CN118251240A

CN118251240A - Muscle targeting complexes for the treatment of facial shoulder humerus muscular dystrophy

Info

Publication number: CN118251240A
Application number: CN202280074761.4A
Authority: CN
Inventors: 内尔松·赫西亚; 罗梅什·R·苏布拉马尼亚; 穆罕默德·T·卡塔纳尼; 蒂莫西·威登; 布伦丹·奎因; 科迪·A·德雅尔丹; 约翰·纳吉姆; 肖恩·斯普林
Original assignee: Dyne Therapeutics Inc
Current assignee: Dyne Therapeutics Inc
Priority date: 2021-11-12
Filing date: 2022-11-10
Publication date: 2024-06-25

Abstract

Some aspects of the disclosure relate to oligonucleotides designed to target DUX4RNA and to targeting complexes for delivering the oligonucleotides to cells (e.g., muscle cells), and uses thereof, particularly in connection with the treatment of diseases (e.g., FSHD). Wherein the complex comprises an anti-transferrin receptor 1 (TfR 1) antibody covalently linked to an oligonucleotide configured to reduce expression or activity of DUX 4.

Description

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

RELATED APPLICATIONS

The present application claims the following benefits in accordance with 35 u.s.c. ≡119 (e): U.S. provisional application No.63/278,882 entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY" filed on 11/12 of 2021; U.S. provisional application No.63/278,993 entitled "TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY" filed on 11/12 of 2021; U.S. provisional application No.63/312,617 entitled "MUSCLE TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY" filed on 22 nd 2 of 2022; and U.S. provisional application No.63/312,633, entitled "TARGETING COMPLEXES AND USES THEREOF FOR TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY," filed on 22, 2, 2022, each of which is incorporated herein by reference in its entirety.

Technical Field

The present application relates to targeting complexes for delivering molecular loads (molecular payload) (e.g., oligonucleotides) to cells and uses thereof, particularly in connection with the treatment of diseases.

Reference electronic sequence Listing

The contents of the electronic sequence Listing (D082470074 WO00-SEQ-CBD. Xml; size: 467,675 bytes; and date of creation: 2022, 11, 3) 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 genes encoding proteins required for the formation of healthy muscle tissue. Facial shoulder muscular dystrophy (facioscapulohumeral muscular dystrophy, FSHD) is a dominant inherited type of MD that affects mainly the muscles of the face, shoulder and upper arms. Other symptoms of FSHD include abdominal muscle weakness, retinal abnormalities, hearing loss, joint pain and inflammation. FSHD is the most common of the 9 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 whose function is unknown. The DUX4 gene encoding the DUX4 protein is located in the D4Z4 repeat region on chromosome 4 and is normally expressed only in fetal development, 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, which accounts for about 95% of cases, is associated with a deletion of the D4Z4 repeat on chromosome 4. Unaffected individuals typically have more than 10 repeats arranged in the subtelomere region of chromosome 4, while 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 distal region of D4Z 4. 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 translated into a stable protein. Type 2 FSHD, which accounts for about 5% of cases, is associated with mutations in the SMCHD1 gene on chromosome 18. Apart from supportive care and treatment for disease symptoms, there is no effective treatment for FSHD.

Disclosure of Invention

In some aspects, the disclosure provides oligonucleotides designed to target DUX4 RNA. In some embodiments, the present disclosure provides oligonucleotides complementary to DUX4 RNA that are useful for reducing the level of DUX4 mRNA and/or protein associated with facial shoulder brachial muscular dystrophy (FSHD) pathology including muscle atrophy, inflammation, and reduced differentiation potential and oxidative stress. In some embodiments, the oligonucleotides provided herein target the 3' utr of DUX4 RNA. In some embodiments, the oligonucleotides provided herein are designed to direct the degradation of DUX4 RNA. In some embodiments, the oligonucleotide is designed to block translation of DUX4 RNA used to produce the DUX4 protein. In some embodiments, the oligonucleotides are designed to have desired bioavailability and/or serum stability characteristics. In some embodiments, the oligonucleotides are designed to have desired binding affinity properties. In some embodiments, the oligonucleotides are designed to have a desired toxicity and/or immunogenicity profile.

According to some aspects, the present disclosure provides complexes that target muscle cells (e.g., primary myoblasts) for delivery of molecular loads (e.g., DUX4 targeting oligonucleotides described herein) to those cellular purposes. In some embodiments, the complexes provided herein are particularly useful for delivering molecular loads that inhibit expression or activity of DUX4, for example in subjects having or suspected of having facial shoulder humeral muscular dystrophy (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 load to the muscle cell. In some embodiments, the complex is taken up into the cell by receptor-mediated internalization, and then the molecular charge 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 of the ligation complex is released by endosomal cleavage of the covalent linker of the oligonucleotide and the muscle targeting agent.

Some aspects of the disclosure provide a complex comprising an anti-transferrin receptor 1 (anti-TRANSFERRIN RECEPTOR 1, tfR 1) antibody covalently linked to an oligonucleotide configured for reducing expression or activity of DUX4, wherein the anti-TfR 1 antibody comprises a 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 1, 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 one of the anti-TfR 1 antibodies listed in tables 2-7, and wherein the oligonucleotide comprises an antisense strand comprising a complementarity region of the DUX4 sequence shown in SEQ ID NO 160 or SEQ ID NO 365.

In some embodiments, the 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 comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 95% identity to SEQ ID No. 76 and/or a light chain variable region (VL) comprising an amino acid sequence having at least 95% identity to SEQ ID No. 75. In some embodiments, 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. In some embodiments, the anti-TfR 1 antibody is a Fab, optionally wherein the Fab comprises the heavy and light chains of any one of the anti-TfR 1 fabs listed in table 5. In some embodiments, the Fab comprises a heavy chain comprising an amino acid sequence having at least 85% identity to SEQ ID NO. 101 and/or a light chain comprising an amino acid sequence having at least 85% identity to SEQ ID NO. 90. In some embodiments, the 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.

In some embodiments, the oligonucleotide is 20 to 30 nucleotides in length. In some embodiments, the oligonucleotide comprises a complementary region of at least 15 consecutive nucleotides of the DUX4 sequence shown in SEQ ID NO. 160 or SEQ ID NO. 365. In some embodiments, the oligonucleotide comprises a complementary region of at least 15 consecutive nucleotides of the DUX4 sequence as set forth in any one of SEQ ID NOS 161-168 or 213-288. In some embodiments, the oligonucleotide comprises at least 15 contiguous nucleotides of any one of SEQ ID NOs 169 to 176 or 289 to 364, wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by T. In some embodiments, the oligonucleotide does not comprise the nucleotide sequence of SEQ ID NO. 151. In some embodiments, the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOS 169 to 176 or 289 to 364.

In some embodiments, the oligonucleotide further comprises a sense strand that hybridizes to the antisense strand to form a double stranded siRNA.

In some embodiments, the oligonucleotide comprises at least one modified internucleoside linkage. In some embodiments, the oligonucleotide comprises one or more modified nucleosides. In some embodiments, the one or more modified nucleosides is a 2' -modified nucleoside. In some embodiments, the oligonucleotide is a diamide morpholino oligomer (phosphorodiamidate morpholino oligomer, PMO) phosphate.

In some embodiments, the antibody is covalently linked to the oligonucleotide through a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker comprises a valine-citrulline sequence.

Further aspects of the disclosure provide methods of reducing DUX4 expression in a muscle cell comprising contacting the muscle cell with an effective amount of a complex described herein for promoting internalization of the oligonucleotide into the muscle cell. In some embodiments, the cell is in vitro. In some embodiments, the cell is in a subject. In some embodiments, the subject is a human.

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 described herein, wherein the subject has abnormal production of DUX4 protein. In some embodiments, the subject has one or more deletions of the D4Z4 repeat in chromosome 4. In some embodiments, the subject has 10 or fewer D4Z4 repeats. In some embodiments, the subject has 9, 8, 7, 6, 5, 4, 3, 2, or 1D 4Z4 repeats. In some embodiments, the subject does not have a D4Z4 repeat.

Also provided herein are oligonucleotides comprising the nucleotide sequence of any one of SEQ ID NOs 169 to 176 or 289 to 364. In some embodiments, the oligonucleotide is a Phosphorodiamidate Morpholino Oligomer (PMO).

Drawings

FIG. 1 shows that a conjugate comprising anti-TfR Fab 3M12 VH4/Vk3 conjugated to a DUX4 targeting oligonucleotide (SEQ ID NO: 151) inhibited the DUX4 transcriptome in C6 (AB 1080) immortalized FSHD1 cells, as shown by reduced mRNA expression by MDB3L2, TRIM43 and ZCAN 4. The conjugates showed superior activity in inhibiting DUX4 transcriptome relative to unconjugated DUX4 targeting oligonucleotides.

Figures 2A to 2B show dose response curves for gene knockdown. FIG. 2A shows MBD3L2 knockdown in C6 (AB 1080) immortalized FSHD1 cells treated with a conjugate comprising anti-TfR Fab 3M12VH4/Vk3 conjugated to a DUX4 targeting oligonucleotide (SEQ ID NO: 151). FIG. 2B shows MBD3L2, TRIM43 and ZCAN 4 knockdown in myotubes of FSHD patients treated with conjugates comprising anti-TfR Fab 3M12VH4/Vk3 conjugated to DUX4 targeting oligonucleotide (SEQ ID NO: 151). Fig. 2B contains MBD3L2 data shown in fig. 2A.

FIG. 3 shows non-human primate plasma levels over time of DUX4 targeting oligonucleotide (SEQ ID NO: 151) after administration of 30mg/kg unconjugated ('naked') oligonucleotide or 3, 10 or 30mg/kg oligonucleotide equivalent of a conjugate comprising anti-TfR 1 Fab 3M12 VH4/Vk3 covalently linked to a DUX4 targeting oligonucleotide ("Fab-oligonucleotide conjugate").

FIG. 4 shows the tissue level of DUX4 targeting oligonucleotide (SEQ ID NO: 151) measured in a non-human primate muscle tissue sample two weeks after administration of 30mg/kg unconjugated ('naked') oligonucleotide or 3, 10 or 30mg/kg oligonucleotide equivalent of a conjugate comprising anti-TfR 1Fab 3M12 VH4/Vk3 covalently attached to a DUX4 targeting oligonucleotide.

FIG. 5 shows tissue levels of DUX4 targeting oligonucleotide (SEQ ID NO: 151) measured in non-human primate muscle tissue samples collected by biopsy (left 5 bars) one week after administration of 30mg/kg of unconjugated oligonucleotide ('oligonucleotide') or 3, 10 or 30mg/kg of oligonucleotide equivalent of conjugate ('conjugate') comprising anti-TfR 1 Fab3M12 VH4/Vk3 covalently linked to a DUX4 targeting oligonucleotide or two weeks after administration thereof by autopsy (right 5 bars).

