino-1-hexanol (NH ₂-(CH₂)₆ -OH) and the 5' phosphate of the sense strand of the oligonucleotide. In some embodiments, the compound of formula NH ₂-(CH₂)₆ -is conjugated to an oligonucleotide (e.g., an oligonucleotide listed in table 9) by a reaction between 6-amino-1-hexanol (NH ₂-(CH₂)₆ -OH) and the 5' phosphate of the sense strand of the oligonucleotide. In some embodiments, the compound of formula NH ₂-(CH₂)₆ -is conjugated to an oligonucleotide (e.g., an oligonucleotide listed in table 8) by a reaction between 6-amino-1-hexanol (NH ₂-(CH₂)₆ -OH) and the 5' phosphate of the antisense strand of the oligonucleotide. In some embodiments, the compound of formula NH ₂-(CH₂)₆ -is conjugated to an oligonucleotide (e.g., an oligonucleotide listed in table 9) by a reaction between 6-amino-1-hexanol (NH ₂-(CH₂)₆ -OH) and the 5' phosphate of the antisense strand of the oligonucleotide. In some embodiments, the compound of formula NH ₂-(CH₂)₆ -is conjugated to the oligonucleotide by reaction between 6-amino-1-hexanol (NH ₂-(CH₂)₆ -OH) and the 3 'phosphate of the oligonucleotide (e.g., the 3' phosphate of the sense strand or the antisense strand). in some embodiments, the compound of formula NH ₂-(CH₂)₆ -is conjugated to an oligonucleotide (e.g., an oligonucleotide listed in table 8) by a reaction between 6-amino-1-hexanol (NH ₂-(CH₂)₆ -OH) and the 3' phosphate of the sense strand of the oligonucleotide. In some embodiments, the compound of formula NH ₂-(CH₂)₆ -is conjugated to an oligonucleotide (e.g., an oligonucleotide listed in table 9) by a reaction between 6-amino-1-hexanol (NH ₂-(CH₂)₆ -OH) and the 3' phosphate of the sense strand of the oligonucleotide. In some embodiments, the compound of formula NH ₂-(CH₂)₆ -is conjugated to an oligonucleotide (e.g., an oligonucleotide listed in table 8) by a reaction between 6-amino-1-hexanol (NH ₂-(CH₂)₆ -OH) and the 3' phosphate of the antisense strand of the oligonucleotide. In some embodiments, the compound of formula NH ₂-(CH₂)₆ -is conjugated to an oligonucleotide (e.g., an oligonucleotide listed in table 9) by a reaction between 6-amino-1-hexanol (NH ₂-(CH₂)₆ -OH) and the 3' phosphate of the antisense strand of the oligonucleotide. In some embodiments, the oligonucleotide is conjugated to a targeting agent, such as a muscle targeting agent, e.g., an anti-TfR 1 antibody, e.g., via an amine group.

In some embodiments, the oligonucleotides described herein (e.g., the siRNA molecules listed in table 8 or 9) have reduced off-target effects, e.g., compared to other known DUX4 targeted sirnas.

C. Joint

The complexes described herein generally comprise a linker that covalently links any of the anti-TfR 1 antibodies described herein to the molecular payload. The linker comprises at least one covalent bond. In some embodiments, the linker may be a single bond, such as a disulfide bond or a disulfide bridge, that covalently links the anti-TfR 1 antibody to the molecular payload. However, in some embodiments, the linker may covalently link any of the anti-TfR 1 antibodies described herein to the molecular payload through a plurality of 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, typically the linker does not negatively affect the functional properties of the anti-TfR 1 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,J.R.and Owen,S.C."Antibody Drug Conjugates:Design and Selection of Linker,Payload and Conjugation Chemistry"AAPS J.2015,17:2,339-351.).

The linker will typically comprise two different reactive species that allow for attachment to both the anti-TfR 1 antibody and the molecular payload. In some embodiments, the two different reactive species may be nucleophiles and/or electrophiles. In some embodiments, the linker comprises two different electrophiles or nucleophiles, which are specific for the two different nucleophiles or electrophiles. In some embodiments, the linker is covalently linked to the anti-TfR 1 antibody by conjugation to a lysine residue or a cysteine residue of the anti-TfR 1 antibody. In some embodiments, the linker is covalently linked to the cysteine residue of the anti-TfR 1 antibody through a maleimide-containing linker, wherein optionally the maleimide-containing linker comprises a maleimidocaproyl or maleimidomethylcyclohexane-1-carboxylate group. In some embodiments, the linker is covalently linked to the cysteine residue or thiol-functionalized molecular payload of the anti-TfR 1 antibody through a 3-aryl propionitrile functional group. In some embodiments, the linker is covalently linked to a lysine residue of the anti-TfR 1 antibody. In some embodiments, the linker is independently covalently linked to the anti-TfR 1 antibody and/or (e.g., and) the molecular payload via an amide bond, a urethane bond, a hydrazide, a triazole, a thioether, and/or a disulfide bond.

I. Cutting joint

The cleavable linker may be a protease-sensitive linker, a pH-sensitive linker or a glutathione-sensitive linker. These linkers are generally cleavable only intracellularly, and are preferably stable in the extracellular environment, e.g., the extracellular environment of a muscle cell.

Protease-sensitive linkers can be cleaved by protease activity. These linkers typically comprise peptide sequences and may be 2 to 10 amino acids, about 2 to 5 amino acids, about 5 to 10 amino acids, about 5 amino acids, about 3 amino acids, or about 2 amino acids in length. In some embodiments, the peptide sequence may comprise naturally occurring amino acids (e.g., cysteine, alanine) or non-naturally occurring or modified amino acids. Non-naturally occurring amino acids include beta-amino acids, homoamino acids, proline derivatives, 3-substituted alanine derivatives, linear core amino acids, N-methyl amino acids, and other amino acids known in the art. In some embodiments, the protease-sensitive linker comprises a valine-citrulline or an alanine-citrulline sequence. In some embodiments, the protease-sensitive linker can be cleaved by a lysosomal protease (e.g., cathepsin B (cathepsin B)) and/or (e.g., and) an endosomal protease.

PH sensitive linkers are covalent linkages that degrade readily in high or low pH environments. In some embodiments, the pH-sensitive linker may be cleaved at a pH in the range of 4 to 6. In some embodiments, the pH-sensitive linker comprises a hydrazone or a cyclic acetal. In some embodiments, the pH sensitive linker is cleaved in endosomes or lysosomes.

In some embodiments, the glutathione-sensitive linker comprises a disulfide moiety. In some embodiments, the glutathione-sensitive linker is cleaved by disulfide exchange reaction with glutathione species within the cell. In some embodiments, the disulfide moiety further comprises at least one amino acid, such as a cysteine residue.

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:

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

In some embodiments, prior to conjugation, the linker comprises a structure of formula (a):

Wherein n is any number from 0 to 10. In some embodiments, n is 3.

In some embodiments, the linker comprises a structure of formula (H):

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

In some embodiments, the linker comprises a structure of formula (I):

Non-cleavable linker

In some embodiments, non-cleavable linkers may be used. Generally, non-cleavable linkers are not readily degraded in a cellular or physiological environment. In some embodiments, the non-cleavable linker comprises an optionally substituted alkyl group, wherein the substitution may include halogen, hydroxy, oxygen species, and other common substitutions. In some embodiments, the linker may comprise an optionally substituted alkyl, an optionally substituted alkylene, an optionally substituted arylene, a heteroarylene, a peptide sequence comprising at least one unnatural amino acid, a truncated glycan, one or more saccharides that are not enzymatically degradable, an azide, an alkyne-azide, a peptide sequence comprising an LPXT sequence, a thioether, biotin, biphenyl, polyethylene glycol, or a repeat unit of an equivalent compound, an acidic ester, an amide, a sulfonamide, and/or an alkoxy-amine linker. In some embodiments, sortase-mediated ligation can be used to covalently join an anti-TfR 1 antibody comprising an LPXT sequence to a molecular payload comprising a (G) _n sequence (see, e.g. Proft T.Sortase-mediated protein ligation:an emerging biotechnology tool for protein modification and immobilization.Biotechnol Lett.2010,32(1):1-10.).

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

Linker conjugation

In some embodiments, the linker is covalently linked to the anti-TfR 1 antibody and/or (e.g., and) the molecular payload through a phosphate, thioether, ether, carbon-carbon, carbamate, or amide linkage. In some embodiments, the linker is covalently attached to the oligonucleotide through 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-TfR 1 antibody through a lysine or cysteine residue present on the anti-TfR 1 antibody.

In some embodiments, the linker or a portion thereof is covalently linked to the anti-TfR 1 antibody and/or (e.g., and) the molecular payload by a cycloaddition reaction between the azide and the alkyne to form a triazole, wherein the azide or alkyne can be located on the anti-TfR 1 antibody, the molecular payload, or the linker. In some embodiments, the alkyne can be a cycloalkyne, such as cyclooctyne. In some embodiments, the alkyne can be a bicyclononene (also known as a bicyclo [6.1.0] nonyne or BCN) or a substituted bicyclononene. In some embodiments, cyclooctane is as described in International patent application publication WO2011136645, published 11/3/2011 under the heading "Fused Cyclooctyne Compounds And Their Use In Metal-FREE CLICK 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 azide-containing sugar or carbohydrate molecule is disclosed in International patent application publication WO2016170186, published on day 10, 27 of 2016, and is 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, where the azide or alkyne can be located on an anti-TfR 1 antibody, molecular payload, or linker, as described in International patent application publication WO2014065661, published on day 5, 2014, entitled "Modified antibody, anti-conjugate and process for the preparation thereof", or International patent application publication WO2016170186, published on day 10, 27 of 2016, and is entitled "anti-TfR 1 antibody "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase".