FIG. 6 shows that conjugates comprising anti-TfR Fab 3M12 VH4/Vk3 conjugated to a DUX4 targeting oligonucleotide (Table 8, #1 or #2, corresponding to SEQ ID NOS: 176, 169, 170, respectively) and a control DUX4 targeting oligonucleotide (corresponding to SEQ ID NO: 151) reduced the expression level of the DUX4 transcriptome marker (MBD 3L2, TRIM43, ZSCAN 4), indicating that the conjugates reduced the expression level of DUX4 in FSHD patient cells in vitro.

Detailed Description

In some aspects, the present disclosure provides complexes comprising a muscle targeting agent covalently linked to a DUX4 targeting oligonucleotide for effective delivery of the oligonucleotide to a muscle cell. In some embodiments, the complexes are particularly useful for delivering molecular loads that inhibit the expression or activity of a target gene in a muscle cell (e.g., in a subject having or suspected of having a rare muscle disease). For example, 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.

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

I. Definition of the definition

And (3) application: the term "administering" 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 "approximately" 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).

Antibody: 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. nos. 5,693,780 and Kabat E Aet 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).

CDR: the term "CDR" as used herein refers to complementarity determining regions within an antibody variable sequence. 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 carboxyl-terminus in the following 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 informationhttp://www.imgt.org,Lefranc,M.-P.et al.,Nucleic Acids Res.,27:209-212(1999);Ruiz,M.et al.,Nucleic Acids Res.,28:219-221(2000);Lefranc,M.-P.,Nucleic Acids Res.,29:207-209(2001);Lefranc,M.-P.,Nucleic Acids Res.,31:307-310(2003);Lefranc,M.-P.et al.,In Silico Biol.,5,0006(2004)[Epub],5:45-60(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,33:D593-597(2005);Lefranc,M.-P.et al.,Nucleic Acids Res.,37:D1006-1012(2009);Lefranc,M.-P.et al.,Nucleic Acids Res.,43:D413-422(2015);Chothia et al.,(1989)Nature 342:877;Chothia,C.et al.(1987)J.Mol.Biol.196:901-917;Al-lazikani et al(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

the international ImMunoGeneTics informatioimgt.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 antibody (CDR-grafted antibody): the term "CDR-grafted antibody" refers to an antibody comprising heavy and light chain variable region sequences from one species but in which 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 in which 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.

Complementary: the term "complementary" as used herein refers to the ability to pair precisely between two nucleosides or two groups of nucleosides. In particular, complementarity is a term that characterizes the degree of hydrogen bond pairing that causes the binding between two nucleosides or 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" refer to amino acid substitutions that do not alter the relative charge or dimensional characteristics of the protein in which they are made. Variants can be prepared according to methods known to those of ordinary skill in the art for altering polypeptide sequences, such as can be found in references compiling such methods: such as Molecular Cloning:A Laboratory Manual,J.Sambrook,et al.,eds.,Fourth Edition,Cold Spring Harbor Laboratory Press,Cold Spring Harbor,New York,2012, or Current Protocols in Molecular Biology, f.m. ausubel, et al, eds., john Wiley & Sons, inc. Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

Covalent attachment: 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 context of a targeting agent (e.g., an antibody), the term "cross-reactive" 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-001306068.2, NM-001363820.1).

Facial shoulder brachial muscular dystrophy (FSHD): the term "facial shoulder humerus muscular dystrophy (FSHD)" as used herein refers to a genetic disease caused by a mutation in the DUX4 gene or SMCHD1 gene, which is characterized by decreased muscle mass and muscle atrophy mainly in the facial, shoulder humerus 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 comprising the DUX4 gene on chromosome 4 allelic variant 4 qA. Type 2 is associated with a mutation in the SMCHD1 gene. Both type 1 and type 2 FSHD are characterized by abnormal production of DUX4 protein other than testis after fetal development. The facial shoulder brachial 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.

A frame: 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 antibodies: 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 receptor antibodies and antigen-binding portions are provided. Such antibodies can be produced by obtaining murine anti-TfR 1 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.

Internalizing cell surface receptors: the term "internalized cell surface receptor" as used herein refers to a cell surface receptor that is internalized by a cell, for example, under an 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 antibodies: 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 loading: the term "molecular cargo" as used herein refers to a molecule or substance that plays a role in regulating biological outcomes. In some embodiments, the molecular load is linked or otherwise associated with a muscle targeting agent. In some embodiments, the molecular cargo is a small molecule, protein, peptide, nucleic acid, or oligonucleotide. In some embodiments, the molecular cargo functions to regulate transcription of the DNA sequence, regulate expression of the protein, or regulate activity of the protein. In some embodiments, the molecular cargo is an oligonucleotide comprising a strand having a complementary region of the target gene.

Muscle targeting agents: 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 load) 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 cargo.

Muscle targeting antibodies: the term "muscle targeting antibody" as used herein refers to a muscle targeting agent 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 load) 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.

An oligonucleotide: the term "oligonucleotide" as used herein 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 human immunoglobulin gene sequences to other DNA sequences).

Complementary region: the term "complementary region" as used herein 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).

The object is: 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 or 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-dimethylaminoethyl-oxyethyl (2 ' -O-DMAEOEE), 2' -O-N-methylacetamido (2 ' -O-NMA), locked nucleic acids (locked nucleic acid, LNA, methylene 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:

II. Complex

Also provided herein are complexes comprising a targeting agent (e.g., an antibody) covalently linked to a molecular cargo. 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 load present in the complex is responsible for the modulation of genes, proteins, and/or (e.g., sum) nucleic acids. The molecular cargo 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 cargo is an oligonucleotide that targets DUX4 in a muscle cell or CNS cell.

In some embodiments, the complex comprises a muscle targeting agent, such as an anti-TfR 1 antibody, covalently linked to a molecular load (e.g., an antisense oligonucleotide targeting DUX 4).

A. muscle targeting agents

Some aspects of the present disclosure provide muscle targeting agents, for example, for delivering molecular loads to muscle cells. In some embodiments, such muscle targeting agents are capable of binding to muscle cells, for example, by specifically binding to an antigen on the muscle cells, and delivering an associated molecular load to the muscle cells. In some embodiments, the molecular cargo 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 is understood that a variety of types of muscle targeting agents may be used in accordance with the present disclosure, and that any muscle target (e.g., muscle surface protein) may be targeted by any of the types of muscle targeting agents described herein. For example, the muscle targeting agent may comprise or consist of: small molecules, nucleic acids (e.g., DNA or RNA), peptides (e.g., antibodies), lipids (e.g., microbubbles (microvesicle)), or sugar moieties (e.g., polysaccharides). 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 molecular loads 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 load 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 loads (e.g., oligonucleotides) in the muscle while reducing toxicity associated with effects in other tissues. In some embodiments, the muscle targeting agent concentrates the bound molecular load in the muscle cells as compared to another cell type within the subject. In some embodiments, the muscle targeting agent concentrates the bound molecular load 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 load 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"JBiol 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 present 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 the transferrin receptor is internalized into the muscle cell along with any bound molecular load. 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 of: corresponding to amino acids 90 to 96 of the human transferrin receptor (as shown in SEQ ID NO: 105), which is not in the apical domain of the transferrin receptor.

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 as set forth 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 as set forth 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 as set forth 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 as 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. Antibodies can also be produced by screening a protein expression library (e.g., phage display library) expressing the Antibodies in some embodiments (see, e.g., U.S. Pat. No. 5,223,409, "Directed evolution of novel binding proteins" submitted by month 1 in 1991; WO 1992/18619, "Heterodimeric receptor libraries using phagemids" submitted by month 1 in 1992; WO 1991/17271, "Recombinant library screening methods" submitted by month 15 in 1992; WO 1992/20791, "Methods for producing members of specific binding pairs" submitted by month 28 in 1992; in some embodiments, phage display library designs can also be used (see, e.g., U.S. Pat. No. 5,223,409, "Directed evolution of novel binding proteins" submitted by month 1 in 1992; see, e.g., U.S. Pat. No. 5,409; in FIG. 4 in 19942; WO 1991/17271, "Recombinant library screening methods" submitted by month 15 in 1992; DNA) and the antigen can be used to immunize animals or other animals, e.g., to obtain Antibodies from humans by way of non-human, and optionally, by way of example, recombinant methods (see, e.g., hartie-in 19842).

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 (e.g., to CNS cells). 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 aspects, provided herein are humanized antibodies that bind to transferrin receptor with high specificity and affinity. In some embodiments, the humanized anti-TfR 1 antibodies described herein specifically bind to any extracellular epitope of a transferrin receptor or epitope that becomes exposed to the antibody. In some embodiments, the humanized anti-TfR 1 antibodies provided herein specifically bind to transferrin receptor from humans, non-human primates, mice, rats, and the like. In some embodiments, a humanized anti-TfR 1 antibody provided herein binds to a human transferrin receptor. In some embodiments, the humanized anti-TfR 1 antibodies described herein bind 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, the humanized anti-TfR 1 antibodies described herein bind to such amino acid segments: corresponding to amino acids 90 to 96 of the 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 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 humanized 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 comprising CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR 1 antibodies provided in table 3, and comprising 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 comprising CDR-L1, CDR-L2, and CDR-L3 of any one of the anti-TfR 1 antibodies provided in table 3, and comprising 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 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. 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 any one of the VLs listed in table 3 or any variant thereof and a light chain constant region having at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity to SEQ ID No. 83. In some embodiments, an anti-TfR 1 antibody described herein comprises a light chain comprising any one of the VLs listed in table 3 or any variant thereof and a light chain constant region comprising NO more than 25 amino acid variations (e.g., NO more than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3,2, or 1 amino acid variations) as compared to SEQ ID NO: 83. In some embodiments, an anti-TfR 1 antibody described herein comprises a light chain comprising any one of the VL's set forth in Table 3, or any variant thereof, and a light chain constant region set forth in SEQ ID NO. 83.

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

* CDRs according to the Kabat numbering system are bolded; the VH/VL sequence is 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's set forth 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 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. 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 present disclosure comprisesNO: 126) CDR-L3 (according to Kabat and Chothia definition System) orID NO: 127) CDR-L3 (according to the Contact definition System). In some embodiments, an anti-TfR 1 antibody of the present disclosure comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, and CDR-L2 identical to CDR-H1, CDR-H2, and CDR-H3 shown in table 7, and comprisesCDR-L3 (according to Kabat and Chothia definition System) orCDR-L3 (according to the Contact definition system).

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 the following: such as Smith P et al, (2012) PNAS 109:6181-6186, U.S. Pat. No.6,737,056, and International publication Nos. WO 02/060919, WO 98/23289, and WO 97/34631, which are incorporated herein by reference.

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 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.,etal.,"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.

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 an amino acid sequence

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 the following: such as Smith P et al, (2012) PNAS109:6181-6186, U.S. Pat. No.6,737,056, and International publication Nos. WO 02/060919, WO 98/23289, and WO 97/34631, which are incorporated herein by reference.

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.

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-targeting peptides have been described below in U.S. patent 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" at 12/11/2001; 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 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 loads. 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 SIRNA DELIVERY," 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, it has an amino acid sequenceIs bound in vitro to C2C12 murine myotubes and in vivo to mouse muscle tissue. Thus, in some embodiments, the muscle targeting agent comprises an amino acid sequenceThe 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, it was identified that there is a sequence Is a 12 amino acid peptide and the muscle targeting peptide is relative toPeptides showed improved binding to C2C12 cells.

Another method for identifying peptides that are selective for muscle (e.g., skeletal muscle) relative to other cell types includes in vitro selection, which has been described in Ghosh D.,et al.,"Selection of muscle-binding peptides from context-specific peptide-presenting phage libraries for adenoviral vector targeting"J Virol 2005;79:13667-72; the entire contents of which are incorporated herein by reference. 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 Most frequently. Thus, in some embodiments, the muscle targeting agent comprises an amino acid sequence

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; 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, linolenyl (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."RNA aptamers and their therapeutic and diagnostic applications."Int.J.Biochem.Mol.Biol.2013;4:27–40.). in some embodiments, muscle targeting aptamers have been previously disclosed (see, e.g., Phillippou,S.et al."Selection and Identification of Skeletal-Muscle-Targeted RNA Aptamers."Mol Ther Nucleic Acids.2018,10:199-214.;Thiel,W.H.et al."Smooth Muscle Cell-targeted RNA Aptamer Inhibits Neointimal Formation."Mol Ther.2016,24:4,779-87.). exemplary muscle targeting aptamers include a01B RNA aptamer and RNA Apt 14 in some embodiments, the aptamers are nucleic acid based, oligonucleotide, or peptide aptamers in some embodiments, the aptamers 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 myomembranes: (1) Adenosine triphosphate (adenosine triphosphate, ATP) binding cassette (adenosine triphosphate binding cassette, ABC) superfamily, which promotes outflow from skeletal muscle tissue and (2) solute carrier (solute carrier, SLC) superfamily, which can promote substrate inflow 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 load (e.g., an oligonucleotide load). In some embodiments, the muscle targeting agent is covalently linked to the molecular cargo. In some embodiments, the muscle targeting agent is non-covalently linked to the molecular cargo.

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 load (e.g., an oligonucleotide load).

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 loading

Some aspects of the disclosure provide molecular loading, e.g., oligonucleotides designed to target DUX4RNA to modulate expression or activity of DUX 4. In some embodiments, modulating expression or activity of DUX4 comprises reducing the level of DUX4RNA and/or (e.g., and) protein. In some embodiments, the DUX4 targeting oligonucleotide is linked or otherwise associated with a muscle targeting agent described herein. In some embodiments, such oligonucleotides are capable of targeting DUX4 in a muscle cell, for example, by specifically binding to a DUX4 sequence in a muscle cell after delivery to the muscle cell by an associated muscle targeting agent. It should be understood that various types of muscle targeting agents may be used in accordance with the present disclosure. In some embodiments, the oligonucleotide comprises a strand having a complementary region of DUX4 sequence. Exemplary DUX4 RNA-targeting oligonucleotides are also described in detail herein, however, it should be understood that the exemplary molecular loadings provided herein are not meant to be limiting.

I. oligonucleotides

In some embodiments, the oligonucleotide may be designed to cause degradation of the mRNA (e.g., the oligonucleotide may be a spacer, siRNA, ribozyme, or aptamer that causes degradation). In some embodiments, the oligonucleotide may be designed to block translation of mRNA. In some embodiments, the oligonucleotide may be designed to cause degradation of the mRNA and block translation of the mRNA. In some embodiments, the oligonucleotide may be designed to result in reduced expression of DUX4 RNA. In some embodiments, the oligonucleotide may be designed to result in reduced expression of the DUX4 protein. Further examples of oligonucleotides are provided herein. It will be appreciated that in some embodiments, oligonucleotides of one form (e.g., antisense oligonucleotides) may be suitably adapted to another form (e.g., siRNA oligonucleotides) by incorporating functional sequences (e.g., antisense strand sequences) from one form to another.

Any suitable oligonucleotide may be used as a molecular charge, as described herein. Some examples of oligonucleotides that can be used to target DUX4 are provided below: U.S. patent No. 9,988,628, published 2/2017, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; U.S. patent No. 9,469,851, published 10/30 in 2014, entitled "RECOMBINANT VIRUS PRODUCTS AND METHODS FOR INHIBITING EXPRESSION OF DUX"; U.S. patent application publication 20120225034, published 9/6 in 2012, entitled "AGENTS USEFUL IN TREATING FACIOSCAPULOHUMERAL MUSCULAR DYSTROPHY"; PCT patent application publication No. WO 2013/120038, published on 15, 8, 2013, each of which is entitled "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;, the contents of each of which are incorporated herein in their 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 may have a complementary region of the sequence as set forth in: human DUX4, corresponding to NCBI sequences NM-001293798.1 (SEQ ID NO: 186), NM-001293798.2 (SEQ ID NO: 187) and/or (e.g., and) NM-001306068.3 (SEQ ID NO: 188); and/or (e.g., and) mouse DUX4, corresponding to NCBI sequence NM-001081954.1 (SEQ ID NO: 189) as follows. In some embodiments, the oligonucleotide may have a hypomethylated, compact D4Z4 repeat complementary region, 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 siRNAsuggests 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-001293798.1) (SEQ ID NO: 186):

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: 187):

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: 188):

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: 189) (NM-001081954.1):

In some embodiments, the oligonucleotides may have complementary regions of DUX4 gene sequences of multiple species (e.g., selected from human, mouse, and non-human species).

In some embodiments, a DUX4 targeting oligonucleotide described herein comprises a nucleotide sequence comprising a region of complementarity of at least 12 consecutive nucleotides (e.g., 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, at least 26, or more consecutive nucleotides) of a DUX4 sequence set forth in any one of SEQ ID NOs 186 to 189.

In some embodiments, the DUX 4-targeting oligonucleotides described herein comprise a nucleotide sequence comprising a region of complementarity to the DUX4 sequence corresponding to nucleotides 1519 to 1553 of SEQ ID NO. 187. In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence comprising a region of complementarity corresponding to at least 12 consecutive nucleotides (e.g., 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, at least 26, or more consecutive nucleotides) of the DUX4 sequence of nucleotides 1519 to 1553 in SEQ ID NO: 187. In some embodiments, a DUX4 targeting oligonucleotide described herein is 15 to 30 nucleotides (e.g., 15 to 30, 18 to 28, 20 to 26, 22 to 27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28.29, or 30 nucleotides) in length and comprises a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, at least 26, or more consecutive nucleotides) of a DUX4 sequence corresponding to nucleotides 1519 to 1553 in SEQ ID No. 187.

In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence comprising SEQ ID No. 160: the complementary region of the DUX4 sequence shown in (A). In some embodiments, a DUX4 targeting oligonucleotide described herein comprises a nucleotide sequence comprising a complementary region of at least 12 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) of a DUX4 sequence set forth in SEQ ID No. 160. In some embodiments, a DUX4 targeting oligonucleotide described herein is 15 to 30 nucleotides (e.g., 15 to 30, 18 to 28, 20 to 26, 22 to 27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28.29, or 30 nucleotides) in length and comprises a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) of the DUX4 sequence set forth in SEQ ID NO: 160.

Some non-limiting examples of DUX4 targeting oligonucleotides are provided in table 8.

TABLE 8 some non-limiting examples of DUX4 targeting oligonucleotides

Each thymine base (T) in any one of the oligonucleotides and/or target sequences provided in table 8 may be independently and optionally replaced by a uracil base (U), and/or each U may be independently and optionally replaced by a T. The target sequences listed in table 8 comprise T, but binding of DUX4 targeting oligonucleotides to RNA and/or DNA is contemplated.

In some embodiments, a DUX 4-targeting oligonucleotide described herein comprises a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) of any one of SEQ ID NOs 161 to 168. In some embodiments, the DUX4 targeting oligonucleotides described herein are 15 to 30 nucleotides (e.g., 15 to 20, 20 to 30, 22 to 27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) in length and comprise a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) of any of SEQ ID NOs 161 to 168. In some embodiments, the DUX4 targeting oligonucleotides described herein do not compriseThe 25 nucleotide complementary region of the DUX4 target sequence.

In some embodiments, a DUX 4-targeting oligonucleotide described herein comprises at least 15 consecutive nucleosides (e.g., 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, at least 26, or more consecutive nucleosides) of the nucleotide sequence of any one of SEQ ID NOs 169 to 176, wherein each thymine base (T) can be independently and optionally replaced by a uracil base (U), and each U can be independently and optionally replaced by T. In some embodiments, the DUX4 targeting oligonucleotide is a Phosphodiamide Morpholino Oligomer (PMO). In some embodiments, the DUX4 targeting oligonucleotides described herein do not comprise a nucleotide sequence

In some embodiments, the DUX4 targeting oligonucleotides described herein comprise the nucleotide sequence of any one of SEQ ID NOS: 169-176, wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by T.

In some embodiments, any of the DUX4 targeting oligonucleotides described herein is a Phosphodiamide Morpholino Oligomer (PMO).

In some embodiments, the DUX 4-targeting oligonucleotides described herein comprise a nucleotide sequence comprising a region of complementarity to the DUX4 sequence corresponding to nucleotides 1474 to 1574 of SEQ ID NO. 187. In some embodiments, a DUX4 targeting oligonucleotide described herein comprises a nucleotide sequence comprising a region of complementarity corresponding to at least 12 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) of a DUX4 sequence of nucleotides 1474 to 1574 in SEQ ID No. 187. In some embodiments, a DUX4 targeting oligonucleotide described herein is 15 to 30 nucleotides (e.g., 15 to 30, 18 to 28, 20 to 26, 22 to 27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28.29, or 30 nucleotides) in length and comprises a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) of a DUX4 sequence corresponding to nucleotide 1474 to nucleotide 1574 in SEQ ID No. 187.