In some embodiments, the linker comprises a spacer, such as a polyethylene glycol spacer or an acyl/carbamoyl sulfonamide spacer, such as HYDRASPACE ^TM spacer. In some embodiments, the spacer is as described in Verkade,J.M.M.et al.,"A Polar Sulfamide Spacer Significantly Enhances the Manufacturability,Stability,and Therapeutic Index of Antibody-Drug Conjugates",Antibodies,2018,7,12.

In some embodiments, the linker is covalently linked to the anti-TfR 1 antibody and/or (e.g., and) the molecular payload by a Diels-Alder reaction between the dienophile and the diene/heterodiene, wherein the dienophile or diene/heterodiene may be located on the anti-TfR 1 antibody, the molecular payload, or the linker. In some embodiments, the linker is covalently linked to the anti-TfR 1 antibody and/or (e.g., and) the molecular payload by other circumferential reactions (PERICYCLIC REACTION), such as an alkene reaction. In some embodiments, the linker is covalently linked to the anti-TfR 1 antibody and/or (e.g., and) the molecular payload by an amide, thioamide, or sulfonamide linkage reaction. In some embodiments, the linker is covalently linked to the anti-TfR 1 antibody and/or (e.g., and) molecular payload by a condensation reaction to form an oxime, hydrazone, or semicarbazide group that is present between the linker and the anti-TfR 1 antibody and/or (e.g., and) molecular payload.

In some embodiments, the linker is covalently linked to the anti-TfR 1 antibody and/or (e.g., and) the molecular payload by a conjugate addition reaction between a nucleophile (e.g., an amine or hydroxyl group) and an electrophile (e.g., a carboxylic acid, carbonic acid, or aldehyde). In some embodiments, the nucleophile may be present on the linker and the electrophile may be present on the anti-TfR 1 antibody or molecular payload prior to performing the reaction between the linker and the anti-TfR 1 antibody or molecular payload. In some embodiments, before the reaction between the linker and the anti-TfR 1 antibody or molecular payload is performed, an electrophile may be present on the linker and a nucleophile may be present on the anti-TfR 1 antibody or molecular payload. In some embodiments, the electrophile may be an azide, pentafluorophenyl, a silicon center, a carbonyl, a carboxylic acid, an anhydride, an isocyanate, a thioisocyanate, a succinimidyl ester, a sulfosuccinimidyl ester, a maleimide, an alkyl halide, an alkyl pseudohalide, an epoxide, an episulfide, an aziridine, an aryl, an activated phosphorus center, and/or an activated sulfur center. In some embodiments, the nucleophile may be an optionally substituted alkene, an optionally substituted alkyne, an optionally substituted aryl, an optionally substituted heterocyclyl, a hydroxy, an amino, an alkylamino, an anilino, and/or a thiol group.

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

Wherein n is any number from 0 to 10. In some embodiments, n is 3.

In some embodiments, a linker comprising the structure of formula (a) (e.g., optionally through an additional chemical moiety) is covalently attached to a molecular payload (e.g., an oligonucleotide). In some embodiments, the molecular payload is a double-stranded siRNA oligonucleotide, and the linker comprising the structure of formula (a) (e.g., optionally through an additional chemical moiety) is covalently attached to the sense strand (e.g., at the 5' end) of the siRNA oligonucleotide. In some embodiments, the molecular payload is a double-stranded siRNA oligonucleotide, and the linker comprising the structure of formula (a) (e.g., optionally through an additional chemical moiety) is covalently attached to the sense strand (e.g., at the 3' end) of the siRNA oligonucleotide. In some embodiments, a linker comprising the structure of formula (a) is covalently attached to an oligonucleotide, e.g., by nucleophilic substitution with an amine-L1-oligonucleotide to form a urethane linkage, thereby producing a compound comprising the structure of formula (B):

Wherein n is any number from 0 to 10. In some embodiments, n is 3. In some embodiments, the oligonucleotide comprising a compound of formula (B) comprises the sense strand of an siRNA oligonucleotide. In some embodiments, the antisense strand anneals to the sense strand of the siRNA oligonucleotide (e.g., at an annealing temperature of 25 ℃ to 105 ℃). In some embodiments, the annealing step is performed at an annealing temperature of 25 ℃ to 150 ℃ (e.g., 25 ℃ to 150 ℃,25 ℃ to 100 ℃,25 ℃ to 75 ℃,25 ℃ to 50 ℃,25 ℃ to 40 ℃,25 ℃ to 30 ℃, 30 ℃ to 150 ℃, 30 ℃ to 100 ℃, 30 ℃ to 75 ℃, 30 ℃ to 50 ℃, 30 ℃ to 40 ℃, 75 ℃ to 150 ℃, 75 ℃ to 125 ℃, or 75 ℃ to 100 ℃). In some embodiments, the annealing step is performed at an annealing temperature of 100 ℃. For example, the annealing reaction mixture can be incubated at 100 ℃ (e.g., 3,4, 5, 6, 7, 8, 9, 10 minutes or longer) and allowed to cool at room temperature to anneal the sense strand and the antisense strand. In some embodiments, the annealing step is performed at an annealing temperature of 20 ℃, 21 ℃,22 ℃, 23 ℃, 24 ℃,25 ℃, 26 ℃, 27 ℃, 28 ℃, 29 ℃, 30 ℃,31 ℃,32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃,37 ℃, 38 ℃, 39 ℃, 40 ℃. In some embodiments, the annealing step is performed at an annealing temperature of 30 ℃. For example, the annealing reaction mixture can be incubated (e.g., 1 to 24 hours, such as1 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) at 30 ℃ to anneal the sense strand and the antisense strand. In some embodiments, the oligonucleotide comprising a compound of formula (B) 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.

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

Wherein m is any number from 0 to 10. In some embodiments, m is 4. In some embodiments, in a click reaction between a compound comprising a structure of formula (B) and a compound comprising a 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 a click reaction between a compound comprising a structure of formula (B) and a compound comprising a 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).

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

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 isolated prior to further covalent attachment to the lysine of the anti-TfR 1 antibody. In some embodiments, when the compound comprising the structure of formula (D) is isolated prior to further covalent attachment to the lysine of the anti-TfR 1 antibody, the molar ratio between the compound comprising the structure of formula (C) and the compound comprising the structure of formula (B) in the click reaction between the compound comprising the structure of formula (B) and the compound comprising the structure of formula (C) is greater than 1, e.g., 3:1 or 4:1.

In some embodiments, the compound comprising the structure of formula (D) is not isolated prior to further covalent attachment to the lysine of the anti-TfR 1 antibody. In some embodiments, when the compound comprising the structure of formula (D) is not isolated prior to further covalent attachment to the lysine of the anti-TfR 1 antibody, the molar ratio between the compound comprising the structure of formula (C) and the compound comprising the structure of formula (B) in the click reaction between the compound comprising the structure of formula (B) and the compound comprising the structure of formula (C) is less than 1, e.g., 0.9:1 or 0.85:1.

In some embodiments, the compound of structure (D) is further covalently linked to a lysine of an anti-TfR 1 antibody, thereby forming a complex comprising the structure of formula (E):

Wherein n is any number from 0 to 10, wherein m is any number from 0 to 10. In some embodiments, n is 3 and/or (e.g., and) m is 4. It is understood that the amide shown in formula (E) adjacent to the anti-TfR 1 antibody is produced by reaction with an amine of the anti-TfR 1 antibody, such as lysine epsilon amine. In some embodiments, the oligonucleotide of formula (E) 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 (E) 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 of formula (C) is further covalently linked to a lysine of an anti-TfR 1 antibody, thereby forming a compound comprising the structure of formula (F):

Wherein m is 0 to 15 (e.g., 4). It is understood that the amide shown in formula (F) adjacent to the anti-TfR 1 antibody is produced by reaction with an amine of the anti-TfR 1 antibody (e.g., lysine epsilon amine).

In some embodiments, the azide of the compound of structure (B) forms a triazole by a click reaction with the alkyne of the compound of structure (F), thereby forming a complex comprising the structure of formula (E):

wherein n is any number from 0 to 10, wherein m is any number from 0 to 10. In some embodiments, n is 3 and/or (e.g., and) m is 4. It is understood that the amide shown in formula (E) adjacent to the anti-TfR 1 antibody is generated from a reaction with an amine (e.g., lysine epsilon amine) of the anti-TfR 1 antibody. In some embodiments, the oligonucleotide of formula (E) 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 (E) 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 azide of the compound of structure (a) forms a triazole by a click reaction with the alkyne of the compound of structure (F), thereby forming a compound comprising the structure of formula (G):

Wherein n is any number from 0 to 10, wherein m is any number from 0 to 10. In some embodiments, n is 3 and/or (e.g., and) m is 4. In some embodiments, the oligonucleotide is covalently linked to a compound comprising the structure of formula (G), thereby forming a complex comprising the structure of formula (E). It is understood that the amide shown in formula (G) adjacent to the anti-TfR 1 antibody is produced by reaction with an amine (e.g., lysine epsilon amine) of the anti-TfR 1 antibody.