In some embodiments, the DUX4 targeting oligonucleotides described herein comprise a nucleotide sequence comprising SEQ ID No. 365: The complementary region of the DUX4 sequence shown in (A). In some embodiments, a DUX 4-targeting oligonucleotide described herein comprises a nucleotide sequence comprising a complementary region of at least 12 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) of a DUX4 sequence set forth in SEQ ID NO: 365. In some embodiments, a DUX4 targeting oligonucleotide described herein is 15 to 30 nucleotides (e.g., 15 to 30, 18 to 28, 20 to 26, 22 to 27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28.29, or 30 nucleotides) in length and comprises a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, at least 26, at least 27, at least 28, at least 29, at least 30, or more consecutive nucleotides) of a DUX4 sequence set forth in SEQ ID NO 365.

Some non-limiting examples of DUX4 targeting oligonucleotides are provided in table 9.

TABLE 9 some non-limiting examples of DUX4 targeting oligonucleotides

Each thymine base (T) in any one of the oligonucleotides and/or target sequences provided in table 9 may be independently and optionally replaced by a uracil base (U), and/or each U may be independently and optionally replaced by a T. The target sequences listed in table 9 comprise T, but binding of DUX4 targeting oligonucleotides to RNA and/or DNA is contemplated.

In some embodiments, a DUX 4-targeting oligonucleotide described herein comprises a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, or more consecutive nucleotides) of any one of SEQ ID NOS: 213 to 288. In some embodiments, a DUX4 targeting oligonucleotide described herein is 15 to 30 nucleotides (e.g., 15 to 20, 20 to 30, 22 to 27, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) in length and comprises a region of complementarity of at least 15 consecutive nucleotides (e.g., 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, or more consecutive nucleotides) of any of SEQ ID NOs 213 to 288. In some embodiments, the DUX4 targeting oligonucleotides described herein do not compriseThe 25 nucleotide complementary region of the DUX4 target sequence.

In some embodiments, a DUX 4-targeting oligonucleotide described herein comprises at least 15 consecutive nucleosides (e.g., 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, or more consecutive nucleosides) of the nucleotide sequence of any one of SEQ ID NOS: 289 to 364, wherein each thymine base (T) can be independently and optionally replaced with a uracil base (U), and each U can be independently and optionally replaced with T. In some embodiments, the DUX4 targeting oligonucleotide is a Phosphodiamide Morpholino Oligomer (PMO). In some embodiments, the DUX4 targeting oligonucleotides described herein do not comprise a nucleotide sequence

In some embodiments, the DUX 4-targeting oligonucleotides described herein comprise the nucleotide sequence of any one of SEQ ID NOS: 289-364, wherein each thymine base (T) may be independently and optionally replaced with a uracil base (U), and each U may be independently and optionally replaced with T.

In some embodiments, any of the oligonucleotides may be in salt form, e.g., as a sodium, potassium, or magnesium salt.

In some embodiments, the 5 'or 3' nucleoside (e.g., terminal nucleoside) of any of the oligonucleotides described herein 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 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 substituted or unsubstituted alkyl. In certain embodiments, the spacer is a substituted or unsubstituted alkylene, a substituted or unsubstituted heterocyclylene, a substituted or unsubstituted heteroarylene, -O-, -N (R ^A) -or-C (=o) N (R ^A)₂), or a combination thereof.

In some embodiments, the 5 'or 3' nucleoside of any one of the oligonucleotides described herein 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, a phosphodiester linkage is present between a compound of formula NH ₂-(CH₂)_n -and a 5 'or 3' nucleoside of an 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 5' phosphate 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 antibody), for example, via an amine group.

A. Oligonucleotide size/sequence

Oligonucleotides may be of a variety of different lengths, e.g., 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 30 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, 15 to 20 nucleotides in length, 20 to 25 nucleotides in length, 20 to 30 nucleotides in length, and the like.

In some embodiments, an oligonucleotide for purposes of the present disclosure is "complementary" to a target nucleic acid when its nucleic acid sequence is specifically hybridizable to the target nucleic acid. In some embodiments, oligonucleotides that hybridize to a target nucleic acid (e.g., mRNA or pre-mRNA molecule) result in modulation of activity or expression of the target (e.g., reduced mRNA translation, altered pre-mRNA splicing, exon skipping, target mRNA degradation, etc.). In some embodiments, the nucleic acid sequence of an oligonucleotide has a sufficient degree of complementarity to its target nucleic acid such that it does not hybridize to a non-target sequence under conditions where it is desired to avoid non-specific binding, e.g., under physiological 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 nucleotide sequence to which it is targeted to specifically hybridize or be specific to the target nucleic acid. In certain embodiments, the oligonucleotide comprises one or more nucleobases mismatched relative to the target nucleic acid. In certain 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 oligonucleotide comprises a complementary region of the target nucleic acid that is 8 to 15, 8 to 30, 8 to 40, or 10 to 50, or 5 to 50, 15 to 20, 20 to 25, or 5 to 40 nucleotides in length. 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 12 consecutive nucleotides of the target nucleic acid. In some embodiments, an oligonucleotide may comprise 1, 2, or 3 base mismatches as compared to a portion of consecutive nucleotides of a 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, 20, 21, 22, 23, 24, 25, 26, or 27 contiguous nucleotides comprising the sequence of any one of SEQ ID NOs 169 to 176 or 289 to 364. In some embodiments, the oligonucleotide comprises a sequence comprising any one of SEQ ID NOs 169 to 176 or 289 to 364. 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 (e.g., 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, at least 26) consecutive nucleotides of any of SEQ ID NOs 169 to 176 or 289 to 364. In some embodiments, the DUX 4-targeting oligonucleotide does not comprise a sequence

In some embodiments, the oligonucleotide comprises a region complementary to the nucleotide sequence set forth in any one of SEQ ID NOS.161 to 168 or 213 to 288. In some embodiments, the oligonucleotide comprises at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides (e.g., consecutive nucleotides) that are complementary to the nucleotide sequence set forth in any one of SEQ ID NOs 161 to 168 or 213 to 288. In some embodiments, the oligonucleotide comprises a sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or 100% complementary to at least 12 or at least 15 consecutive nucleotides of any one of SEQ ID NOs 161 to 168 or 213 to 288. In some embodiments, the DUX 4-targeting oligonucleotide does not compriseThe 25 nucleotide complementary region of the DUX4 target sequence.

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 or 9). In some embodiments, such target sequences are 100% complementary to the oligonucleotide sequences listed in table 8 or 9.

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 a thymine nucleotide or nucleoside.

In some embodiments, any one or more thymine bases (T) in any one of the oligonucleotides provided herein (e.g., an oligonucleotide listed in table 8 or 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. Additionally, in some embodiments, the oligonucleotides may exhibit one or more of the following properties: does not mediate alternative splicing; not immunostimulatory; resistance to nucleases; has improved cellular uptake compared to the unmodified oligonucleotide; is nontoxic to cells or mammals; internal excretion of endosomes in cells is improved; minimizing TLR stimulation; or avoid 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 into which they are incorporated more resistant to nuclease digestion than the native oligodeoxynucleotide or oligoribonucleotide molecule; 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 of the oligonucleotide are modified nucleotides/nucleoside. 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-dimethylaminoethyl oxyethyl (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'-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 ENAs are provided in the following: international patent publication No. WO2005/042777, published on month 5 and 12 of 2005, and entitled "APP/ENA ANTISENSE"; morita et al, nucleic Acids Res, journal 1:241-242,2001;Surono etal.,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 disclosure of which is incorporated herein by reference in its entirety. Some examples of cets are provided in the following: 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 oligonucleotide comprises a modified nucleoside disclosed in one of the following U.S. patents or patent application publications: U.S. patent 7,399,845, granted on month 7 and 15 of 2008, entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. patent 7,741,457, which was issued on month 6 and 22 of 2010, and titled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. patent 8,022,193, which was granted on day 20, 9, 2011, and entitled "6-Modified Bicyclic Nucleic Acid Analogs"; U.S. patent 7,569,686, issued 8/4/2009, and entitled "Compounds And Methods For Synthesis Of Bicyclic Nucleic Acid Analogs"; U.S. patent 7,335,765, which was granted on month 2, 26 of 2008, and titled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. patent 7,314,923, issued on 1/2008, and entitled "Novel Nucleoside And Oligonucleotide Analogues"; U.S. patent 7,816,333, which was issued on 10/19 of 2010 and entitled "Oligonucleotide Analogues And Methods Utilizing The Same" and U.S. publication 2011/0009471, now U.S. patent 8,957,201, which was issued on 17 of 2015, 2, and entitled "Oligonucleotide Analogues And Methods Utilizing The Same", each of which is incorporated herein by reference in its entirety for all purposes.

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 at least one modified nucleoside. The oligonucleotide may 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 more for the oligonucleotide 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.

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, and chiral phosphonates, phosphinates, phosphoramidates comprising 3' -aminophosphamidates and aminoalkyl phosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphates and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those of opposite polarity wherein 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; amide backbone (see DE MESMAEKER ET al. Ace. Chem. Res.1995, 28:366-374); morpholino backbone (see Summerton AND WELLER, U.S. Pat. No.5,034,506); or a peptide nucleic acid (peptide nucleic acid, PNA) backbone (wherein the phosphodiester backbone of the oligonucleotide is replaced by a polyamide backbone, the nucleotide 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 modulated by a configuration based on 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 that comprise nucleoside units linked together by substantially all Sp or substantially all Rp phosphorothioate intersugar 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 in 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).

G. Peptide Nucleic Acid (PNA)

In some embodiments, both the sugar and the internucleoside linkage (backbone) of the nucleotide unit of the oligonucleotide are replaced by new groups. In some embodiments, the maintenance base unit is used to hybridize to an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is known as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar backbone of the oligonucleotide is replaced with an amide containing backbone (e.g., an aminoethylglycine backbone). The nucleobase is retained and is bound directly or indirectly to the aza nitrogen atom of the amide moiety of the backbone. Representative publications reported for the preparation of PNA compounds include, but are not limited to, U.S. Pat. nos. 5,539,082, 5,714,331 and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in NIELSEN ET al, science,1991,254,1497-1500.

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 a 2 '-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 nucleotides, 5 to 12 nucleotides, or 6 to 10 nucleotides in length.