In some embodiments, in any of the complexes described herein, the anti-TfR 1 antibody is covalently linked to the molecular payload (e.g., oligonucleotide) via a linker comprising the structure of formula (H) by a lysine of the anti-TfR 1 antibody:

In some embodiments, in any of the complexes described herein, the anti-TfR 1 antibody is covalently linked to the molecular payload (e.g., oligonucleotide) via a linker comprising the structure of formula (I) by a lysine of the anti-TfR 1 antibody:

In some embodiments, in formulas (B), (D), (E) and (I), L1 is a spacer that is a substituted or unsubstituted aliphatic, a substituted or unsubstituted heteroaliphatic, a substituted or unsubstituted carbocyclylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted arylene, a substituted or unsubstituted heteroarylene 、-O-,-N(R^A)-,-S-,-C(＝O)-,-C(＝O)O-,-C(＝O)NR^A-,-NR^AC(＝O)-,-NR^AC(＝O)R^A-,-C(＝O)R^A-,-NR^AC(＝O)O-,-NR^AC(＝O)N(R^A)-,-OC(＝O)-,-OC(＝O)O-,-OC(＝O)N(R^A)-,-S(O)₂NR^A-,-NR^AS(O)₂-, or a combination thereof, wherein each R ^A is independently hydrogen or a substituted or unsubstituted alkyl. In some embodiments, L1 is

Wherein L2 is

Wherein a marks the site of direct linkage to the carbamate moiety of formulae (B), (D), (E) and (I), and B marks the site of covalent linkage to the oligonucleotide (either directly or through another chemical moiety).

In some embodiments, L1 is:

In some embodiments, L1 is

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

In some embodiments, L1 is attached to the 3' phosphate of the oligonucleotide. In some embodiments, the linkage of L1 to the 3' phosphate of the oligonucleotide forms a phosphodiester bond between L1 and the oligonucleotide.

In some embodiments, L1 is optional (e.g., not necessarily present).

In some embodiments, any of the complexes described herein have the structure of formula (J):

Where n is 0 to 15 (e.g., 3) and m is 0 to 15 (e.g., 4). It is understood that the amide shown in formula (J) adjacent to the anti-TfR 1 antibody is produced by reaction with an amine (e.g., lysine epsilon amine) of the anti-TfR 1 antibody.

In some embodiments, any of the complexes described herein have the structure of formula (K):

Where n is 0 to 15 (e.g., 3) and m is 0 to 15 (e.g., 4).

In some embodiments, the oligonucleotide is modified to include an amine group at the 5 'end, 3' end, or internal (e.g., as an amine functionalized nucleobase) prior to attachment to a compound (e.g., a compound of formula (a) or formula (G)).

Although linker conjugation is described in the context of anti-TfR 1 antibodies and oligonucleotide molecule payloads, it is to be understood that such linker conjugation is contemplated for use on other muscle targeting agents (e.g., other muscle targeting antibodies) and/or other molecular payloads.

D. some examples of antibody-molecule payload complexes

Also provided herein are some non-limiting examples of complexes comprising any of the anti-TfR 1 antibodies described herein covalently linked to any of the molecular payloads (e.g., oligonucleotides) described herein. In some embodiments, an anti-TfR 1 antibody (e.g., any one of the anti-TfR 1 antibodies provided in tables 2-7) is covalently linked to a molecular payload (e.g., an oligonucleotide comprising at least 12 (e.g., 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 nucleotide sequence of any one of SEQ ID NOs 174-266) through a linker. In some embodiments, an anti-TfR 1 antibody (e.g., any one of the anti-TfR 1 antibodies provided in tables 2-7) is covalently linked to a molecular payload (e.g., an oligonucleotide, such as the oligonucleotide provided in table 8) through a linker. In some embodiments, an anti-TfR 1 antibody (e.g., any one of the anti-TfR 1 antibodies provided in tables 2-7) is covalently linked to a molecular payload (e.g., an oligonucleotide, such as the oligonucleotide provided in table 9) through a linker. Any of the linkers described herein may be used. In some embodiments, if the molecular payload is an oligonucleotide, the linker is attached to the 5 'end, the 3' end, or the interior of the sense strand and the antisense strand. In some embodiments, the molecular payload is an siRNA, and the linker is attached to the 5' end of the sense strand. In some embodiments, the molecular payload is an siRNA, and the linker is attached to the 3' end of the sense strand. In some embodiments, the linker is linked to the anti-TfR 1 antibody by a thiol-reactive linkage (e.g., through a cysteine in the anti-TfR 1 antibody). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is linked to the antibody (e.g., an anti-TfR 1 antibody described herein) through an amine group (e.g., through a lysine in the antibody). In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide listed in table 8). In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide listed in table 9). In some embodiments, the molecular payload is the sense strand of the siRNA. In some embodiments, the molecular payload is the antisense strand of the siRNA. In some embodiments, the molecular payload is an siRNA comprising a sense strand and an antisense strand.

One example of the structure of a complex comprising an anti-TfR 1 antibody covalently linked to a molecular payload through a linker is provided below:

Wherein the linker is linked to the antibody by a thiol-reactive linkage (e.g., through a cysteine in the antibody). In some embodiments, the molecular payload is an oligonucleotide comprising at least 12 (e.g., 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 nucleotide sequence of any one of SEQ ID NOs 174 to 266. In some embodiments, the molecular payload is an oligonucleotide comprising the nucleotide sequence of any one of SEQ ID NOs 174 to 266. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

Another example of the structure of a complex comprising an anti-TfR 1 antibody covalently linked to a molecular payload through a linker is provided below:

Wherein n is a number from 0 to 10, wherein m is a number from 0 to 10, wherein the linker is attached to the antibody via an amine group (e.g., on a lysine residue), and/or (e.g., and) wherein the linker is attached to the sense strand or the antisense strand (e.g., at the 5 'terminus, the 3' terminus, or internally). In some embodiments, the linker is attached to the 5' end of the sense strand. In some embodiments, the linker is attached to the 3' end of the sense strand. In some embodiments, the linker is attached to the antibody via lysine, the linker is attached to the oligonucleotide at the 5' end, n is 3 and m is 4. In some embodiments, the molecular payload is an oligonucleotide comprising at least 12 (e.g., 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) nucleotides of the nucleotide sequence of any one of SEQ ID NOs 174 to 266. In some embodiments, the molecular payload is an oligonucleotide comprising the nucleotide sequence of any one of SEQ ID NOs 174 to 266. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, L1 is any one of the spacers described herein. It is understood that the amide shown in formula (E) adjacent to the anti-TfR 1 antibody is produced by reaction with an amine of the anti-TfR 1 antibody (e.g., lysine epsilon amine).

It is understood that antibodies can be linked to molecular payloads having different stoichiometries, a property which can be referred to as a drug-to-antibody ratio (drug to antibody ratio, DAR), where "drug" is the molecular payload. In some embodiments, one molecular payload is linked to one antibody (dar=1). In some embodiments, two molecular payloads are linked to one antibody (dar=2). In some embodiments, three molecular payloads are linked to one antibody (dar=3). In some embodiments, four molecular payloads are linked to one antibody (dar=4). In some embodiments, a mixture of different complexes is provided, each complex having a different DAR. In some embodiments, the average DAR for the complexes in such mixtures may be in the range of 1 to 3, 1 to 4, 1 to 5, or more. DAR can be enhanced by conjugating the molecular payload to different sites on the antibody and/or (e.g., and) by conjugating the multimer to one or more sites on the antibody. For example, DAR of 2 can be achieved by conjugating a single molecular payload to two different sites on an antibody or by conjugating a dimer molecular payload to a single site on an antibody.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody described herein (e.g., an antibody provided in tables 2-7) covalently linked to a molecular payload. In some embodiments, a complex described herein comprises an anti-TfR 1 antibody described herein (e.g., an antibody provided in tables 2-7) covalently linked to a molecular payload through a linker (e.g., a linker comprising a valine-citrulline sequence). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is linked to the antibody (e.g., an anti-TfR 1 antibody described herein) by a thiol-reactive linkage (e.g., through a cysteine in the antibody). In some embodiments, the linker (e.g., a linker comprising a valine-citrulline sequence) is linked to the antibody (e.g., an anti-TfR 1 antibody described herein) through an amine group (e.g., through a lysine in the antibody). In some embodiments, the molecular payload is an oligonucleotide comprising at least 12 (e.g., 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) nucleotides of the nucleotide sequence of any one of SEQ ID NOs 174 to 266. In some embodiments, the molecular payload is an oligonucleotide comprising the nucleotide sequence of any one of SEQ ID NOs 174 to 266. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 in any one of the antibodies listed in table 2. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a VH comprising the amino acid sequence of SEQ ID No. 69, SEQ ID No. 71, or SEQ ID No. 72 and a VL comprising the amino acid sequence of SEQ ID No. 70. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a VH comprising the amino acid sequence of SEQ ID No. 73 or SEQ ID No. 76 and a VL comprising the amino acid sequence of SEQ ID No. 74. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a VH comprising the amino acid sequence of SEQ ID No. 73 or SEQ ID No. 76 and a VL comprising the amino acid sequence of SEQ ID No. 75. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a VH comprising the amino acid sequence of SEQ ID No. 77 and a VL comprising the amino acid sequence of SEQ ID No. 78. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a VH comprising the amino acid sequence of SEQ ID No. 77 or SEQ ID No. 79 and a VL comprising the amino acid sequence of SEQ ID No. 80. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:154 and a VL comprising the amino acid sequence of SEQ ID NO: 155. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide listed in table 8). In some embodiments, the molecular payload is an oligonucleotide (e.g., an oligonucleotide listed in table 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 84, SEQ ID No. 86, or SEQ ID No. 87 and a light chain comprising the amino acid sequence of SEQ ID No. 85. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 88 or SEQ ID No. 91 and a light chain comprising the amino acid sequence of SEQ ID No. 89. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 88 or SEQ ID No. 91 and a light chain comprising the amino acid sequence of SEQ ID No. 90. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 92 or SEQ ID No. 94 and a light chain comprising the amino acid sequence of SEQ ID No. 95. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 92 and a light chain comprising the amino acid sequence of SEQ ID No. 93. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 156 and a light chain comprising the amino acid sequence of SEQ ID No. 157. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 97, SEQ ID No. 98, or SEQ ID No. 99 and a VL comprising the amino acid sequence of SEQ ID No. 85. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 100 or SEQ ID No. 101 and a light chain comprising the amino acid sequence of SEQ ID No. 89. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 100 or SEQ ID No. 101 and a light chain comprising the amino acid sequence of SEQ ID No. 90. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:102 and a light chain comprising the amino acid sequence of SEQ ID NO: 93. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 102 or SEQ ID No. 103 and a light chain comprising the amino acid sequence of SEQ ID No. 95. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular payload, wherein the anti-TfR 1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID No. 158 or SEQ ID No. 159 and a light chain comprising the amino acid sequence of SEQ ID No. 157. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 8), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 8, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand. In some embodiments, the molecular payload is an oligonucleotide comprising an antisense strand comprising a region of complementarity of at least 16 nucleotides to a target sequence in the DUX4 mRNA (e.g., a target sequence listed in table 9), optionally wherein the antisense strand comprises at least 16 consecutive nucleotides of any one of the antisense sequences listed in table 9, optionally wherein the oligonucleotide further comprises a sense strand hybridized to the antisense strand.