In some embodiments, the spacer region of the spacer oligonucleotide may comprise modified nucleosides, such as C4' -substituted nucleosides, acyclic nucleosides, and nucleosides of the arabinose (arabino) configuration, that are known to be acceptable for efficient rnase H action, in addition to DNA nucleosides. In some embodiments, the spacer comprises one or more unmodified internucleoside. 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 5'-X-Y-Z-3'：5-10-5,4-12-4.3-14-3.2-16-2,1-18-1,3-10-3,2-10-2,1-10-1,2-8-2,4-6-4,3-6-3,2-6-2,4-7-4,3-7-3,2-7-2,4-8-4,3-8-3,2-8-2,1-8-1,2-9-2,1-9-1,2-10-2,1-10-1,1-12-1,1-16-1,2-15-1,1-15-2,1-14-3,3-14-1,2-14-2,1-13-4,4-13-1,2-13-3,3-13-2,1-12-5,5-12-1,2-12-4,4-12-2,3-12-3,1-11-6,6-11-1,2-11-5,5-11-2,3-11-4,4-11-3,1-17-1,2-16-1,1-16-2,1-15-3,3-15-1,2-15-2,1-14-4,4-14-1,2-14-3,3-14-2,1-13-5,5-13-1,2-13-4,4-13-2,3-13-3,1-12-6,6-12-1,2-12-5,5-12-2,3-12-4,4-12-3,1-11-7,7-11-1,2-11-6,6-11-2,3-11-5,5-11-3,4-11-4,1-18-1,1-17-2,2-17-1,1-16-3,1-16-3,2-16-2,1-15-4,4-15-1,2-15-3,3-15-2,1-14-5,5-14-1,2-14-4,4-14-2,3-14-3,1-13-6,6-13-1,2-13-5,5-13-2,3-13-4,4-13-3,1-12-7,7-12-1,2-12-6,6-12-2,3-12-5,5-12-3,1-11-8,8-11-1,2-11-7,7-11-2,3-11-6,6-11-3,4-11-5,5-11-4,1-18-1,1-17-2,2-17-1,1-16-3,3-16-1,2-16-2,1-15-4,4-15-1,2-15-3,3-15-2,1-14-5,2-14-4,4-14-2,3-14-3,1-13-6,6-13-1,2-13-5,5-13-2,3-13-4,4-13-3,1-12-7,7-12-1,2-12-6,6-12-2,3-12-5,5-12-3,1-11-8,8-11-1,2-11-7,7-11-2,3-11-6,6-11-3,4-11-5,5-11-4,1-19-1,1-18-2,2-18-1,1-17-3,3-17-1,2-17-2,1-16-4,4-16-1,2-16-3,3-16-2,1-15-5,2-15-4,4-15-2,3-15-3,1-14-6,6-14-1,2-14-5,5-14-2,3-14-4,4-14-3,1-13-7,7-13-1,2-13-6,6-13-2,3-13-5,5-13-3,4-13-4,1-12-8,8-12-1,2-12-7,7-12-2,3-12-6,6-12-3,4-12-5,5-12-4,2-11-8,8-11-2,3-11-7,7-11-3,4-11-6,6-11-4,5-11-5,1-20-1,1-19-2,2-19-1,1-18-3,3-18-1,2-18-2,1-17-4,4-17-1,2-17-3,3-17-2,1-16-5,2-16-4,4-16-2,3-16-3,1-15-6,6-15-1,2-15-5,5-15-2,3-15-4,4-15-3,1-14-7,7-14-1,2-14-6,6-14-2,3-14-5,5-14-3,4-14-4,1-13-8,8-13-1,2-13-7,7-13-2,3-13-6,6-13-3,4-13-5,5-13-4,2-12-8,8-12-2,3-12-7,7-12-3,4-12-6,6-12-4,5-12-5,3-11-8,8-11-3,4-11-7,7-11-4,5-11-6,6-11-5,1-21-1,1-20-2,2-20-1,1-20-3,3-19-1,2-19-2,1-18-4,4-18-1,2-18-3,3-18-2,1-17-5,2-17-4,4-17-2,3-17-3,1-16-6,6-16-1,2-16-5,5-16-2,3-16-4,4-16-3,1-15-7,7-15-1,2-15-6,6-15-2,3-15-5,5-15-3,4-15-4,1-14-8,8-14-1,2-14-7,7-14-2,3-14-6,6-14-3,4-14-5,5-14-4,2-13-8,8-13-2,3-13-7,7-13-3,4-13-6,6-13-4,5-13-5,1-12-10,10-12-1,2-12-9,9-12-2,3-12-8,8-12-3,4-12-7,7-12-4,5-12-6,6-12-5,4-11-8,8-11-4,5-11-7,7-11-5,6-11-6,1-22-1,1-21-2,2-21-1,1-21-3,3-20-1,2-20-2,1-19-4,4-19-1,2-19-3,3-19-2,1-18-5,2-18-4,4-18-2,3-18-3,1-17-6,6-17-1,2-17-5,5-17-2,3-17-4,4-17-3,1-16-7,7-16-1,2-16-6,6-16-2,3-16-5,5-16-3,4-16-4,1-15-8,8-15-1,2-15-7,7-15-2,3-15-6,6-15-3,4-15-5,5-15-4,2-14-8,8-14-2,3-14-7,7-14-3,4-14-6,6-14-4,5-14-5,3-13-8,8-13-3,4-13-7,7-13-4,5-13-6,6-13-5,4-12-8,8-12-4,5-12-7,7-12-5,6-12-6,5-11-8,8-11-5,6-11-7, or 7-11-6 of

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-dimethylaminoethyl oxyethyl (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-ALALBEBE-(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; wherein "a" represents a 2' -modified nucleoside; "B" represents a 2'-4' bicyclic nucleoside; "K" represents constrained ethyl nucleoside (cEt); "L" represents LNA nucleoside; and "E" represents a 2' -MOE modified ribonucleoside; "D" represents 2' -deoxyribonucleoside; "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.

I. Mixed polymer

In some embodiments, the oligonucleotides described herein may be mixed-mer or comprise mixed-mer sequence patterns. In general, a mixed mer is an oligonucleotide that contains both natural and non-naturally occurring nucleosides or, in general, two different types of non-naturally occurring nucleosides in an alternative mode. The hybrid polymers generally have higher binding affinities than unmodified oligonucleotides and can be used to specifically bind to target molecules, e.g., to block binding sites on target molecules. Generally, the mixed multimer does not recruit RNase to the target molecule and thus does not promote cleavage of the target molecule. Such oligonucleotides that are incapable of recruiting RNase H have been described, for example, see WO2007/112754 or WO2007/112753.

In some embodiments, the hybrid polymer comprises or consists of: repeating patterns of nucleoside analogs and naturally occurring nucleosides, or one type of nucleoside analog and a second type of nucleoside analog. However, the hybrid polymer need not contain a repeating pattern and may alternatively contain any arrangement of modified nucleosides and naturally occurring nucleosides, or any arrangement of one type of modified nucleoside and a second type of modified nucleoside. The repeating pattern can be, for example, every second or every third nucleoside is a modified nucleoside (e.g., LNA), and the remaining nucleosides are naturally occurring nucleosides (e.g., DNA), or 2' substituted nucleoside analogs (e.g., 2' -MOEs or 2' fluoro analogs), or any other modified nucleoside described herein. It is recognized that a repeating pattern of modified nucleosides, such as LNA units, can be combined with the modified nucleosides at fixed positions, such as at the 5 'or 3' ends.

In some embodiments, the hybrid polymer does not comprise more than 5, more than 4, more than 3, or more than 2 contiguous regions of naturally occurring nucleosides (e.g., DNA nucleosides). In some embodiments, the hybrid polymer comprises at least one region consisting of at least two consecutive modified nucleosides, e.g., at least two consecutive LNAs. In some embodiments, the hybrid polymer comprises at least one region consisting of at least three consecutive modified nucleoside units, e.g., at least three consecutive LNAs.

In some embodiments, the hybrid polymer does not comprise more than 7, more than 6, more than 5, more than 4, more than 3, or more than 2 regions of contiguous nucleoside analogs (e.g., LNAs). In some embodiments, the LNA units may be replaced with other nucleoside analogs (e.g., those mentioned herein).

The hybrid polymers can be designed to include a mixture of affinity-enhanced modified nucleosides (e.g., LNA nucleosides and 2' -O-Me nucleosides in a non-limiting example). In some embodiments, the hybrid polymer comprises modified 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 nucleosides.

Any suitable method may be used to produce the hybrid polymer. Representative U.S. patents, U.S. patent publications, and PCT publications that teach the preparation of hybrid polymers include U.S. patent publication nos. US 20060184646, US20090209748, US20090298916, US20110077288, and US20120322851, and U.S. patent No.7687617.

In some embodiments, the hybrid polymer comprises one or more morpholino nucleosides. For example, in some embodiments, the mixed polymers can include morpholino nucleosides mixed (e.g., mixed in an alternating fashion) with one or more other nucleosides (e.g., DNA, RNA nucleosides) or modified nucleosides (e.g., LNA, 2' -O-Me nucleosides).

In some embodiments, the hybrid polymers may be used for splice correction or exon skipping, for example, as reported in volume ：Touznik A.,et al.,LNA/DNA mixmer-based antisense oligonucleotides correct alternative splicing of the SMN2 gene and restore SMN protein expression in type 1SMA fibroblasts Scientific Reports,, volume 7, article No. :3672(2017),Chen S.et al.,Synthesis of a Morpholino Nucleic Acid(MNA)-Uridine Phosphoramidite,and Exon Skipping Using MNA/2′-O-Methyl Mixmer Antisense Oligonucleotide,Molecules 2016,21,1582,, the respective contents of which are incorporated herein by reference.

RNA interference (RNAi)

In some embodiments, the oligonucleotides provided herein may be in the form of 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, and 21 to 23 base pairs in length.

After selection of the appropriate target RNA sequence, an siRNA molecule comprising a nucleotide sequence (i.e., an antisense sequence) that is 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 may be double stranded (i.e., dsRNA molecules comprising an antisense strand and a complementary sense strand that hybridizes to form dsRNA) 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 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 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 siRNA molecule comprises an antisense strand comprising a region complementary to a target region in a target 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 target mRNA. In some embodiments, the target region is a region of contiguous nucleotides in the target 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 region of complementarity of a target RNA sequence, and the region of complementarity is 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、37、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 target RNA sequence. In some embodiments, the siRNA molecule comprises a nucleotide sequence comprising no more than 1,2, 3, 4, or 5 base mismatches with a portion of consecutive nucleotides of the target RNA sequence. In some embodiments, the siRNA molecule 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 molecules comprise an antisense strand comprising a nucleotide sequence complementary (e.g., at least 85%, at least 90%, at least 95%, or 100%) to a target RNA sequence of an oligonucleotide provided herein. 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 provided herein. In some embodiments, the siRNA molecule comprises an antisense strand comprising 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 contiguous nucleotides of an oligonucleotide provided herein.