In any of the exemplary complexes described herein, in some embodiments, the anti-TfR 1 antibody is covalently linked to the molecular payload through a linker comprising the structure of formula (I):

wherein n is 3 and m is 4.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 8) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 in any one of the antibodies set forth in table 2, wherein the complex has the structure of formula (E):

Where n is 3 and m is 4. It is understood that the amide shown in formula (E) adjacent to the anti-TfR 1 antibody is produced by reaction with an amine of the anti-TfR 1 antibody (e.g., lysine epsilon amine). In some embodiments, an anti-TfR 1 antibody is covalently linked to the 5' end of the sense strand of an oligonucleotide described herein. In some embodiments, an anti-TfR 1 antibody is covalently linked to the 3' end of the sense strand of an oligonucleotide described herein.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 9) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of any one of the antibodies set forth in table 2, wherein the complex has the structure of formula (E):

Where n is 3 and m is 4. It is understood that the amide shown in formula (E) adjacent to the anti-TfR 1 antibody is produced by reaction with an amine of the anti-TfR 1 antibody (e.g., lysine epsilon amine). In some embodiments, an anti-TfR 1 antibody is covalently linked to the 5 'end or the 3' end of the sense strand of an oligonucleotide described herein. In some embodiments, an anti-TfR 1 antibody is covalently linked to the 5' end of the sense strand of an oligonucleotide described herein. In some embodiments, an anti-TfR 1 antibody is covalently linked to the 3' end of the sense strand of an oligonucleotide described herein.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 8) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises the VH and VL of any one of the antibodies set forth in table 3, wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 9) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises the VH and VL of any one of the antibodies set forth in table 3, wherein the complex has the structure of formula (E):

Where n is 3 and m is 4. It is understood that the amide shown in formula (E) adjacent to the anti-TfR 1 antibody is produced by reaction with an amine of the anti-TfR 1 antibody (e.g., lysine epsilon amine). In some embodiments, an anti-TfR 1 antibody is covalently linked to the 5 'end or the 3' end of the sense strand of an oligonucleotide described herein. In some embodiments, an anti-TfR 1 antibody is covalently linked to the 5' end of the sense strand of an oligonucleotide described herein. In some embodiments, an anti-TfR 1 antibody is covalently linked to the 3' end of the sense strand of an oligonucleotide described herein. In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 8) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises the heavy and light chains of any one of the antibodies set forth in table 4, wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 9) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises the heavy and light chains of any one of the antibodies set forth in table 4, wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody that is a Fab covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 8) through a lysine in the anti-TfR 1 Fab, wherein the anti-TfR 1 Fab comprises the heavy and light chains of any one of the antibodies set forth in table 5, wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody that is a Fab covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 9) through a lysine in the anti-TfR 1 Fab, wherein the anti-TfR 1 Fab comprises the heavy and light chains of any one of the antibodies set forth in table 5, wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 8) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises:

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

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

Wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 9) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises:

Wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 8) via a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 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, wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 9) via a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 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, wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody that is a Fab covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 8) through a lysine in the anti-TfR 1 Fab, wherein the anti-TfR 1 Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90, wherein the complex has the structure of formula (E):

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody that is a Fab covalently linked to the 3 'or 5' end of an oligonucleotide described herein (e.g., the sense strand or the antisense strand of an oligonucleotide set forth in table 9) through a lysine in the anti-TfR 1 Fab, wherein the anti-TfR 1 Fab comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:101 and a light chain comprising the amino acid sequence of SEQ ID NO:90, wherein the complex has the structure of formula (E):

In some embodiments, in any one of the examples of complexes described herein, L1 is:

Wherein L2 is

Wherein a marks the site of direct linkage to the carbamate moiety of formulae (B), (D), (E) and (I) and B marks the site of covalent linkage to the oligonucleotide (either directly or via a further chemical moiety).

In some embodiments, L1 is:

In some embodiments, L1 is

In some embodiments, L1 is optional (e.g., need not be present).

III. preparation

The complexes provided herein may be formulated in any suitable manner. In general, the complexes provided herein are formulated in a manner suitable for pharmaceutical use. For example, the complex may be delivered to the subject using a formulation that minimizes degradation, facilitates delivery, and/or (e.g., and) uptake or provides additional beneficial properties to the complex in the formulation. In some embodiments, provided herein are compositions comprising a complex and a pharmaceutically acceptable carrier. Such compositions may be suitably formulated so that when administered to a subject, either in the immediate environment of administration to the target cells or systemically, a sufficient amount of the complex is able to enter the target muscle cells. In some embodiments, the complex is formulated in a buffer solution such as phosphate buffered saline solution, liposomes, micelle structures, and capsids.

It is to be understood that in some embodiments, the compositions may each comprise one or more components of the complexes provided herein (e.g., muscle targeting agents, linkers, molecular payloads, or precursor molecules of any of them).

In some embodiments, the complex is formulated in water or an aqueous solution (e.g., water adjusted with pH). In some embodiments, the complex is formulated in an alkaline buffered aqueous solution (e.g., PBS). In some embodiments, the formulations disclosed herein comprise an excipient. In some embodiments, the excipient imparts improved stability, improved absorption, improved solubility, and/or therapeutic enhancement (e.g., sum) of the active ingredient to the composition. In some embodiments, the excipient is a buffer (e.g., sodium citrate, sodium phosphate, tris base, or sodium hydroxide) or a carrier (e.g., buffer solution, petrolatum (petrolatum), dimethyl sulfoxide, or mineral oil).

In some embodiments, the complex or a component thereof (e.g., an oligonucleotide or antibody) is lyophilized for extended shelf life and then made into a solution prior to use (e.g., administration to a subject). Thus, the excipient in a composition comprising a complex or component thereof described herein may be a lyoprotectant (e.g., mannitol, lactose, polyethylene glycol, or polyvinylpyrrolidone) or a disintegration temperature regulator (e.g., dextran, ficoll, or gelatin).

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

Pharmaceutical compositions suitable for injectable use comprise sterile aqueous solutions (when water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The carrier may be a solvent or dispersion medium comprising, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycols, and the like), and suitable mixtures thereof. In some embodiments, the formulation comprises isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Sterile injectable solutions may be prepared by incorporating the required amount of the compound in the selected solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.

In some embodiments, the composition may comprise at least about 0.1% of the complex or component thereof, or more, although the percentage of active ingredient may be from about 1% to about 80% or more by weight or volume of the total composition. Those skilled in the art will consider factors such as solubility, bioavailability, biological half-life, route of administration, product shelf life, and other pharmacological considerations in preparing such pharmaceutical formulations, and thus a variety of dosages and therapeutic regimens may be desirable.

Methods of use/treatment

Complexes comprising a muscle targeting agent covalently linked to a molecular payload as described herein are useful in the treatment of FSHD. In some embodiments, the complex is effective in treating FSHD type 1. In some embodiments, the complex is effective in treating FSHD type 2. In some embodiments, FSHD is associated with a deletion in the D4Z4 repeat region on chromosome 4 that comprises the DUX4 gene. In some embodiments, FSHD is associated with a mutation in the SMCHD1 gene. In some embodiments, FSHD is associated with a mutation in the DNMT3B gene. In some embodiments, FSHD is associated with a mutation in the LRIF gene. In some embodiments, the subject may be a human subject, a non-human primate subject, a rodent subject, or any suitable mammalian subject. In some embodiments, the subject may have myotonic muscular dystrophy. In some embodiments, the subject has elevated DUX4 gene expression outside of fetal development and testes. In some embodiments, the subject has type 1 or type 2 facial shoulder brachial muscular dystrophy. In some embodiments, the subject with FSHD has a mutation in the SMCHD1 gene. In some embodiments, the subject with FSHD has a mutation in the DNMT3B gene. In some embodiments, the subject with FSHD has a mutation in the LRIF gene. In some embodiments, the subject with FSHD has a deletion mutation in the D4Z4 repeat region on chromosome 4.

One aspect of the present disclosure includes methods involving administering an effective amount of a complex described herein to a subject. In some embodiments, an effective amount of a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently linked to a molecular payload may be administered to a subject in need of treatment. In some embodiments, the pharmaceutical composition comprising a complex as described herein may be administered by a suitable route, which may include intravenous administration, for example as a bolus or by continuous infusion over a period of time. In some embodiments, intravenous administration may be by intramuscular, intraperitoneal, intracerebroventricular, subcutaneous, intra-articular, intrasynovial, or intrathecal routes. In some embodiments, the pharmaceutical composition may be in solid form, aqueous form, or liquid form. In some embodiments, the aqueous or liquid form may be atomized or lyophilized. In some embodiments, the atomized or lyophilized form can be reconstituted with an aqueous or liquid solution.

Compositions for intravenous administration may comprise a variety of carriers, such as vegetable oils, dimethylacetamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycols, and the like). For intravenous injection, the water-soluble antibody may be administered by an instillation method by which a pharmaceutical formulation comprising the antibody and physiologically acceptable excipients is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, ringer's solution, or other suitable excipients. An intramuscular formulation, e.g. a sterile formulation in the form of a suitable soluble salt of an antibody, may be dissolved in a pharmaceutically acceptable excipient, e.g. water for injection, 0.9% saline or 5% dextrose solution, and administered.

In some embodiments, the pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently linked to a molecular payload is administered by a site-specific or local delivery technique. Some examples of these techniques include implantable reservoir sources of the complex, local delivery catheters, site-specific carriers, direct injection, or direct application.

In some embodiments, a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently linked to a molecular payload is administered at an effective concentration that imparts a therapeutic effect to a subject. As recognized by those of skill in the art, the effective amount will vary depending on the severity of the disease, the unique characteristics of the subject being treated (e.g., age, physical condition, health or weight), the duration of the treatment, the nature of any concurrent treatment, the route of administration, and related factors. These relevant factors are known to those skilled in the art and can be solved by only routine experimentation. In some embodiments, the effective concentration is the maximum dose considered safe for the patient. In some embodiments, the effective concentration will be the lowest possible concentration that provides the greatest efficacy.

Empirical considerations (e.g., the half-life of the complex in the subject) will generally help determine the concentration of the pharmaceutical composition used for treatment. The frequency of administration can be determined and adjusted empirically to maximize therapeutic efficacy.

Generally, for administration of any of the complexes described herein, the initial candidate dose may be about 1 to 100mg/kg or higher, depending on factors such as safety or efficacy. In some embodiments, the treatment will be administered once. In some embodiments, the treatment will be administered daily, every two weeks, weekly, every two months, monthly, or at any time interval that minimizes the safety risk to the subject while providing maximum efficacy. Generally, efficacy and treatment as well as safety risks can be monitored throughout the course of treatment.

The efficacy of the treatment may be assessed using any suitable method. In some embodiments, the efficacy of the treatment may be assessed by evaluating observations of symptoms associated with FSHD, including reduced muscle mass and muscle atrophy primarily in facial, shoulder and upper arm muscles.

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

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

In some embodiments, the pharmaceutical composition may comprise more than one complex comprising a muscle targeting agent covalently linked to a molecular payload. In some embodiments, the pharmaceutical composition may further comprise any other suitable therapeutic agent for treating a subject (e.g., a human subject suffering from FSHD). In some embodiments, other therapeutic agents may enhance or supplement the efficacy of the complexes described herein. In some embodiments, other therapeutic agents may function to treat different symptoms or diseases as compared to the complexes described herein.

Examples

Example 1 targeting Gene expression Using transfected antisense oligonucleotides

Sirnas targeting hypoxanthine phosphoribosyl transferase (hypoxanthine phosphoribosyltransferase, HPRT) were tested in vitro for their ability to reduce expression levels of HPRT in immortalized cell lines. Briefly, hepa1-6 cells were transfected with control siRNA (siCTRL; 100 nM) formulated using lipofectamine 2000 or HPRT-targeted siRNA (siHPRT; 100 nM). HPRT expression levels were assessed 48 hours after transfection. Control experiments were also performed in which vehicle (phosphate buffered saline) was delivered to the Hepa1-6 cells in culture and the cells were maintained for 48 hours. As shown in fig. 1, HPRT SIRNA was found to reduce HPRT expression levels by about 90% compared to the control. Table 10 provides the sequences of the sirnas used. HPRT expression levels are represented by HPRT mRNA expression levels.

Table 10. Sequences of siHPRT and siCTRL

	Sequence(s)	SEQ ID NO:
			SiHPRT sense strand	5'-UcCuAuGaCuGuAgAuUuUaU-(CH₂)₆NH₂-3′	163
SiHPRT antisense strand	5'-aUaAaAuCuAcAgUcAuAgGasAsu-3′	164
			SiCTRL sense strand	5'-UgUaAuAaCcAuAuCuAcCuU-(CH₂)₆NH₂-3′	165
SiCTRL antisense strand	5’-aAgGuAgAuAuGgUuAuUaCasAsa-3′	166

* Lower case-2 'O-Me ribonucleoside, upper case-2' fluororibonucleoside, s-phosphorothioate linkage

Example 2 targeting HPRT with muscle targeting Complex

A muscle targeting complex was generated comprising HPRT SIRNA (siHPRT) used in example 1 covalently linked to the anti-transferrin receptor antibody DTX-a-002 through a non-cleavable N-gamma-maleimidobutyryl-oxysuccinimidyl ester (GMBS) linker. DTX-A-002 is RI7217 anti-TfR 1 Fab.

Briefly, GMBS linkers were dissolved in anhydrous DMSO and coupled to the 3' end of the siHPRT sense strand by amide bond formation under aqueous conditions. Completion of the reaction was verified by the Kaiser test. Excess linker and organic solvent were removed by gel permeation chromatography. The purified siHPRT maleimide functionalized sense strand was then coupled to a DTX-a-002 antibody using a michael addition reaction.

The product of the antibody coupling reaction was then purified by hydrophobic interaction chromatography (hydrophobic interaction chromatography, HIC-HPLC). The anti-TfR 1-siHPRT complex comprising one or two siHPRT molecules covalently linked to a DTX-a-002 antibody was purified. Densitometry determined that the average siHPRT to antibody ratio of the purified complex sample was 1.46.SDS-PAGE analysis showed that >90% of the purified complex samples contained DTX-A-002 linked to one or two siHPRT molecules.

Using the same method as described above, a control IgG2a-siHPRT complex was generated, comprising HPRT SIRNA (siHPRT) used in example 1 covalently linked to an IgG2a (Fab) antibody (DTX-A-003) via a GMBS linker. Densitometry determined that the average siHPRT to antibody ratio of DTX-C-001 (IgG 2a-siHPRT complex) was 1.46, and SDS-PAGE showed that >90% of the purified control complex samples contained DTX-a-003 linked to one or two siHPRT molecules.

The anti-TfR 1-siHPRT complex was then tested for internalization and inhibition of intracellular HPRT. Hepa 1-6 cells with relatively high levels of transferrin receptor expression were incubated for 72 hours in the presence of vehicle (phosphate buffered saline), igG2a-siHPRT (100 nM), anti-TfR 1-siCTRL (100 nM) or anti-TfR 1-siHPRT (100 nM). After 72 hours incubation, cells were isolated and the expression level of HPRT was determined (fig. 2). Cells treated with anti-TfR 1-siHPRT exhibited about 50% reduction in HPRT expression relative to cells treated with vehicle control and those treated with IgG2a-siHPRT complex. Meanwhile, cells treated with IgG2a-siHPRT or anti-TfR 1-siCTRL showed comparable levels of HPRT expression to vehicle controls (HPRT expression was not reduced). These data indicate that anti-transferrin receptor antibodies against TfR1-siHPRT are capable of internalizing the complex, thereby allowing siHPRT to inhibit expression of HPRT. HPRT expression levels are represented by HPRT mRNA expression levels.

Example 3 targeting HPRT in mouse muscle tissue with muscle targeting Complex

The muscle targeting complex described in example 2 was tested against TfR1-siHPRT for inhibition of HPRT in mouse tissue. C57BL/6 wild-type mice were injected intravenously with a single dose of vehicle control (phosphate buffered saline), siHPRT (2 mg/kg siRNA), igG2a-siHPRT (2 mg/kg siRNA, corresponding to 9mg/kg antibody complex) or anti-TfR 1-siHPRT (2 mg/kg siRNA, corresponding to 9mg/kg antibody complex). Each experimental condition was repeated in four separate C57BL/6 wild type mice. After a period of 3 days following injection, mice were euthanized and divided into isolated tissue types. The HPRT expression levels of individual tissue samples were then determined (fig. 3A-3B and 4A-4E).

Mice treated with the anti-TfR 1-siHPRT complex exhibited reduced expression of HPRT in the gastrocnemius muscle (31% reduction; p < 0.05) and heart (30% reduction; p < 0.05) relative to mice treated with siHPRT controls (fig. 3A-3B). Meanwhile, the level of HPRT expression in mice treated with IgG2a-siHPRT complex was comparable to siHPRT control (little or no reduction in HPRT expression) for all muscle tissue types assayed.