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 individual oligonucleotides into a stem-loop structure, wherein the self-complementary sense and antisense regions of the siRNA molecule are linked by: nucleic acid-based or non-nucleic acid-based linkers, and circular single stranded RNAs having two or more loop structures and stems comprising self-complementary sense and antisense strands, wherein the circular RNAs can be processed in vivo or in vitro to produce active siRNA molecules 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 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 nucleotides on the antisense strand. In some embodiments, the siRNA molecule comprises a 3' overhang of about 1 to about 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 internucleotide linkages. In some embodiments, the modified nucleotide comprises 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-dimethylaminoethyl oxyethyl (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 phosphodiamide morpholinos. In some embodiments, each nucleotide of the siRNA molecule is a phosphodiamide morpholino.

In some embodiments, the siRNA molecule comprises phosphorothioate or other modified internucleotide linkages. In some embodiments, the siRNA molecule comprises phosphorothioate internucleoside 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 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 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, 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, and chiral phosphonates, phosphinates, phosphoramidates comprising 3' -aminophosphamidates and aminoalkyl phosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphates and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those of opposite polarity wherein adjacent pairs of nucleoside units are linked at 3'-5' to 5'-3' or 2'-5' to 5 '-2'; see U.S. Pat. Nos. 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-dimethylaminoethyl oxyethyl (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 phosphodiamide morpholinos. In some embodiments, the antisense strand is a Phosphodiamide Morpholino Oligomer (PMO).

In some embodiments, the antisense strand comprises phosphorothioate linkages or other modified internucleotide linkages. In some embodiments, the antisense strand comprises phosphorothioate internucleoside 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 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 internucleoside linkages of the 5 'or 3' terminus of the siRNA molecule. In some embodiments, the modified internucleotide 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, and chiral phosphonates, phosphinates, phosphoramidates comprising 3' -aminophosphamidates and aminoalkyl phosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphates and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those of opposite polarity wherein adjacent pairs of nucleoside units are linked at 3'-5' to 5'-3' or 2'-5' to 5 '-2'; see U.S. Pat. Nos. 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 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-dimethylaminoethyl oxyethyl (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 phosphodiamide morpholinos. In some embodiments, the antisense strand is a Phosphodiamide Morpholino Oligomer (PMO). In some embodiments, the sense strand comprises phosphorothioate or other modified internucleotide linkages. In some embodiments, the sense strand comprises phosphorothioate internucleoside 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 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 internucleoside linkages of the 5 'or 3' terminus of the sense strand.

In some embodiments, the modified internucleotide 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, and chiral phosphonates, phosphinates, phosphoramidates comprising 3' -aminophosphamidates and aminoalkyl phosphoramidates, thiocarbonylphosphoramidates, thiocarbonylalkylphosphonates, thiocarbonylalkylphosphates and borane phosphates with normal 3'-5' linkages, 2'-5' linked analogs of these, and those of opposite polarity wherein 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.

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 modifications that increase or decrease the load of the RNA-induced silencing complex (RNA-induced silencing complex, RISC). 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 a 2 '-O-methoxyethyl (2' -MOE) modification. The addition of a 2 '-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 Commun 5:3459, 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 acic, UNA) modification that reduces RISC loading of the sense strand and improves silencing efficacy of the antisense strand, as described in Sneadet 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: nucleic acids (e.g., DNA or RNA), peptides (e.g., antibodies), lipids (e.g., microvesicles), or sugar moieties (e.g., polysaccharides). 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 of the anti-TfR antibodies provided herein). 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 attached 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 linked to the 5' end of the antisense strand of the siRNA molecule. In some embodiments, the muscle targeting agent can be attached 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.

K. micro RNA (miRNA)

In some embodiments, the oligonucleotide may be a microrna (miRNA). Micrornas (referred to as "mirnas") are small non-coding RNAs that belong to a class of regulatory molecules that control gene expression by binding to complementary sites on target RNA transcripts. Generally, mirnas are produced from large RNA precursors, known as primary mirnas (pri-mirnas), which are processed in the nucleus to precursor mirnas of about 70 nucleotides, which fold into an imperfect stem-loop structure. These precursor mirnas are typically subjected to additional processing steps within the cytoplasm, where mature mirnas of 18 to 25 nucleotides in length are excised from one side of the precursor miRNA hairpin by the rnase III enzyme Dicer.

Mirnas as used herein include primary mirnas, precursor mirnas, mature mirnas, or fragments of variants thereof that retain the biological activity of the mature mirnas. In one embodiment, the miRNA may range in size from 21 nucleotides to 170 nucleotides. In one embodiment, the size of the miRNA is in the range of 70 to 170 nucleotides in length. In another embodiment, mature mirnas of 21 to 25 nucleotides in length may be used.

Aptamer

In some embodiments, the oligonucleotides provided herein may be in the form of an aptamer. In general, an aptamer is any nucleic acid that specifically binds to a target (e.g., small molecule in a cell, protein, nucleic acid) under molecular loading. In some embodiments, the aptamer is a DNA aptamer or an RNA aptamer. In some embodiments, the nucleic acid aptamer is single-stranded DNA or RNA (ssDNA or ssRNA). It is understood that single stranded nucleic acid aptamers may form a helical and/or (e.g., sum) loop structure. The nucleic acid forming the nucleic acid aptamer may comprise naturally occurring nucleotides, modified nucleotides, naturally occurring nucleotides with a hydrocarbon linker (e.g., alkylene) or polyether linker (e.g., PEG linker) interposed between one or more nucleotides, modified nucleotides with a hydrocarbon or PEG linker interposed between one or more nucleotides, or a combination thereof. Exemplary publications and patents describing aptamers and methods of producing aptamers include, for example, lorsch and Szostak,1996; jayasena,1999; U.S. patent No.5,270,163;5,567,588;5,650,275;5,670,637;5,683,867;5,696,249;5,789,157;5,843,653;5,864,026;5,989,823;5,569,630;8,318,438 and PCT application WO 99/31275, each of which is incorporated herein by reference.

M. multimers

In some embodiments, the molecular charge may comprise a multimer (e.g., a concatemer) of 2 or more oligonucleotides linked by a linker. In some embodiments, in this way, the oligonucleotide loading of the complex/conjugate can be increased beyond the available ligation sites on the targeting agent (e.g., available thiol sites on the antibody), or otherwise adjusted to achieve a particular loading capacity. The oligonucleotides in the multimer may be the same or different (e.g., targeting different genes or different sites on the same gene or product thereof).

In some embodiments, the multimer comprises 2 or more oligonucleotides linked together by a cleavable linker. However, in some embodiments, the multimer comprises 2 or more oligonucleotides linked together by a non-cleavable linker. In some embodiments, the multimer comprises 2,3, 4, 5, 6, 7, 8, 9, 10 or more oligonucleotides linked together. In some embodiments, the multimer comprises 2 to 5, 2 to 10, or 4 to 20 oligonucleotides linked together.

In some embodiments, the multimer comprises 2 or more oligonucleotides that are end-to-end (in a linear arrangement). In some embodiments, a multimer comprises 2 or more oligonucleotides joined end-to-end by an oligonucleotide-based linker (e.g., a poly-dT linker, an abasic linker). In some embodiments, the multimer comprises a5 'end of one oligonucleotide linked to a 3' end of another oligonucleotide. In some embodiments, the multimer comprises a3 'end of one oligonucleotide linked to a 3' end of another oligonucleotide. In some embodiments, the multimer comprises a5 'end of one oligonucleotide linked to a 5' end of another oligonucleotide. Nonetheless, in some embodiments, a multimer may comprise a branching structure comprising multiple oligonucleotides linked together by a branching linker.

Further examples of multimers that can be used in the complexes provided herein are disclosed in the following: for example, U.S. patent application No. 2015/0315588A1, entitled Methods of delivering multiple targeting oligonucleotides to a cell using cleavable linkers, which was published on month 11, 5 of 2015; U.S. patent application No. 2015/0247241 A1, titled Multimeric Oligonucleotide Compounds, published on month 9, 3 of 2015; U.S. patent application No. US2011/0158937A1, titled Immunostimulatory Oligonucleotide Multimers, published in 2011, month 6, 30; and U.S. patent No. 5,693,773, entitled Triplex-Forming Antisense Oligonucleotides Having Abasic Linkers Targeting Nucleic Acids Comprising Mixed Sequences Of Purines And Pyrimidines,, entitled 12/2/1997, the respective contents of which are incorporated herein by reference in their entirety.

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 load. 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 load. However, in some embodiments, the linker may covalently link any of the anti-TfR 1 antibodies described herein to the molecular load through multiple covalent bonds. In some embodiments, the linker may be a cleavable linker. However, in some embodiments, the linker may be a non-cleavable linker. The linker is generally stable in vitro and in vivo, and may be stable in certain cellular environments. In addition, typically the linker does not negatively affect the functional properties of the anti-TfR 1 antibody or molecular load. 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 ofLinker,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 load. 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 attached to the cysteine residue or thiol-functionalized molecular load 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 load 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 of a muscle cell or CNS 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 link an anti-TfR 1 antibody comprising an LPXT sequence to a molecular load 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, an optionally substituted alkenylene, an optionally substituted alkynylene, an optionally substituted cycloalkylene, an optionally substituted cycloalkenylene, an optionally substituted arylene, an optionally substituted heteroarylene further comprising at least one heteroatom selected from N, O and S; an optionally substituted heterocyclylene group 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 load via 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 charge 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 charge, 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 as described in international patent application publication WO2016170186, which is published on 10/27 of 2016, and is titled "Process For The Modification Of A Glycoprotein Using A Glycosyltransferase That Is Or Is Derived From Aβ(1,4)-N-Acetylgalactosaminyltransferase". in some embodiments, a cycloaddition reaction is performed between an azide and an alkyne to form a triazole, wherein the azide or alkyne can be on an anti-TfR 1 antibody, molecular load, or linker, as described in: international patent application publication WO2014065661, published on 5 months 1 of 2014, entitled "Modified antibody, anti-conjugate and process for the preparation thereof"; or International patent application publication WO2016170186, published 10/27/2016, entitled "Process For The Modification Of A Glycoprotein Using AGlycosyltransferase 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 load 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 load, or the linker. In some embodiments, the linker is covalently linked to the anti-TfR 1 antibody and/or (e.g., and) the molecular load 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 load 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 load 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 load.