Mice treated with the anti-TfR 1-siHPRT complex showed no change in HPRT expression in non-muscle tissues such as brain, liver, lung, kidney and spleen tissues (fig. 4A-4E).

These data indicate that anti-transferrin receptor antibodies against TfR1-siHPRT complexes are capable of internalizing the complexes into muscle-specific tissues in an in vivo mouse model, allowing siHPRT to inhibit expression of HPRT. These data also demonstrate that the anti-TfR 1-oligonucleotide complexes of the present disclosure are capable of specifically targeting muscle tissue. The reduction of HPRT mRNA indicates inhibition of HPRT.

EXAMPLE 4 Activity of DUX 4-targeted siRNA conjugates in myotubes of FSHD patients

The activity of a conjugate comprising anti-TfR 1 Fab 3M12VH4/vκ3 covalently linked to a DUX 4-targeted siRNA was tested in AB1080 immortalized FSHD patient-derived myotubes. In the conjugate, anti-TfR 1 Fab is covalently linked to the 3' end of each siRNA sense strand through a linker, and the corresponding antisense strand anneals to the sense strand. The siRNA tested was siRNA-A (with the region complementary to SEQ ID NO 174), siRNA-B (with the region complementary to SEQ ID NO 175), or siRNA-C (with the region complementary to SEQ ID NO 176).

AB1080 (C6) immortalized FSHD patient-derived myotubes were treated with siRNA conjugates at concentrations equivalent to 1pM, 1nM or 100nM siRNA for 10 days. cDNA was obtained from cells using TAQMAN FAST ADVANCED CELLS-to-Ct kit (Thermo FISHER SCIENTIFIC) and levels of three DUX4 transcriptome markers MBD3L2 (Hs 00544743 _m1), TRIM43 (Hs 00299174 _m1), ZSCAN4 (Hs 00537549 _m1) and RPL13A (Hs 04194366 _g1) were analyzed by qPCR with a specific TaqMan assay (Thermo FISHER SCIENTIFIC). Two-step amplification reactions and fluorescence measurements for determining cycle threshold (Ct) were performed on QuantStudio a 7 instrument (Thermo FISHER SCIENTIFIC). FSHD comprehensive scores in FSHD1 cells were calculated using three DUX4 transcriptome markers (MBD 3L2, TRIM43 and ZSCAN 4), where Δct= (average Ct of 3 DUX4 markers) - (average Ct of RPL 13A), ΔΔct = Δct (treated) - Δct (vehicle), FSHD complex = 2- ΔΔct x 100 (%) (see fig. 5). The results showed that siRNA conjugates achieved a reduction in the tested DUX4 transcriptome markers in FSHD patient-derived myotubes and that treatment with conjugates equivalent to 100nM siRNA achieved about 50% reduction in the tested DUX4 transcriptome markers.

Example 4 siRNA and antibody conjugation protocol

The following protocol was used to prepare the siRNA conjugates tested in example 4. Conjugates comprising siRNA-A, siRNA-B, siRNA-C covalently linked to anti-TfR 1 Fab 3M12 VH4/V.kappa.3 were generated. The conjugates can be produced by the reactions shown below.

Materials:

Azide-PEG 3-vc-PABC-PFP linker MW= 773.79

Azido-PEG 8-PFP ester linker mw= 633.6

BCN-PEG4-PFP linker mw=607.6

Anti-TfR 1 Fab 3m12 VH4/vk3:mw=47968

SiRNA-A, siRNA-B and siRNA-C

Anti-TfR 1 Fab 3M12 VH4/V.kappa.3 buffer was exchanged into 50mM EPPS pH 8.0 and concentrated to a concentration of about 10 mg/ml.

The sense strand of each siRNA tested was dissolved in water to a concentration of 50mg/ml (concentration confirmed by UV absorbance). 4 Xazide-linker (20 mg/ml in DMA), 50 Xtributylamine (4.2M) and 70% DMA were added to the sense strand solution and incubated overnight at room temperature to yield azido-sense strand intermediates. After incubation, 1/10 volume of 3M NaCl and 3 volumes of cold isopropanol (isopropanol alcohol, IPA) were added to the reaction mixture. The reaction mixture was placed in a freezer at-80 ℃ for 30 minutes and then rotated at 4500rpm for 25 minutes. The pellet containing the azido-sense strand intermediate was washed twice with 70% ethanol (the pellet was aspirated with a pipette tip during washing) and dissolved in PBS to a final concentration of about 40mg/ml (the concentration was determined by UV absorbance).

To generate azide-siRNA duplex intermediates, the antisense strand of each siRNA tested was dissolved in PBS to a concentration of 50mg/ml (concentration confirmed by UV absorbance). The azide-sense strand intermediate and the corresponding antisense strand were mixed in a 1:1 ratio. For annealing, 300 to 500ml of water was heated to boiling in a glass beaker and the tube containing the azide-sense strand intermediate and antisense strand mixture was placed in a boiling water bath for 5 minutes and allowed to stand in the water bath until it cooled to room temperature on a bench. Annealing efficiency of each siRNA was measured using a UPLC SEC column.

To generate BCN-azide-siRNA duplex intermediate, azide-siRNA duplex intermediate solution in 30mm mes, ph 5.0 was slowly added to the same volume of DMA on ice. The 4 XBCN-PEG 4-PFP linker (20 mg/ml in DMA) was then slowly added to the azide-siRNA duplex intermediate/DMA mixture and incubated at room temperature for 3 to 3.5 hours to produce the BCN-azide-siRNA duplex intermediate. Alternatively, a3 XBCN-PEG 4-PFP linker (20 mg/ml in DMA) may be used in this step. The completion of the reaction was checked using a UPLC C18 column.

The crude reaction mixture was then purified prior to conjugation with anti-TfR 1 Fab 3m12 VH4/vκ3. To isolate the BCN-azide-siRNA duplex intermediate, 1/10 volume of 3M NaCl and 3 volumes of cold IPA were added to the reaction mixture and placed in a-80 ℃ refrigerator for 30 minutes followed by 25 minutes of rotation at 4500 rpm. The pellet containing BCN-azide-siRNA duplex intermediate was washed twice with 70% ethanol (pellet lifted with pipette tips during washing) and dissolved in 20mM MES, pH 5.0 to a concentration of about 20mg/ml (confirmed by UV absorbance).

Alternatively, the crude reaction mixture may be immediately subjected to the conjugation reaction without any purification. In this case, a reaction mixture of 40% DMA, BCN-PEG4-PFP linker and 2 to 3mM azide-siRNA duplex intermediate solution in 25mM MES at pH5.0 was established and incubated in a 30℃water bath for 3 to 4 hours to produce BCN-azide-siRNA duplex intermediate. The molar ratio between azide-siRNA duplex and BCN-PEG4-PFP linker was 1:0.85.

Finally, anti-TfR 1Fab 3m12 VH4/vκ3 was mixed with 1× (targeting DAR 1) BCN-azide-siRNA duplex intermediate and incubated overnight at room temperature to produce anti-TfR 1Fab-siRNA conjugate.

Alternative conjugation methods may also be used. anti-TfR 1 Fab 3M12 VH4/Vκ3 was buffer exchanged into 50mM EPPS pH 8.0 and concentrated to a concentration of about 10 mg/ml. The sense strand of each siRNA tested was dissolved in water to a concentration of 100mg/ml (concentration was confirmed by UV absorbance). 4 Xazide-linker (20 mg/ml in DMA), 100 Xtributylamine (4.2M) and 70% DMA were added to the sense strand solution and incubated overnight at room temperature to yield azido-sense strand intermediates. After incubation, 1/10 volume of 3M NaCl and 5 volumes of cold isopropyl alcohol (IPA) were added to the reaction mixture. The reaction mixture was placed in a freezer at-80 ℃ for 30 minutes, followed by 25 minutes of rotation at 4500 rpm. The precipitate containing the azido-sense strand intermediate was washed twice with 80% ethanol and dissolved in water to a final concentration of about 40mg/ml (confirmed by UV absorbance).

To generate the azide-siRNA duplex intermediates, the antisense strand of each siRNA tested was dissolved in water to a concentration of 50 mg/ml. The azide-sense strand intermediate and the corresponding antisense strand were mixed in a 1:1 ratio and incubated in a 30 ℃ water bath for more than 4 hours for annealing.

To generate the BCN-azide-siRNA duplex intermediate, a reaction mixture of 40% DMA, 4 molar excess BCN-PEG4-PFP linker, and 2 to 3mM azide-siRNA duplex intermediate solution in 25mM MES at pH 5.0 was established and incubated in a 30 ℃ water bath for 2 to 3 hours to generate the BCN-azide-siRNA duplex intermediate. Alternatively, a3 XBCN-PEG 4-PFP linker (20 mg/ml in DMA) may be used in this step. The completion of the reaction was checked using a UPLC C18 column.

The crude reaction mixture was then purified prior to conjugation with anti-TfR 1 Fab 3m12 VH4/vκ3. To isolate the BCN-azide-siRNA duplex intermediate, 1/10 volume of 3M NaCl and 3 volumes of cold IPA were added to the reaction mixture and placed in a-80 ℃ freezer for 30 minutes followed by 25 minutes of rotation at 4500 rpm. The precipitate containing BCN-azide-siRNA duplex intermediate was washed twice with 80% ethanol and dissolved in water to a concentration of about 20mg/ml.

Alternatively, the crude reaction mixture may be immediately subjected to the conjugation reaction without any purification. In this case, a reaction mixture of 40% DMA, molar excess BCN-PEG4-PFP linker and 2 to 3mM azide-siRNA duplex intermediate solution in 25mM MES at ph5.0 was established and incubated in a 30 ℃ water bath for 3 to 4 hours to produce BCN-azide-siRNA duplex intermediate. The molar ratio between azide-siRNA duplex and BCN-PEG4-PFP linker was 1:0.85.

Finally, 5 to 10mg/ml of anti-TfR 1 Fab 3M12VH4/vκ3 in 50mM EPPS, ph8.5 was mixed with 1× (DAR 1 targeted) BCN-azide-siRNA duplex intermediate and incubated overnight at room temperature to produce anti-TfR 1 Fab-siRNA conjugates.

Alternative conjugation methods can also be used and used to prepare siRNA conjugates tested in examples 5 to 7 below. In an alternative method, anti-TfR 1 Fab 3M12VH4/vκ3 (5 to 6 mg/ml), 1 xbcn (20 mg/ml in DMA, 32.9 mM) and 10% DMA in PBS are mixed and incubated at room temperature for at least 5 hours to produce an anti-TfR 1 Fab 3M12VH4/vκ3-BCN intermediate. The antibody-BCN intermediate was then purified using TFF and buffer exchanged into 50mM EPPS at pH 8.0.

Azide-siRNA duplex intermediates were generated as described above.

Anti-TfR 1 Fab 3m12 VH4/vκ3-BCN intermediate (concentration in EPPS between 6 and 7mg/ml, pH 8.0) was mixed with 1.0 molar excess of azide-siRNA duplex intermediate and incubated overnight at room temperature to produce anti-TfR 1 Fab-siRNA conjugate.

Purification by TSKgel superQ-5PW column:

the anti-TfR 1Fab-siRNA conjugates produced using the two-step or one-step reactions were purified using the following methods.

The crude conjugation reaction product in 50mM EPPS at pH8.0 was diluted with 5 column volumes (cv) of 10mM Tris at pH 8.0. The sample was loaded onto a 1ml TSKgel superQ-5PW column at 0.5 ml/min, with less than 10mg conjugate per ml resin. The column was washed with buffer A (20 mM Tris, pH 8.0) at 1 ml/min for 5 to 6cv. The conjugate was then eluted at 1 ml/min with 15 to 20cv of elution buffer containing 79% buffer A (20 mM Tris, pH 8.0) and 21% buffer B (20mM Tris,pH8.0+1.5M NaCl). The eluted conjugate was then buffer exchanged into PBS and concentrated to a concentration of >5 mg/ml.

In an alternative purification method, the crude conjugation reaction product was diluted with 1 volume of 10mM Tris pH 8.0 and loaded onto a 5ml TSKgel superQ-5PW column. The column was eluted with a gradient of 15% buffer B (buffer B: 20mM Tris+1.5M NaCl pH 8.0; buffer A: 20mM Tris pH 8.0)) to 40% buffer B at 1.5 ml/min over 25 Column Volumes (CV). The DAR 1 conjugate fraction was collected, buffer exchanged into PBS and concentrated to a concentration of >5 mg/ml.

Example 5 in vitro Activity of DUX 4-targeting siRNA

At both siRNA concentrations (0.2 nM and 20 nM), 48 DUX 4-targeted siRNAs were tested for their activity in knocking down the DUX4 transcriptome in the FSHD patient-derived C6 myotubes. The sirnas tested were siRNA 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. SIRNA SIRNA 7 targeting exon 1 was used as positive control. The siRNA numbers correspond to those in table 8. The calculated composite scores using the average mRNA levels of the three DUX4 transcriptome markers MBD3L2, TRIM43 and ZSCAN4 in FSHD patient derived C6 myotubes are shown in fig. 6.

The results show that most of the tested sirnas achieved a comparable level of knockdown of the tested DUX4 transcriptome markers in FSHD patient-derived C6 myotubes compared to the positive control. Some sirnas have more pronounced knockdown compared to positive controls.

In addition, 16 of the 48 siRNAs (siRNA 136, 137, 74, 77, 78, 104, 107, 106, 86, 89, 133, 97, 128, 92, 95 and 94) were also tested in Hepa1-6 cells at a concentration of 20 nM. DualGlo reporter plasmids were designed for use in screening siRNA. The plasmid contains the coding sequence of human DUX4 mRNA in the 3' -UTR of the reporter luciferase. Hepa1-6 cells were transfected with DualGlo reporter plasmids and 20nM of 16 DUX-targeted siRNAs. siRNA7 was also used as a positive control in Hepa1-6 cells. After transfection, cells were maintained for 1 day. DUX4 knockdown activity of each of the 16 sirnas is shown in table 10, along with DUX4 transcriptome knockdown activity of the same 16 sirnas tested above in FSHD patient-derived C6 myotubes.

The results showed that the tested sirnas achieved a comparable or more significant level of DUX4 knockdown in Hepa1-6 cells and DUX4 transcriptome knockdown in FSHD patient-derived C6 myotubes compared to the positive control, indicating robust efficacy in multiple in vitro FSHD models.

The combined scores calculated using the average mRNA levels of the human MBD3L2, TRIM43 and ZSCAN4 markers at a range of concentrations (2 pM to 40 nM) of the 16 tested sirnas with the positive control also produced dose response curves and they showed knockdown of the DUX4 transcriptome markers (see figures 7A to 7C).

TABLE 10 in vitro Activity of DUX-4 targeted siRNAs

Example 5 Activity of siRNA complexes targeting DUX4 in myotubes of FSHD patients

Seven complexes comprising anti-TfR 1 Fab3M12 VH4/vκ3 covalently linked to DUX 4-targeted siRNA through a linker were tested for activity in AB1080 immortalized FSHD patient-derived myotubes. For each siRNA complex, two forms were tested, one in which anti-TfR 1 Fab was covalently attached to the 3 'end of the siRNA sense strand, and the other in which anti-TfR 1 Fab was covalently attached to the 5' end of each siRNA sense strand. The sirnas tested were sirnas 142 through 148. The siRNA numbers correspond to those in table 9. siRNA targeting exon 1 was used as a positive control.

The AB1080 (C6) immortalized FSHD patient-derived myotubes were treated with siRNA complexes at concentrations equivalent to 10nM, 100nM or 1000nM for 10 days. cDNA was obtained from cells using TAQMAN FAST ADVANCED CELLS-to-Ct kit (Thermo FISHER SCIENTIFIC) and levels of three DUX4 transcriptome markers MBD3L2 (Hs 00544743 _m1), TRIM43 (Hs 00299174 _m1), ZSCAN4 (Hs 00537549 _m1) and RPL13A (Hs 04194366 _g1) were analyzed by qPCR with a specific TaqMan assay (Thermo FISHER SCIENTIFIC). Two-step amplification reactions and fluorescence measurements for determining the cycle threshold (Ct) were performed on QuantStudio instrument (Thermo FISHER SCIENTIFIC). The calculated composite scores using the average mRNA levels of the three DUX4 transcriptome markers MBD3L2, TRIM43 and ZSCAN4 in FSHD patient derived C6 myotubes are shown in fig. 8.

The results showed that the tested siRNA achieved significant level of knockdown of the tested DUX4 transcriptome markers in FSHD patient-derived C6 myotubes as compared to the positive control.

Example 6 Activity of siRNA complexes targeting DUX4 in primary cells derived from FSHD patients

The activity of complexes comprising anti-TfR 1 Fab 3m12 VH4/vκ3 covalently linked to DUX 4-targeted siRNA was tested in FSHD1 primary cells (MB 010, MB015, MB016, MB018, and MB 020). In this complex, anti-TfR 1 Fab is covalently linked to the 5' end of the sense strand of each siRNA via a linker, and the corresponding antisense strand anneals to the sense strand. The siRNA tested was siRNA145. The siRNA numbers correspond to those in table 9. Primary cells were treated with siRNA complexes at a concentration equivalent to 100nM siRNA for 7 days. cDNA was obtained from cells using TAQMAN FAST ADVANCED CELLS-to-Ct kit (Thermo FISHER SCIENTIFIC) and levels of three DUX4 transcriptome markers MBD3L2 (Hs 00544743 _m1), TRIM43 (Hs 00299174 _m1), ZSCAN4 (Hs 00537549 _m1) and RPL13A (Hs 04194366 _g1) were analyzed by qPCR with a specific TaqMan assay (Thermo FISHER SCIENTIFIC). Two-step amplification reactions and fluorescence measurements for determining the cycle threshold (Ct) were performed on QuantStudio instrument (Thermo FISHER SCIENTIFIC).

The results (see table 11) show that siRNA achieved a significant reduction of the tested DUX4 transcriptome markers in FSHD patient-derived primary cells.

TABLE 11

Example 7 Activity of DuX 4-targeting siRNA complexes in mouse muscle tissue

The activity of a complex comprising anti-TfR 1Fab 3M12 VH4/Vκ3 covalently linked to DUX 4-targeting siRNA through a linker was tested in eight to nine week old hTfR1/FLExDUX4/ACTA mice. For each siRNA complex, two forms were tested, one in which anti-TfR 1Fab was covalently attached to the 3 'end of the siRNA sense strand, and one in which anti-TfR 1Fab was covalently attached to the 5' end of each siRNA sense strand. The sirnas tested were siRNA145, siRNA148, siRNA144, siRNA143 and siRNA142. The siRNA numbers correspond to those in table 9.