In some embodiments, the linker is covalently attached to the anti-TfR 1 antibody and/or (e.g., and) the molecular cargo 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, a nucleophile may be present on the linker and an electrophile may be present on the anti-TfR 1 antibody or molecular load prior to performing a reaction between the linker and the anti-TfR 1 antibody or molecular load. In some embodiments, before the reaction between the linker and the anti-TfR 1 antibody or molecular load is performed, an electrophile may be present on the linker and a nucleophile may be present on the anti-TfR 1 antibody or molecular load. 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, the linker comprising the structure of formula (a) is covalently attached (e.g., optionally via an additional chemical moiety) to the molecular cargo (e.g., an 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) to produce 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 compound of formula (B) is also covalently linked to an additional moiety through a triazole, wherein the triazole is formed by a click reaction between an azide of formula (a) or formula (B) and an alkyne provided on the bicyclononene. 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, the azide of the compound of structure (B) forms a triazole by a click reaction with the alkyne of the compound of structure (C) to form 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 compound of structure (D) is also covalently linked to a lysine of an anti-TfR 1 antibody to form 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 compound of formula (C) is also covalently linked to a lysine of an anti-TfR 1 antibody to form 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) to form a complex comprising the structure of formula (E):

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) to form 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 load (e.g., oligonucleotide) via a linker comprising the structure of formula (H) through 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 load (e.g., oligonucleotide) via a linker comprising the structure of formula (I) through 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-,-NRAC(＝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 labeling the site of covalent attachment (directly or through another chemical moiety) to the oligonucleotide.

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, 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 molecular loading, 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 loading.

D. Some examples of antibody-molecule loading 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 molecular load (e.g., an oligonucleotide) 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 load (e.g., an oligonucleotide, such as an oligonucleotide provided in table 8 or 9) through a linker. Any of the linkers described herein may be used. In some embodiments, if the molecular charge is an oligonucleotide, the linker is attached to the 5 'end of the oligonucleotide, the 3' end of the oligonucleotide, or an internal site of the oligonucleotide. 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

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

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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

Another example of the structure of a complex comprising an anti-TfR 1 antibody covalently linked to a molecular load 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 oligonucleotide (e.g., at the 5 'terminus, the 3' terminus, or internally). 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9). 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.

It is understood that antibodies can be linked to molecular loads 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 load. In some embodiments, one molecular load is linked to one antibody (dar=1). In some embodiments, two molecular loads are linked to one antibody (dar=2). In some embodiments, three molecular loads are linked to one antibody (dar=3). In some embodiments, four molecular loads 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 a molecular load to different sites on an antibody and/or (e.g., and) by conjugating a multimer to one or more sites on an antibody. For example, DAR of 2 can be achieved by conjugating a single molecular load to two different sites on an antibody or by conjugating a dimeric molecular load 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 load. 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 load 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 listed in table 2. In some embodiments, the molecular cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to a molecular load, 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 light chain comprising the amino acid sequence of SEQ ID No. 85. In some embodiments, the molecular cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In some embodiments, the complexes described herein comprise an anti-TfR 1 antibody covalently linked to a molecular load, 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 cargo is a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide listed in table 8 or 9).

In any of the exemplary complexes described herein, in some embodiments, the anti-TfR 1 antibody is covalently linked to the molecular load 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 5' end of a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide set forth in table 8 or 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 generated from a reaction with an amine (e.g., lysine epsilon amine) of the anti-TfR 1 antibody.

In some embodiments, a complex described herein comprises an anti-TfR 1 antibody covalently linked to the 5' end of a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide set forth in table 8 or 9) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises a VH and a 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 5' end of a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide set forth in table 8 or 9) through a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 antibody comprises a heavy chain and a light chain of any one of the antibodies set forth in table 4, wherein the complex has the structure of formula (E):

In some embodiments, the complexes described herein comprise an anti-TfR 1 Fab covalently linked to the 5' end of a DUX4 targeting oligonucleotide (e.g., a DUX4 targeting oligonucleotide set forth in table 8 or table 9) via a lysine in the anti-TfR 1 antibody, wherein the anti-TfR 1 Fab comprises

The heavy and light chains of any one of the antibodies listed in table 5, wherein the complex has the structure of formula (E):

In some embodiments, in any of the examples of complexes described herein, 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-,-NRAS(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 formula (E); and b labeling the site of covalent attachment (directly or through another chemical moiety) to the oligonucleotide.

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, L1 is optional (e.g., not necessarily 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 such that when administered to a subject, whether administered in the immediate environment of the target cell or systemically, a sufficient amount of the complex enters the target cell (e.g., a muscle cell or CNS cell). 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 loads, 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 formulation as disclosed herein comprises 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 in the composition comprises isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride. 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 cargo 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, 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 deletion mutation in the D4Z4 repeat region on chromosome 4.

One aspect of the present disclosure includes a method involving administering an effective amount of a complex as 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 site-specific or local delivery techniques. Some examples of these techniques include implantable reservoir sources of the complex, local delivery catheters, site-specific carriers, direct injection, or direct application.

In some embodiments, a pharmaceutical composition comprising a complex comprising a muscle targeting agent covalently linked to a molecular cargo is administered at an effective concentration to confer 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.

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 the muscles of the face, shoulder and upper arms.

In some embodiments, a pharmaceutical composition comprising a complex described herein comprising a muscle targeting agent covalently linked to a molecular load 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., a baseline level of gene expression prior to treatment).

Examples

Example 1 action of conjugate comprising anti-TfR Fab conjugated to DUX4 targeting oligonucleotide in immortalized myoblasts derived from FSHD patients

Anti-TfR Fab 3M12 VH4/VK3 was conjugated to DUX4 targeting oligonucleotide (SEQ ID NO: 151) via cleavable Val-Cit linker to achieve muscle delivery of the enhancing oligonucleotide. The oligonucleotides are PMOs and target polyadenylation signals of DUX4 transcripts. The activity of the conjugates was evaluated in a C6 (AB 1080) immortalized FSHD1 cell line with significant levels of surface TfR1 expression and activation of DUX4 transcriptome markers (MBD 3L2, TRIM43, ZSCAN 4). It was shown that at a PMO concentration of 8nM, delivery of PMO mediated by anti-TfR Fab receptor (SEQ ID NO: 151) into muscle cells resulted in approximately 75% reduction of DUX4 transcriptome biomarker, whereas equivalent unconjugated PMO did not show significant biomarker reduction compared to vehicle treated cells (fig. 1). The results indicate that conjugation to anti-TfR Fab enhances delivery of therapeutic oligonucleotides to muscle cells for use in the treatment of FSHD.

The term "unconjugated" as used in this example means that the oligonucleotide was not conjugated to an antibody.

In addition, the dose response curves for MBD3L2 mRNA reduction are shown in fig. 2A. The half maximal concentration (IC 50) value required for inhibition of the conjugate was 189pM. Dose response curves for MBD3L2, TRIM43, and ZSCAN4 mRNA reduction are shown in fig. 2B. The IC50 values for the conjugates for inhibition of MBD3L2, TRIM43 and ZCAN 4 were 200pM, 50pM and 200pM, respectively.

Experimental procedure of example 1

Cell culture and sample treatment

C6 (AB 1080) immortalized FSHD myoblasts were inoculated at a density of 45,000 cells/well into bone growth medium (CAT#C-23060, promocell) with a supplemented mixture (C-39365, promocell) and 1% Penstrep (15140-122, gibco) on 96-well plates (ThermoFisher Scientific). After 24 hours, the growth medium was replaced with differentiation medium: nbActiv4 (Brainbits) and 1% Pen/Strep (Gibco). Cells were treated repeatedly with unconjugated DUX4 targeting oligonucleotide, conjugate at PMO concentration of 8nM, or vehicle for 4 hours with technique, then rinsed with 1 x PBS (10010023, gibco). Immediately after the conditioned differentiation medium was added back to the wells, cells were harvested after 5 days for downstream analysis.

Dose response curves for MBD3L2, TRIM43 and ZSCAN4 knockdown, C6 (AB 1080) immortalized FSHD myoblasts as described above but with different concentrations of conjugate.

RNA extraction and qPCR

Total RNA was extracted from cell monolayers using the RNeasy 96 kit (Qiagen) according to the manufacturer's instructions. RNA was quantified using a Biotek plate reader and diluted to 50 ng/sample with nuclease free water (Qiagen) and reverse transcribed with qScript cDNASuperMix (QuantaBio). Gene expression was analyzed by qPCR with a specific TaqMan assay (ThermoFisher) by measuring the levels of transcripts of TRIM43 (Hs 00299174 _m1), MBD3L2 (Hs 00544743 _m1), ZSCAN4 (Hs 00537549 _m1) and RPL13A (Hs 04194366 _g1). Two-step amplification reactions and fluorescence measurements for Ct determination were performed on QuantStudio instrument (Thermo Scientific). Log fold changes in expression of the transcripts of interest were calculated according to the 2- ^ΔΔCT method using RPL13A as reference gene and vehicle-exposed cells as control. Data are expressed as mean ± s.d.

Example 2 pharmacokinetic Properties of antibody-oligonucleotide conjugates in non-human primate

DUX4 targeting oligonucleotide (SEQ ID No. 151), either naked or conjugated to anti-TfR 1 antibody (3 m12 VH4/Vk3 Fab), was administered intravenously to non-human primates. The naked oligonucleotide was administered at a dose of 30mg/kg, while the conjugate was administered at a dose of 3mg/kg, 10mg/kg or 30mg/kg oligonucleotide equivalents. The plasma levels of the oligonucleotides measured over time are shown in figure 3. The results indicate that systemic exposure of the antibody-oligonucleotide conjugate shows dose-dependent pharmacokinetic properties and achieves higher exposure relative to the bare oligonucleotide. Plasma measurements also indicated that the antibody-oligonucleotide conjugate had a long serum half-life of about 60 hours. Furthermore, at an oligonucleotide equivalent dose of 30mg/kg, the antibody-oligonucleotide conjugate showed a 58-fold increase in area under the curve (area under the curve, AUC) and a 3-fold increase in C _max compared to the naked oligonucleotide. These results are summarized in table 16.

TABLE 16 pharmacokinetic values calculated from plasma concentration measurements

Two weeks after administration of the oligonucleotide or antibody-oligonucleotide conjugate, necropsy was performed and muscle tissue from non-human primates was collected and the oligonucleotide level was measured. In each of the muscle tissues tested (heart, orbicularis (orbicularius oris), zygomatic large (zygomaticus major), diaphragm, trapezius, deltoid, gastrocnemius, biceps, quadriceps, and tibialis anterior), each dose (3, 10, or 30mg/kg oligonucleotide equivalent) of antibody-oligonucleotide conjugate had higher tissue oligonucleotide levels than the naked oligonucleotide (30 mg/kg) (fig. 4). As a control, tissue oligonucleotide levels were also measured in tissues collected from vehicle treated animals, and no oligonucleotides were detected in any of the muscle tissues tested. These results indicate that the antibody-oligonucleotide conjugate achieves high DUX4 targeted oligonucleotide exposure to muscle tissue and is significantly higher than the naked oligonucleotide administered. The concentration of oligonucleotides in each muscle tested was 26 to 139 times higher in animals treated with antibody-oligonucleotide conjugates relative to bare oligonucleotides at an oligonucleotide equivalent dose of 30 mg/kg.