The hTfR1/FLExDUX4/ACTA mice were injected intravenously with a single dose of vehicle control (phosphate buffered saline) or siRNA complex at a concentration equivalent to 10mg/kg siRNA. Each experimental condition was repeated in three to fifteen individual hTfR1/FLExDUX4/ACTA mice. Mice were euthanized twenty-eight days after injection. Gastrocnemius and quadriceps were collected and flash frozen in RNAlater ^TM stabilizing solution (Thermo FISHER SCIENTIFIC). By usingRSC miRNA tissue kit (Promega) total mirnas were isolated from gastrocnemius and quadriceps femoris and real-time quantitative RT-PCR was performed.

Three murine downstream DUX4 transcriptome markers Wfdc3 (Mm 01243777 _m1), sord (Mm 00455377 _g1), serpinb6c (Mm 00655242 _m1) and Rpl37A (Mm 00782745 _s1) were analyzed by qPCR with a specific TaqMan assay (Thermo FISHER SCIENTIFIC). Two-step amplification reactions and fluorescence measurements for determining the cycle threshold (Ct) were performed on QuantStudio instrument (Thermo FISHER SCIENTIFIC). FSHD composite scores were calculated using three DUX4 transcriptome markers (Wfdc, sord, serphinb 6 c), where ΔCt= (average Ct of 3 DUX4 markers) - (average Ct of Rpl 37A), ΔΔct=Δct (treated delta Ct (vehicle), FSHD synthesis=2- ΔΔct×100 (%) (see table 12, fig. 9A and 9B).

The results showed that many siRNA complexes achieved significant reduction of the tested DUX4 mouse transcriptome markers in the gastrocnemius and quadriceps of hTfR1/FLExDUX4/ACTA mice.

Table 12

Additional embodiments

1. A complex comprising a muscle targeting agent covalently linked to an oligonucleotide targeting double homologous box 4 (DUX 4) RNA, wherein the oligonucleotide comprises an antisense strand of 18 to 25 nucleotides in length and comprises a complementary region of the target sequence set forth in SEQ ID NOs 174 to 235, and wherein the complementary region is at least 16 contiguous nucleosides in length.

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

3. The complex of embodiment 1 or embodiment 2, wherein the oligonucleotide is an RNAi oligonucleotide.

4. The complex of embodiment 1 or embodiment 2, wherein the antisense strand comprises the nucleotide sequence of any one of SEQ ID NOs 236 to 266.

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

6. The complex of embodiment 5, wherein the sense strand comprises the nucleotide sequence of any one of SEQ ID NOs 205 to 235.

7. The complex of any one of embodiments 1 to 6, wherein the oligonucleotide comprises one or more modified nucleosides.

8. The complex of embodiment 7, wherein the one or more modified nucleosides is a2 'modified nucleotide, optionally wherein the one or more 2' modified nucleosides are selected from the group consisting of 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 '-O-N-methylacetamido (2' -O-NMA)).

9. The complex of embodiment 8, wherein each 2' modified nucleotide is 2' -O-methyl (2 ' -O-Me) or 2' -fluoro (2 ' -F).

10. The complex of any one of embodiments 1 to 9, wherein the oligonucleotide comprises one or more phosphorothioate internucleoside linkages.

11. The complex of embodiment 10, wherein the one or more phosphorothioate internucleoside linkages are present on the antisense strand of the oligonucleotide.

12. The complex of embodiment 11, wherein the two internucleoside linkages at the 3' terminus of the antisense strand are phosphorothioate internucleoside linkages.

13. The complex of embodiment 11 or embodiment 12, wherein the two internucleoside linkages at the 5' terminus of the antisense strand are phosphorothioate internucleoside linkages.

14. The complex of any one of embodiments 1 to 13, wherein one or more cytidine of the oligonucleotide is a2 'modified 5-methylcytidine, optionally wherein the 2' modified 5-methylcytidine is a 2'-O-Me modified 5-methylcytidine or a 2' -F modified 5-methylcytidine.

15. The complex of any of embodiments 1 to 14, wherein the antisense strand is selected from the modified forms of SEQ ID NOs 236 to 266 (e.g., MAS1 to MAS 4) set forth in table 8.

16. The complex of any of embodiments 5 to 15, wherein the sense strand is selected from the modified forms of SEQ ID NOs 205 to 235 (e.g., MS1 to MS 6) set forth in table 8.

17. The complex of embodiments 1 through 16, wherein the oligonucleotide is an siRNA molecule selected from the group consisting of the sirnas listed in table 8.

18. The complex of any one of embodiments 2 to 17, wherein the anti-TfR 1 antibody comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), light chain complementarity determining region 3 (CDR-L3) of any of the anti-TfR 1 antibodies listed in table 2.

19. The complex of any one of embodiments 2 to 17, wherein the anti-TfR 1 antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL) of any of the anti-TfR 1 antibodies listed in table 3.

20. The complex of any one of embodiments 2 to 17, wherein the anti-TfR 1 antibody is a Fab, optionally wherein the Fab comprises the heavy and light chains of any anti-TfR 1 Fab listed in table 5.

21. The complex of any one of embodiments 2 to 20, wherein the anti-TfR 1 antibody comprises:

22. The complex of embodiment 21, wherein the anti-TfR 1 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.

23. The complex of embodiment 22, wherein said anti-TfR 1 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.

24. The complex of any one of embodiments 1 to 23, wherein the muscle targeting agent and antisense oligonucleotide are covalently linked by a linker, optionally wherein the linker comprises a valine-citrulline sequence.

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

26. The method of embodiment 25, wherein reducing DUX4 expression comprises reducing DUX4 protein and/or mRNA levels.

27. A method of treating facial shoulder humeral muscular dystrophy (FSHD), comprising administering to a subject in need thereof an effective amount of the complex of any of embodiments 1-24.

28. The method of embodiment 27, wherein said subject has abnormal production of DUX4 protein.

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

30. A method of producing a complex comprising an anti-transferrin receptor 1 (TfR 1) 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) 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 an anti-TfR 1 antibody to said compound comprising the structure of formula (D) with a muscle targeting agent to obtain a compound comprising the structure of formula (E).

31. The method of embodiment 30, wherein the annealing of step (ii) is performed at 30 ℃.

32. The method of embodiment 30 or embodiment 31, further comprising isolating the compound comprising the structure of formula (D) after step (iii) and before step (iv).

33. The method of embodiment 30 or embodiment 31, wherein the compound comprising the structure of formula (D) is not isolated prior to step (iv).

34. A method of producing a complex comprising an anti-transferrin receptor 1 (TfR 1) antibody covalently linked to an oligonucleotide, the method comprising:

(ii) Annealing the antisense strand to the sense strand of the siRNA oligonucleotide;

(iii) Covalently linking an anti-TfR 1 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).

35. The method of any one of embodiments 30 to 34, wherein the antisense strand is 18 to 25 nucleotides in length and comprises a region of complementarity of the target sequences set forth in SEQ ID NOs 189, 191, 200, 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.

36. The method of any one of embodiments 30-35, wherein the anti-TfR 1 antibody comprises heavy chain complementarity determining region 1 (CDR-H1), heavy chain complementarity determining region 2 (CDR-H2), heavy chain complementarity determining region 3 (CDR-H3), light chain complementarity determining region 1 (CDR-L1), light chain complementarity determining region 2 (CDR-L2), light chain complementarity determining region 3 (CDR-L3) of any of the anti-TfR 1 antibodies listed in table 2.

37. The method of any one of embodiments 30 to 36, wherein said anti-TfR 1 antibody is covalently linked to the 5' end of the sense strand of an siRNA oligonucleotide targeting DUX4 RNA.

Equivalent and terminology

The disclosure illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, in each instance herein, any of the terms "comprising," "consisting essentially of," and "consisting of" can be replaced with any of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the disclosure. Thus, it should be understood that although the present disclosure has been specifically disclosed by some preferred embodiments, optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this disclosure.

In addition, where features or aspects of the present disclosure are described in terms of Markush groups (Markush groups) or other alternative groups, those skilled in the art will recognize that the present disclosure is also thus described in terms of any individual member or subgroup of members of the Markush group or other group.

It is understood that in some embodiments, reference may be made to the sequences shown in the sequence listing in describing the structure of an oligonucleotide or other nucleic acid. In such embodiments, the actual oligonucleotide or other nucleic acid may have one or more alternative nucleotides or nucleosides (e.g., RNA counterparts of DNA nucleosides or DNA counterparts of RNA nucleosides) and/or (e.g., and) one or more modified nucleotides/nucleosides and/or (e.g., and) one or more modified internucleoside linkages and/or (e.g., and) one or more other modifications as compared to the specified sequence, while retaining substantially the same or similar complementary properties as the specified sequence.

The use of nouns without quantitative word modifications in the context of describing the invention (especially in the context of the appended claims) will be interpreted as one or more than one unless otherwise indicated herein or clearly contradicted by context. Unless otherwise indicated, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to"). Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Some embodiments of the invention are described herein. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description.

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

Claims

1. 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 comprises 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)-vinyl phosphonate.

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'-O-N-methylacetamido (2'-O-NMA)).

11. 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;

(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);

Also 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:

(iii) covalently linking the anti-TfR1 antibody to the 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,