To evaluate tissue accumulation of DUX4 targeting oligonucleotides over time, tissue oligonucleotide levels were measured in gastrocnemius biopsy samples collected one week after administration and compared to values measured in necropsy samples collected two weeks after administration. The level of oligonucleotides in the gastrocnemius biopsy samples collected from animals administered 3, 10 or 30mg/kg of oligonucleotide equivalent of antibody-oligonucleotide conjugate was significantly higher than in the biopsy samples collected from animals administered 30mg/kg of naked oligonucleotide and even higher in tissues collected two weeks after administration (fig. 5). No oligonucleotides were detected in tissue samples from vehicle treated animals. These results indicate that the antibody-oligonucleotide conjugate achieves high DUX4 targeted oligonucleotide exposure to muscle tissue when compared to bare oligonucleotides, and that the conjugate accumulates continuously over time.

Example 3 action of conjugate comprising anti-TfR Fab conjugated to DUX4 targeting oligonucleotide in immortalized myoblasts derived from FSHD patients

Anti-TfR Fab 3m12 VH4/VK3 was conjugated to DUX4 targeting oligonucleotides (oligonucleotides #8, #1 or #2 as listed in table 8, corresponding to SEQ ID NOs: 176, 169, 170, respectively) via cleavable Val-Cit linkers to achieve muscle delivery of the enhancing oligonucleotides. Control conjugates were also generated by conjugating anti-TfR Fab 3M12 VH4/VK3 via the same cleavable Val-Cit linker to a control DUX4 targeting oligonucleotide (SEQ ID NO: 151). The activity of the conjugates was evaluated in a C6 (AB 1080) immortalized FSHD1 cell line with significant levels of surface TfR1 expression and activation of DUX4 transcriptome markers (MBD 3L2, TRIM43, ZSCAN 4).

C6 (AB 1080) immortalized FSHD myoblasts were inoculated at a density of 410,000 cells/well into bone growth medium (CAT#C-23060, promocell) with a supplemented mixture (C-39365, promocell) and 1% Penstrep (15140-122, gibco) on 384-well plates (ThermoFisher Scientific). After 24 hours, the growth medium was replaced with differentiation medium: nbActiv4 (Brainbits) and 1% Pen/Strep (Gibco). Cells were treated with conjugates at concentrations equivalent to 10pM, 1nM or 100nM oligonucleotides for 10 days and harvested afterwards for downstream analysis.

As shown in fig. 6, inclusion of the anti-TfR Fab 3m12 VH4/Vk3 conjugate conjugated to the DUX4 targeting oligonucleotide (# 8, #1 or #2 in table 8, corresponding to SEQ ID NOs: 176, 169, 170, respectively), and the control conjugate reduced the expression level of the DUX4 transcriptome marker in FSHD patient cells. These results indicate that the conjugates reduced DUX4 expression levels in FSHD patient cells in vitro.

Other embodiments

1. A complex comprising an anti-transferrin receptor 1 (TfR 1) antibody covalently linked to an oligonucleotide configured for reducing expression or activity of DUX4, 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 one of the anti-TfR 1 antibodies listed in tables 2 through 7, and wherein the oligonucleotide comprises an antisense strand comprising a complementary region of a DUX4 sequence shown in SEQ ID No. 160 or SEQ ID No. 365.

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

3. The complex of any of embodiment 1 or embodiment 2, wherein the anti-TfR 1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 95% identity to SEQ ID NO. 76 and/or a light chain variable region (VL) comprising an amino acid sequence having at least 95% identity to SEQ ID NO. 75,

Optionally, 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.

4. The complex of embodiment 1 or embodiment 2, wherein the anti-TfR 1 antibody is a Fab, optionally wherein the Fab comprises the heavy and light chains of any one of the anti-TfR 1 fabs listed in table 5.

5. The complex of embodiment 4, wherein the Fab comprises a heavy chain comprising an amino acid sequence with at least 85% identity to SEQ ID NO. 101 and/or a light chain comprising an amino acid sequence with at least 85% identity to SEQ ID NO. 90,

Optionally, wherein the 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.

6. The complex of any one of embodiments 1 to 5, wherein the oligonucleotide is 20 to 30 nucleotides in length.

7. The complex of any one of embodiments 1 to 6, wherein the oligonucleotide comprises a complementary region of at least 15 consecutive nucleotides of the DUX4 sequence shown in SEQ ID No. 160 or SEQ ID No. 365.

8. The complex of any one of embodiments 1 to 7, wherein the oligonucleotide comprises a complementary region of at least 15 consecutive nucleotides of the DUX4 sequence as set forth in any one of SEQ ID NOs 161 to 168 or 213 to 288.

9. The complex of any one of embodiments 1 to 8, wherein the oligonucleotide comprises at least 15 consecutive nucleotides of any one of SEQ ID NOs 169 to 176 or 289 to 364, wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by T.

10. The complex of any one of embodiments 1 to 9, wherein the oligonucleotide does not comprise the nucleotide sequence of SEQ ID NO. 151.

11. The complex of any one of embodiments 1 to 9, wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs 169 to 176 or 289 to 364.

12. The complex of any one of embodiments 1 to 11, wherein the oligonucleotide further comprises a sense strand that hybridizes to an antisense strand to form a double stranded siRNA.

13. The complex of any one of embodiments 1 to 12, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

14. The complex of any one of embodiments 1 to 13, wherein the oligonucleotide comprises one or more modified nucleosides, optionally wherein the one or more modified nucleosides is a 2' -modified nucleoside.

15. The complex of any one of embodiments 1 to 12, wherein the oligonucleotide is a diamide morpholino phosphate oligomer (PMO).

16. The complex of any one of embodiments 1 to 15, wherein the antibody is covalently linked to the oligonucleotide through a linker.

17. The complex of claim 16, wherein the linker is a cleavable linker, optionally wherein the linker comprises a valine-citrulline sequence.

18. 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 embodiments 1 to 17 for promoting internalization of the oligonucleotide into the muscle cell.

19. The method of embodiment 18, wherein the cell is in vitro.

20. The method of embodiment 18, wherein the cell is in a subject.

21. The method of embodiment 20, wherein the subject is a human.

22. 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 1-17, wherein the subject has abnormal production of DUX4 protein.

23. The method of any one of embodiments 20 to 22, wherein the subject has one or more deletions of a D4Z4 repeat in chromosome 4.

24. The method of embodiment 23, wherein the subject has 10 or fewer D4Z4 repeats.

25. The method of embodiment 24, wherein the subject has 9, 8, 7, 6, 5, 4, 3, 2, or 1D 4Z4 repeats.

26. The method of any one of embodiments 20 to 22, wherein the subject does not have a D4Z4 repeat.

27. An oligonucleotide comprising the nucleotide sequence of any one of SEQ ID NOs 169 to 176 or 289 to 364, optionally wherein said oligonucleotide is a diamide morpholino oligomer of Phosphate (PMO).

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 (e.g., RNA counterparts of DNA nucleotides or DNA counterparts of RNA nucleotides) and/or (e.g., and) one or more modified nucleotides and/or (e.g., and) one or more modified internucleotide 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

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

3. The complex of any one of claim 1 or claim 2, wherein the anti-TfR 1 antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence having at least 95% identity to SEQ ID NO. 76 and/or a light chain variable region (VL) comprising an amino acid sequence having at least 95% identity to SEQ ID NO. 75,

4. The complex of claim 1 or claim 2, wherein the anti-TfR 1 antibody is a Fab, optionally wherein the Fab comprises the heavy and light chains of any one of the anti-TfR 1 fabs listed in table 5.

5. The complex of claim 4, wherein the Fab comprises a heavy chain comprising an amino acid sequence with at least 85% identity to SEQ ID NO. 101 and/or a light chain comprising an amino acid sequence with at least 85% identity to SEQ ID NO. 90,

6. The complex of any one of claims 1 to 5, wherein the oligonucleotide is 20 to 30 nucleotides in length.

7. The complex of any one of claims 1 to 6, wherein the oligonucleotide comprises a region of complementarity of at least 15 consecutive nucleotides of the DUX4 sequence set forth in SEQ ID No. 160 or SEQ ID No. 365, optionally wherein the oligonucleotide comprises a region of complementarity of at least 15 consecutive nucleotides of the DUX4 sequence set forth in any one of SEQ ID nos. 161 to 168 or 213 to 288.

8. The complex of any one of claims 1 to 7, wherein the oligonucleotide comprises at least 15 consecutive nucleotides of any one of SEQ ID NOs 169 to 176 or 289 to 364, wherein each thymine base (T) may be independently and optionally replaced by a uracil base (U), and each U may be independently and optionally replaced by T, optionally wherein the oligonucleotide comprises the nucleotide sequence of any one of SEQ ID NOs 169 to 176 or 289 to 364.

9. The complex of any one of claims 1 to 8, wherein the oligonucleotide does not comprise the nucleotide sequence of SEQ ID NO: 151.

10. The complex of any one of claims 1 to 9, wherein the oligonucleotide further comprises a sense strand that hybridizes to the antisense strand to form a double-stranded siRNA.

11. The complex of any one of claims 1 to 10, wherein the oligonucleotide comprises at least one modified internucleoside linkage.

12. The complex of any one of claims 1 to 11, wherein the oligonucleotide comprises one or more modified nucleosides, optionally wherein the one or more modified nucleosides is a 2' -modified nucleoside.

13. The complex of any one of claims 1 to 12, wherein the antibody is covalently linked to the oligonucleotide through a linker, optionally wherein the linker is a cleavable linker, further optionally wherein the linker comprises a valine-citrulline sequence.

14. 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 13 for promoting internalization of the oligonucleotide into the muscle cell.

15. The method of claim 14, wherein the cell is in vitro.

16. The method of claim 14, wherein the cell is in a subject, optionally wherein the subject is a human.

17. 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 claims 1-13, wherein the subject has abnormal production of DUX4 protein, optionally wherein the subject is a human.

18. The method of any one of claims 16 to 17, wherein the human subject has one or more deletions of the D4Z4 repeat in chromosome 4.

19. The method of claim 18, wherein the subject has 10, 9, 8, 7, 6, 5, 4, 3, 2, 1D 4Z4 repeat or no D4Z4 repeat.

20. An oligonucleotide comprising the nucleotide sequence of any one of SEQ ID NOs 169 to 176 or 289 to 364.